METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
 Autoimmunity

Methods and Protocols

Second Edition

Edited by

Andras Perl
Departments of Medicine, Microbiology and Immunology, Biochemistry and Molecular Biology,
State University of New York, Upstate Medical University, College of Medicine, Syracuse, NY, USA
Editor
Andras Perl
Departments of Medicine
Microbiology and Immunology
Biochemistry and Molecular Biology
State University of New York
Upstate Medical University
College of Medicine
Syracuse, NY, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-60761-719-8 ISBN 978-1-60761-720-4 (eBook)
DOI 10.1007/978-1-60761-720-4
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2012943612

Springer Science+Business Media New York 2012

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for
the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.
Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the
Publishers location, in its current version, and permission for use must always be obtained from Springer. Permissions
for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution
under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of publication, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be
made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

Humana Press is a brand of Springer

Springer is part of Springer Science+Business Media (www.springer.com)
Dedication

To my parents, Ibolya and Miklos, and family, Katalin, Annmarie, Marcel, and Daniel, for
their inspiration, support, and love in my pursuit of medicine and science.
Preface

Research is to see what everybody else has seen,

and to think what nobody else has thought
Albert Szentgyrgyi

The first edition of Autoimmunity: Methods and Protocols, published in 2005, has been
initiated to provide a ready-to-use guide to establish and interrogate human and animal
models of autoimmune diseases. The second edition contains several updated chapters and
many new ones. Due to continued refinement of and widespread access to transgenic tech-
nology, perhaps hundreds of new animal models have been developed that exhibit features
of autoimmune disease. Alternatively, the increasing resolution of whole genome typing
oligonucleotide chips and full genome sequencing identified many new pathways that can
lead to autoimmunity.
The first chapter, Pathogenesis and Spectrum of Autoimmunity, discusses major hypoth-
eses driving this most tantalizing area of research since the concept of autoimmunity has
been proposed by Paul Ehrlich in 1900. Considering the great diversity and ever-changing
spectrum of autoimmunity, it has not been possible to include models and experimental
protocols for each known clinical disorder. Rather, several chapters have been devoted to
the most prevalent and complex diseases, such as systemic lupus erythematosus (SLE),
rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (IDDM), scleroderma or
progressive systemic sclerosis (PSS), and multiple sclerosis (MS). The chapters are contrib-
uted by laboratories actively using the presented models. Each chapter contains an intro-
ductory section that discusses relevance of the model for a particular disease and autoimmunity
in general.
The first set of eight chapters contains methods and protocols used to assess immuno-
logical and biochemical pathways of diseases pathogenesis in human samples. Chapters in
this section focus on methods to identify susceptibility genes, intercellular signaling via
cytokines, intracellular signaling through the T-cell receptor and signal processing via pro-
tein kinases, identification and enumeration of autoantigen-specific T cells and autoanti-
bodies, dysregulation of apoptosis and its role in modification of self antigens, and epigenetic
control of gene expression via DNA methylation. The second section, Chapters 923, con-
tains protocols to establish, to investigate, and to treat inflammatory arthritis, experimental
allergic encephalomyelitis (EAE), IDDM, scleroderma, and uveitis in animal models. The
methods focus on assessment of genetic, immunological, and biochemical parameters
underlying spontaneous or exogenous antigen-induced diseases. Although the individual
protocols focus on a specific disease, they can be adapted to investigate additional signaling
pathways or pathogenic autoantigens.
This book does not replace laboratory manuals with recipes for standard cell and molec-
ular biology and immunology techniques, such as cell culture, gene cloning, sequencing,
and amplification by polymerase chain reaction, vector design for generation of transgenic
and knockout animals, flow cytometry, fluorescence microscopy, electrophoresis, and gene

vii
viii Preface

and protein microarray and sequencing methods. Although these general methods are not
described in detail, they are appropriately referenced in each section. With both my col-
leagues in the field and newcomers in mind, step-by-step protocols and detailed trouble-
shooting guides supplement all chapters.
I am thankful to Professor John Walker for inviting me to organize the 2nd edition and
Dr. Paul Phillips for continued encouragement and support. I am grateful to the distin-
guished authors for their time, expertise, and devotion that made this book possible. If the
reader feels that a particularly relevant disease or model is missing, I should be held respon-
sible. Refining and extracting new meaning of old models and developing new ones is a
constantly ongoing process. Therefore, we invite our readers to approach the authors with
questions and comments or offer new models and protocols for a future volume of this
endeavor.

Syracuse, NY, USA Andras Perl

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Pathogenesis and Spectrum of Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . 1

Andras Perl
2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus. . . . . . . . . . . . 11
R. Hal Scofield and Kenneth M. Kaufman
3 Methods and Protocols to Study T Cell Signaling Abnormalities
in Human Systemic Lupus Erythematosus. . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Vaishali R. Moulton, Mindy S. Lo, and George C. Tsokos
4 Assessment of Mitochondrial Dysfunction in Lymphocytes
of Patients with Systemic Lupus Erythematosus. . . . . . . . . . . . . . . . . . . . . . . . 61
Andras Perl, Robert Hanczko, and Edward Doherty
5 The Role of Endocytic Recycling in Autoimmunity . . . . . . . . . . . . . . . . . . . . . 91
Tiffany Telarico and Andras Perl
6 Multiparameter Flow Cytometry and Bioanalytics for B Cell
Profiling in Systemic Lupus Erythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Denise A. Kaminski, Chungwen Wei, Alexander F. Rosenberg,
F. Eun-Hung Lee, and Ignacio Sanz
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus . . . . . . 135
Stanford L. Peng
8 Murine Models of Lupus Induced by Hypomethylated T Cells
(DNA Hypomethylation and Lupus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Bruce Richardson, Amr H. Sawalha, Donna Ray,
and Raymond Yung
9 Aspects of CNS Lupus: Mouse Models of Anti-NMDA Receptor
Antibody Mediated Reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Czeslawa Kowal and Betty Diamond
10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE . . . . . . . 207
Ramalingam Bethunaickan, Ranjit Sahu, and Anne Davidson
11 A Murine Autoimmune Model of Rheumatoid Arthritis
and Systemic Lupus Erythematosus Associated with Deregulated
Production of IL-17 and IL-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Partha S. Biswas, Kyuho Kang, Sanjay Gupta, Govind Bhagat,
and Alessandra B. Pernis

ix
x Contents

12 The Parent-into-F1 Murine Model in the Study of Lupus-Like

Autoimmunity and CD8 Cytotoxic T Lymphocyte Function . . . . . . . . . . . . . . 253
Kateryna Soloviova, Maksym Puliaiev, Anthony Foster,
and Charles S. Via
13 Genetic Approach to Study Lupus Glomerulonephritis . . . . . . . . . . . . . . . . . . 271
Yan Ge, Michael G. Brown, Hongyang Wang, and Shu Man Fu
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods . . . . . . . . 291
Patrick S.C. Leung, Guo Xiang Yang, Amy Dhirapong,
Koichi Tsuneyama, William M. Ridgway, and M. Eric Gershwin
15 Modeling Innate Immunity in Murine Skin:
Utilization of Subcutaneous Osmotic Pumps for Inflammatory
and Fibrotic Skin Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Michael Dimarzio, Giuseppina Farina, and Robert Lafyatis
16 Flow Cytometric Identification of Fibrocytes
in Scleroderma Lung Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Thomas M. Russell, Erica L. Herzog, and Richard Bucala
17 Oxidative Stress and Beta Cell Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Yama L. Lightfoot, Jing Chen, and Clayton E. Mathews
18 Experimental Autoimmune Encephalomyelitis. . . . . . . . . . . . . . . . . . . . . . . . . 363
Praveen Rao and Benjamin M. Segal
19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune
Encephalomyelitis and Theilers Virus-Induced
Demyelinating Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Derrick P. McCarthy, Maureen H. Richards,
and Stephen D. Miller
20 Pathogenesis of Multiple Sclerosis: What Can We Learn
from the Cuprizone Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Peter Acs and Bernadette Kalman
21 Assessing Inflammatory Disease at Mucosal Surfaces
in Murine Genetic Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
R.W. Engelman and William G. Kerr
22 Rodent Models of Experimental Autoimmune Uveitis . . . . . . . . . . . . . . . . . . . 443
Rajeev K. Agarwal, Phyllis B. Silver, and Rachel R. Caspi
23 Tolerance Induction via B-Cell Delivered Gene Therapy . . . . . . . . . . . . . . . . . 471
Robert J. Rossi, Belinda M. Jackson, Ai-Hong Zhang,
and David W. Scott
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Contributors

PETER ACS Department of Neurology, SUNY Upstate Medical University,

VA Medical Center, Syracuse, NY, USA
RAJEEV K. AGARWAL Laboratory of Immunology, National Eye Institute, NIH,
Bethesda, MD, USA
RAMALINGAM BETHUNAICKAN Center for Autoimmunity and Musculoskeletal Diseases,
Feinstein Institute for Medical Research, Manhasset,New York, NY, USA
GOVIND BHAGAT Department of Pathology and Cell Biology, Columbia University
Medical Center and New York Presbyterian Hospital, New York, NY, USA
PARTHA S. BISWAS Autoimmunity & Inflammation Program, Hospital for Special
Surgery, New York, NY, USA
MICHAEL G. BROWN Department of Medicine and Microbiology and Center
of Immunity, Inflammation and Regenerative Medicine, University of Virginia
School of Medicine, Charlottesville, VA, USA
RICHARD BUCALA Yale University, New Haven, CT, USA
RACHEL R. CASPI Section on Immunoregulation, Laboratory of Immunology,
National Eye Institute, Bethesda, MD, USA
JING CHEN Departments of Pathology, Immunology and Laboratory Medicine,
The University of Florida College of Medicine, Gainesville, FL, USA
ANNE DAVIDSON Center for Autoimmunity and Musculoskeletal Diseases,
Feinstein Institute for Medical Research, Manhasset, NY, USA
AMY DHIRAPONG Division of Rheumatology, Allergy and Clinical Immunology,
School of Medicine, University of California, Davis, CA, USA
BETTY DIAMOND The Center for Autoimmune and Musculoskeletal Disease,
The Feinstein Institute for Medical Research, North Shore-Long Island
Jewish Health System, Manhasset, NY, USA
MICHAEL DIMARZIO Rheumatology Section, Boston University School of Medicine,
Boston, MA, USA
EDWARD DOHERTY Departments of Medicine, Microbiology and Immunology,
State University of New York, Upstate Medical University, College of Medicine,
Syracuse, NY, USA
R.W. ENGELMAN Departments of Pathology & Cell Biology and Pediatrics,
Lee Moffitt Comprehensive Cancer Center and Research Institute,
University of South Florida, Tampa, FL, USA
GIUSEPPINA FARINA Rheumatology Section, Boston University School of Medicine,
Boston, MA, USA
ANTHONY FOSTER Department of Pathology, Uniformed Services University
of Health Sciences, Bethesda, MD, USA
SHU MAN FU Department of Medicine and Microbiology and Center of Immunity,
Inflammation and Regenerative Medicine, University of Virginia School of Medicine,
Charlottesville, VA, USA

xi
xii Contributors

YAN GE Department of Medicine and Microbiology and Center of Immunity,

Inflammation and Regenerative Medicine, University of Virginia School of Medicine,
Charlottesville, VA, USA
M. ERIC GERSHWIN Division of Rheumatology, Allergy and Clinical Immunology,
School of Medicine, University of California, Davis, CA, USA
SANJAY GUPTA Autoimmunity and Inflammation Program, Hospital for Special
Surgery, New York, NY, USA
ROBERT HANCZKO Departments of Medicine, Microbiology and Immunology,
State University of New York, Upstate Medical University, College of Medicine,
Syracuse, NY, USA
ERICA L. HERZOG Section of Pulmonary and Critical Care Medicine,
Department of Medicine, Yale University, New Haven, CT, USA
BELINDA M. JACKSON Department of Medicine, Uniformed Services University
of the Health Sciences, Bethesda, MD, USA
BERNADETTE KALMAN Department of Neurology, SUNY Upstate Medical University,
VA Medical Center, Syracuse, NY, USA
DENISE A. KAMINSKI Division of Allergy, Immunology, and Rheumatology,
Department of Medicine, University of Rochester Medical Center, Rochester,
NY, USA
KYUHO KANG Graduate Program in Immunology and Microbial Pathogenesis,
Weill Cornell Graduate School of Medical Sciences, New York, NY, USA
KENNETH M. KAUFMAN Arthritis and Clinical Immunology Program,
Oklahoma Medical Research Foundation, Oklahoma City, OK, USA;
Department of Medicine, College of Medicine, University of Oklahoma Health
Sciences Center, Oklahoma City, OK, USA; Department of Veterans Affairs
Medical Center, Oklahoma City, OK, USA
WILLIAM G. KERR Department of Microbiology & Immunology, SUNY Upstate
Medical University, Syracuse, NY, USA; Department of Pediatrics, SUNY Upstate
Medical University, Syracuse, NY, USA; Department of Chemistry,
Syracuse University, Syracuse, NY, USA
CZESLAWA KOWAL The Center for Autoimmune and Musculoskeletal Disease,
The Feinstein Institute for Medical Research, North Shore-Long Island Jewish
Health System, New York, NY, USA
ROBERT LAFYATIS Rheumatology Section, Boston University School of Medicine,
Boston, MA, USA
F. EUN-HUNG LEE Division of Allergy, Immunology, and Rheumatology,
Department of Medicine, University of Rochester Medical Center,
Rochester, NY, USA
PATRICK S.C. LEUNG Division of Rheumatology, Allergy and Clinical
Immunology, School of Medicine, University of California, Davis, CA, USA
YAIMA L. LIGHTFOOT Departments of Pathology, Immunology and Laboratory
Medicine, University of Florida College of Medicine, Gainesville, FL, USA
MINDY S. LO Division of Rheumatology, Department of Medicine, Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, MA, USA;
Division of Immunology, Department of Medicine, Childrens Hospital Boston,
Harvard Medical School, Boston, MA, USA
 Contributors xiii

CLAYTON E. MATHEWS Departments of Pathology, Immunology, and Laboratory

Medicine, The University of Florida College of Medicine, Gainesville, FL, USA
DERRICK P. MCCARTHY Department of Microbiology-Immunology
and Interdepartmental Immunobiology Center, Northwestern University
Feinberg School of Medicine, Chicago, IL, USA
STEPHEN D. MILLER Department of Microbiology-Immunology,
and Interdepartmental Immunobiology Center, Northwestern
University Feinberg Medical School, Chicago, IL, USA
VAISHALI R. MOULTON Division of Rheumatology, Department of Medicine,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
STANFORD L. PENG Rheumatology Clinical Research Unit, Benaroya Research
Institute at Virginia Mason, Seattle, WA, USA
ANDRAS PERL Departments of Medicine, Microbiology and Immunology, Biochemistry
and Molecular Biology, State University of New York, Upstate Medical University,
College of Medicine, Syracuse, NY, USA
ALESSANDRA B. PERNIS Autoimmunity & Inflammation Program,
Hospital for Special Surgery, New York, NY, USA; Graduate Program
in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School
of Medical Sciences, New York, NY, USA
MAKSYM PULIAIEV Department of Pathology, Uniformed Services University
of Health Sciences, Bethesda, MD, USA
PRAVEEN RAO Department of Neurology, University of Michigan Multiple Sclerosis
Center and Holtom-Garrett Program in Neuroimmunology, University of Michigan,
Ann Arbor, MI, USA
DONNA RAY Ann Arbor Veterans Affairs Hospital, University of Michigan,
Ann Arbor, MI, USA
MAUREEN H. RICHARDS Department of Microbiology-Immunology
and Interdepartmental Immunobiology Center, Northwestern University
Feinberg School of Medicine, Chicago, IL, USA
BRUCE RICHARDSON University of Michigan and the Ann Arbor Veterans Affairs
Hospital, Ann Arbor, MI, USA
WILLIAM M. RIDGWAY Division of Immunology, Allergy and Rheumatology,
University of Cincinnati College of Medicine, Cincinnati, OH, USA
ALEXANDER F. ROSENBERG Division of Allergy, Immunology, and Rheumatology,
Department of Medicine, University of Rochester Medical Center, Rochester,
NY, USA
ROBERT J. ROSSI Department of Medicine, Uniformed Services University
of the Health Sciences, Bethesda, MD, USA
THOMAS M. RUSSELL Section of Pulmonary and Critical Care Medicine,
Department of Medicine, Yale University, New Haven, CT, USA
RANJIT SAHU Center for Autoimmunity and Musculoskeletal Diseases,
Feinstein Institute for Medical Research, Manhasset, NY, USA
IGNACIO SANZ Division of Allergy, Immunology, and Rheumatology,
Department of Medicine, University of Rochester Medical Center,
Rochester, NY, USA
xiv Contributors

AMR H. SAWALHA University of Michigan and the Ann Arbor Veterans Affairs
Hospital, Ann Arbor, MI, USA
R. HAL SCOFIELD Arthritis and Clinical Immunology Program, Oklahoma Medical
Research Foundation, Oklahoma City, OK, USA; Department of Medicine,
College of Medicine, University of Oklahoma Health Sciences Center,
Oklahoma City, OK, USA; Department of Veterans Affairs Medical Center,
Oklahoma City, OK, USA
DAVID W. SCOTT Department of Medicine, Uniformed Services University
of the Health Sciences, Bethesda, MD, USA
BENJAMIN M. SEGAL Department of Neurology, University of Michigan
Multiple Sclerosis Center and Holtom-Garrett Program in Neuroimmunology,
University of Michigan, Ann Arbor, MI, USA
PHYLLIS B. SILVER Laboratory of Immunology, National Eye Institute, NIH,
Bethesda, MD, USA
KATERYNA SOLOVIOVA Department of Pathology, Uniformed Services University
of Health Sciences, Bethesda, MD, USA
TIFFANY TELARICO Departments of Medicine, Microbiology and Immunology,
Biochemistry and Molecular Biology, College of Medicine, State University
of New York, Upstate Medical University, Syracuse, NY, USA
GEORGE C. TSOKOS Division of Rheumatology, Department of Medicine,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
KOICHI TSUNEYAMA Department of Pathology, University of Toyama, Toyama, Japan
CHARLES S. VIA Department of Pathology, Uniformed Services University of Health
Sciences, Bethesda, MD, USA
HONGYANG WANG Department of Medicine and Microbiology and Center
of Immunity, Inflammation and Regenerative Medicine, University of Virginia
School of Medicine, Charlottesville, VA, USA
CHUNGWEN WEI Division of Allergy, Immunology, and Rheumatology,
Department of Medicine, University of Rochester Medical Center, Rochester,
NY, USA
GUO XIANG YANG Division of Rheumatology, Allergy and Clinical Immunology,
School of Medicine, University of California, Davis, CA, USA
RAYMOND YUNG Ann Arbor Veterans Affairs Hospital, University of Michigan,
Ann Arbor, MI, USA
AI-HONG ZHANG Department of Medicine, Uniformed Services University
of the Health Sciences, Baltimore, MD, USA
 Chapter 1

Pathogenesis and Spectrum of Autoimmunity

Andras Perl

Abstract
The immune system specifically recognizes and eliminates foreign antigens and, thus, protects integrity of
the host. During maturation of the immune system, tolerance mechanisms develop that prevent or inhibit
potentially harmful reactivities to self-antigens. Autoreactive B and T cells that are generated during
immune responses are eliminated by apoptosis in the thymus, lymph nodes, or peripheral circulation or
actively suppressed by regulatory T cells. However, autoreactive cells may survive due to failure of apopto-
sis or molecular mimicry, i.e., presentation and recognition of cryptic epitopes of self-antigens, or aberrant
lymphokine production. Preservation of the host requires the development of immune responses to for-
eign antigen and tolerance to self-antigens. Autoimmunity results from a breakdown of tolerance to self-
antigens through an interplay of genetic and environmental factors.
One of the basic functions of the immune system is to specifically recognize and eliminate foreign
antigens and, thus, protect integrity of the host. Through rearrangements and somatic mutations of vari-
ous gene segments encoding T and B cell receptors and antibody molecules, the immune system acquires
tremendous diversity. During maturation of the immune system, recognition of self-antigens plays an
important role in shaping the repertoires of immune receptors. Tolerance mechanisms develop that pre-
vent or inhibit potentially harmful reactivities to self-antigens. These self-defense mechanisms are mediated
on the levels of central and peripheral tolerance, i.e., autoreactive T cells are either eliminated by apoptosis
in the thymus, lymph nodes, or peripheral circulation or actively suppressed by regulatory T cells. Likewise,
autoreactive B cells are eliminated in the bone marrow or peripheral lymphoid organs. However, immune
responses triggered by foreign antigens may be sustained by molecular mimicry, i.e., presentation and
recognition of cryptic epitopes of self-antigens. Further downstream, execution of immune responses
depends on the functioning of intracellular signaling networks and the cooperation of many cell types com-
municating via surface receptors, cytokines, chemokines, and antibody molecules. Therefore, autoimmu-
nity represents the end result of the breakdown of one or multiple basic mechanisms of immune tolerance
(Table 1).

Key words: Autoimmunity, Autoimmune diseases, Genetics, Environment, Tolerance, Autoantigens,

Biomarkers, Treatment

Autoimmunity may occur in normal individuals with a higher

frequency in older people. Infectious diseases often elicit
autoreactivities based on similarity between exogenous and self-
antigens. Infection-induced autoimmunity is usually self-limited by

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_1, Springer Science+Business Media New York 2012

1
 2
A. Perl

Table 1
Exogenous and endogenous factors involved in autoimmunity

Exogenous Agent Example Disease Mechanism Reference

Virus Coxsackie Diabetes Mimicry (39)
Bacterium Klebsiella Reactive arthritis Mimicry (40)
Drug, chemical, ultraviolet light 5-Azacytidine Lupus Demethylation (41) (42)

Endogenous System Gene Disease Mechanism Reference

MHC locus MHC class II Thyroiditis Antigen presentation (11, 43)
Complement Lupus Immune complex disposal (10)
Hormone Estrogen Lupus Gene expression (44)
Cytokine IL-10 Lupus B and T cell dysfunction (28, 45, 46)
Transcription factor NF-B Lupus T cell dysfunction (47)
Transcription factor FoxP3 IPEX Treg (4)
Dysregulated apoptosis Fas mutation ALPS Defective apoptosis (23, 24)
Dysregulated apoptosis Caspase 10 mutation ALPS Defective apoptosis (25)
 1 Pathogenesis and Spectrum of Autoimmunity 3

elimination of the antigen-producing cells or organisms.

However, self-reactivity may be sustained through molecular
mimicry (1), i.e., homology between exogenous and endogenous
epitopes, and the inability of the immune system to destroy self-
reactive B or T cells via apoptosis (2), anergy (3), or active regula-
tory mechanisms (4). Nevertheless, autoimmunity does not
necessarily lead to tissue injury. Autoantibodies, such as rheuma-
toid factor or antinuclear antibodies, occur in >5 % of normal sub-
jects, and may have protective roles (5), without ever resulting in
rheumatoid arthritis (RA) (6) or systemic lupus erythematosus
(SLE) characterized by the very presence of such antibody reactivi-
ties within the context of clinical phenotypes (7) (Table 1).
Autoimmunity can damage nearly every tissue or cell type of
the body. The spectrum, severity, and duration of disease vary
widely. Depending on the organ systems involved, systemic and
organ-specific autoimmune diseases have been delineated. Systemic
autoimmune diseases include SLE, RA, scleroderma, granuloma-
tosis with polyangiitis (GPA; formerly Wegeners granulomatosis),
Goodpasture syndrome, Sjogrens syndrome, dermatomyositis,
psoriasis, ankylosing spondylitis, and inflammatory bowel diseases
(Table 2). While SLE can involve almost any tissue of the body,

Table 2
Systemic of autoimmune diseases

Disease Organ system involvement and immunopathology

SLE
RA
Ankylosing spondylitis
Scleroderma
Psoriasis
Sjogren syndrome
Dermatomyositis
Inflammatory bowel disease
Granulomatosis with
polyangiitis
Goodpasture syndrome
Periarteritis nodosa
All, primarily joints, skin, blood vessels, serous membranes, kidney,
lung, heart; antinuclear antibodies
Joints, blood vessels, serous membranes, lung; anti-IgG IgM
rheumatoid factor
Axial > peripheral joints, uveitis, aortitis
Skin, blood vessels, gut, lung, heart, kidney
Skin, joints
Salivary and lacrimal glands, pancreas, lung, kidney; antibodies SSA
and SSB, lymphocytic infiltration of involved tissues
Skin, muscle, blood vessels
Small and/or large intestine, joint, uvea; perinuclear anti-neutrophil
cytoplasmic antibodies directed to myeloperoxidase
Blood vessel inflammation in kidney, lung, skin; cytoplasmic
anti-neutrophil cytoplasmic antibodies directed to proteinase 3
Kidney, lung, antibody to basement membrane
Blood vessel inflammation in all tissues (typically kidney, skin,
intestines) sparing lung
4 A. Perl

Table 3
Organ-specific autoimmune diseases

Disease Typical involvement and immunopathology

Insulin-dependent diabetes mellitus Pancreas, anti-insulin and anti-glutamic acid
decarboxylase antibodies
Multiple sclerosis Central nervous system, anti-myelin T cell and antibody
reactivities
Myasthenia gravis Peripheral nervous system, antibody to acetylcholine
receptor
Thyroiditis Thyroid gland, anti-thyroid antibodies
Uveitis Uvea, antibody, and T cell mediated
Pernicious anemia Stomach, antibody to intrinsic factor required for
absorption of vitamin B12
Pemphigus Skin, antibody to intercellular adhesion molecule
desmoglein-3
Pemphigoid Skin
Vitiligo Skin
Myocarditis Heart
Autoimmune hemolytic anemia Erythrocytes; antibody mediated
Autoimmune thrombocytopenia Platelets; antibody mediated
Acquired thrombotic thrombopenic Antibody to von Willebrand factor-cleaving
purpura metalloprotease
Autoimmune hearing loss Inner ear; cochlin-specific T cell-mediated disease
Primary biliary cirrhosis Liver; antibody mediated targeting pyruvate
dehydrogenase
Autoimmune hepatitis Liver; antibody mediated targeting cytochrome p450

inflammatory bowel diseases extend to fewer tissues, the gut, the joints,
and the eye. Organ-specific diseases include insulin-dependent dia-
betes mellitus, multiple sclerosis, uveitis, thyroiditis, pernicious
anemia, autoimmune hemolytic anemia, thrombocytopenia, hepa-
titis, primary biliary cirrhosis, pemphigus, pemphigoid, and vitiligo
(Table 3). Individual patients may have more than one autoimmune
disorder, concurrently, and subsequently.
While the causes of autoimmune diseases have not been clearly
defined, independent lines of evidence have implicated environ-
mental factors and genetic determinants of the host (8). As shown
in Table 1, polymorphisms of HLA molecules that regulate antigen
presentation (9), complement deficiency states (10), inactivation
of the transcription factor FoxP3 (4) have been identified as
inherited factors influencing disease susceptibility. Concordance
 1 Pathogenesis and Spectrum of Autoimmunity 5

rates for autoimmune diseases, such as SLE, IDDM, RA, and MS,
are approximately 25 % in monozygotic twins. The latest approaches
and successes to map lupus susceptibility genes are described in
Chapter 2. Alternatively, the discordance rate may be as high as
70 % among monozygotic twins (11), suggesting a significant role
for exogenous agents.
The concept of autoimmunity, designated as horror autotoxi-
cus, was first proposed by Paul Ehrlich in 1900 (12). The clonal
selection theory and specific elimination of self-reactive forbid-
den clones, as means of preventing autoimmunity, were hypoth-
esized by McFarlane Burnet (12). The first organ-specific
autoimmune disease, experimental thyroiditis, was described by
Noel Rose and Ernest Witebsky in 1956 (13). Rose and Vladutiu
recognized the influence of the major histocompatibility (MHC)
gene locus on development of autoimmunity. Similar to the
consequences of microbial infections, autoimmune diseases are
characterized by polyclonal T cell expansions and antibody pro-
duction, suggesting antigen-driven process (1, 14). During the
past century, tremendous efforts have been made to identify self-
antigens and infectious agents capable of inducing autoimmunity
in humans and animal models. Such studies led to the discovery of
disease-specific autoantigens that have become instrumental in
clinical diagnosis (15). As examples, antinuclear antibodies, rheu-
matoid factor, cyclic citrullinated peptide antibodies, proteinase
3-specific cytoplasmic anti-neutrophil cytoplasmic antibodies
(cANCAs), anti-glomerular basement membrane antibodies, thy-
roid-stimulating hormone receptor, thyroid peroxidase, and thyro-
globulin antibodies are routinely used to establish diagnosis of
SLE, RA, GPA, Goodpasture syndrome, Graves disease, and
Hashimotos thyroiditis, respectively. Various antigens from tissues
targeted by organ-specific autoreactivities have been used to gener-
ate useful animal models. Joint cartilage-derived antigens, such as
collagen (16) and proteoglycan (17), can induce inflammatory
arthritis in mice. Myelin-derived antigens, myelin basic protein,
myelin oligodendrocyte glycoprotein, proteolipid protein (Chapters
18 and 19), as well as mimicking viral antigens can trigger enceph-
alomyelitis resembling MS (Chapter 19). Certain animal strains
spontaneously develop insulin-dependent diabetes mellitus
(IDDM) characterized by autoreactivities to pancreas-derived
antigens, such as insulin (Chapter 17) or glutamic acid
decarboxylase (18). These animal models provide new information
on pathogenesis of autoimmunity: identify and confirm relevant
autoantigens, and delineate critical checkpoints of signaling net-
works that can be targeted for therapeutic interventions. As an
example, tumor necrosis factor (TNF) antagonists reduced
severity of collagen-induced arthritis in animal models and became
a major breakthrough in treatment of RA (19). Along the same
line, TNF has been shown to protect against lupus (20) and
6 A. Perl

experimental allergic encephalomyelitis (EAE) in animal models

(21, 22). Such clinical and experimental observations indicate
significant differences in pathogenesis between autoimmune dis-
eases. TNF triggers programmed cell death; therefore its block-
ade is thought to hinder elimination of autoreactive cells via this
mechanism. Defects in apoptosis underlie the pathology of human
autoimmune lymphoproliferative syndromes (2325) and animal
models of SLE (26) (Table 1). Methods to assess apoptosis are
described in Chapter 4. Development of autoimmunity is also
influenced by the cytokine milieu (19). While MS patients over-
produce Th1 type cytokines (27), lupus is characterized by pre-
dominance of Th2 cytokines, such as IL-10 (28). IL-17 and IL-21
play important roles both in RA and SLE (Chapter 11). Signaling
through the T cell receptor, adaptor molecules, and kinases
(Chapters 35) and activation of B cells are distinctly altered in
patients with SLE (Chapter 6). Chapters 713 present murine
models of SLE, the prototypic multisystem autoimmune disease.
Chapter 7 reviews spontaneous and chemically inducible animal
models of SLE. Chapter 8 focuses on demethylation-induced auto-
immunity, which accounts for the pathogenicity of SLE detectable
in a significant subset of patients treated with the antihypertensive
hydralazine and anti-arrhythmia medication procainamide.
Chapter 9 presents a novel model of central nervous system (CNS)
lupus triggered by anti-DNA antibodies cross-reactive with the
N-methyl-D-aspartate receptor. Chapters 10 and 13 focus on the
pathogenesis of often fatal kidney manifestations of SLE, and the
role of intra-renal macrophages and genetic factors underlying
acute and chronic forms of inflammation in the kidneys, respec-
tively. Chapter 12 presents graft-versus-host disease models of
lupus, which mimic the auto-antibody profile and acute and chronic
forms of glomerulonephritis noted in patients with SLE. Chapter
14 focuses on autoimmune liver diseases, elaborating the role of
key xenobiotics, target antigens, and modulatory cytokines.
Chapter 15 presents a highly novel model of scleroderma, a rare
but invariably therapy-resistant autoimmune disease, utilizing
osmotic pumps to induce the innate immune system. Chapter 16
provides flow cytometry methods to identify fibrocytes, effector
cells of pulmonary inflammation; fibrosis in systemic sclerosis; and
possibly other autoimmune diseases. Chapter 17 provides method-
ologies to investigate oxidative stress in the pancreas, the target
organ of autoimmune diabetes. Chapters 1820 describe murine
models of multiple sclerosis, focusing on myelin antigen-specific
CD4 T cell-mediated oligodendrocyte damage (Chapter 18),
Theilers murine encephalitis virus-induced demyelinating disease
(Chapter 19), and the pro-oxidant chemical cuprizone-induced
oligodendrocyte damage (Chapter 20). Chapter 21 presents a
novel model of inflammatory bowel disease caused by the deletion
Scr homology domain 2-containing inositol-5-phosphatase.
 1 Pathogenesis and Spectrum of Autoimmunity 7

Chapter 22 provides an authoritative review of rodent models of

the often devastating and therapy-resistant eye disease, autoim-
mune uveitis. Chapter 23 provides a highly innovative B cell-medi-
ated retroviral vaccination approach for the treatment on
mono-antigenic autoimmunity. Although these chapters only cover
a limited number of diseases and experimental models, each pres-
ents a state-of-the-art approach to autoimmunity research.
Genome-wide genetic association studies invariably confirmed
importance of the MHC locus in all autoimmune diseases and
identified additional genomic loci that influence susceptibility to
SLE (Chapter 1), MS (29), and IDDM (30). Gene expression
profiling has emerged as a key tool for pathway analysis and bio-
marker discovery, with promising relevance for clinical diagnosis.
Microarray analyses have revealed an upregulation of interferon
-inducible genes (31), suggestive of infectious etiology in SLE (32).
Ongoing clinical trials of mTOR blockade through reversing gluta-
thione depletion with N-acetylcysteine (33) or treatment with
rapamycin improved disease outcomes in patients with SLE (34) as
well as in animal models of lupus (35, 36) and multiple sclerosis
(37, 38). Coordinated efforts aimed at delineating the triggering
antigens, underlying environmental and genetic factors and signal-
ing networks, are required to understand the pathogenesis, promote
the diagnosis, and develop safe and effective, i.e., biomarker-driven,
specific treatments for each autoimmune disease.

References
1. Oldstone MBA (1987) Molecular mimicry
and autoimmune disease. Cell 50:819820
2. Nagata S (2010) Apoptosis and autoimmune
diseases. Ann N Y Acad Sci 1209:1016
3. Waterman PM, Cambier JC (2010) The
conundrum of inhibitory signaling by ITAM-
containing immunoreceptors: potential molec-
ular mechanisms. FEBS Lett 584:48784882
4. Sakaguchi S (2011) Regulatory T cells: history
and perspective. Methods Mol Biol 707:317
5. Silverman GJ (2011) Regulatory natural
autoantibodies to apoptotic cells: pallbearers
and protectors. Arthritis Rheum 63:597602
6. Arnett FC, Edworthy SM, Bloch DA, McShane
DJ, Fries JF, Cooper NS, Healey LA, Kaplan
SR, Liang MH, Luthra HS (1988) The
American Rheumatism Association 1987
revised criteria for the classification of rheuma-
toid arthritis. Arthritis Rheum 31:315324
7. Tan EM, Cohen AS, Fries JF, Masi AT,
McShane DJ, Rothfield NF, Schaller JG, Talal
N, Winchester RJ (1982) The 1982 revised
criteria for the classification of systemic
lupus erythematosus. Arthritis Rheum 25:
12711277
8. Perl A (2010) Systems biology of lupus: map-
ping the impact of genomic and environmental
factors on gene expression signatures, cellular
signaling, metabolic pathways, hormonal and
cytokine imbalance, and selecting targets for
treatment. Autoimmunity 43:3247
9. Silverstein AM, Rose NR (1997) On the mys-
tique of the immunological self [review] [91
refs]. Immunol Rev 159:197206, discussion
10. Agnello V (1986) Lupus diseases associated
with hereditary and acquired deficiencies of
complement. Springer Semin Immunpathol
9:183219
11. Arnett FC, Reveille JD (1992) Genetics of sys-
temic lupus erythematosus. Rheum Dis Clin
North Am 18:865892
12. Ehrlich P (1900) On immunity with special
reference to cell life. Proc R Soc Lond (Biol)
66:428448
13. Rose NR, Witebsky E (1956) Studies on organ
specificity. V. Changes in the thyroid glands of
8 A. Perl
rabbits following active immunization with rabbit
thyroid extracts. J Immunol 76:417427
14. Steinberg AD, Gourley MF, Klinman DM,
Tsokos GC, Scott DE, Krieg AM (1991)
Systemic lupus erythematosus. Ann Intern
Med 115:548559
15. Samter M, Talmage DW, Frank MM, Austen
KF, Claman HN (1988) Immunological dis-
eases. Little Brown, Boston, MA
16. Brand DD, Kang AH, Rosloniec EF (2004)
The mouse model of collagen-induced arthri-
tis. In: Perl A (ed) Autoimmunity: methods
and protocols, 102nd edn. Humana, Totowa,
NJ, pp 295312
17. Abu-Hamad S, Sivan S, Shoshan-Barmatz V
(2006) The expression level of the voltage-
dependent anion channel controls life and
death of the cell. PNAS 103:57875792
18. Liu E, Yu L, Moriyama H, Eisenbarth GS
(2004) Animal models of insulin-dependent
diabetes, 102 edn. In: Perl A (ed) Humana,
Totowa, NJ. pp 195212
19. Slavin A, Kelly-Modis L, Labadia M, Ryan K,
Brown ML (2010) Pathogenic mechanisms
and experimental models of multiple sclerosis.
Autoimmunity 43:504513
20. Mageed RA, Isenberg DA (2002) Tumour
necrosis factor alpha in systemic lupus erythe-
matosus and anti-DNA autoantibody produc-
tion [review] [45 refs]. Lupus 11:850855
21. Wang J, Asensio VC, Campbell IL (2002)
Cytokines and chemokines as mediators of pro-
tection and injury in the central nervous system
assessed in transgenic mice. [Review] [146 refs].
Curr Top Microbiol Immunol 265:2348
22. Ghezzi P, Mennini T (2001) Tumor necrosis
factor and motoneuronal degeneration:
an open problem [Review] [51 refs].
Neuroimmunomodulation 9:178182
23. Fisher GH, Rosenberg FJ, Straus SE, Dale JK,
Middleton LA, Lin AY, Strober W, Lenardo
MJ, Puck JM (1995) Dominant interfering
Fas gene mutations impair apoptosis in a
human autoimmune lymphoproliferative syn-
drome. Cell 81:935946
24. Drappa J, Vaishnaw AK, Sullivan KE, Chu J-L,
Elkon KB (1996) Fas gene mutations in the
Canale-Smith syndrome, an inherited lym-
phoproliferative disorder associated with auto-
immunity. N Engl J Med 335:16431649
25. Wang J, Zheng L, Lobito A, Chan FK, Dale J,
Sneller M, Yao X, Puck JM, Straus SE, Lenardo
MJ (1999) (1999) Inherited human Caspase
10 mutations underlie defective lymphocyte
and dendritic cell apoptosis in autoimmune
lymphoproliferative syndrome type II. Cell
98:4758
26. Cohen PL, Eisenberg RA (1991) Lpr and gld:
Single gene models of systemic autoimmunity
and lymphoproliferative disease. Annu Rev
Immunol 9:243269
27. Clerici M, Fusi ML, Caputo D, Guerini FR,
Trabattoni D, Salvaggio A, Cazzullo CL,
Arienti D, Villa ML, Urnovitz HB, Ferrante P
(1999) Immune responses to antigens of
human endogenous retroviruses in patients
with acute or stable multiple sclerosis.
J Neuroimmunol 99:173182
28. Georgescu L, Vakkalanka RK, Elkon KB, Crow
MK (1997) Interleukin-10 promotes activa-
tion-induced cell death of SLE lymphocytes
mediated by Fas ligand. J Clin Invest 100:
26222633
29. Wang JH, Pappas D, De Jager PL, Pelletier D,
de Bakker PI, Kappos L, Polman CH, Chibnik
LB, Hafler DA, Matthews PM, Hauser SL,
Baranzini SE, Oksenberg JR (2011) Modeling
the cumulative genetic risk for multiple
sclerosis from genome-wide association data.
Genome Med 3:3
30. Steck AK, Rewers MJ (2011) Genetics of type
1 diabetes. Clin Chem 57:176185
31. Crow MK, Kirou KA (2004) Interferon-alpha
in systemic lupus erythematosus. [Review] [74
refs]. Curr Opin Rheumatol 16:541547
32. Perl A (1999) Mechanisms of viral pathogen-
esis in rheumatic diseases (Invited Review).
Ann Rheum Dis 58:454461
33. Lai Z-W, Hanczko R, Bonilla E, Caza TN,
Clair B, Bartos A, Miklossy G, Jimah J, Doherty E,
Tily H, Francis L, Garcia R, Dawood M, Yu J,
Ramos I, Coman I, Faraone SV, Phillips PE,
Perl A (2012) N-acetylcysteine reduces disease
activity by blocking mTOR in T cells of lupus
patients. Arth Rheum Accepted Article,
DOI 10.1002/art.34502
34. Fernandez D, Bonilla E, Mirza N, Perl A
(2006) Rapamycin reduces disease activity and
normalizes T-cell activation-induced calcium
fluxing in patients with systemic lupus erythe-
matosus. Arthritis Rheum 54:29832988
35. Warner LM, Adams LM, Sehgal SN (1994)
Rapamycin prolongs survival and arrests
pathophysiologic changes in murine systemic
lupus erythematosus. Arthritis Rheum 37:
289297
36. Wu T, Qin X, Kurepa Z, Kumar KR, Liu K,
Kanta H, Zhou XJ, Satterthwaite AB, Davis
LS, Mohan C (2007) Shared signaling net-
works active in B cells isolated from genetically
 1 Pathogenesis and Spectrum of Autoimmunity
distinct mouse models of lupus [Article].
J Clin Invest 117:21862196
37. Esposito M, Ruffini F, Bellone M, Gagliani N,
Battaglia M, Martino G, Furlan R (2010)
Rapamycin inhibits relapsing experimental
autoimmune encephalomyelitis by both
effector and regulatory T cells modulation.
J Neuroimmunol 220:5263
38. Donia M, Mangano K, Amoroso A, Mazzarino
MC, Imbesi R, Castrogiovanni P, Coco M,
Meroni P, Nicoletti F (2009) Treatment with
rapamycin ameliorates clinical and histological
signs of protracted relapsing experimental
allergic encephalomyelitis in Dark Agouti rats
and induces expansion of peripheral
CD4 + CD25 + Foxp3+ regulatory T cells.
J Autoimmun 33:135140
39. Tian J, Lehmann PV, Kaufman DL (1994) T
cell cross-reactivity between Coxsackievirus
and glutamate decarboxylase is associated with
a murine diabetes susceptibility allele. J Exp
Med 180:19791984
40. Baum H, Davies H, Peakman M (1996)
Molecular mimicry in the MHC: hidden clues
to autoimmunity? Immunol Today 17:6470
41. Richardson BC, Strahler JR, Pivirotto TS,
Quddus J, Bayliss GE, Gross LA, ORourke KS,
Powers D, Hanash SM, Johnson MA (1992)
Phenotypic and functional similarities between
5-azacytidine-treated T cells and a T-cell subset
in patients with active systemic lupus erythema-
tosus. Arthritis Rheum 35:647662
9
42. Reap EA, Roof K, Maynor K, Borrero M,
Booker J, Cohen PL (1997) Radiation and
stress-induced apoptosis: a role for Fas/Fas
ligand interactions. Proc Natl Acad Sci U S A
94:57505755
43. Vladutiu AO, Rose NR (1971) Autoimmune
murine thyroiditis relation to histocompatibil-
ity (H-2) type. Science 174:11371139
44. Lockshin MD (2002) Sex ratio and rheumatic
disease: excerpts from an Institute of Medicine
report [Review] [30 refs]. Lupus 11:
662666
45. Llorente L, Zou W, Levy Y, Richaud-Patin Y,
Wijdenes J, Alcocer-Varela J, Morel-Fourrier
B, Brouet JC, Alarcon-Segovia D, Galanaud P
(1995) Role of interleukin 10 in the B
lymphocyte hyperactivity and autoantibody
production of human systemic lupus erythe-
matosus. J Exp Med 181:839844
46. Gergely PJ, Niland B, Gonchoroff N, Pullmann
R Jr, Phillips PE, Perl A (2002) Persistent
mitochondrial hyperpolarization, increased
reactive oxygen intermediate production, and
cytoplasmic alkalinization characterize altered
IL-10 signaling in patients with systemic lupus
erythematosus. J Immunol 169:10921101
47. Wong HK, Kammer GM, Dennis G, Tsokos
GC (1999) Abnormal NF-kappa B activity in
T lymphocytes from patients with systemic
lupus erythematosus is associated with
decreased p65-RelA protein expression.
J Immunol 163:16821689
 Chapter 2

Mapping Susceptibility Gene in Systemic Lupus

Erythematosus
R. Hal Scofield and Kenneth M. Kaufman

Abstract
Genome-wide association studies have identified many dozen genetic intervals that harbor single-nucleotide
polymorphisms (SNPs) showing statistical association with systemic lupus erythematosus. Despite the
wealth of data produced, there are limitations of these studies. The causal alleles at a given locus are not
identified; only SNP is strong linkage disequilibrium with the putative causative alleles. In order to address
identification of the causative SNPs for lupus susceptibility genes, we have initiated a candidate gene study
for which more than 40 investigators have contributed patient and control samples. In addition, these
investigators have designated SNPs to be placed on a custom array. In this way fine mapping of genetic
association findings can occur in order to identify causal alleles. These efforts have thus far benefitted
greatly from comparisons of different ethnicities. Work on about ten previously identified associations has
been published using this resource. Genome-wide association studies cannot identify rare SNPs or muta-
tions, which may impart greater relative risks than common variants. Much of the genetics of lupus may be
from rare variants or mutations. In order to approach this aspect of lupus genetics, next-generation
sequencing has begun in which all exons will be sequenced in controls and patients. This effort can also be
used to identify causal alleles from association intervals not yet otherwise identified.

Key words: Systemic lupus erythematosus, Genetic association, Single-nucleotide polymorphisms,

Next-generation sequencing

1. Introduction

Nearly 4,000 single-gene human diseases or conditions have been

described. Many of these are exceedingly rare while only a few such
as hemochromatosis, sickle cell disease, or cystic fibrosis are more
common. In contrast, most common human diseases tend to occur
within families but have no discernable pattern of inheritance
among or within families. Some examples are diseases that are
extremely common in the twenty-first century, and include hyper-
tension, coronary artery disease, and type 2 diabetes.

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_2, Springer Science+Business Media New York 2012

11
12 R.H. Scofield and K.M. Kaufman

Another example of a disease with complex inheritance,

although not as common as those diseases cited above, is systemic
lupus erythematosus (SLE). The overall estimated prevalence in
the United States is approximately 1264 cases per 100,000 indi-
viduals (1, 2). Significant gender differences are observed in preva-
lence and incidence (female:male = 9:1). In addition, there are
important racial differences in the prevalence of SLE as well disease
manifestations and severity. For example, at least two to fourfold
higher incidence in non-Caucasian as compared with Caucasian
population has been observed (3). Thus, the population risk for
SLE is about 1 in 1,000 for adult white women and about 1 in 250
for black women in the United States (4). As discussed below, we
have exploited complementary study of African-Americans and
European-Americans with SLE to identify susceptibility alleles.
Siblings of SLE patients have a risk of the disease between 2
and 5 % (5) while the concordance rate among identical twin pairs
is only up to about 25 % (6). Estimates are that about two-thirds
of the risk of SLE is genetic (7). Much of the remaining risk may
be environmental. The susceptibility potential of environmental
exposure may be related to an individuals genetics. That is, there
may be genetic factors impacting susceptibility to environmental
exposure that leads to SLE. The complex pattern of inheritance of
SLE suggests multigenic inheritance, requiring interaction of vari-
ous combinations of contributing genes at multiple loci that is
likely to contribute to clinically diverse phenotypes. Despite the
potential difficulties, genome-wide association studies have shown
this to be the case.
Interestingly, while SLE is generally a complicated genetic ill-
ness, the disease also can rarely be caused by mutations in a single
gene. In point of fact, these mutations occur in the early comple-
ment components C1q, C2, and C4. About three-quarters of indi-
viduals with complete hereditary deficiency of either C1q or C4 are
destined to acquire SLE, while the risk in those with complete
hereditary deficiency of C2 is much lower, perhaps not much
higher than 10 % (8). Risk of SLE with respect to complement
component C4 is related to gene copy number, which can vary
from zero to ten in the population (9). However, we have found
that even among families with two or more patients, genetic com-
plement deficiencies are uncommon with only 2 of 544 multi-case
families having one of these single-gene defects (10). Genetic com-
plement deficiencies are examples of rare variants that impair a high
to moderate risk of disease.
SLE is a clinically heterogeneous disease. Current guidelines
require a set of 4 out of 11 American College of Rheumatology
(ACR) criteria for classification of a patient as having SLE (11).
The unifying feature is the production of autoantibodies against
nuclear components. As a result, antinuclear antibody (ANA) test-
ing is very sensitive for the disease, although not highly specific
2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus 13

since ANAs are sporadically detected in as much as 2 % of the female

population over the age of 40 as well as in the sera of persons with
many other diseases. On the other hand, antibodies to double-
stranded DNA and the Sm protein are very specific for SLE such
that these specificities are part of the classification criteria (11).
While SLE is a clinically complex disease, there are common
themes in its pathogenesis, which in part emerging genetics has
helped establish. At least a portion of the pathophysiology is attrib-
uted to deposition of immune complexes, which are continuously
formed by autoantigens and autoantibodies, in various tissues.
Thus, pathogenesis is related to dysregulation of self-reactive B
cells. B cells may serve a critical role in antigen presentation.
Additionally, immune dysfunction of the T lymphocytes involved
in the adaptive immune system and elements of the innate immune
system, such as complement proteins and toll-like receptors, espe-
cially those that recognize ribonucleoprotein complex, are also
involved in disease expression. Overall, however, the pathogenesis
of SLE is incompletely understood.
For disorders with a poorly known biochemical basis, like SLE,
identification of the susceptibility genes is key to an increased
understanding of the biological basis of the disease and contributes
to understanding the development and pathogenesis of the disease.
Genetic insight may lead to novel therapeutic interventions. Such
information may be valuable in predicting the course of SLE in
individual patients, and this could prove to be an important guide
to therapy and monitoring. Additionally, genetic screening could
be used to identify individuals who are at risk so that they can take
the advantage of early diagnosis and treatment, or even preventive
therapy, if some day available.
Genetics studies in SLE began with examination of the HLA
complex on chromosome 6. Specific HLA types or alleles impart
about a twofold risk of SLE. However, in many cases HLA alleles
are more strongly associated with specific SLE-associated autoanti-
bodies than with the disease itself (12). For example, our group
showed nearly 25 years ago that HLA-DQ1/2 heterozygosity
identified patients with anti-Ro (also known as SS-A) (13). In a
subsequent analysis, we found that addition of certain T cell recep-
tor gene alleles to SLE-DQ1/2 strengthened the association with
anti-Ro (14).
Genetics studies in genetically complex diseases require large
numbers of patients, family members, and controls. In order to
facilitate study of SLE genetics beyond the HLA, we began identi-
fying, collecting, and storing samples on families with more than
one SLE about 20 years ago. This effort, designated the Lupus
Family Registry and Repository, or LFRR, was originally designed
to support genetic linkage studies using DNA microsatellite
repeats. In fact, one of the first SLE genetic linkage papers was
published using this cohort (15). Replicated linkages with SLE
14 R.H. Scofield and K.M. Kaufman

have been reported at 1q23q25, 1q41q42, 2q35q37, 4p16p15,

4q31q33, 6p21.3, 6p22p11, 7p22, 16p12q13, 19q13, 20p13
p12, and 20q12 (1618). Some of these same genetic intervals
have also been found in genetic association studies. Meanwhile, as
discussed below for chromosome 11p13 near the gene for CD44,
other linkages that were either not confirmed or for which statisti-
cal values were only suggestive have been found in association
studies using single-nucleotide polymorphisms (SNPs). Large col-
lections of disease-affected and -unaffected individuals have allowed
genome-wide genetics studies, either linkage or association, to
proceed.
For the LFRR, putative patients have come from all over the
United States as well as from numerous collaborators outside this
country, including Columbia, Korea, and the United Kingdom.
Putative SLE patients, who might be self-referred or referred by a
physician, complete an extensive questionnaire designed to elicit
information about SLE as well as any other health problems.
Subsequent to 1995, the LFRR questionnaire includes a connec-
tive tissue disease screening questionnaire (19). Putative SLE
patients then undergo a phone interview by a physician or nurse to
ascertain the likelihood of SLE. Finally, medical records are
reviewed, again by a physician or a nurse especially trained in SLE,
in order to document that the subject does or does not meet 4 of
the 11 ACR classification criteria (11). Family members willing to
participate in the study fill out a questionnaire. If there is an indica-
tion of possible SLE, these individuals are interviewed by study
personnel in a manner similar to the putative patients. A searchable
database contains nearly 1,000 points of information on each sub-
ject enrolled in the LFRR.
Blood samples are obtained from all subjects. Sera, plasma,
DNA, RNA, and in some cases peripheral blood cells are stored.
Originally, in the genetics linkage era, families had to have at least
two SLE patients and all subjects underwent typing using a panel
of 308 microsatellite markers (15). At present, collection contin-
ues but SLE patients do not need a relative with SLE to be
included in the study. Of course, microsatellite typing is no longer
performed.
We have recently described in full the LFRR (20). At present
this resource holds samples and data on about 3,000 SLE patients,
1,500 age- and sex-matched controls along with more than 7,000
family members. As stated above, SLE is more common among
Black Americans than the majority population. About 40 % of SLE
patients in the LFRR collection self-identify as Black Americans. As
discussed below, the ability to compare genetic association between
that found in White Americans and Black Americans has proven
very useful. LFRR data and samples are available to qualified inves-
tigators for studies directed at SLE.
 2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus 15

2. Methods

2.1. Genome-Wide In the past several years genome-wide genetic association studies
Association Studies (GWAS) have been performed in several large collections of SLE
patients and controls. These genome-wide studies of up to 500,000
SNPs have identified at least 30 and perhaps up to 50 genetic asso-
ciations for SLE (21, 22). A review of this rapidly changing field in
2009 found 20 confirmed results with p values exceeding the
genome-wide significance level of less than 1 108 (23). There are
now replication studies, including in non-white cohorts (2428).
As discussed below, study of these findings across racial/ethnic
groups may be informative, especially in regard to identifying the
causative alleles (27, 28). However, it is of note that all the GWAS
in SLE have been in either European-derived or Asian subjects.
And, while the causative, functional alleles that impart the risk
of SLE were not likely identified in genome-wide association stud-
ies, there are common themes among the genes and the gene
products thus far implicated. For example, B lymphocyte activa-
tion, apoptosis, or the interferon signaling pathway genes can be
identified as commonly represented among those genes thus far
implicated (23, 29). Interestingly, only the IRF5 association is seen
in all four populations that have been currently studied. Many of
the associations cross population boundaries but not always.
Perhaps this is due to population-specific genetic risks for lupus.
Alternatively, these results may be due to the fact that the func-
tional polymorphisms have not been identified. That is, the pres-
ently identified alleles are only in linkage disequilibrium with the
actual causative allele, and this linkage differs between human pop-
ulation groups. As more of the functional polymorphisms are
identified our ability to fully examine the associations across popu-
lation boundaries will be increased.
The genome-wide association study in which our research
group participated was a collaborative effort formed and supported
by the Alliance for Lupus Research that was known as the
International Consortium for Systemic Lupus Erythematosus
Genetics (SLEGEN). For this study, 317,501 SNPs were typed for
alleles in 720 American women of European ancestry with SLE
along with 2,337 racially matched controls. As a replication cohort,
we studied consistently associated SNPs in two additional indepen-
dent sample sets of 1,846 SLE-affected women and 1,825 con-
trols. There was strong genetic association with the HLA region
on chromosome 6p21 as well as the previously confirmed associa-
tion at chromosome 7p32 at the IRF5 gene. In addition, there was
evidence of association at genome-wide statistical significance with
replication at 16p11.2 (ITGAM), 11p15.5 (KIAA1542), 3p14.3
(PXK), and 1q25.1 (rs10798269).
16 R.H. Scofield and K.M. Kaufman

SNPs near or within the genes FCGR2A, PTPN22, and STAT4,

regions previously associated with SLE and other autoimmune dis-
eases, also had evidence of genetic association in this study (21).
While much has been discovered in the last decade concerning
the genetics of SLE, there is much yet to be learned. GWAS has
found only a fraction of the genetic risk. Rare alleles and mutations
that impart a moderate risk of SLE remain undiscovered and can-
not be found by GWAS. Genegene interaction is virtually unex-
plored as is the genetics of SLE phenotypes, although recently this
subject has been approached by genetic association (30, 31).
Furthermore, GWAS has not identified the causative allele that
actually imparts the genetic susceptibility. Yet insights into the
genetic pathogenesis are leading to new therapies, as for example
anti-interferon drugs are in clinical trials (29).

2.2. Large Lupus In order to confirm, consolidate, and extend the findings from the
Association Study 2 various SLE GWASs, we initiated candidate gene study that, similar
to SLEGEN, would be a collaborative effort of many investigators
interested in the genetics of SLE. Collaborators agreed to contrib-
ute DNA samples on at least 50 SLE patients and in return those
contributors could select SNPs to be included in the custom array.
Additional SNPs were included based on the financial contribution
of each collaborator to the project. For production of the SNP typ-
ing array, this project utilized the Illumina Infinium genotyping
assay. Initially, 33,788 custom SNPs were submitted by the 42 col-
laborators (see Table 1). After manufacturing, 32,217 SNPs passed
Illumina Quality control measurements (95.3 %). A total of 3,241
SNPs were removed due to an allele call rate below 90 %. An addi-
tional 3,445 SNPs were removed due to a major allele frequency
less than 1 %. Thus, 27,276 SNPs passed quality control proce-
dure, and were used for analysis. In addition, approximately 3,000
ancestrally informative SNPs were included. Each collaborator was
supplied with the results of the SNPs that he or she had included
in the study as well as any information that others had selected any
of these same SNPs for typing.
For a typical Large Lupus Association Study 2 (LLAS2), more
than 4,000 lupus patients with about 4,000 controls of European
descent, about 1,500 African-American lupus patients with more
than 1,800 African-American controls, about 1,000 Hispanic
patients with approximately 350 Hispanic controls enriched for
Amerindian-European admixture, and about 1,300 Asian lupus
patients with an equal number of Asian controls can be included.
Thus, this study uses samples from multiple racial and ethnic
groups. All SLE cases meet four or more of the 1997 ACR revised
criteria for the classification of SLE (11). Samples from these
patients were provided from multiple sites to the Oklahoma
Medical Research Foundation (OMRF). Each recruiting site had
Institutional Review Board (IRB) approval to recruit subjects and
the overall study was approved by OMRFs IRB.
2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus 17

Table 1
Collaborators and their role in the LLAS2 study

Collaborator Role

Marta Alarcon-Riquelme SamplesC, H, Am

Juan-Manual Anaya SamplesAm
S-C Bae SamplesAs
Susan Boackle SamplesC, AA, As, H
Lindsey Criswell SamplesC
Gary Gilkeson SamplesC, AA, Gullah
Diane Kamen SamplesGullah
Joel Guthridge SamplesAs
Chaim Jacob SamplesC, AA, As, H
Judith James SamplesC, AA, As, H
Kenneth Kaufman Genotyping, quality control
Alan Adler Genotyping
Jennifer Kelly Study design, quality control, data
assembly
Robert Kimberly SamplesC AA, H
E Brown SamplesC, AA, H
Jeff Edberg SamplesC, AA, H
John Reveille SamplesC, AA, H
Luis Vila SamplesC, AA, H
Michelle Petri SamplesC, AA, H
Rosiland Ramsey-Goldman SamplesC, AA, H
Carl Langeford Study design, analysis, quality control
Adwillia Analysis, quality control
Mcomeau Analysis, quality control
Jziegler Analysis, quality control
Mimarion Analysis, quality control
Joan Merrill SamplesC, AA
Kathy Moserk SamplesC
Pat Gaffney Study design; samplesC
Tim Niewold SamplesC, AA
Hal Scofield SamplesC, AA, H
(continued)
18 R.H. Scofield and K.M. Kaufman

Table 1
(continued)

Collaborator Role
Anne Stevens SamplesC, AA, As, H
Betty Tsao SamplesC, AA, As, H
Tim Vyse SamplesC
John Harley Study design; samplesC, AA, H, Am
B Freedman SamplesAA
Stuart Glenn Clinical data organization and quality
control
C = Caucasians, AA = African-Americans, H = Hispanic, As = Asians, Am = Amerindian

All SNP genotyping was performed at the OMRF. Of course,

individual collaborators are responsible for analyses of their own
data, but there will be common themes on how the data are stud-
ied. For example, in several studies SNPs with a genotype success
rate of <90 %, HardyWeinberg equilibrium p value <0.001 in con-
trols, or a minor allele frequency of <0.01 have been excluded from
the analysis. SNPs with a p value for the difference in missing pro-
portions between cases and controls of <0.01 have also been
excluded.
The 42 collaborators took a variety of approaches in the selec-
tion of SNPs. Some opted for haplotype-marking SNPs in a genetic
interval previously showing genetic association in an attempt to
define the true susceptibility allele in this region. That is, in brief,
this approach can be considered fine mapping of a genetic interval,
and has resulted in the desired outcome. For example, a paper
recently published establishes genetic association between SLE and
the IL21 gene (32). In this paper, the ability to compare genetics
across racial lines was critically important. In particular, the smaller
haplotype lengths found among those of African origin compared
to those of European origin was enlightening.
In another LLAS2 project, two separate investigators targeted
the same genetic interval on chromosome 11 at p13. One collabo-
rator was following up a strong genetic linkage in this region (33)
by fine mapping with large numbers of SNPs, while the other col-
laborator was pursuing a suggestive genetic association in the
Minnesota SLE GWAS (22) with only two SNPs in LLAS2. These
investigators, including the authors, worked together, combining
the data from SNPs in this region, and found strong genetic asso-
ciation, which lies in the promoter region of the CD44 gene. As
we have described, this gene has very interesting biology that
 2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus 19

Table 2
Papers published or in press as a result of LLAS2

References Senior author(s) Study/topic

(40) Sawalha, Alarcn-Riquelme Amerindian ancestry
(41) Harley, Sawalha Severe phenotype
(34) Scofield, Gaffney, Moser PDHX and CD44
(39) Harley, Atkinson TREX1
(35) Gaffney TNFAIP3
(32) Nath, Harley, Sawalha IL21
(38) Criswell, Barcellos rs4774 CIITA
(42) Tsao Comp factor H

makes it a promising candidate for an SLE susceptibility gene (34).

Again, comparison of the genetic association found in Black
Americans and White Americans was quite useful because of differ-
ences in haplotype blocks (34). In this way, the interval containing
this SLE susceptibility allele was narrowed considerably. A paper
examining the genetic association of TNFAIP3 gene, which
encodes the ubiquitin-modifying enzyme A20, has also used
LLAS2 data. Again, comparing the association found in different
ethnic/racial groups, this time European-derived American to
Korean SLE patients, was important. A TT-to-A polymorphism
(deletion T followed by a T-to-A transversion) was associated with
SLE in each of these groups. This variant is located in a region of
high genetic conservation and binds to regulatory complexes with
reduced affinity. This results in low TNFAIP3 mRNA expression as
well as lower expression of the gene product, A20 (35). Both these
LLAS2 papers demonstrate the importance of studying distinct
population groups.
Thus far investigators using data from LLAS2 have identified
new regions of genetic association and causative alleles as well as
developed functional data for these alleles. Table 2 lists the papers
that have been published or are in press as a result of this effort.
At least 11 other papers have been submitted and are under con-
sideration. Many collaborators have not yet pursued studies of
regions in which they selected SNPs. So, we anticipate that this
study will continue to provide useful results for many years.

2.3. Next-Generation Genome-wide association studies are technically feasible and pro-
Sequencing ductive as demonstrated by the several that have been performed
in an uncommon disease such as SLE with identification of about
30 risk alleles. Nevertheless, there are significant drawbacks and
20 R.H. Scofield and K.M. Kaufman

limitations to these studies. These studies are able to identify common

population variants that give modest increases in disease risk. In
fact, the relative risks of the 30 or so thus far identified risk alleles
are in the range of 1.21.5. Furthermore, the SNPs showing
genetic association are almost surely not the actual causative sus-
ceptibility allele, but instead only in linkage disequilibrium with
the true susceptibility allele. Finally, calculations indicate that only
a minority of the genetic risk for SLE, as well as other polygenic
human diseases, has been identified by GWAS (36). Rare variants
imparting a moderate risk of disease and copy number variation
cannot be identified by GWAS.
In order to detect the association of rare variants to the dis-
ease, the number of samples will have to be as large, if not larger,
as what has already been done in the LLAS experiments. So, new
approaches will be needed to discover SLE-susceptibility variants
such as these. One such approach is sequencing of the genome.
This could be whole genome sequencing or sequence-capture
sequencing in which DNA or exons from particular genes are
sequenced in their entirety. This idea has recently been reviewed
(37). From a theoretical standpoint, in order to identify the func-
tional variations, additional high-density genotyping will be
required. However, we can only genotype the SNPs that have been
identified. That is (perhaps it goes without saying), SNPs that
remain unknown cannot be tested for genetic association
until they have been discovered. To overcome this issue, next-
generation sequencing will be required to sequence regions of
interest in SLE patients to identify candidate functional polymor-
phisms that truly impart increased susceptibility for SLE. The SNPs
identified in this re-sequencing/deep-sequencing effort will be the
ones typed in the future.
Two members of our research group (Pat Gaffney and Graham
Wiley) are performing next-generation sequencing in the form of
sequence capture technology as well as by full exome sequencing.
In this technique, particular genomic regions are targeted for full
sequencing. Oligonucleotides from targeted sequences are printed
on an array, and then genomic DNA is applied and subjected to
amplification of the targeted sequence. Thus, a selection step is
inserted prior to the amplification step such that only sequences of
interest are amplified. Following amplification, sequencing is per-
formed on the array platform with many thousands of replicates for
any given genomic region. We have performed exon sequence-
capture, next-generation sequencing on SLE and control subjects.
This has identified many new polymorphisms (SNPs and others
such as deletions or insertions). The members of our group per-
forming this work envision finding both the true susceptibility
allele as well as rare variants by using this technique.
 2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus 21

3. Conclusion

Large genome-wide association studies have identified many

common variants that impart a modest increased risk for SLE. At
present we are pursuing several of these associated genetic intervals
through a study designated LLAS2. The goal of these studies is to
identify the allelic differences that truly impart the risk of disease.
Discovering genetic differences that are rare in the population and
increase susceptibility to a moderate degree will require next-gen-
eration sequencing techniques, in which we are actively engaged.
The challenges of continuing to study SLE genetics are several.
New and large collections of patients, especially minority subjects,
need to be sought and maintained. Ensuring that subjects have
SLE and collecting samples are time consuming and expensive. As
genome-sequencing techniques are applied to this problem, even
greater informatics, computational, and data analysis capabilities
will be required.

References
1. Hochberg MC (1997) The epidemiology of
systemic lupus erythematosus. In: Wallace DJ,
Hahn BH (eds) Dubois lupus erythematosus.
Williams and Wilkins, Baltimore, MD, pp
4965
2. Lawrence RC, Helmick CG, Arnett FC, Deyo
RA, Felson DT, Giannini EH, Heyse SP,
Hirsch R, Hochberg MC, Hunder GG, Liang
MH, Pillemer SR, Steen VD, Wolfe F (1998)
Estimates of the prevalence of arthritis and
selected musculoskeletal disorders in the
United States. Arthritis Rheum 41:778799
3. Petri M (1998) The effect of race on incidence
and clinical course in systemic lupus erythema-
tosus: the Hopkins Lupus Cohort. J Am Med
Womens Assoc 53:916
4. Borchers AT, Naguwa SM, Shoenfeld Y,
Gershwin ME (2010) The geoepidemiology of
systemic lupus erythematosus. Autoimmun
Rev 9:A277287
5. Vyse TJ, Todd JA (1996) Genetic analysis of
autoimmune disease. Cell 85:311318
6. Deapen D, Escalante A, Weinrib L, Horwitz
D, Bachman B, Roy-Burman P, Walker A,
Mack TM (1992) A revised estimate of twin
concordance in systemic lupus erythematosus.
Arthritis Rheum 35:311318
7. Harley IT, Kaufman KM, Langefeld CD,
Harley JB, Kelly JA (2009) Genetic suscepti-
bility to SLE: new insights from fine mapping
and genome-wide association studies. Nat Rev
Genet 10:285290
8. Manderson AP, Botto M, Walport MJ (2004)
The role of complement in the development of
systemic lupus erythematosus. Annu Rev
Immunol 22:43156
9. Wu YL, Yang Y, Chung EK, Zhou B, Kitzmiller
KJ, Savelli SL, Nagaraja HN, Birmingham DJ,
Tsao BP, Rovin BH, Hebert LA, Yu CY (2008)
Phenotypes, genotypes and disease susceptibil-
ity associated with gene copy number varia-
tions: complement C4 CNVs in European
American healthy subjects and those with sys-
temic lupus erythematosus. Cytogenet
Genome Res 123:13141
10. Aggarwal R, Sestak AL, DSousa A, Dillon SP,
Namjou B, Scofield RH (2010) Complete
complement deficiency in a large cohort of
familial systemic lupus erythematosus. Lupus
19:5257
11. Hochberg MC (1997) Updating the American
College of Rheumatology revised criteria for
the classification of systemic lupus erythemato-
sus. Arthritis Rheum 40:1725
12. Schur PH (1995) Genetics of systemic lupus
erythematosus. Lupus 4:425437
13. Harley JB, Reichlin M, Arnett FC, Alexander
EL, Bias WB, Provost TT (1986) Gene inter-
action at HLA-DQ enhances autoantibody
production in primary Sjogrens syndrome.
Science 232:11451147
14. Scofield RH, Frank MB, Neas BR, Horowitz
RM, Hardgrave KL, Fujisaku A, McArthur R,
Harley JB (1994) Cooperative association of
22 R.H. Scofield and K.M. Kaufman
T cell beta receptor and HLA-DQ alleles in the
production of anti-Ro in systemic lupus ery-
thematosus. Clin Immunol Immunopathol
72:335341
15. Moser KL, Neas BR, Salmon JE, Yu H, Gray-
McGuire C, Asundi N, Bruner GR, Fox J,
Kelly J, Henshall S, Bacino D, Dietz M, Hogue
R, Koelsch G, Nightingale L, Shaver T, Abdou
NI, Albert DA, Carson C, Petri M, Treadwell
EL, James JA, Harley JB (1998) Genome scan
of human systemic lupus erythematosus: evi-
dence for linkage on chromosome 1q in
African-American pedigrees. Proc Natl Acad
Sci U S A 95:1486914874
16. Tsao BP et al (1997) Evidence for linkage of a
candidate chromosome 1 region to human sys-
temic lupus erythematosus. J Clin Invest
99:725731
17. Forabosco P et al (2006) Meta-analysis of
genome-wide linkage studies of systemic lupus
erythematosus. Genes Immun 7:609614
18. Lee YH, Nath SK (2005) Systemic lupus ery-
thematosus susceptibility loci defined by
genome scan meta-analysis. Hum Genet
118:434443
19. Karlson EW, Sanchez-Guerrero J, Wright EA,
Lew RA, Daltroy LH, Katz JN, Liang MH
(1995) A connective tissue disease screening
questionnaire for population studies. Ann
Epidemiol 5:297302
20. Rasmussen A, Sevier S, Kelly J, Glenn S, Aberle
T, Cooney CM, Grether A, Goodmon E, Ning
J, Tesiram J, Morrisey J, Powe T, Drexel M,
Daniel W, Namjou B, Ojwang JO, Nguyen
KM, Cavett JW, Te JL, James JA, Scofield RH,
Moser KL, Gilkeson GS, Kamen DL, Carson
C, MdC B, Punaro MG, Quintero-del-Rio
AL, Karp DR, Wallace DJ, Weisman M, Merrill
J, Rivera R, Petri MA, Albert DA, Espinoza
LR, Utset TO, Shaver TS, Arthur E, Anaya
JM, Bruner GR, Harley JB (2010) The lupus
family registry and repository. Rheumatology
(Oxford) 50:4759
21. Harley JB, Alarcon-Riquelme ME, Criswell
LA, Jacob CO, Kimberly RP, Moser KL, Tsao
BP, Vyse TJ, Langefeld CD, Nath SK,
Guthridge JM, Cobb BL, Mirel DB, Marion
MC, Williams AH, Divers J, Wang W, Frank
SG, Namjou B, Gabriel SB, Lee AT, Gregersen
PK, Behrens TW, Taylor KE, Fernando M,
Zidovetzki R, Gaffney PM, Edberg JC, Rioux
JD, Ojwang JO, James JA, Merrill JT, Gilkeson
GS, Seldin MF, Yin H, Baechler EC, Li QZ,
Wakeland EK, Bruner GR, Kaufman KM, Kelly
JA (2008) Genome-wide association scan in
women with systemic lupus erythematosus
identifies susceptibility variants in ITGAM,
PXK, KIAA1542 and other loci. Nat Genet
40:204210
22. Hom G, Graham RR, Modrek B, Taylor KE,
Ortmann W, Garnier S, Lee AT, Chung SA,
Ferreira RC, Pant PV, Ballinger DG, Kosoy R,
Demirci FY, Kamboh MI, Kao AH, Tian C,
Gunnarsson I, Bengtsson AA, Rantapaa-
Dahlqvist S, Petri M, Manzi S, Seldin MF,
Ronnblom L, Syvanen AC, Criswell LA,
Gregersen PK, Behrens TW (2008) Association
of systemic lupus erythematosus with C8orf13-
BLK and ITGAM-ITGAX. N Engl J Med
358:900909
23. Graham RR, Hom G, Ortmann W, Behrens
TW (2009) Review of recent genome-wide
association scans in lupus. J Intern Med
265:680688
24. Suarez-Gestal M, Calaza M, Endreffy E,
Pullmann R, Ordi-Ros J, Domenico Sebastiani
G, Ruzickova S, Jose Santos M, Papasteriades
C, Marchini M, Skopouli FN, Suarez A, Blanco
FJ, DAlfonso S, Bijl M, Carreira P, Witte T,
Migliaresi S, Gomez-Reino JJ, Gonzalez A,
European Consortium of SLE DNA Collections
(2009) Replication of recently identified sys-
temic lupus erythematosus genetic associa-
tions: a case-control study. Arthritis Res Ther
11:R69
25. Gateva V, Sandling JK, Hom G, Taylor KE,
Chung SA, Sun X, Ortmann W, Kosoy R,
Ferreira RC, Nordmark G, Gunnarsson I,
Svenungsson E, Padyukov L, Sturfelt G,
Jonsen A, Bengtsson AA, Rantapaa-Dahlqvist
S, Baechler EC, Brown EE, Alarcon GS,
Edberg JC, Ramsey-Goldman R, McGwin G
Jr, Reveille JD, Vila LM, Kimberly RP, Manzi
S, Petri MA, Lee A, Gregersen PK, Seldin MF,
Ronnblom L, Criswell LA, Syvanen AC,
Behrens TW, Graham RR (2009) A large-scale
replication study identifies TNIP1, PRDM1,
JAZF1, UHRF1BP1 and IL10 as risk loci for
systemic lupus erythematosus. Nat Genet
41:12281233
26. Han S, Kim-Howard X, Deshmukh H,
Kamatani Y, Viswanathan P, Guthridge JM,
Thomas K, Kaufman KM, Ojwang J, Rojas-
Villarraga A, Baca V, Orozco L, Rhodes B,
Choi CB, Gregersen PK, Merrill JT, James JA,
Gaffney PM, Moser KL, Jacob CO, Kimberly
RP, Harley JB, Bae SC, Anaya JM, Alarcon-
Riquelme ME, Matsuda K, Vyse TJ, Nath SK
(2009) Evaluation of imputation-based asso-
ciation in and around the integrin-alpha-M
(ITGAM) gene and replication of robust asso-
ciation between a non-synonymous functional
variant within ITGAM and systemic lupus
erythematosus (SLE). Hum Mol Genet
18:11711180
 2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus
27. Ito I, Kawasaki A, Ito S, Hayashi T, Goto D,
Matsumoto I, Tsutsumi A, Hom G, Graham
RR, Takasaki Y, Hashimoto H, Ohashi J,
Behrens TW, Sumida T, Tsuchiya N (2009)
Replication of the association between the
C8orf13-BLK region and systemic lupus ery-
thematosus in a Japanese population. Arthritis
Rheum 60:553558
28. Molineros JE, Kim-Howard X, Deshmukh H,
Jacob CO, Harley JB, Nath SK (2009)
Admixture in Hispanic Americans: its impact
on ITGAM association and implications for
admixture mapping in SLE. Genes Immun
10:539545
29. Perl A (2009) Emerging new pathways of
pathogenesis and targets for treatment in sys-
temic lupus erythematosus and Sjogrens syn-
drome. Curr Opin Rheumatol 21:443447
30. Kariuki SN, Franek BS, Kumar AA, Arrington
J, Mikolaitis RA, Utset TO, Jolly M, Crow
MK, Skol AD, Niewold TB (2010) Trait-
stratified genome-wide association study
identifies novel and diverse genetic associations
with serologic and cytokine phenotypes in sys-
temic lupus erythematosus. Arthritis Res Ther
12:R151
31. Taylor KE, Chung SA, Graham RR, Ortmann
WA, Lee AT et al (2011) Risk alleles for sys-
temic lupus erythematosus in a large case-con-
trol collection and associations with clinical
subphenotypes. PLoS Genet 7:e1001311
32. Hughes T, Kim X, Kelly JA, Kaufman KM,
Langefeld CD, Ziegler J, Kimberly RP, Edberg
JC, Ramsey-Goldman R, Petri M, Reveille JD,
Brown EE, Vila LM, James JA, Gilkeson GS,
Moser KL, Gaffney PM, Merrill JT, Vyse TJ,
Alarcon-Riquelme ME, Nath SK, Harley JB,
Sawalha AH (2011) Fine mapping and trans-
ethnicity genotyping in IL2/IL21 establish
the genetic association between IL21 and sys-
temic lupus erythematosus. Arthritis Rheum
63:16891697
33. Scofield RH, Bruner GR, Kelly JA, Kilpatrick
J, Bacino D, Nath SK, Harley JB (2003)
Thrombocytopenia identifies a severe familial
phenotype of systemic lupus erythematosus
and reveals genetic linkages at 1q22 and
11p13. Blood 101:9927
34. Lessard CJ, Adrianto I, Kelly JA, Kaufman
KM, Grundahl KM, Adler A, Williams AH,
Gallant C, Alarcn-Riquelme ME for the
BIOLUPUS and GENLES Networks, Anaya
J-M, Bae S-C, Boackle SA, Brown EE, Chang
D-M, Criswell LA, Edberg JC, Freedman BI,
Gregersen PK, Gilkeson GS, Jacob CO, James
JA Kamen DL, Kimberly RP, Martin J, Merrill
JT, Niewold TB, Park S-Y, Petri MA, Pons-
Estel BA, Rosalind Ramsey-Goldman R,
Reveille JD, Song YW, Stevens AM, Tsao BP,
23
Vila LM, Vyse TJ, Yu C-Y, Guthridge JM,
Bruner GR, Langefeld CD, Montgomery C,
Harley JB, Scofield RH, Gaffney PM, Moser
KL (2011) Identification of a systemic lupus
erythematosus susceptibility locus at 11p13
between PDHX and CD44 in a multi-ethnic
study. Am J Hum Genet 88:8391
35. Adrianto I, Wen F, Templeton A, Wiley G,
King JB, Lessard CJ, Bates JS, Hu Y, Kelly JA,
Kaufman KM, Guthridge JM, Alarcon-
Riquelme ME for the BIOLUPUS and
GENLES Networks, Anaya JM, Bae SC, Bang
SY, Boackle SA, Brown EE, Petri MA, Gallant
C, Ramsey-Goldman R, Reveille JD, Vila LM,
Criswell LA, Edberg JC, Freedman BI,
Gregersen PK, Gilkeson GS, Jacob CO, James
JA, Kamen DL, Kimberly RP, Martin J, Merrill
JT, Niewold TB, Park SY, Pons-Estel BA,
Scofield RH, Stevens AM, Tsao BP, Vyse TJ,
Langefeld CD, Harley JB, Moser KL, Webb
CF, Humphrey MB, Montgomery CG,
Gaffney PM (2011) Association of a functional
variant downstream of TNFAIP3 with systemic
lupus erythematosus. Nat Genet 43:253258
36. Manolio TA, Collins FS, Cox NJ, Goldstein
DB, Hindorff LA, Hunter DJ, McCarthy MI,
Ramos EM, Cardon LR, Chakravarti A, Cho
JH, Guttmacher AE, Kong A, Kruglyak L,
Mardis E, Rotimi CN, Slatkin M, Valle D,
Whittemore AS, Boehnke M, Clark AG,
Eichler EE, Gibson G, Haines JL, Mackay TF,
McCarroll SA, Visscher PM (2009) Finding
the missing heritability of complex diseases.
Nature 461:747753
37. Cirulli ET, Goldstein DB (2010) Uncovering
the roles of rare variants in common disease
through whole-genome sequencing. Nat Rev
Genet 11:415425
38. Bronson PG, Goldstein BA, Ramsay PP,
Beckman KM, Noble JA, Lane JA, Sledin MF,
Kelly JA, Harley JB, Moser KL, Gaffney PM,
Behrens TW, Criswell LA, Barcellos LF (2011)
The rs4774 CIITA missense variant is associ-
ated with risk of systemic lupus erythematosus.
Genes Immun 12:667671
39. Namjou B, Kothari PH, Kelly JA, Glenn SB,
Ojwang JO, Adler A, Alarcon-Riquelme ME,
Gallant CJ, Boackle SA, Criswell LA, Kimberly
RP, Brown E, Edberg J, Stevens AM, Jacob
CO, Tsao BP, Gilkeson GS, Kamen DL, Merrill
JT, Petri M, Goldman RR, Vila LM, Anaya
JM, Niewold TB, Martin J, Pons-Estel BA,
Sabio JM, Callejas JL, Vyse TJ, Bae SC, Perrino
FW, Freedman BI, Scofield RH, Moser KL,
Gaffney PM, James JA, Langefeld CD,
Kaufman KM, Harley JB, Atkinson JP (2011)
Evaluation of the TREX1 gene in a large
multi-ancestral lupus cohort. Genes Immun
12:2709
24 R.H. Scofield and K.M. Kaufman
40. Sanchez E, Webb RD, Rasmussen A, Kelly JA,
Riba L, Kaufman KM, Torre IG, Moctezuma
JF, Maradiaga-Cecea MA, Cardiel-Rios MH,
Acevedo E, Cucho-Venegas M, Garcia MA,
Gamron S, Pons-Estel BA, Vasconcelos C,
Martin J, Tusi-Luna T, Harley JB, Richardson
B, Sawalha AH, Alarcn-Riquelme ME (2011)
Genetically determined Amerindian ancestry
correlates with increased frequency of risk
alleles for systemic lupus erythematosus.
Arthritis Rheum 62:37223729
41. Webb R, Kelly JA, Somers EC, Hughes T,
Kaufman KM, Sanchez E, Nath SK, Bruner G,
Alarcon-Riquelme ME, Gilkeson GS, Kamen
DL, Richardson BC, Harley JB, Sawalha AH
(2011) Early disease onset is associated with a
more severe phenotype in lupus patients and is
predicted by a higher genetic risk for lupus.
Ann Rheum Dis 70:151156
42. Zhao J, Wu H, Khosravi M, Cui H, Qian X,
Kelly JA, Kaufman KM, Langefeld CD,
Williams AH, Comeau ME, Ziegler JT, Marion
MC, Adler A, Glenn SB, Alarcon-Riquelme
ME, Pons-Estel BA, Harley JB, Bae SC, Bang
SY, Cho SK, Jacob CO, Vyse TJ, Niewold TB,
Gaffney PM, Moser KL, Kimberly RP, Edberg
JC, Brown EE, Petri MA, Ramsey-Goldman
R, Vila LM, Reveille JD, James JA, Gilkeson
GS, Kamen DL, Freedman BI, Anaya JM,
Merrill JT, Criswell LA, Scofield RH, Stevens
AM, Boackle SA, Guthridge JM, Chang DM,
Song YW, Park JA, Lee EY, Grossman JM,
Hahn BH, Goodship T, Cantor RM, Yu CY,
Shen N, Tsao BP (2011) Association of genetic
variants in complement factor H and factor
H-related genes with systemic lupus
erythematosus susceptibility. PLoS Genet
7(5):e1002079
 Chapter 3

Methods and Protocols to Study T Cell Signaling

Abnormalities in Human Systemic Lupus Erythematosus
Vaishali R. Moulton, Mindy S. Lo, and George C. Tsokos

Abstract
Abnormal expression of key signaling molecules and defective functions of T lymphocytes play a significant
role in the pathogenesis of systemic lupus erythematosus (SLE). T cell receptor (TCR/CD3)-mediated
stimulation of SLE T cells show increased protein tyrosine phosphorylation of cellular proteins with faster
kinetics, heightened calcium flux response, and decreased IL-2 production. The molecular mechanisms of
T cell signaling abnormalities in SLE T cells are complex. Current research has been directed towards
investigating various factors that contribute to abnormal tyrosine phosphorylation, intracellular calcium
response, and cytokine production. Central to this dysfunction is the aberrant expression and function of
the TCR/CD3 chain. Latest developments suggest multiple explanations are involved, including altered
receptor structure, supramolecular assembly, modulation of membrane clustering, aberrant cellular
distribution, and pre-compartmentalization with lipid-rafts. The methods and protocols described here
pertaining to T cell signaling abnormalities in SLE T cells are optimized in many ways and are derived by
the combined task and continuous efforts of many researchers in the lab over a long period of time. These
simplified protocols can be readily applied to study T cell signaling abnormalities in SLE to identify the
genetic, molecular, and biochemical factors contributing to aberrant immune cell function and unravel
the pathophysiology of SLE.

Key words: T cell isolation, Cell signaling, Calcium response, Tyrosine phosphorylation,
Immunoblotting, Cell proliferation, Cytokines, Autoimmune disease, Systemic lupus erythematosus,
T cell receptor, CD3

1. Introduction

1.1. TCR Signaling
One of the earliest steps in the signal transduction after TCR/CD3
engagement is the phosphorylation of the tyrosine residues within
the three immunoreceptor tyrosine-based activation motifs
(ITAMs) of the chain by Lck and Fyn, leading to the association
and activation of ZAP-70 (14). Once activated, Fyn, Lck, and

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_3, Springer Science+Business Media New York 2012

25
26 V.R. Moulton et al.

ZAP-70 cooperate in the tyrosine phosphorylation, activation, and

juxtaposition of downstream signal transducers that contribute to
the initiation of MAP kinase cascades, PI3-kinase activation, and
Ca2+ flux. Increase in the intracellular Ca2+ after T cell activation
gives rise to sequential activation of sets of genes that in turn
initiate proliferation, differentiation, and effector functions.
Basically, in addition to the complete investigation on the expres-
sion of various signaling molecules, T lymphocyte signaling is
analyzed at four stages to compare the (a) early (1, 2, and 3 min)
tyrosine phosphorylation of the cellular proteins, (b) intracellular
calcium response, (c) expression of cytokines, and (d) cell prolif-
eration. In normal T cells, the intensity of the T cell signaling
directly correlates with the level of expression of critical T cell
signaling molecules such as TCR chain. TCR chain is the limiting
factor in T cell receptor assembly, transport, surface expression,
and receptor function (5, 6). However, in SLE the cell signaling
remains abnormal and appears to correlate inversely with the level
of TCR chain. The exact nature of the mechanism that triggers
the inverse correlation has currently become a topic of intense
interest to many researchers.
The -chain exists in multiple forms and membrane fractions
with distinct functions. In addition to the soluble 16-kDa unphos-
phorylated form, the detergent-soluble fraction of TCR-chain
includes the phosphorylated p21- and p23-kDa forms and mono-
and polyubiquitinated forms. The detergent-insoluble membrane
fraction includes the actin cytoskeleton-bound form and lipid raft-
associated forms of the TCR-chain. Systemic lupus erythematosus
(SLE) T cells display lower levels of p16, p21, and 23-kDa and
detergent-insoluble membrane-bound forms of the TCR-chain
compare to normal T cells. In contrast, ubiquitinated forms of the
-chain are high in SLE T cells. We reported that membrane clus-
tering of the TCR-chain is significantly increased in SLE T cells.
A majority of patients with SLE also display decreased expression
of TCR-chain messenger RNA (mRNA) (7, 8). Aberrant splicing
of the 3 untranslated region and poor stability of the TCR mes-
sage (9, 10) are important contributors to this observed decrease
in mRNA expression.

1.2. Applications Signaling studies have several applications in SLE and other auto-
immune disorders. The precise pathologic mechanisms behind
abnormal T cell functions in SLE remain incompletely understood.
Identification of underlying genetic, molecular, and biochemical
mechanisms that is responsible aberrant T cell signaling will con-
tribute to our understanding of the pathogenesis of SLE as well as
provide novel targets for future pharmacological intervention.

1.3. Study Design Normal T cells or cells from patients with other autoimmune
diseases such as rheumatoid arthritis, Sjogrens syndrome, anti-
phospholipid syndrome and dermatomyositis can be used as
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 27

controls for SLE studies. Preferably, control samples should be

matched for age, race, and gender. Samples on different dates can
be compared by normalizing the data against proper internal con-
trol; however, it is preferable that samples be prepared in identical
fashion and stored for similar length of time before use. T cells can
be stored in liquid nitrogen indefinitely, although cell recovery and
viability diminishes significantly over time. It is important to distin-
guish between TCR/CD3-mediated signaling from non-receptor-
mediated signaling abnormalities in SLE T cells by comparing
TCR/CD3 activation using antibodies to stimulation with
phorbol-12-myristate 13-acetate (PMA)/ionomycin, which
bypasses the TCR. Studies on T cell signaling abnormalities should
be correlated with SLE disease activity index (SLEDAI) to distin-
guish abnormalities that are intrinsic to the disease from that are
associated with disease activity. It is also important to consider the
effects of patient medications, including both current and previous
medications, since the effect of some cytotoxic drugs are presumed
to last longer. To further confirm the results, the study should be
designed to follow up the patients over a period of 34 years dur-
ing which the SLE disease activity will improve considerably. If
possible, it is also a good idea to request the patients withhold their
medications (particularly corticosteroids) at least 24 h before draw-
ing the blood sample to reduce the effects of medication on in vitro
studies.

2. Materials

2.1. General Materials 1. RPMI-1640 (with L-glutamine) store at 4 C (Quality

and Supplies Biological Inc., Gaithersburg, MD).
2. Fetal bovine serum (FBS), heat inactivated, endotoxin tested
(Quality Biological Inc., Gaithersburg, MD).
3. Ficoll-Hypaque, LymphoprepTM (Axis-Shield PoC AS, Oslo,
Norway).
4. PBS 10-pH 7.4 (Quality Biological Inc., Gaithersburg, MD).
5. Bovine serum albumin (BSA)-Fraction V (Sigma Aldrich, St.
Louis, MO).
6. Trypan blue 0.4 % (Sigma Aldrich, St. Louis, MO).
7. DMSO (Sigma Aldrich, St. Louis, MO).
8. Ethylenediaminetetraacetic acid (EDTA), 0.5 M (Biofluids
Inc, Rockville, MD).
9. HEPES buffer 1 M (Sigma Aldrich, St. Louis, MO).
10. Blotto nonfat dry milk (Santa Cruz Biotechnology, Santa
Cruz, CA).
28 V.R. Moulton et al.
2.2. Antibiotics
2.3. Antibodies
2.3.1. Activating Antibodies
2.3.2. Cross-linking
Antibody
2.3.3. Isotype Controls
2.3.4. Staining Antibodies
11. RNase-free water (Sigma Aldrich, St. Louis, MO).
12. 15 and 50 ml disposable polypropylene conical tubes (Fisher
Scientific, Suwanee, GA).
13. 24-Well and 96-well flat bottom tissue culture polystyrene
plates with lids (Corning Inc., Corning, NY).
14. Disposable syringe filter assembly0.45 or 0.22 m(USA/
Scientific plastics, Ocala, FL).
15. Polyallomer centrifuge tubes 14 89 mm (Beckman, Palo
Alto, CA).
1. Penicillin/streptomycin (10,000 units/ml penicillin and
10,000 units/ml streptomycin) (Quality Biological Inc.,
Gaithersburg, MD). Dilute 1:100 in media for use.
1. Anti-CD3 IgG, clone OKT3 (Orthobiotech, Raritan, NJ).
Available as 5 ml ampules of 0.5 mg/ml. After opening the
ampule the antibody should be aliquoted into 100 l under
sterile conditions and stored at 4 C.
2. Anti-CD3 IgG, clone UCHT1 (BD Pharmingen, San Diego,
CA).
3. Anti-CD3 IgM, clone 2Ad2a2 was a generous gift from Dr.
Ellis Reinherz, Dana Farber Cancer Institute, Boston, MA.
4. Anti-CD28 IgG (NA/LETM), clone-CD 28.2 (BD Pharmingen,
San Diego, CA).
Goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
1. Mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
2. Rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
3. Goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
4. Human IgG (Sigma Aldrich, St. Louis, MO) used for blocking
antibodies.
1. Anti-phosphotyrosine, HRP-conjugated or unconjugated,
clone 4G10 (Upstate Biotechnology (now part of Millipore),
Billerica, MA).
2. Anti-CD3, clone M-20 (Santa Cruz Biotechnology).
3. Anti-CD3, carboxy terminal binding antibody, clone C-20
(Santa Cruz Biotechnology).
4. Anti-FcR (Upstate Biotechnology (now part of Millipore),
Billerica, MA).
5. Anti-actin, clone I-19 (Santa Cruz Biotechnology).
6. Anti-LAT, clone FL-233 (Santa Cruz Biotechnology).
7. Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology).
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 29

2.3.5. Fluorescent-Labeled All the immunoglobulins have minimum crossreactivity with

Antibodies bovine, chicken, goat, guinea pig, Syrian hamster, horse, human,
mouse (except three), rat, and sheep IgG.
1. TRITC-labeled donkey anti-rabbit IgG (H + L) (Jackson
ImmunoResearch Lab, West Grove, PA).
2. TRITC-labeled donkey anti-goat IgG (H + L) (Jackson
ImmunoResearch Lab).
3. FITC-labeled anti-mouse IgG (H + L) (Jackson Immuno-
Research Lab).
4. FITC-labeled donkey anti-rabbit IgG (H + L) (Jackson
ImmunoResearch Lab).

2.4. Kits 1. Magnetic column separation (MACS) Pan human T cell isola-
tion kit (Miltenyi Biotech, Auburn, CA).
2. RosetteSep-human T cell enrichment Cocktail (StemCell
Technologies Inc., Vancouver BC, Canada).
3. Quantikine human IL-2 Immunoasssay kit (R&D Systems Inc,
Minneapolis, MN).
4. Protein assay kit (BioRad, Hercules, CA).
5. RNeasy Mini kit (Qiagen, Valencia, CA).

2.5. Electrophoresis, 1. Novex precast gels 412 % BisTris NuPageTM gels (Invitrogen,
Western Transfer, Carlsbad, CA).
Immunoblotting, and 2. Whatman filter papers (Fisher Scientific, Suwanee, GA).
Immunopreciptation
3. PVDF membrane, Immobilon-P, Pore size: 0.45 m (Sigma
Reagents Aldrich, St. Louis, MO).
4. NuPageTM-reducing agent (10) (Invitrogen Corporation,
Carlsbad, CA).
5. NuPageTM-transfer buffer (20) (Invitrogen Corporation,
Carlsbad, CA).
6. NuPageTM-LDS (laureal dodecyl sulfate) sample buffer (4)
(Invitrogen, Carlsbad, CA).
7. SeeBlue Plus 2 protein ladder (Invitrogen, Carlsbad, CA).
8. Protein A/G plus-agarose-2 ml 50 % slurry (Santa Cruz
Biotechnology, Santa Cruz, CA).
9. Phenyl phosphate disodium salt (Sigma Aldrich, St. Louis, MO).
10. Kodak X-ray film (Sigma Aldrich, St. Louis, MO).
11. Tween-20 (Sigma Aldrich, St. Louis, MO).
12. Triton-X-100 (Fisher Scientific, Suwanee, GA).

2.6. Flow Cytometry 1. FACS tubes, 12 75 mm polypropylene test tubes without cap
and Fluorescence (Fisher Scientific, Suwanee, GA).
Microscopy Reagents 2. Polylysine-coated Poly Prep slides (Sigma Aldrich).
30 V.R. Moulton et al.
2.7. RT-PCR, Gene
Expression Analysis
2.8. Density Gradient
Centrifugation and
Protein Concentration
2.9. Instruments
10. NuPage gel casts, and transfer casts (Invitrogen, Carlsbad, CA).
3. Pap pen with normal large tip (Electron Microscopy Sciences,
Washington, PA).
4. Cover glasses that are 22 22 mm by 0.130.17 mm thick
(VWR Scientific, Buffalo Grove, IL).
5. Gel/MountTM (Biomedia Corp., Hayward, CA).
6. Cytoseal mounting medium (Fisher Scientific).
7. Saponin (Sigma Aldrich).
8. Digitonin (Sigma Aldrich).
9. Glycine (Bio-Rad).
1. Ethidium bromide (Sigma Aldrich).
2. UltraPure agarose (Invitrogen, Carlsbad, CA).
3. Taq polymerase (Promega Corp.).
4. PCR beads (Amersham Pharmacia, Piscataway, NJ).
5. X-gal (Sigma-Aldrich).
1. Sucrose (Bio-Rad, Hercules, CA).
2. Trichloroacetic acid (TCA) (Sigma Aldrich).
3. Sodium chloride (Sigma Aldrich).
4. Morpholino ethane sulfonic acid (MES) (Sigma Aldrich).
5. Brij58 (Sigma Aldrich).
6. Triton X-100 (Fisher Scientific, Suwanee, GA).
7. Acetone (Fisher Scientific).
1. Table top centrifuge-Sorvall-RT 6000D, rotor: H-1000 B
(Kendro Laboratory products, Newtown, CT).
2. Table top micro-tube centrifugerefrigeratedBiofuge-
Fresco (Kendro Laboratory products, Newtown, CT).
3. Non-refrigerated, Micromax centrifuge (International
Equipment Company, Needham Heights, MA).
4. Hemocytometer and coverslips (Fisher Scientific, Suwanee, GA).
5. Flow cytometer, model FACSCalibur (Becton Dickinson, San
Jose, CA).
6. Epics Altra flow cytometer (Coulter Beckmann, Hailegh, FL).
7. Spectrophotometer (UVSpec) and software (Pharmacia-
Amersham, Piscataway, NJ).
8. Thermomax plate reader (Molecular Devices, Sunnyvale, CA,
Software-SoftPro10).
9. Gel docking station and software (Software, Quantity One,
Bio-Rad, Hercules, CA).
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 31

11. Power supplies, BioRad Power Pac 300 (Bio-Rad, Hercules, CA).
12. Light microscope, Olympus (OPELCO, Dulles, VA).
13. Dounce homogenizer (Fisher Scientific).
14. Beckman ultracentrifuge and SW41 rotor (Palo Alto, CA).
15. PCR machine, T3 thermocycler (Biometra, Heidelberg,
Germany).
16. Lightcycler 480 real-time PCR instrument (Roche,
Germany).
17. Confocal microscope, Bio-Rad Radiance 2100 (Bio-Rad; Laser
Sharp 2000 software).
18. Olympus model IX70 fluorescence microscope (OPELCO,
Dulles, VA).
19. Nucleoporator (Lonza, Cologne, Germany).
20. Sonicator (Fisher Scientific).
21. Nanodrop 2000 spectrophotometer (Thermo Scientific,
Wilmington, DE).

3. Methods

3.1. Isolation The blood should be collected in heparin green top blood collec-
and Cell Culture tion tubes and kept at room temperature (see Note 1). Peripheral
of T Lymphocytes blood mononuclear cells (PBMCs) are isolated by density gradient
centrifugation over Ficoll-Hypaque and the whole process is done
3.1.1. Separation
at room temperature in a sterile hood.
of Peripheral Blood
Mononuclear Cells 1. Aliquot blood into 50 ml conical disposable centrifuge tubes,
10 ml per tube.
2. Add 30 ml RPMI 1640 medium with Pen-Strep, cap, and mix
by inverting.
3. Carefully underlay the sample with 10 ml Lymphoprep.
4. Centrifuge in a Sorvall table top centrifuge at 650g for 45 min.
Turn off brake during centrifugation to prevent mixing of
the density gradient layers during deceleration process.
5. Remove the interface of white ring or buffy coat containing
PBMCs and transfer into new 50 ml conical tubes.
6. Dilute the PBMCs with 3 volumes of RPMI-1640 with Pen-
Strep, mix, and centrifuge at 370g for 5 min.
7. Discard the supernatant and resuspend the cells in appropriate
volume of RPMI-1640.
8. Count the cells using a hemocytometer and light microscope by
diluting 10 l of cell suspension into 200 l trypan blue. The
32 V.R. Moulton et al.

cells should be counted from all four corner squares and the
total number obtained should be more than 200 for accurate
results. Total number of cells = cell count/4 20 104 cells/ml.
Note: If the PBMCs are contaminated with red blood cells (RBCs)
and the sample appears red it can be hemolyzed using ACK lysis
buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA).
PBMCs should be maintained in RPMI containing Pen-Strep and
10 % FBS at 37 C in a 5 % CO2 incubator. If going directly on to
T cell isolation, the serum can be omitted from the medium.

3.1.2. Isolation of T Magnetic separation and RosetteSep methods routinely yield >97 %
Lymphocytes from PBMCs CD3-positive T lymphocytes and have distinct advantages.
Rosetting using sheep RBCs is inexpensive but is less preferred due
to less pure yields (see Note 2).

Magnetic Separation Using T cells can be isolated by negative depletion by magnetic separation
MicroBeads using a kit from Miltenyi Biotech. This procedure is simple and
involves incubation of the PBMCs with a cocktail of hapten-
conjugated antibodies that bind to non-T lymphocytes followed
by removal of the antibody bound non-T lymphocyte by a magnetic
bead coated with secondary anti-hapten antibody. The non-T
lymphocytes bound to magnetic beads are retained in a column
under the influence of a powerful magnet. The flow-through
containing purified T cells are used for the studies. Although the
manufacturers protocol suggests that PBMCs may be stored in
the refrigerator overnight in PBS containing 2 mM EDTA supple-
mented with 10 % autologous serum after the last washing step
before separation using MACS column, we found that storing SLE
PBMCs under these conditions sometimes results in large clumps
that fail to resuspend and later clog the separation column.
Overnight incubation of SLE PBMCs also leads to decreased
viability of the cells and cell lysis.
1. Count the PBMCs and wash them in 10 ml MACS buffer
(1 PBS, 1 % FBS or 0.5 % BSA, 2 mM EDTA).
2. Remove the supernatant very carefully and resuspend the cells
in 80 l (per 107 cells) ice cold MACS buffer.
3. Add 20 l hapten-antibody cocktail per 107 cells, mix well, and
incubate at 6 C for 10 min.
4. Wash two times with MACS buffer (20 times the labeling
volume).
5. Resuspend the cells in 80 l MACS buffer and add 20 l MACS
anti-hapten MicroBeads per 107 cells, mix well, and incubate at
6 C for 15 min.
6. Wash the cells one time as in step 4 and resuspend in 500 l
MACS buffer.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 33

7. Set up the MACS column in between the magnet and wash the
column with 3 ml of MACS buffer.
8. Load the cells to the column and collect the flow-through as
well as washing with 10 ml of MACS buffer.
9. Centrifuge the T-lymphocytes at 200g for 5 min in Sorvall
table-top centrifuge and discard the supernatant.
10. Resupend the cells in appropriate volume of RPMI-1640
containing 10 % FBS and Pen-Strep.

RosetteSep T lymphocytes can be directly isolated from the SLE blood samples
using this protocol. The method of T cell isolation by RosetteSep
is essentially based on cross-linking the non-T lymphocytes with
antibodies coated to latex beads that separate with RBCs in Ficoll-
Hypaque density gradient centrifugation. This negative selection
protocol takes less time than MACS separation. However, the yield
of purified T lymphocytes is slightly lower than MACS isolation
from PBMCs.
1. Aliquot blood into 50 ml conical disposable centrifuge tubes,
10 ml per tube.
2. Add 250 l StemCell T cell isolation antibody to each tube.
Mix well using a 25 ml pipette and incubate at room tempera-
ture for 30 min.
3. Add equal volume of PBS with 10 % FBS; mix by gentle
pipetting so as not to disturb the already-formed rosettes.
4. Underlay the sample with 10 ml of Lymphoprep and centrifuge
at 1000g for 30 min at room temperature, with the brake
turned off to prevent layers from mixing during deceleration.
5. Carefully collect the white interphase layer of cell and transfer
to fresh tubes.
6. Wash cells by adding 10 ml of PBS with 10 % FBS, followed by
centrifugation for 10 min at 650g (brake may be turned on
again).
7. Repeat wash step.
8. Resuspend the purified T cells in 1 ml of 10 % FBS plus Pen-
Strep. Count cells as per Subheading 3.1.1.
9. If not being used immediately, T cells may stored in liquid
nitrogen as cell suspension in 90 % FBS with 10 % DMSO. Cell
density should not exceed 106 cells/ml. Cells should be frozen
first at 80 C for 24 h before transfer to liquid nitrogen tank.

3.1.3. Cell Culture 1. Prepare culture media with RPMI-1640 containing 10 % FBS
of T Lymphocytes and Pen-Strep.
2. Add 1 g/ml phytohemagglutinin.
34 V.R. Moulton et al.

3. Resuspend the T cells at a concentration of 1 106 cells/ml in

the above medium and transfer to 37 C 5 % CO2 incubator.
4. After 2 days, centrifuge the cells at 200g for 5 min and resu-
pend in fresh culture medium containing 1 g/ml phytohe-
magglutinin and 25 units/ml interleukin-2.
5. Change the medium when the color becomes yellow (usually
every 25 days). Always resuspend the cells at 1 106 cells/ml.
Note: SLE T cells grown under these conditions and can be main-
tained up to 40 days. It should be always kept in mind that growth
under these conditions likely selects out healthier T cells that may
be less likely to reflect the SLE phenotype.

3.2. Analysis of TCR/ Intracellular calcium flux response is measured by flow cytometry
CD3-Mediated after labeling the cells with INDO-1 (Molecular Probes, Eugene,
Signaling in OR) (11). INDO-1 loaded cells are analyzed using an Epics Altra
Lymphocytes (Coulter Beckmann, Hialeah, FL) flow cytometer equipped with a
high power dual wavelength (365 and 488 nm) argon laser and
3.2.1. Measurement UV source. In each run, the cells are first run unstimulated to
of TCR/CD3-Mediated record the baseline fluorescence ratio, which represents the resting
Intracellular Calcium [Ca2+]i levels. After 40 s either antibody OKT3 or other activating
Response in SLE T Cells antibodies or the isotype control mIgG2a, is added to the tube
followed by cross-linking with the goat anti-mouse IgG at 130 s
and the calcium response is recorded for 10 min. The mean
fluorescence ratio calculated by the software MultiTime (version 3,
Phoenix Flow Systems, San Diego, CA) is directly proportional to
the free cytosolic Ca2+.
It is very important to perform the calcium analysis as soon as
possible after blood collection to be representative of signaling
in vivo. Isolated T lymphocytes (5 106/ml) should be recovered
in RPMI 1640 containing 10 % FBS and Pen-Strep at 37 C in 5 %
CO2 for 46 h for an optimal response and should not be
maintained more than 16 h. Longer periods of incubation of the
lymphocytes in culture medium may decrease the effective calcium
response qualitatively and quantitatively. When comparing the
intracellular calcium response of SLE T cells to normal T cells,
experiments should be done simultaneously on the same day to
avoid instrumental variations.
1. Prepare 100 ml of RPMI-1640 medium containing 1 % FBS,
10 mM HEPES, pH 7.4, and Pen-Strep and warm to 37 C.
2. Take 1 ml of SLE T cells maintained in RPMI-1640
(1 106 cells/ml) in a flow cytometry tube. Add 1 l INDO-1
(add 50 l DMSO to one vial of 50 g INDO-1, keep pro-
tected from light) and incubate at 37 C for 1 h.
3. Dilute 200 l INDO-1 loaded T lymphocytes into 2 ml in pre-
warmed culture medium and load onto EPICs Altra flow
cytometer.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 35

4. The sample should be run at a flow rate of approximately

150 events/s to increase the efficiency. Flow rates that are too
fast can result in clogging the tube. As the analysis time is long,
the slower flow rate is also helpful to prevent using up the
sample too quickly.
5. After recording the baseline for 40 s, add 2.5 g OKT3, mix
the samples, and allow to run for another 50 s.
6. Add 20 g/ml goat anti-mouse antibody, mix, and allow to
run for 10 min with intermittent shaking.
7. Data can be analyzed using MultiTime software and plotted as
mean ratio or percent responding cells.
Note: Mean fluorescence ratio calculated by the software MultiTime
is directly proportional to the free cytosolic Ca2+ (Fig. 1a). The
intracellular calcium response can also be represented as percent-
age of responding cells. A positive responding cell is one whose
[Ca2+]i is increased by two standard deviations above mean back-
ground levels. Intracellular calcium response data can be inter-
preted by calculating the total area under the response curve or by
taking the peak mean ratio at a selected time point. After the peak

Fig. 1. TCR/CD3-induced intracellular calcium response in SLE T cells. 1 106 SLE T cells
were resuspended in 1 ml pre-warmed culture medium and loaded with 1 L (1 g/ml in
DMSO) INDO-1 (Molecular Probes, Eugene OR) for 60 min at 37 C. INDO-1 loaded cells
were diluted ten times (0.22 ml) with pre-warmed filtered RPMI containing 1 % FBS.
After establishing the baseline fluorescence ratio which represents the resting [Ca2+]i lev-
els for 40 s, the cells were stimulated with either OKT3 (2.5 g/ml), or the isotype control
mIgG2a followed by 10 g/ml of goat anti-mouse Ab at 130 s. The intracellular calcium
response was continuously monitored for 10 min with intermittent shaking.
36 V.R. Moulton et al.
3.2.2. Tyrosine
Phosphorylation of Cellular
Protein Substrates
Cell Activation and Lysis
Protein Assay
response, lack of gradual decrease in the intracellular calcium
response to the baseline suggests a sustained response and can be
interpreted as abnormal termination of T cell signaling. More than
1 l INDO-1 per 1 ml of cells should not be used because the
DMSO can reduce the calcium response. When running at high
flow-rate a 10 min recording may require more than 2 ml of cells.
As an alternative to OKT3, an anti-CD3 IgM antibody can be
used; this does not require use of a secondary cross-linking anti-
body. Activation with PMA/ionomycin can also be used to evalu-
ate non-TCR-mediated activation.
1. Transfer 5 106 SLE T cells in 500 l serum-free RPMI-1640
medium to a 1.5 ml microfuge tube.
2. Incubate the cells at 37 C for 10 min in a water bath.
3. Stimulate the cells with the addition of 1 g/ml OKT3 for 0,
1 or 2 min at 37 C. (Antibody 2Ad2a2-IgM 1:200 dilution or
UCHT1 10 g/ml antibody can be substituted for OKT3.
When using OKT3 Ab, it can be cross linked by adding 10 g/
ml goat anti-mouse Ab immediately to increase the level of
tyrosine phosphorylation).
4. Stop the reaction by adding 0.5 ml ice-cold 2 stop buffer
(50 mM TrisHCL, 100 mM NaCl, 100 mM NaF, 2 mM
Na3VO4, 10 mM EDTA, 10 mM sodium pyrophosphate,
2 mM PMSF, 20 g/ml leupeptin and 20 g/ml aprotinin).
5. Pellet cells by centrifugation, discard supernatant, and resus-
pend pellet in lysis buffer (working solution for 1 ml: 2 stop
buffer 500 l; water 483 l; 0.5 M EDTA, 5 l; aprotinin,
1 l; 5 mg/ml leupeptin, 1 l; 0.2 M PMSF 5 l; 200 mM
Na3VO4, 5 l) containing 1 % Nonidet P-40 (NP-40) (Sigma
Aldrich, St. Louis, MO).
6. Incubate the cells at 4 C for 1 h in a shaker in the cold room.
7. Centrifuge the cells at 18,000g or top speed for 10 min at 4 C
and collect the supernatant. The pellet that can be used to eval-
uate tyrosine phosphorylation of proteins in detergent-insolu-
ble membrane fractions should be washed twice with 100 l
lysis buffer.
8. Both the pellet and the cytoplasmic protein extract should be
stored at 80 C.
Protein concentrations can be measured with any of the standard
commercially-available kits such as the Bradford assay systems from
BioRad (Hercules, CA) or Sigma (St. Louis, MO). Normal T lympho-
cytes yield approximately 25 g/l protein if five million cells are
lysed in 100 l lysis buffer. Jurkat T lymphoma cell lines yield
approximately 20 g/l protein if five million cells are lysed in
150 l lysis buffer. Generally, electrophoresis of 1520 g of the
protein sample is required for easy detection by immunoblotting.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 37

Protein Separation by Depending on the molecular weight of the protein under investigation,
Polyacrylamide Gel different gel types can be used (see Note 3). Upper molecular
Electrophoresis weights above 100 kDa can be better resolved with a 6 % gel,
50100 kDa can be better resolved with a 10 % gel and to visualize
lower molecular weight proteins a 12 % gel can be used. Sometimes
a gradient gel such as a 412 % BisTris gel may be necessary to see
the whole spectrum of proteins. The Invitrogen-Novex pre-cast
gel is set up after removing the tape and comb in the electrophore-
sis apparatus according to manufacturers instructions and both
inner and outer chambers are filled with SDS running buffer. The
samples are denatured in SDS loading buffer and reduced using
DTT or 2-mercaptoethanol (12).
1. Pipette 1015 g protein lysate from different samples in a
1.5 ml microfuge tube.
2. Add equal volume of 4 loading buffer (Invitrogen-Novex,
Carlsbad, CA) containing bromophenol blue, SDS (sodium
dodecyl sulfate) and 2 mercaptoethanol. The total volume of
the sample is limited by the size of the well, and in general
should not exceed 2530 l.
3. Boil for 5 min in a water bath and cool to room temperature.
4. Assemble the pre-cast gel in the electrophoresis apparatus
according to manufacturers instructions.
5. Fill inner and outer chambers with running buffer. Gently flush
the wells with running buffer using a pipette.
6. Load samples carefully into each well.
7. Load protein ladder (SeeBlue Protein Plus, Invitrogen).
8. Run gel for 1 h at constant voltage of 200 V until the dye front
reaches the bottom of the gel. Remove the gel and continue
with staining or transfer to membrane.
Note: Care should be taken to avoid spillage of samples into adja-
cent wells. The voltage should be reduced if smiling of the gel
occurs.

Transfer to Membrane 1. Wearing gloves, cut the PVDF membrane to a rectangle

12 mm larger than the gel.
2. Wet membrane in methanol for 2030 s; rinse with distilled
water and soak in transfer buffer.
3. Soak pads, filter paper and membranes for 15 min in transfer
buffer (for 1 l, 50 ml of 20 transfer buffer, 849 ml distilled
water, and 100 ml of methanol).
4. Disassemble gel from plastic casing and soak for 5 min in
transfer buffer.
5. Carefully assemble transfer sandwich in the following order:
negative electrode/pad/pad/filter/gel/membrane/filter/pad/
pad/positive electrode, ensuring that there are no bubbles.
38 V.R. Moulton et al.

6. Transfer to electrophoresis apparatus.

7. Fill the inner chamber with transfer buffer until the pads just
covered. Fill outer chamber with distilled water.
8. Run at 30 V constant for 1 h. Alternatively, run at 1015 V
overnight at 4 C.
9. Disassemble the sandwich and mark front side of the mem-
brane with a pen.
Note: The membrane can be stained with Ponceau S (0.3 % Ponceau
S in 5 % TCA) to confirm the transfer. Place the membrane in
Ponceau S solution for 5 min at room temperature, destain with
water, photograph it and completely destain with water by soaking
for 10 min. Over transfer and loss of proteins may occur if transfer
is prolonged beyond 1 h.

Immunoblotting 1. Rinse the membrane in 1 TBS and transfer it to a plastic box.

2. Block the membrane with 35 % BSA in TBS for 1 h. If not
using anti-phosphotyrosine antibody, dry milk (35 %) may be
substituted.
3. Discard solution and add primary antibody diluted in fresh
blocking solution. Anti-phosphotyrosine antibody-HRP is
used at 1:1,500 dilution; anti-TCR antibody is diluted 1:1,000
in blocking solution. The membrane is incubated for 12 h at
room temperature or overnight at 4 C with agitation using a
rocker.
4. Wash the membrane three times in 15 ml of TBS-T (1 TBS,
0.1 % Tween 20), 10 min each.
5. If the antibody used is not conjugated with HRP, then a
second incubation for 1 h with anti-mouse HRP (1:2,000 in
fresh blocking solution) is required. This is followed by another
three washes of TBS-T before development.

Detection 1. Mix equal volumes of ECL reagents 1 and 2 (Amersham-

Pharmacia); typically 2 ml total volume is sufficient for one
blot.
2. Pipet mix over the blot, ensuring that reagent completely cov-
ers the membrane. Incubate for 1 min in the dark.
3. Drain the ECL reagent from the membrane, dry the mem-
brane gently and then put in between plastic envelope or saran
wrap and place it on a detection cassette.
4. Cut Kodak X-ray film (Sigma Aldrich, St. Louis, MO) to
appropriate size, bend upper right hand corner to identify the
position and put the film on the membrane and expose for 10 s
and a second film for 1 min.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities
3.2.3. Kinetics of Tyrosine
Phosphorylation of Cellular
Proteins
3.2.4. Western Blot
Stripping and Reprobing
3.2.5. Immunoprecipitation
39
5. Develop the film in Kodak automatic processor. If necessary
increase or decrease the exposure time.
Note: Immunoblotting for phosphotyrosine should be done first
before stripping and re-blotting for other proteins (see Note 3).
Do not let the blot to dry out during any step of immunoblotting
as this will increase background staining.
The kinetics of early tyrosine phosphorylation of cellular proteins
can be studied by activation of SLE T cells for 0, 1, 2, and 3 min,
and analysis of tyrosine phosphorylated proteins as mentioned
above. The kinetics of phosphorylation of cellular protein sub-
strates in faster in SLE T cells compared normal T cells (Fig. 2).
1. After ECL detection, wash membrane with TBS-T for 5 min
before incubating with Immunopure stripping buffer (Pierce
Chemical Co., Rockford, IL) for 90 min at room temperature.
2. Wash membrane three times with TBS-T as described in
Subheading Immunoblotting.
3. The membrane can then be reprobed for another protein by
following the same protocol as in Subheading Immunoblotting.
Membranes can be stripped and re-probed at least 34 times,
but sensitivity will diminish significantly with each stripping.
Immunoprecipitation is used to increase detection of low quantity
proteins, or to detect interactions between proteins. In this method,
an antibody against the TCR is used to pull out the CD3 and
Fig. 2. Increased phosphorylation of TCR/CD3-mediated phosphorylation of TCR chain
with faster kinetics in SLE T cells. Magnetically purified SLE T cells were stimulated
through TCR/CD3 with 10 g/ml of OKT3 and 20 g/ml goat anti-mouse Ab for 0, 1, 2 min
and lysed. Proteins (15 g) were analyzed on NuPAGE under reducing conditions and
immunoblotted with horseradish peroxidase-conjugated anti-phosphotyrosine antibody,
4G10. SLE T cells show supranormal phosphorylation of cellular protein substrates with
faster kinetics compared normal T cells.
40 V.R. Moulton et al.

associated proteins in complex with CD3. The extracted proteins

can then be run on a gel and detected by immunoblotting.
1. Lyse 20 million cells in 250 l lysis buffer and prepare the
cytoplasmic extract as described above.
2. Preclear the cell lysate by adding protein A/G agarose/
Sepharose by adding 25 l of 50 % slurry and incubate at 4 C
for 10 min on a rocker or orbital shaker.
3. Remove the protein A/G agarose beads by centrifugation at
18,000g or top speed at 4 C for 10 min and transfer the
supernatant to a fresh tube.
4. Add 10 g of anti-CD3 antibody and mix for 2 h at RT or
overnight at 4 C on an orbital shaker.
5. Capture the immunocomplex by adding 25 l protein A or
protein G agarose/Sepharose bead slurry (50 % slurry) by
gently rocking in a rocker or orbital shaker for 2 h or over-
night at 4 C.
6. Collect the agarose/Sepharose beads by centrifugation for 15 s
at 18,000g.
7. Discard the supernatant and wash the beads three times with
250 l lysis buffer.
8. Resuspend the beads in 2 SDS sample buffer, mix, and boil
for 5 min. The supernatant can then be loaded onto electro-
phoresis gel as described in Subheading Protein Separation by
Polyacrylamide Gel Electrophoresis.
9. Electro-transfer the samples to PVDF membranes and immu-
noblot with TCR-chain antibody or antibodies to other
downstream signaling molecule.

3.2.6. Estimation of Lipid The functionally important lipid raft-associated form of the
Raft-Associated TCR- TCR-chain is quantitated by treating the SLE T cells with cho-
Chain lesterol-solubilizing agent methyl--cyclodextrin, which disrupts
lipid rafts. The increase in the amount of TCR-chain in the
detergent-soluble fraction after treatment corresponds to the lipid
raft-associated form of TCR-chain (see Note 4). Application of
this method has shown that in contrast to normal controls, the
TCR-chain is more segregated within lipid rafts in SLE T cells
(7, 13) (Fig. 3a). The membrane-bound linker for activation of T
cells (LAT) is used to demonstrate co-localization of the TCR
within lipid rafts.
1. Pipette 5 106 T cells into two microfuge tubes and resuspend
in 1 ml of RPMI-1640 without serum.
2. T cells are treated with or without 30 mM methyl--
cyclodextrin (Sigma Aldrich) for 30 min at room temperature.
3. Lyse cells in 1 % NP-40 lysis buffer as described in Subheading
Cell Activation and Lysis and centrifuge at 18,000g for
10 min at 4 C.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities
3.2.7. Lipid Raft Isolation
41
Fig. 3. Analysis of the membrane lipid raft-associated and actin cytoskeleton associated
forms of the TCR-chain in SLE T cells. (a) SLE T cells were treated with the lipid raft-
disrupting agent methyl--cyclodextrin for 30 min. The cells were lysed, and the
detergent-soluble and detergent-insoluble fractions were collected by centrifugation,
separated by SDS-PAGE, transferred, and blotted with the TCR-chain C-terminal mono-
clonal antibody (mAb). On treatment with methyl--cyclodextrin, the increased level of the
p16 and p21 23-kDa forms of the TCR-chain in the detergent-soluble fraction represent
lipid raft-associated TCR-chain. Densitometric analysis showed that, in SLE T cells, the lipid
raft-associated TCR-chain is increased compared to normal donors. (b) Sucrose density
gradient analysis showing distribution of LAT in raft and nonraft fractions; 100 106 cells
were lysed with a lysis buffer containing 1 % Brij58 and fractionated by discontinuous
sucrose gradient. Immunoblot analysis of LAT was performed with an equal volume of cell
lysate from each fraction. Raft and nonraft fractions were as indicated.
4. Run both detergent-soluble and insoluble fractions on a 12 %
gel and transfer to PVDF membranes (Millipore, Bedford,
MA). Immunoblot membranes with TCR-chain C-terminal
antibody and other relevant antibodies and develop as
described above.
Lipid raft and associated proteins can be isolated from T cells using
density gradient centrifugation in an ultracentrifuge (14, 15). This
allows more comprehensive evaluation of the lipid raft components.
1. T cells (25 million) are pelleted and lysed in 1 ml of 1 % Brij58
or 0.2 % Triton X-100 lysis buffer (0.2 % Triton X-100 or 1 %
Brij58 in MBS buffer [25 mM MES, 150 mM NaCl, pH 6.5],
supplemented with 1 mM sodium orthovanadate, 2 mM
EDTA, 1 mM PMSF, and 1 g/ml aprotinin). The lysis buf-
fer is prepared as follows for 200 ml stock solution: 2 g 1 %
Brij58 or 400 l 0.2 % Triton X-100 400, 1.0665 g 25 mM
MES, 1.743 g 150 mM NaCl. Freshly add 800 l 2 mM
EDTA of 0.5 M, 1 mM Na3VO4, 1 mM PMSF, 1 g/ml
aprotinin.
42 V.R. Moulton et al.

2. Homogenize the cell lysate with 15 strokes of loose-fitting

Dounce homogenizer and incubate on ice for 30 min.
3. Mix the lysate with an equal volume of 85 % sucrose and trans-
fer to the bottom of a 12-ml ultracentrifuge tube. Sucrose gra-
dient solutions are 85 % w/v sucrose in MBS (42.5 g in 50 ml
MBS), 35 % w/v sucrose in MBS (17.5 g in 50 ml MBS), and
5 % w/v sucrose in MBS (2.5 g in 50 ml MBS).
4. Place a step gradient of 6 ml of 35 % sucrose and 4 ml of 5 %
sucrose solution containing 1 mM Na3VO4 and 2 mM EDTA
on top of the lysate.
5. Centrifuge samples for 1822 h at 200,000 g (39,000 rpm)
at 4 C using a SW-41 rotor in a Beckmann ultracentrifuge.
6. Collect 1 ml fractions starting from the top with a 1-ml pipet
(12 fractions). The 1-ml samples can also be collected from the
bottom by carefully piercing the bottom of tube using a syringe
needle.
7. When 1 ml samples are collected from the top, fractions 4 and
5 contain the cloudy band represent the raft fractions, and frac-
tions 11 and 12 are soluble membrane fractions (Fig. 3b),
identified by LAT staining.

Concentration This step is optional and may be required to concentrate proteins

of Protein in Lipid Raft from the fractions, especially if using fewer cells.
Fractions by TCA
1. Add 250 l of 100 % TCA (add 350 ml water to 500 g TCA;
Precipitation
store at room temperature).
2. Mix and incubate on ice for 10 min.
3. Centrifuge in a microfuge at 18,000g or top speed for 5 min.
4. Remove the supernatant carefully, leaving behind the white
pellet.
5. Wash the pellet twice with 200 l cold acetone by resuspend-
ing and spinning in the microfuge at 18,000g for 5 min.
6. Air-dry the pellet for 510 min to dry residual acetone.
7. Add 2 SDS-PAGE sample buffer and reducing agent (5 %
2-mercaptoethanol or 0.1 M DTT), boil for 10 min, and load
on SDS-PAGE.

3.2.8. Cell Proliferation SLE T cells do not proliferate as well as cells from normal con-
Assay trols. This is measured most commonly by assaying thymidine
incorporation using radioactive-labeled thymidine. This protocol
also uses immobilized stimulating antibodies, obviating need for a
cross-linker.
1. Pre-coat a 96-well culture plate with 100 l per well of anti-
CD3 antibody (OKT3, 110 g/ml) and anti-CD28 antibody
(12.5 g/ml), diluted in PBS. Wrap plate in plastic wrap and
 3 Methods and Protocols to Study T Cell Signaling Abnormalities
3.2.9. Cytokine Expression
3.3. Gene Expression
Studies of Signaling
Molecules in SLE T
Cells
3.3.1. Reverse
Transcriptase Polymerase
Chain Reaction
Isolation of mRNA
43
incubate overnight at 4 C. Remove antibody solution before
using.
2. Aliquot 1 105 SLE T cells to each well (triplicates) in RPMI-
1640 containing 10 % FBS, setting up triplicate wells for each
condition.
3. Incubate cells at 37 C for 3 days.
4. Add 1 Ci of [3H] thymidine/well (6.7 Ci/mmol), and
incubate for 18 h.
5. Harvest the cells using a Tomtec 96-well plate harvester
(Wallac, Gaithersburg, MD).
6. Quantitate incorporated radioactivity using a Microbeta Tri-
luxe plate scintillation counter (Wallac, Gaithersburg, MD).
1. Plate 5 106 SLE T cells in 1 ml medium in a 24 well culture
plate.
2. Activate cells by adding 10 g/ml anti-CD3 antibody plus
2.5 g/ml anti-CD28 antibody for 24 or 48 h.
3. Transfer cell suspension to a microfuge tube and centrifuged at
18,000g for 2 min at 4 C.
4. Harvest supernatants and use immediately or freeze at 80 C.
5. Measure IL-2 concentration using 100 l of the culture super-
natant in triplicates by Quantikine ELISA kit (R&D systems,
Minneapolis, MN). A standard curve should always be estab-
lished for reliability and reproducibility.
6. The levels of IFN- and IL-4 or other cytokines in culture
supernatants can be measured similarly using other commer-
cially available kits.
7. Read the absorbance of the color reactions at 450 nm in an
ELISA microplate reader (Bio-Rad, Hercules, CA).
Aberrant protein expression of signaling molecules, including
TCR, can be analyzed in further detail through molecular biology
techniques (see Note 5).
1. Lyse 5 106 cells in 350 l RLT buffer supplied with Qiagen
RNeasy minikit (Qiagen Inc.). If not used immediately, the
sample can be frozen at 80 C for several months.
2. Homogenize the samples by centrifugation at 18,000g for
2 min in a Qiashredder.
3. Add 350 l of 70 % ethanol to the homogenized lysate and mix
well by pipetting.
4. Apply 700 l of the sample to RNeasy minicolumn placed in a
2-ml collection tube and spin at 10,000g for 15 s. Discard the
filtrate, reload the remaining sample, and spin as above.
44 V.R. Moulton et al.

5. Add 700 l of buffer RW1 to the RNeasy column and spin at

10,000g for 15 s. Discard the flowthrough and collection
tube.
6. Transfer RNeasy column to a new 2-ml collection tube and
pipet 500 l buffer RPE; centrifuge the tube at 18,000g for
15 s. Discard the flowthrough.
7. Add 500 l RPE buffer to the RNeasy column and centrifuge
at 18,000g for 1 min.
8. Transfer the RNeasy column to a new 1.5-ml collection tube.
Pipet 3050 l RNasefree water directly onto the RNeasy silica
gel membrane. Centrifuge at 10,000g for 1 min. Keep the
3040 l RNA recovered from the column and immediately
place on ice. Store at 80 C.

Estimation of the Amount 1. Dilute 2 l RNA into 100 l of RNase-free water.

of Total mRNA 2. Transfer 100 l of the diluted RNA to a quartz cuvette and
read the absorbance ratio at 260280 nm in an Amersham
Pharmacia UVSpec spectrophotometer.
3. Optical density (OD) value 40 (factor for RNA) 50 (dilu-
tion) = g/ml of RNA.
4. Alternatively, RNA concentration can be measured faster and
without need for sample dilution, using a Nanodrop 2000
(Thermo Scientific, Wilmington, DE) spectrophotometer as
follows: Open the ND-2000 software on the computer. Clean
the arm and pedestal with RNAse-free water and wipe dry. Load
12 ml of RNAse free water to initialize the instrument. Select
RNA option. Wipe sample port, and load 12 l of RNAse
free water and make Blank measurement. Wipe sample port,
and load 12 ml of RNA sample and measure concentration.

Reverse Transcription 1. Use between 200 ng to 1 g RNA in a volume of 9.9 l diluted

in RNase-free water. Denature the RNA at 70 C for 10 min
and cool at 4 C.
2. Prepare a 10.1 l RT mix by adding 4 l of 25 mM MgCl2, 2 l
of 10 reverse transcription buffer, 2 l 10 mM deoxyribonu-
cleotide triphosphates, 1 l oligodT primers, 0.5 l recombi-
nant RNasin ribonuclease inhibitor, and 0.6 l AMV reverse
transcriptase (15 units). Add the mix to the denatured RNA
sample. For multiple samples, a master mix should be prepared
excluding the RNA, which has to be added at the end.
3. Incubate the samples at 42 C for 15 min followed by 95 C
for 5 min and immediately cool on ice. The single-stranded
cDNA can be used immediately for PCR or stored at 80 C if
necessary.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 45

Polymerase Chain Reaction 1. On ice, prepare 47 l reaction in a 0.5-ml PCR tube by

combining 5 l 10 PCR buffer, 3 l 25 mM MgCl2 (final
concentration 1.5 mM), 2 l 10 dNTPs, 1 l 10 pmol TCR-
chain forward primer and 1 l 10 pmol TCR-chain reverse
primer (see Notes), 34 l water, and 1 l Taq DNA polymerase
(2.5 units). Add 2 l of the cDNA.
2. For amplification of the TCR chain, set up PCR conditions in
a BioRad thermocycler as follows: 94 C denaturation for
5 min, followed by 36 cycles at 94 C for 1 min, 67 C anneal-
ing for 1 min, 72 C extension for 2 min, and a final extension
at 72 C for 7 min.

Agarose gel 1. Run 12 l of samples mixed with 3 l of 5 loading buffer in

Electrophoresis 1.2 % Ultrapure agarose gel using 1 Tris-borateEDTA
buffer.
2. Stain the sample with 5 l of ethidium bromide (10 mg/ml)
and visualize under ultraviolet illuminator or Bio-Rad gel
imaging station. Ethidium bromide can also be included in the
gel just before casting. Alternatively, the gels can be stained
with 5 l SYBRGreen in 50 ml TE buffer (Molecular Probes,
Eugene, OR), which is 100-fold more sensitive and less muta-
genic than ethidium bromide.

TA Cloning To identify mutations in the TCR-chain gene, the PCR products

can be cloned in a TOPO TA cloning vector (Invitrogen).
Recombinant clones (510) are then sequenced to identify mutations.
Each clone should be sequenced from both the orientations to
verify mutations. The presence of the same mutations in more than
one clone distinguishes artifacts from mutations. A patient sample
obtained a second time in a follow-up study can also be used to
confirm mutations.
1. Take 1 l PCR product and clone to TOPO TA cloning kit
(Invitrogen) according to manufacturers instructions.
2. Transform 2 l of the PCR product to competent cell supplied
in the kit as suggested and plate 50- and 100-l samples in two
LB agar plates containing 100 g/ml ampicillin and X-gal.
3. Grow 12 white recombinant colonies in 5 ml LB medium con-
taining 100 g/ml ampicillin.
4. Prepare miniprep plasmids from 12 white recombinant colo-
nies using Qiagen plasmid miniprep kit.
5. Digest 1 g of the purified plasmid with EcoRI and analyze by
agarose gel electrophoresis and ethidium bromide staining to
verify the presence of inserts.
46 V.R. Moulton et al.

3.3.2. Real-Time PCR
1. The reaction is set up in a 20 l volume in a 96-well plate using
the SYBR green master mix from Roche (Germany).
2. Add 10 l of SYBR Green Master mix, 6 l PCR grade water,
1 l each forward and reverse primers, and 2 l of single-
stranded cDNA.
3. Pipette the sample onto a 96-well plate, seal plate with sealing
foil, and centrifuge at 1,500g for 2 min.
4. Load plate onto a Roche Lightcycler 480 instrument.
5. Run the PCR under the following conditions: denaturation at
94 C for 1 min, followed by 40 cycles of amplification: 94 C
for 15 s, 67 C annealing for 15 s, 72 C extension for 30 s,
followed by 1 cycle of melting curve at 95 C for 15 s, 65 C
for 2 min, and 97 C continuous and a final cooling step at
37 C.
6. Analyze data based on the fluorescence cycle threshold (Ct)
differences between target genes compared to housekeeping
genes using the Ct relative quantification method. If abso-
lute quantification is desired, run serial dilutions of cDNA to
generate a standard curve and calculate absolute values for
unknown genes.
7. The reaction products can also be run on agarose gel electro-
phoresis to verify for specific products.

3.3.3. mRNA Stability T cells can be treated with 10 g/ml actinomycin D to arrest RNA
Assay synthesis and can be incubated for different times. At the end of
each period, the total RNA is isolated, and RT-PCR or real-time
PCR is carried out as described above. The decrease in the level of
TCR-chain mRNA at different times compared to zero time gives
an indication of mRNA stability.

3.3.4. Transfection After several modifications of the electroporation method, we estab-

lished this method to transfect primary T cells routinely with 2530 %
efficiency (16, 17). The technique is mainly based on making the T
cells more electrocompetent by incubating the PBMCs overnight at
37 C in culture medium in the presence of 1 g/ml phytohemag-
glutinin. T cells are isolated from the PBMCs on the next day as
described in Subheading 2, and transfected (see Note 6).
1. Incubate the PBMCs overnight at a density of 3 106/ml in
RPMI-1640 containing penicillin/streptomycin and 10 % FBS
or autologous serum and 1 g/ml phytohemagglutinin for
1820 h.
2. Isolate T cells by negative selection of non-T cells using MACS
separation as described in Subheading 3.1.2.
3. Resuspend 5 106 to 20 106 of the purified T cells in 500 l
Opti-MEM medium and transfer to a Gene Pulser cuvette
(Bio-Rad).
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 47

4. Add plasmid DNA (1015 g) and mix gently.

5. Electroporate the cells at 300 V/1,000 F in a Bio-Rad
electroporator at room temperature.
6. Transfer the cells to a 15-ml conical tube; wash with prewarmed
culture medium and resuspend in AIM-V medium.
Electroporated cells are incubated in the presence of 10 units
interleukin 2/ml at 37 C for 4872 h for optimum gene
expression.

3.3.5. Nucleofection Nucleofection is a technique that introduces the DNA or RNA

directly to the nucleus using the Nucleofector electroporation device
and solution (Lonza, Germany). It has a great advantage in that it is
not necessary to activate or manipulate the cells before nucleofec-
tion. Nucleofection works well with 15 g of nucleic acid.
1. Add 0.5 ml supplement to 2.25 ml of the Nucleofector solution
and mix gently (stable for 3 months at 4 C). Prewarm the
nucleofector solution to room temperature.
2. Prepare 6-well plates by pipetting 3.5 ml of culture medium
and preincubate the plates at 37 C.
3. Transfer 5 106cells to an Eppendorf tube, centrifuge, and
discard the supernatant so that no residual medium covers the
cell pellet.
4. Resuspend the pellet in 100 l Nucleofector solution at room
temperature.
5. Immediately add required amount of DNA. The cell number
can be increased up to 20 106 cells, but the DNA should be
kept to a maximum of 5 g. Too much DNA can lead to
decreased transfection efficiency.
6. Transfer the sample into a Lonza cuvette, avoid air bubbles,
and close the cuvette with cap.
7. Insert the cuvette into the cuvette holder of the Nucleofector.
8. Select program U-014 and press the X key to start the pro-
gram; once transfection is complete (few seconds), the display
should show OK.
9. Remove the samples from the cuvette immediately after the
program has finished using the plastic pipettes provided. To
transfer the cells, add 0.5 ml prewarmed culture medium to the
cuvette and then pipette the cells to the prepared 6-well plate.
10. Incubate the cells in a humidified 37 C, 5 % CO2 incubator.

3.3.6. Chromatin The chromatin immunoprecipitation assay (ChIP) is a powerful tech-

Immunoprecipitation Assay nique to analyze the amount of transcription factors or acetylated
histones bound to the promoter DNA in live SLE T cells. In this
protocol, we describe the methodology for evaluating the amount of
Elf-1 transcription factor binding to the TCR-chain promoter.
48 V.R. Moulton et al.
Preparation of Salmon
Sperm DNA-Blocked
Sepharose Protein A/G
Cross-linking of the Cells
and Preparation of Cell
Extract
Chromatin Immuno-
precipitation
1. Incubate 10 g/l sonicated salmon sperm DNA at 45 C for
10 min.
2. Wash 50 l (50 % slurry) of Sepharose protein A/G two times
with blocking buffer (10 mM TrisHCl, 150 mM NaCl, 1 mM
EDTA, 0.05 % NaN3 at pH 7.5).
3. Resuspend the beads in 100 l blocking buffer and add 1.2 l
of 10 g/l salmon sperm DNA and 3 l of 10 mg/ml BSA.
4. Incubate in the cold room overnight in an orbital or rotating
shaker.
1. Take 1 106 T cells. Activate the cells as preferred.
2. Add 1/37 volume of 37 % formaldehyde to the cells and mix
immediately. Close the cap and incubate the cells 20 min at
37 C.
3. Add 12 volumes of ice-cold PBS to cells to stop the reaction.
Spin down the cells at 650g for 5 min.
4. Wash cells twice with ice-cold PBS.
5. Lyse the cells in an Eppendorf tube at room temperature in
lysis buffer (50 mM TrisHCl, 10 mM EDTA, 1 % SDS at pH
8.1; just before use, add 1 mM PMSF, 1 g/ml aprotinin, and
1 g/ml leupeptin) at 1 106 cells/200 l. Incubate on ice for
10 min.
6. Sonicate samples on ice five times, with the duration of each
burst 10 s at continuous output and with a 30 s pause between
each burst.
7. Spin cells at 300g for 15 min at 4 C. Transfer the supernatant
to another tube and discard the pellet.
8. Freeze the sample at 80 C or proceed with immuno-
precipitation.
1. Thaw the frozen sample and add 1.8 ml of ChIP dilution buf-
fer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM
TrisHCl at pH 8.1, 167 mM NaCl; just before use, add the
protease inhibitors).
2. To reduce nonspecific background, preclear the 2 ml diluted
sample with 80 l of salmon sperm DNA-blocked protein A/G
Sepharose bead (50 % slurry). Incubate the samples 1 h at
4 C, shaking in a nutator.
3. Pellet the protein A/G agarose by centrifugation at 18,000g or
top speed for 30 s and transfer the supernatant to a new tube.
4. Add TCR-chain transcription factor, Elf-1 antibody
(612 g/2 ml) and incubate overnight with shaking at 4 C.
For a negative control, use a no antibody or isotype control
antibody immunoprecipitation by incubating the supernatant
fraction with 60 l PBS or with isotype control antibody.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities
3.3.7. ChIP to Detect the
Binding of Phosphorylated
Transcription Factors
3.3.8. Oligonucleotide
Pulldown Assay
49
5. Add 60 l of salmon sperm-blocked protein A/G Sepharose
bead (50 % slurry) to each sample and incubate at 4 C for 2 h
or overnight.
6. Centrifuge the samples at 18,000g for 30 s and discard the
supernatant.
7. Wash the protein A/G Sepharose/antibody/Elf-1 complex for
5 min on a rotating platform twice with 1 ml of low-salt
immune complex wash buffer.
8. Wash the protein A/G Sepharose/antibody/Elf-1 complex for
5 min on a rotating platform once with 1 ml of lithium chlo-
ride wash buffer.
9. Wash the protein A/G Sepharose/antibody/Elf-1 complex for
5 min on a rotating platform twice with 1 ml of 1 TrisEDTA
buffer.
10. Resuspend in 100 l TrisEDTA buffer containing 0.5 %
SDS.
11. Digest the sample with 0.5 l of proteinse K (20 mg/ml) for
2 h at 45 C.
12. Reverse cross-link the protein from DNA by incubating at
65 C for 46 h.
13. Extract the DNA using a Qiagen DNA purification kit.
14. Amplify the DNA by PCR using the TCR-chain promoter
forward primer 5 CCA TCG AGA ACT TGT ATT TG 3,
307 to 288 sense bps, and reverse primer 5 GCC CTA CCT
GTA ATC GG 3, +129 to +113 antisense bps, according to
the numbering of Rellahan et al. (18).
The ChIP protocol can also be exploited to analyze the level of
phosphorylated transcription factor bound to promoter DNA by
first immunoprecipitation with anti-phosphotyrosine antibody and
then with the antibody of interest.
1. Immunoprecipitate the cell lysates with anti-phosphotyrosine
antibody 4 G10 (12 l per 2 ml) or anti-phosphoserine and
anti-phosphothreonine for 1 h and capture with 80 l (50 %
slurry) of protein A/G Sepharose.
2. Elute the anti-phosphotyrosine immunoprecipitates from the
protein A/G Sepharose beads by incubating with 10 mM phe-
nyl phosphate in lysis buffer containing 1 % NP-40 at 4 C for
15 min.
3. Reprecipitate phosphotyrosine-immunoprecipitated samples
with the antibody of interest, and the ChIP assay is continued
as described above.
This assay identifies proteins bound to a known sequence of
nucleic acid (DNA or RNA in the form of a short 2530 base
50 V.R. Moulton et al.

oligonucleotide). The experiment described herein aims to identify

proteins binding to an AU rich element (ARE) within the 3-UTR
of the CD3 mRNA. ARE2 refers to the second of three ARE
sites within the CD3 3-UTR (10).
1. Custom-synthesized 25-base biotin-labeled ARE-bearing RNA
oligonucleotides and random scrambled control RNA oligo-
nucleotides can be purchased from Integrated DNA
Technologies (Coralville, IA).
2. Wash streptavidin magnetic beads (PureBiotech LLC,
Middlesex, NJ) three times in Wash buffer (Hepes-buffered
saline (HBS-P) buffer: 0.01 M Hepes buffer, pH 7.4, 0.15 M
NaCl, 0.005 % (v/v) Surfactant P-20, plus 5 mM MgCl2).
3. Incubate 40 pmol of biotinylated RNA oligonucleotides with
30 l of washed streptavidin magnetic beads in binding buffer
[Wash buffer supplemented with 0.2 mg/ml BSA and protease
inhibitors 2 mM 4-2-aminoethyl benzenesulfonyl fluoride
(AEBSF), 10 g/ml aprotinin, 10 g/ml leupeptin, and 1 mM
dithiothreitol (DTT)] for 30 min at room temperature.
4. Select magnetic bead-bound oligonucleotides using a Magnetic
Particle Separator (MPS, Pure-Biotech, LLC).
5. Jurkat cell nuclear lysates (~700 g) were pretreated with
polyinosine-cytosine (dI-dC) (50 g/400 g of lysate) and
16 units of RNasin/100 g lysate in binding buffer at room
temperature for 15 min.
6. 100 l of bead oligonucleotide was added to the nuclear lysate
and incubated at room temperature for 1 h.
7. The MPS was used to selectively bind the oligo-protein com-
plexes, whereas unbound proteins were washed three times
with binding buffer.
8. Each wash fraction was collected and labeled Wash 1, 2 (Fig. 4,
lanes 4, 5, 9, and 10), and 3. Binding buffer with 0.3 M NaCl
was used for a pre-elute wash.
9. Bound proteins were eluted from the oligonucleotides using
10 mM glycine/HCl, pH 2.0 (Biacore, Inc., Piscataway, NJ).
10. Three eluate fractions were collected and labeled Elute 1, 2,
and 3 (Fig. 4, lanes 13 and 68). Washes and eluates (13 l)
were prepared for SDS PAGE and loaded onto a 12-well 1.0-
mm 412 % BisTris NuPAGE precast gel and run at 200 V for
35 min.
11. Silver staining of the gel was carried out according to instruc-
tions in the Silver Express staining kit (Invitrogen). Specific
bands (indicated by arrows in Fig. 4) were excised, washed
twice in 50 % acetonitrile, and sent for mass spectrometry
identification of proteins.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 51

Fig. 4. Oligonucleotide pulldown Assay: Identification of proteins binding to CD3 3-UTR

defined ARE2 RNA oligonucleotide. Biotinylated oligonucleotides were bound to
streptavidin-labeled magnetic beads and incubated with Jurkat T cell nuclear extracts.
Using a magnetic particle separator, unbound proteins were washed three times with
binding buffer containing 0.15 M NaCl. Washes were collected and labeled Wash 1, 2, and
3. Oligonucleotide-bound proteins were eluted with a glycine/HCl, pH 2.0, solution, and
eluates were labeled Elute1, 2, and 3. Elutes 13 and Washes 1 and 2 were resolved on
a 412 % BisTris denaturing gel followed by silver staining. Nucleotide sequences of the
ARE2 oligo and the scrambled control oligonucleotide are indicated.

3.4. Cellular Expression of signaling molecules can be determined using

Immunology Studies fluorescence labeling and flow cytometry technologies. Similarly,
of Signaling Molecules distribution of a particular signaling molecule in the cell can be
in SLE T Cells analyzed by direct or indirect fluorescence microscopy. These tech-
niques can be applied to nonpermeable and permeabilized cells to
distinguish surface expression and intracellular expression,
respectively.

3.4.1. Flow Cytometry The technique can be applied to molecules that have extracellular
domains, and fluorescent-conjugated antibodies directed to the
Cell Surface Staining
extracellular domain are readily available. For each assay, a sample
with an isotype control of the primary antibody is required to
determine the background binding. Before adding the antibodies,
the nonspecific Fc receptor binding should be blocked using
52 V.R. Moulton et al.

excess human IgG. It is important to perform all steps of the

experiment at 4 C. When aspirating the solution after centrifuga-
tion, be careful not to disturb the small pellet, which may not be
seen very clearly.
1. Transfer 12 million cells to flow cytometry tubes.
2. Centrifuge the cells at 350g at 4 C in a Sorvall tabletop cen-
trifuge for 5 min.
3. Discard the media, resuspend the cells in staining buffer 200 l
PBS + 1 % FBS, and centrifuge as in step 2.
4. Centrifuge the sample again as in step 2 and discard the
supernatant.
5. Resuspend the cells in 200 l staining buffer and add 2 g of
the fluorescent-conjugated primary antibody (or unconjugated
primary antibody) and 50-fold excess human IgG. Similarly, in
a second tube, add isotype control antibody instead of primary
Ab. Incubate 30 min on ice in the dark.
6. Wash cells twice with 1 ml ice-cold staining buffer as in step 2.
7. If using direct fluorescent-conjugated primary Ab, proceed
directly to step 9. If using unconjugated primary Ab, resus-
pend the cells in 200 l staining buffer, add 50-fold excess
human IgG to block the Fc receptor binding, and incubate
with 2 l of goat anti-mouse fluorescent-conjugated antibod-
ies for 30 min on ice in the dark.
8. Wash twice with 1 ml staining buffer and resuspend in 200 l
fixing buffer (PBS + 1 % paraformaldehyde) and run on the
flow cytometer. Levels of expression can be quantitated by
either percentage of cells expressing the signaling molecule or
by mean fluorescence intensity. If the cells are analyzed by flow
cytometry using the same settings, the level of expression on
different samples on different days can be normalized by
adjusting against the readings of an untreated control (19).

Intracellular Staining Flow cytometry using permeabilized cells can be applied to the
TCR-chain or other signaling molecules that do not have sub-
stantial extracellular domains or to detect cytokine expression.
Flow cytometry using permeabilized cells will provide an estimate
of the total level of expression of the TCR-chain. For each assay,
a sample with an isotype control of the primary antibody is required
to determine the background binding. Before adding the antibod-
ies, the nonspecific Fc receptor binding should be blocked using
excess human IgG. It is important to precool the centrifuge and to
perform the experiment at 4 C, except for permeabilization with
detergents, which is done at room temperature. When discarding
 3 Methods and Protocols to Study T Cell Signaling Abnormalities 53

the solution after centrifugation, be careful not to disturb the

pellet, which is tiny and may not be seen very clearly.
1. Transfer 12 million cells to flow cytometry tubes.
2. Centrifuge the cells at 350g at 4 C in a Sorvall tabletop cen-
trifuge for 5 min.
3. Discard the media, resuspend the cells in 200 l staining buf-
fer, and centrifuge as in step 2.
4. Resuspend the cells in 200 l digitonin or saponin permeabili-
zation buffer and incubate at room temperature for 10 min.
5. Centrifuge as in step 2.
6. Aspirate the supernatant, resuspend the cells in permeabiliza-
tion buffer, add 2 g of the murine anti-human TCR-(6B1.02)
or isotype control and 50-fold excess human IgG, and incu-
bate for 30 min on ice.
7. Wash twice with 1 ml ice-cold staining buffer as in step 2.
8. If using direct fluorescent-conjugated primary Ab, proceed
directly to step 12. If using unconjugated primary Ab, resus-
pend the sample in 200 l permeabilization buffer, add 2 l of
goat anti-mouse fluorescence-conjugated antibodies and
50-fold excess human IgG to block the Fc receptor binding,
and incubate for 30 min on ice in the dark.
9. Wash three times with 1 ml staining buffer and resuspend in
200 l PBS containing 1 % paraformaldehyde and loaded onto
the flow cytometer. Levels of expression can be quantitated by
either percentage of cells expressing the signaling molecule or
by mean fluorescence intensity. If the cells are analyzed by flow
cytometry using the same settings, the level of expression on
different samples on different days can be normalized by
adjusting against the readings of an untreated control (19).

3.4.2. Fluorescence The distribution of the TCR-chain in lymphocytes is visualized by

Microscopy fluorescence microscopy. Although not very quantitative,
fluorescence microscopic images will also give a broad idea of the
gross level of expression of the TCR-chain (Fig. 5a). The cells can
be permeabilized and stained in the tube or can be adhered to
slides and stained. The staining procedure in the tubes results in
increased recovery of the cells because T cells of patients with SLE
do not attach well to the polylysine-coated slides. Appropriate
markers like LAT or cholera toxin B subunit can be used for local-
izing the TCR-chain to lipid rafts. When double labeling, the
TCR-chain can be stained with PE-labeled secondary antibody,
and markers can be labeled with FITC-labeled antibody. Isotype
control is necessary to ensure specific staining.
54 V.R. Moulton et al.

Fig. 5. Fluorescence microscopy of the TCR-chain in SLE T cells showed increased

clustering. (a) Two million T cells of patients with SLE or normal donors were fixed with
0.25 % paraformaldehyde and permeabilized with digitonin. Cells were incubated with
antimouse TCR-chain C-terminal monoclonal antibody for 1 h and then washed in stain-
ing buffer. Rhodamine-labeled anti-mouse goat IgG secondary antibody was added, and
the sample was incubated for 1 h. The cells were washed and mounted on slides, and the
samples were visualized in an Olympus fluorescence microscope at 100 magnification.
Staining of the TCR-chain was enhanced in SLE T cells to reveal the distribution.
(b) Confocal microscopy of the TCR-chain in SLE T cells. SLE T cells adhered on
polylysine-coated slides by incubating for 1 h at room temperature. The cells were acti-
vated with anti-CD3-IgM (clone 2Ad2A2) at 37 C for 2 min. The reaction was stopped
with 3 % paraformaldehyde for 15 min, and cells were permeabilized with a buffer con-
taining 0.05 % saponin and the blocking antibody human IgG 1 g. Cells were stained
with anti-TCR (clone C-20) and anti-LAT (clone FL-233) for 1 h at room temperature and
counterstained with anti-goat TRITC and anti-rabbit FITC, respectively, for 30 min. Cells
were washed; air-dried, and mounted using Gel/Mount. Coverslips were applied, and the
edges were sealed with cytoseal. Samples were analyzed with a laser scanning confocal
fluorescence microscope with a 1 70 scope, Bio-Rad Lasersharp 2000 software. Unlike
the normal T cells, preformed membrane clustering of the TCR-chain in SLE T cells led
to TCR capping within 2 min of activation.

1. Transfer 12 million cells to flow cytometry tubes.

2. Centrifuge the cells at 350 g at 4 C in a Sorvall tabletop cen-
trifuge for 5 min.
3. Discard the media, resuspend the cells in 100 l PBS, and cen-
trifuge as in step 2.
4. Mildly fix the cell by adding 100 l 0.25 % paraformaldehyde
in PBS and incubate on ice for 10 min.
5. Centrifuge the sample again as in step 2 and discard the
supernatant.
6. Resuspend the cells in 100 l digitonin or saponin permeabili-
zation buffer and incubate at room temperature for 10 min;
centrifuge as in step 2.
 3 Methods and Protocols to Study T Cell Signaling Abnormalities
3.4.3. Confocal Microscopy
55
7. Resuspend the cells in permeabilization buffer, add 50-fold
excess human IgG, and incubate for 30 min.
8. Incubate the sample with 2 g of the murine anti-human TCR-
(6B1.02) or isotype control antibody for 30 min on ice.
9. Wash twice with 2 ml ice-cold staining buffer as in step 2.
10. Resuspend the sample in 100 l permeabilization buffer, add
50-fold excess human IgG to block the Fc receptor binding,
and incubate with 2 l of goat anti-mouse FITC-conjugated
antibodies for 30 min on ice in the dark.
11. Wash three times with 2 ml staining buffer, resuspend in 20 l
Gel/Mount, and apply a drop on a glass slide; carefully place
the coverslip, avoiding air bubbles, and seal the sides with
Cytoseal. The slide is ready to view under a fluorescence micro-
scope. Keeping the stained cells for a long time may lead to
leakage of the stain.
Confocal microscopy can be used to determine the distribution of
molecules such as TCR-chain in three dimensions in SLE T cells
before and after activation (Fig. 5b). The microscope will take sec-
tioned images of the stained cells, and the overall distribution of
the TCR-chain can be analyzed. Also, there is better resolution of
the specimen by confocal microscopy because the laser is focused
on a single plane, as opposed to the diffuse direction of the laser
beam in fluorescence microscopy. An isotype control is required
for both nonactivated and activated samples.
1. Make two circles on a polylysine-coated slide using a pap pen
and add 1 million cells in 100 l RPMI-1640 containing
1 % FBS.
2. Place the slides in a box and keep the box at room temperature
for 1 h to adhere the cells.
3. Activate the cells by OKT3 or anti-CD3 IgM antibody at 37 C
for the time required. Clustering requires a short time activa-
tion of 23 min, whereas capping requires activation for
10 min.
4. Remove the medium by suction and fix the cells in 3.7 %
paraformaldehyde (dissolve 3.7 % paraformaldehyde in approx.
50 ml PBS, increase the pH using NaOH to dissolve, and read-
just the final pH to 7.0 using 6 N HCl and make up the volume
to 100 ml) for 30 min. Alternatively, the cells can also be
activated in a microfuge tube and then adhered to the
polylysine-coated glass slides.
5. Wash with 200 l PBS.
6. Permeabilize using 100 l permeabilization buffer, add 50 g
of human IgG to block Fc receptor binding, and incubate at
room temperature for 15 min.
56 V.R. Moulton et al.

7. Remove the permeabilization buffer and stain with anti-TCR-

chain antibody in 100 l permeabilization buffer for 1 h along
with excess human IgG at room temperature.
8. Wash three times, 5 min each, with 200 l permeabilization
buffer.
9. Stain with secondary FITC- or PE-labeled goat anti-mouse
IgG in 100 l permeabilization buffer containing excess human
IgG for 30 min at room temperature.
10. Wash four times, 5 min each, with PBS at room temperature.
This step is critical to remove any residual FBS, which can oth-
erwise give high background. Dry the samples for 15 s.
11. Add a drop of Gel/Mount and place the coverslips carefully to
avoid air bubble trapping. Seal the sides of the coverslips with
Cytoseal and view the slide with an Olympus confocal micro-
scope (1 70 scope, software, Bio-Rad LaserSharp 2000).
Slides may be stored at 28 C for up to 5 days if necessary, and
initial signals will be strong enough.

4. Notes

1. Keeping the SLE blood at 4 C is not advisable because of

cryoglobulins present in many patients with SLE. Storing the
blood for longer duration reduces the yield of PBMCs because
of possible aggregation between lymphocytes or with other
blood cells, forming clumps as well as lysis. Also, prolonged
incubation of blood results in increased adhesion of RBCs to
PBMCs. If this happens, RBCs need to be lysed before pro-
ceeding further; otherwise, the protein and the proteolytic
activity from RBC may bias the results. Repeated or prolonged
ACK exposure will damage the lymphocytes and also decrease
yield. Same day use of the SLE blood sample is advised for
maximum yield of viable PBMCs and best reproducible results.
If the cells are used for transfection, purification of lympho-
cytes on the same day significantly improves the transfection
efficiency. PBMC yields can be quite variable and are often
decreased from SLE patients, as many patients are leukopenic,
particularly during times of active disease.
2. The negatively selected lymphocytes obtained by MACS sepa-
ration and RosetteSep are untouched and thus this tech-
nique is superior to positive selection methods. To improve the
yield of lymphocytes by MACS separation, elute the column
with three times the column volume of buffer. Also failure to
de-gas the buffer each time impedes the speed of elution
and decreases the yield of elution. The purity of the isolated
3 Methods and Protocols to Study T Cell Signaling Abnormalities 57

T lymphocytes can be determined by CD3 staining and flow

cytometry.
3. The N-terminal TCR-chain antibody recognizes 16-kDa and
ubiquitinated forms of the TCR-chain. We demonstrated that
the C-terminal antibody recognizes the phosphorylated p21-
and p23-kDa form of the -chain in addition to 16-kDa and
ubiquitinated forms. Both the antibodies were able to detect a
novel 14-kDa form of the TCR-chain (7). It is important to
run the detergent soluble cytosolic fraction along with the
detergent-insoluble membrane fraction to confirm -chain
deficiency rather than a translocation between compartments.
Membrane translocation is one of the causes for decreased
expression of the TCR-chain in heat-shocked T cells (20).
The membranes should be stripped and reprobed with control
actin antibody to confirm equal loading and the integrity of the
samples. Stripping and reprobing the blot is also important to
confirm ubiquitination, phosphorylation, or other modification
of the molecule of interest. After a few experiments, immuno-
blotting can be done with two antibodies together if the molec-
ular weight of the proteins is significantly different.
4. The increase in the amount of TCR-chain in the detergent-
soluble fraction after incubating with methyl--cyclodextrin
was considered a lipid raft-associated fraction. Similar to lipid
raft-bound TCR-chain, the actin-bound TCR-chain can be
estimated by treating the cells with or without 10 M cytocha-
lasin D, which disrupts actin polymerization, leading to accu-
mulation of the TCR-chain in the detergent-soluble cytoplasmic
fraction. An increase in the level of the TCR-chain in the
detergent-soluble fraction after 3 h treatment with cytochalasin
D will provide an estimate of the actin-bound form of the
TCR-chain (2123). If activation of cells is necessary for the
experiments, it should be done before lysis of cells. A minimum
of 25 106 cells is required for proper isolation of lipid rafts.
Very fine balancing of the tubes before centrifugation is required
for good results. The buckets of rotors should be properly tight-
ened; otherwise, the tube will flatten, and the vacuum will aspi-
rate off the samples during centrifugation, resulting in loss of
sample. Collection of the 1-ml fraction should be accurate; oth-
erwise, it may lead to cross contamination.
5. Five million SLE T cells yield approx. 3 g total RNA. If the
OD 260 is very low, then decrease the dilution of the total
RNA. A 260/280 OD ratio of 1.82.0 represents pure RNA.
A control reaction using -actin or another housekeeping gene
is necessary to evaluate the integrity and equal loading of the
RNA. The level of TCR-chain mRNA should be normalized
against the level of actin for comparison between samples. The
primers for PCR amplification of TCR-chain coding region
58 V.R. Moulton et al.

Fig. 6. RT-PCR analysis of the expression of TCR-chain mRNA in T lymphocytes from

normal and SLE donor. (a) Total RNA was isolated from 5 106 T lymphocytes and reverse
transcribed; the TCR-chain was amplified by specific primers. The PCR products (15 l)
were electrophoresed in 1.2 % agarose gel and visualized by ethidium bromide staining.
M is the 100-bp DNA ladder molecular weight marker; N is the normal donor. (b) PCR
amplification of TCR-chain promoter from T cells of SLE and normal healthy control
subjects. Genomic DNA was isolated from T lymphocytes or granulocytes, and the pro-
moter region was PCR amplified using specific primers. The PCR products (10 l) were
electrophoresed on a 1.2 % agarose gel and visualized by ethidium bromide staining.
(c) RT-PCR analysis of the expression of TCR-chain mRNA 3 untranslated region (UTR)
in SLE T lymphocytes. Total RNA was reverse transcribed, and the normal or alternatively
spliced TCR-chain mRNA 3-UTR or -actin was amplified by specific primers. The PCR
products (15 l) were electrophoresed in 1.2 % agarose gel and visualized by ethidium
bromide staining. M is the 100-bp DNA ladder molecular weight marker; N indicates
normal donor.

are: forward 5 AGC CTC TGC CTC CCA GCC TCT TTC
TGA G 3 (3562 sense bps) and reverse 5 TCA GTG GCT
GAG AAG AGT GAA CCG GGT TG 3 (669641 antisense
bps according to numbering of Weissman et. al. (24)) (Fig. 6a).
Primers for amplifying the TCR-chain promoter are as fol-
lows: forward 5 CCA TCG AGA ACT TGT ATT TG 3, 307
to 288 sense bps; and reverse 5 GCC CTA CCT GTA ATC
GG 3, +129 to +113 antisense bps according to the number-
ing of Rellahan et al. (18) (Fig. 6b). The primers for amplify-
ing the TCR-chain 3 untranslated region are as follows:
forward 5 CAG CCA GGG GAT TTC CAC CAC TCA AAG
3 (567592 sense base pairs); and reverse 5 CCC TAG TAC
ATT GAC GGG TTT TTC CTG 3 (14721443 antisense
bps) (Fig. 6c). The -actin primers used in these experiments
are as follows: forward 5 CAT GGG TCA GAA GGA TTC
CT 3; and reverse 5 AGC TGG TAG CTC TTC TCC A 3.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 59

Fig. 7. Transfection and expression of TCR-chain by nucleofection in SLE T cells.

(a) Analysis of transfection efficiency by nucleofection in SLE T cells. SLE T cells were
transfected by nucleofection with a GFP expression plasmid or control -galactosidase
plasmid. After 18 h, the cells were directly analyzed for GFP expression by flow cytometry.
A representative of five experiments using cells from different patients is shown. The
transfection efficiency was 7075 %. (b) Analysis of TCR-chain expression by Western
blotting in nucleofected SLE T cells. After 18 h, transfected cells (5 106) were lysed, and
the detergent-soluble and detergent-insoluble fractions were collected. Protein from the
detergent-soluble fraction (10 g) was analyzed on 412 % NuPAGE gel under reducing
conditions, transferred to PVDF membrane, and immunoblotted with TCR-chain
C-terminal mAb. The membrane was stripped and reprobed with CD3 mAb and -actin
antibody.

For the quantitative real-time PCR, addition of excess SYBR

green will give false-positive results.
6. Enhanced green fluorescence protein cloned in pIRES2 vector
(pIRES2-EGFP, Clontech, Mountain View, CA) can be used
as control to monitor transfection efficiency (Fig. 7a). The
nucleofection protocol routinely gives 7075 % transfection
efficiency. Following nucleofection, the TCR-chain is
expressed after 3 h, and an approximate tenfold increase in
expression was observed after 12 h (Fig. 7b). More than 8 h
incubation of purified T cells before transfection reduces the
efficiency of transfection. If the cell number is decreased, then
the culture volume can be reduced accordingly. Reduction in
the volume of Nucleofector solution significantly reduces the
transfection efficiency. To reuse the electroporation cuvettes,
wash with bleach and rinse six times with water. Rinse with
95 % ethanol twice, replace the cap, and dry upside down in
the hood.
60 V.R. Moulton et al.
References
1. Irving BA, Chan AC, Weiss A (1993) Functional
characterization of a signal transducing motif
present in the T cell antigen receptor zeta
chain. J Exp Med 177:10931103
2. Zenner G, Vorherr T, Mustelin T, Burn P
(1996) Differential and multiple binding of
signal transducing molecules to the ITAMs of
the TCR-zeta chain. J Cell Biochem
63:94103
3. Kersh EN, Shaw AS, Allen PM (1998) Fidelity
of T cell activation through multistep T cell
receptor zeta phosphorylation. Science (N Y)
281:572575
4. Kersh EN, Kersh GJ, Allen PM (1999) Partially
phosphorylated T cell receptor zeta molecules
can inhibit T cell activation. J Exp Med
190:16271636
5. Weiss A, Littman DR (1994) Signal transduc-
tion by lymphocyte antigen receptors. Cell
76:263274
6. Wange RL, Samelson LE (1996) Complex
complexes: signaling at the TCR. Immunity
5:197205
7. Nambiar MP, Enyedy EJ, Fisher CU, Krishnan
S, Warke VG, Gilliland WR, Oglesby RJ, Tsokos
GC (2002) Abnormal expression of various
molecular forms and distribution of T cell recep-
tor zeta chain in patients with systemic lupus
erythematosus. Arthritis Rheum 46:163174
8. Takeuchi T, Tsuzaka K, Pang M, Amano K,
Koide J, Abe T (1998) TCR zeta chain lacking
exon 7 in two patients with systemic lupus ery-
thematosus. Int Immunol 10:911921
9. Moulton VR, Tsokos GC (2010) Alternative
splicing factor/splicing factor 2 regulates the
expression of the zeta subunit of the human T
cell receptor-associated CD3 complex. J Biol
Chem 285:1249012496
10. Moulton VR, Kyttaris VC, Juang YT,
Chowdhury B, Tsokos GC (2008) The RNA-
stabilizing protein HuR regulates the expres-
sion of zeta chain of the human T cell
receptor-associated CD3 complex. J Biol Chem
283:2003720044
11. Liossis SN, Ding XZ, Dennis GJ, Tsokos GC
(1998) Altered pattern of TCR/CD3-mediated
protein-tyrosyl phosphorylation in T cells from
patients with systemic lupus erythematosus.
Deficient expression of the T cell receptor zeta
chain. J Clin Invest 101:14481457
12. Laemmli UK (1970) Cleavage of structural
proteins during the assembly of the head of
bacteriophage T4. Nature 227:680685
13. Abe T, Toguchi T, Takeuchi T, Kiyotaki M,
Homma M (1981) Mitogenic responses to
lipopolysaccharide by B lymphocytes from
patients with systemic lupus erythematosus.
Scand J Immunol 15:475482
14. Darlington PJ, Baroja ML, Chau TA, Siu E, Ling
V, Carreno BM, Madrenas J (2002) Surface cyto-
toxic T lymphocyte-associated antigen 4 partitions
within lipid rafts and relocates to the immunologi-
cal synapse under conditions of inhibition of T cell
activation. J Exp Med 195:13371347
15. Xavier R, Brennan T, Li Q, McCormack C,
Seed B (1998) Membrane compartmentation
is required for efficient T cell activation.
Immunity 8:723732
16. Herndon TM, Juang YT, Solomou EE,
Rothwell SW, Gourley MF, Tsokos GC (2002)
Direct transfer of p65 into T lymphocytes from
systemic lupus erythematosus patients leads to
increased levels of interleukin-2 promoter activ-
ity. Clin Immunol 103:145153
17. Nambiar MP, Fisher CU, Kumar A, Tsokos CG,
Warke VG, Tsokos GC (2003) Forced expres-
sion of the Fc receptor gamma-chain renders
human T cells hyperresponsive to TCR/CD3
stimulation. J Immunol 170:28712876
18. Rellahan BL, Jensen JP, Howcroft TK, Singer
DS, Bonvini E, Weissman AM (1998) Elf-1
regulates basal expression from the T cell
antigen receptor zeta-chain gene promoter.
J Immunol 160:27942801
19. Valensin S, Paccani SR, Ulivieri C, Mercati D,
Pacini S, Patrussi L, Hirst T, Lupetti P, Baldari
CT (2002) F-actin dynamics control segrega-
tion of the TCR signaling cascade to clustered
lipid rafts. Eur J Immunol 32:435446
20. Nambiar MP, Fisher CU, Enyedy EJ, Warke VG,
Krishnan S, Tsokos GC (2000) Heat stress down-
regulates TCR zeta chain expression in human T
lymphocytes. J Cell Biochem 79:416426
21. Caplan S, Baniyash M (1996) Normal T cells
express two T cell antigen receptor popula-
tions, one of which is linked to the cytoskeleton
via zeta chain and displays a unique activation-
dependent phosphorylation pattern. J Biol
Chem 271:2070520712
22. Rozdzial MM, Malissen B, Finkel TH (1995)
Tyrosine-phosphorylated T cell receptor zeta
chain associates with the actin cytoskeleton
upon activation of mature T lymphocytes.
Immunity 3:623633
23. Rozdzial MM, Pleiman CM, Cambier JC,
Finkel TH (1998) pp 56Lck mediates TCR
zeta-chain binding to the microfilament
cytoskeleton. J Immunol 161:54915499
24. Weissman AM, Baniyash M, Hou D, Samelson
LE, Burgess WH, Klausner RD (1988) Molecular
cloning of the zeta chain of the T cell antigen
receptor. Science (N Y) 239:10181021
 Chapter 4

Assessment of Mitochondrial Dysfunction in Lymphocytes

of Patients with Systemic Lupus Erythematosus
Andras Perl, Robert Hanczko, and Edward Doherty

Abstract
Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within
the immune system. Activation, proliferation, or death of cells of the immune system is dependent on
controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mito-
chondrial transmembrane potential (ym) reflects the energy stored in the electrochemical gradient across
the inner mitochondrial membrane which, in turn, is used by F0F1-ATPase to convert ADP to ATP during
oxidative phosphorylation. Mitochondrial hyperpolarization (MHP) and transient ATP depletion repre-
sent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of patients
with SLE exhibit elevated ym, i.e., persistent mitochondrial hyperpolarization (MHP), cytoplasmic alka-
linization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP.
Increased production of nitric oxide has been identified as a cause of MHP and increased mitochondrial
biogenesis. Oxidative stress affects signaling through the T cell receptor as well as activity of redox-
sensitive caspases. ATP depletion causes diminished activation-induced apoptosis and sensitizes lupus
T cells to necrosis. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a
key sensor of MHP and mediator of enhanced Ca2+ flux in lupus T cells.

Key words: Systemic lupus erythematosus, Mitochondrial hyperpolarization, Reactive oxygen inter-
mediates, Cytoplasmic alkalinization, Caspases, Glutathione depletion, ATP depletion, Apoptosis,
Necrosis, mTOR

1. Introduction

Systemic lupus erythematosus (SLE) is a chronic inflammatory

disease characterized by T and B cell dysfunction and production
of antinuclear antibodies. Abnormal T cell activation and cell death
underlie the pathology of SLE (1, 2). Potentially autoreactive
T and B lymphocytes during development (3) and after completion

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_4, Springer Science+Business Media New York 2012

61
62 A. Perl et al.

of an immune response are removed by apoptosis (4). Paradoxically,

lupus T cells exhibit both enhanced spontaneous apoptosis and
defective activation-induced cell death. Increased spontaneous
apoptosis of peripheral blood lymphocytes (PBL) has been linked
to chronic lymphopenia (5) and compartmentalized release of
nuclear autoantigens in patients with SLE (6). By contrast, defec-
tive CD3-mediated cell death may be responsible for persistence of
autoreactive cells (7).

1.1. Mitochondrial Both cell proliferation and apoptosis are energy-dependent pro-
Checkpoints of T Cell cesses. Energy in the form of ATP is provided through glycolysis
Activation and and oxidative phosphorylation. The mitochondrion, the site of
Apoptosis oxidative phosphorylation, has long been identified as a source of
energy and cell survival (8). The synthesis of ATP is driven by an
electrochemical gradient across the inner mitochondrial membrane
maintained by an electron transport chain and the membrane
potential (negative inside and positive outside). A small fraction of
electrons react directly with oxygen and form reactive oxygen
intermediates (ROI). Disruption of the mitochondrial membrane
potential has been proposed as the point of no return in apoptotic
signaling (911). Mitochondrial membrane permeability is subject
to regulation by an oxidationreduction equilibrium of ROI, pyri-
dine nucleotides (NADH/NAD + NADPH/NADP) and GSH
levels (12). Regeneration of GSH by glutathione reductase from
its oxidized form, GSSG, depends on NADPH produced by the
pentose phosphate pathway (PPP) (13). ROI levels and ym are
regulated by the supply of reducing equivalents from PPP (14, 15).
While ROI have been considered as toxic by-products of aerobic
existence, evidence is now accumulating that controlled levels of
ROI modulate various aspects of cellular function and are neces-
sary for signal-transduction pathways, including those mediating
T cell activation and apoptosis (16).
Increased production of ROI was demonstrated in TNF
(1719) and Fas-mediated cell death (9, 14, 2023). Disruption of
the mitochondrial membrane potential (ym) has been proposed
as the point of no return in apoptotic signaling (911). Interestingly,
elevation of ym, mitochondrial hyperpolarization (MHP), and
ROI production precede phosphatidylserine (PS) externalization
and a disruption of ym in Fas- (15) and H2O2-induced apoptosis
of Jurkat human leukemia T cells and normal human peripheral
blood lymphocytes (24). These observations were extended to p53
(25), tumor necrosis factor a (26), staurosporin (27), camptothe-
cin (28), and nitric oxide-induced apoptosis (29). Elevation of ym
is independent from activation of caspases and represents an early
event in apoptosis (15, 25). Pretreatment with caspase inhibitors,
DEVD, Z-VAD, and Boc-Asp, completely abrogated Fas-induced
PS externalization, indicating that activation of caspase-3, caspase-8,
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 63

and related cysteine proteases were absolutely required for cell

death (3033). ROI levels were partially inhibited in DEVD-treated
Jurkat cells, suggesting that caspase-3 activation, perhaps through
damage of mitochondrial membrane integrity, contributes to ROI
production and serves as a positive feedback loop at later stages of
the apoptotic process. Nevertheless, ROI levels remained
significantly elevated after pretreatment with caspase inhibitors.
This suggested that activation of caspase-3 or caspase-8 was not
required for increased ROI production and ym hyperpolarization.
By contrast, DEVD, Z-VAD, and Boc-Asp blocked PS externaliza-
tion and decline of ym in annexin V-positive Jurkat cells, suggest-
ing that disruption of ym (1) was a relatively late event with
respect to ROI production and ym hyperpolarization and (2)
depended on activation of caspase-3 and related proteases. The
precise mechanism by which Fas and TNF signaling leads to
changes in ym and ROI levels remains to be defined. Cleavage of
cytosolic bid by caspase-8 generates a p15 carboxyterminal frag-
ment that translocates to mitochondria. This may represent the
initial insult to mitochondria in the Fas/TNF pathway (34).
MHP appears to be the earliest change associated with Fas
(15), H2O2 (24), HIV-1 (35), p53 (25), TNFa (26), staurosporin
(27), camptothecin (28), and NO-induced apoptosis (29).
Elevation of ym is also triggered by activation of the CD3/CD28
complex (36) or stimulation with Con A (15), IL-10, IL-3, IFN-g,
or TGFb (37). Therefore, elevation of ym or MHP represents an
early but reversible switch not exclusively associated with apopto-
sis. With ym hyperpolarization and extrusion of H+ ions from the
mitochondrial matrix, the cytochromes within the electron trans-
port chain become more reduced which favors generation of ROI
(38). MHP is caused by exposure to nitric oxide (NO) which is
produced during T cell activation (39). Reduced glutathione
(GSH) is profoundly depleted in lymphocytes of SLE patients (36)
(Table 1), which may predispose to persistent MHP via
S-nitrosylation of complex I upon exposure to NO (40). Thus, the
effect of NO on MHP is tightly related to GSH levels. With MHP
and extrusion of H+ ions from the mitochondrial matrix, the cyto-
chromes within the electron transport chain become more reduced
which promotes ROI production and generates oxidative stress (8).
Diminished production of GSH in face of MHP and increased
ROI production are suggestive of a severe metabolic defect in
lupus T cells. The activation of the mammalian target of rapamycin
(mTOR) has recently emerged as a key sensor of MHP (41) and
mediator of enhanced Ca2+ flux in lupus T cells (42). mTOR
activation may influence Ca2+ flux through interaction with
HRES-1/Rab4, a regulator of endocytic recycling of CD3 and
CD4 (Fig. 1) (43).
64 A. Perl et al.

1.2. Pharmacological
Targeting
of Mitochondrial
Dysfunction in SLE
MHP predisposes for increased ROI production (38). Oxidative
stress affects activity of transcription factors AP-1 and NF-kB
(44, 45), and, further downstream, may lead to the skewed
expression of IL-2, TNF, and IL-10 (46). Increased spontaneous
apoptosis of lymphocytes has been linked to increased IL-10 pro-
duction, release of Fas ligand, and overexpression of Fas receptor
in SLE (47). Since increased ROI levels confer sensitivity to H2O2,
NO, TNF, and Fas-induced cell death (14, 15), elevated baseline

Table 1
Signaling abnormalities of T cell death in patients with SLE

Signal Effect Reference

ym ROI , ATP (36)
ROI Spontaneous apoptosis , IL-10 production (36, 37)
GSH ROI , Spontaneous apoptosis (14, 36)
Spontaneous apoptosis Compartmentalized autoantigen release, disease (5, 6, 36, 76)
activity
H2O2 Apoptosis , necrosis (36)
CD3/CD28 AICD , necrosis (37)
ATP Predisposes for necrosis (36, 64)
Necrosis Inflammation (36), (77)
AICD Persistence of autoreactive cells (7, 37)
FasR Spontaneous apoptosis (76)
FasL Spontaneous apoptosis (47)
IL-10 Selective induction of apoptosis in SLE (37, 47, 78)
NO MHP, mTOR (39, 43, 50)
IL-10 blockade Spontaneous apoptosis , ROI (37, 47)
IL-12 Spontaneous apoptosis , ROI (37)
: increase; : decrease

Fig. 1. (continued) space (34). Phosphorylation of BAD by mitochondria-anchored PKA results in anti-apoptotic sequestration
of BAD into the cytosol (85). Signaling through cell death receptors, such as Fas (15), CD3/CD28 co-stimulation (36, 37),
ROS (24), NO (29), as well as lymphokines, IL-3, IL-10, IFN-g, and TGF-b1 influence ym, ATP synthesis and susceptibility
to apoptosis (37). MHP and mitochondrial biogenesis is mediated via production of NO by eNOS or nNOS (39) and
up-regulation of transcription factors PGC-1a, Tfam, and ALAS (86). NO production by eNOS may be compartmentalized to
the T cell synapse (87). NO causes transient MHP via reversible inhibition of complex IV/cytochrome c oxidase (29) and
persistent MHP via S-nitrosylation of complex I of the ETC in a state of GSH depletion (40). mTOR senses ym (41), interacts
with the small GTPase HRES-1/Rab4 (43), and regulates Ca2+ release (42).
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 65

Fig. 1. Overview of mitochondrial redox and metabolic checkpoints of T cell activation and apoptosis signals. Antigen
binding-initiated signaling through the T cell receptor complex/CD3 and the CD28 co-stimulatory molecule activate phso-
phatidylinositol 3-kinase (PI3K) and protein tyrosine kinases (PTK). Increased cytosolic Ca2+ concentration activates the
serine/threonine phosphatase calcineurin which dephosphorylates the NFAT. Dephosphorylated NFAT can translocate to the
nucleus where it promotes transcription of IL-2 in concert with AP-1, NF6B, and Oct-1. Ca2+ flux into mitochondria increases
production of ROS and NF-6B activation (7981). Mitochondrial membrane integrity is maintained by a balance of mem-
brane-stabilizing bcl-2 and bcl-XL and pore-inducing bax and bad (34) as well as the metabolic capacity to synthesize
reducing equivalents, NADPH, GSH, and TRX. Controlled increase of ROS levels activates NF-6B and promotes cell growth.
Excess ROS production and disruption of ym lead to activation-induced cell death executed by caspase 3 (digesting vitally
important proteins PARP, 70K U1RNP, lamin, and actin) and caspase 3-dependent DNase (CAD, causing nuclear DNA frag-
mentation). Cleavage by caspase 3 is thought to expose cryptic epitomes and cause autoantigenicity of self antigens (82).
Activity of redox-sensitive transcription factors NF-6B, p53, AP-1, and Sp1 is regulated through release from inhibitor
complexes and conformational changes in their active sites. Intracellular antioxidants reduced glutathione (GSH) and thi-
oredoxin (TRX-DT) are regenerated at the expense of NADPH supplied primarily through metabolism of glucose via the
pentose phosphate pathway (PPP) (83). Among PPP products, ribose 5-phosphate is required for nucleotide and DNA syn-
thesis and support cell growth, C3C7 sugars influence mitochondrial function and ROS production, inositol and ADP-ribose
serve as precursors for second messengers, inositol phosphates and cADP-ribose, respectively. Dehydroascorbate (DHA) is
imported through GLUT1. DHA is metabolized through the PPP, thereby enhancing GSH levels. DHA also increases surface
expression of Fas-R (84). Glutathione reductase and TRX reductase synthesize GSH and TRX-DT at the expense of NADPH.
Formulation of the PPP and its efficiency to provide NADPH is dependent on the expression of G6PD and TAL (14, 15). ym
is controlled by intracellular GSH/NADH/NADPH levels, integrity of the permeability transition pore complex largely com-
prised of adenine nucleotide translocator (ANT, inner membrane), voltage-dependent anion channel (VDAC, outer mem-
brane), and translocation and dimerization of pro- and anti-apoptotic bcl-2 family members in the intermembrane
66 A. Perl et al.

ym, ROI production, and pHi may have key roles in altered
activation and death of lupus T cells. Although MHP was not
affected, IL-10 antibody or IL-12 normalized ROI production
and intracellular alkalinization in lupus PBL (37). Therefore, IL-10
antagonists may partially correct signaling dysfunction in lupus.
Recent studies showed diminished GSH/GSSG ratios in the
kidneys of 8-month-old vs. 4-month-old (NZB NZW) F1 mice;
treatment with N-acetylcysteine (NAC), a precursor of GSH and
stimulator of its de novo biosynthesis, prevented the decline of
GSH/GSSG ratios, reduced autoantibody production and devel-
opment of glomerulopnephritis (GN) and prolonged the survival
of (NZB NZW) F1 mice (48). Oral NAC has been used to treat
oxidative stress in patients with idiopathic pulmonary fibrosis (IPF)
(49). In a 1-year study of IPF patients treated with prednisone and
azathioprine, addition of NAC (3 600 mg/day) improved vital
capacity and reduced myelotoxicity in comparison to placebo.
Therefore, prospective clinical studies appear justified to assess
whether NAC treatment can reverse GSH depletion, correct T cell
signaling defects and provide clinical benefit to patients with
lupus.
NO production is a particularly interesting target because it
provides a link between seemingly dissociated features of T cell
activation and mitochondrial function. NO induces MHP and
mitochondrial biogenesis, increases Ca2+ in the cytosol and mito-
chondria of normal T cells, and recapitulates the enhanced CD3/
CD28-induced Ca2+ fluxing of lupus T cells (50). NO contributes
to the development of GN in the MRL/lpr lupus mouse model
(51). Inactivation of iNOS does not block the development of
lupus (52), suggesting a role for eNOS and nNOS isoforms
expressed in T cells. However, given the widespread expression of
these isoforms in vascular smooth muscle and brain, it will be nec-
essary to develop T-cell-specific approaches for inhibiting NOS to
avoid potentially deleterious side effects.

2. Materials

1. Ficoll-Paque Plus (Amersham-Pharmacia, Uppsala, Sweden).

2. RPMI 1640 medium, fetal calf serum, penicillin, streptomycin,
amphotericin B (Life Technologies, Grand Island, NY).
3. OKT3 monoclonal antibody (CRL 8001 from ATCC,
Rockville, MD).
4. CD28.2 monoclonal antibody (Pharmingen, San Diego, CA).
5. Cytokines: IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15,
TNF-a, TGF-b1, and IFN-g (PeproTech, Rocky Hill, NJ).
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 67

6. Polyclonal goat anti-human IL-10 neutralizing antibody (R&D

Systems, Minneapolis, MN).
7. Annexin binding buffer: 10 mM HEPES pH 7.4, 140 mM
NaCl, and 2.5 mM CaCl2.
8. Phosphate-buffered saline (PBS): 8 g of NaCl, 0.2 g of KCl,
1.44 g of Na2HPO4, 0.24 g of KH2PO4 dissolved in 1 l of H2O
with pH adjusted to 7.4.
9. Fluorescein-conjugated annexin V (annexin V-FITC) and phy-
coerythrin-conjugated annexin V (annexin V-PE, R & D
Systems, Minneapolis, MN).
10. Propidium iodide (R&D Systems).
11. Triton X-100 (Sigma, St. Louis, MO).
12. Hydroethidine (HE, Molecular Probes, Eugene, OR).
13. Quantum Red/Cy5-conjugated monoclonal antibodies
directed to CD3, CD4, CD8, CD14 (Sigma, St. Louis, MO),
CD45RA, and CD45RO (Pharmingen, San Diego, CA).
14. Fluorescence microscope: Nikon Eclipse E800 camera (Nikon
Corporation, Tokyo, Japan). Equipped with SPOT digital
camera (Diagnostic Instruments, Sterling Heights, MI).
15. Flow cytometer: Becton Dickinson FACStar Plus flow cytom-
eter equipped with an argon ion laser delivering 200 mW of
power at 488 nm.
16. Oxidation-sensitive fluorescent probes 5,6-carboxy-2,7-
dichlorofluorescein-diacetate (DCFH-DA), dihydrorhodamine
123 (DHR) and hydroethidine (HE, Molecular Probes,
Eugene, OR).
17. Cationic lipophilic dyes with high binding affinity to
mitochondria: 3,3-dihexyloxacarbocyanine iodide
(DiOC6), 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimida
zolocarbocyanine iodide (JC-1), tetramethylrhodamine,
methyl ester, perchlorate (TMRM), all from Molecular Probes
(Eugene, OR).
18. Carbonyl cyanide m-chlorophenylhydrazone (mClCCP,
Sigma).
19. Luminometer: AutoLumat LB953 (Berthold GmbH, Wildbad,
Germany).
20. ATP determination kit (Molecular Probes, Eugene, OR).
21. ApoGlow kit (Lumitech, Nottingham, UK).
22. Carboxy SNARF-1-acetoxymethyl ester acetate (SNARF-1,
Molecular Probes, Eugene, OR) 23. DMSO (Sigma).
23. High K+ buffers of varying pH values (120 mM KCl, 30 mM
NaCl, 0.5 mM MgSO4, 1 mM CaCl2, 1 mM NaHPO4, 5 mM
glucose and 10 mM HEPES).
68 A. Perl et al.

24. Nigericin (Sigma, St. Louis, MO; diluted from a stock solution
of 500 mg/ml in ethanol).
25. Deproteinizing buffer for glutathione (GSH) assay: 70 % per-
chloric acid and 15 mM bathophenanthrolinedisulfonic acid
(BPDS, Sigma).
26. g-Glutamyl glutamate (g-Glu-Glu, Sigma), internal standard
for GSH assay.
27. After repeated freezing and thawing, samples were centrifuged
at 15,000 g for 3 min. 50 ml of 100 mM mono-iodo-acetic
acid in 0.2 mM m-cresol purple was added to 500 ml superna-
tant. Samples were neutralized by addition of 480 ml of 2 M
KOH and 2.4 M KHCO3 and incubated in the dark at room
temperature for 10 min. Then, 1 ml of 1 % fluoro-dinitro-
benzene was added and the samples were incubated in the dark
at 4 C overnight. After centrifugation and filtering, 100 ml of
supernatants were injected into the HPLC Model 2690 (Waters
Alliance System, Milford, MA) equipped with a Model 996
photodiode array detector and Spherisorb NH2 column
(4.6 250 mm; 10 mm; Waters).
28. 7-Amino-4-trifluoromethyl-coumarin (AFC, Sigma).
29. Caspase substrate peptides: DEVD-AFC, Z-IETD-AFC, where
Z represents a benzyloxycarbonyl group; caspase inhibitor
peptides Z-Val-Ala-Asp(Ome).fmk (Z-VAD), Boc-Asp.fmk
(Boc-Asp) as well as non-caspase cysteine protease inhibitor,
Z-Phe-Ala.fmk (Z-FA) can be obtained from Enzyme Systems
Products (Livermore, CA).
30. Caspase assay buffer: 250 mM sucrose, 20 mM HEPESKOH
pH 7.5, 50 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol.
31. Versene (Life Technologies).
32. Concanavalin A (Con A, Sigma).
33. Goat anti-mouse IgG (ICN, Aurora OH).
34. Tritiated thymidine, 3HTdR (ICN).
35. CH-11 IgM monoclonal antibody to Fas/Apo-1/CD95
(Upstate Biotechnology, Saranac Lake, NY).
36. Complete RPMI medium: RPMI 1640 supplemented with
10 % fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicil-
lin, 100 mg/ml gentamicin, and 10 mg/ml amphotericin B.
37. Plastic tissue culture dishes (Becton Dickinson, Franklin
Lake, NJ).
38. Phenol, chloroform, isoamyl alcohol, proteinase K, agarose
(all RNase and DNase-free. molecular biology grade, from
Sigma).
39. Spectrophotometer.
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 69

40. Luminometer.
41. Carbonyl cyanide m-chlorophenylhydrazone (mClCCP,
Sigma).
42. Folin & Ciocalteus Phenol Reagent Solution (Sigma).
43. 4 mm diameter 0.45 mm polypropylene filter (Whatman,
Mainstead, England).
44. Monoclonal antibody to poly(ADP-ribose) polymerase (PARP)
C-2-10 (53).
45. Monoclonal antibody 5F7 directed to C-terminal amino acids
176-460 of human FLICE/Mch5/caspase-8 (Panvera,
Madison, WI).
46. Monoclonal antibody 31A1067 directed to caspase 3 (Gene
Therapy Systems, San Diego, CA).
47. Monoclonal antibody C4 directed to human b actin
(Boehringer, Indianapolis, IN).
48. Biotinylated secondary antibodies and horseradish peroxidase-
conjugated avidin (Jackson Laboratories, West Grove, PA).
49. 4-Chloronaphthol (Sigma).
50. Enhanced chemiluminescence detection kit (Western Lightning
Chemiluminescence Reagent Plus, PerkinElmer Life Sciences,
Boston, MA).
51. Kodak Image Station 440CF equipped with Kodak 1D
Image Analysis Software (Eastman Kodak Company,
Rochester, NY).

3. Methods

The methods described below outline (1) in vitro lymphocyte cul-

ture, activation, and apoptosis assays, flow cytometric analysis of
(2) ym and (3) ROI production and (4) intracellular pH, (5)
measurement of intracellular ATP and ADP, (6) HPLC analysis of
reduced (GSH) and oxidized forms of glutathione (GSSG), and
(7) caspase enzyme assays (see Note 1).

3.1. Lymphocyte 1. Collect peripheral blood in sterile tubes containing 50 U hepa-

Culture, Activation,
and Viability Assays
3.1.1. Separation
of Peripheral Blood
Mononuclear Cells
rin (Sigma) per ml of blood.
2. Layer blood diluted 1:1 with PBS on Ficoll-Paque. Typically
layer 10 ml diluted blood over 5 ml of Ficoll-Paque.
3. Centrifuge cells at 500 g for 30 min with centrifuge
brake off.
70 A. Perl et al.

4. Remove peripheral blood mononuclear cells (PBMC) from

interface between Ficoll-Paque and plasma with pipettor.
5. Wash PBMC three times in PBS by centrifugation at 300 g
for 10 min.
6. PBMC are resuspended at 106 cells/ml in RPMI 1640 medium,
supplemented with 10 % fetal calf serum, 2 mM L-glutamine,
100 IU/ml penicillin, and 100 mg/ml gentamicin (complete
RPMI medium) and incubated for experiments at 37 C in a
humidified atmosphere with 5 % CO2.

3.1.2. Separation
of Monocytes and
Peripheral Blood
Lymphocytes
1. Precoat Petri dishes with autologous serum for 30 min
at 37 C.
2. Add 5 ml of PBMC (maximum 5 106/ml) to serum-pre-
treated dishes and incubate for 1 h at 37 C.
3. Remove nonadherent cells by washing three times with 5 ml of
warm (37 C) complete RPMI medium.
4. To obtain a monocyte-enriched cell fraction wash dishes vigor-
ously with warm medium.
5. Add 4 ml of ice-cold 0.05 % Versene and 1 ml autologous
serum to each dish for 15 min at room temperature.
6. Scrape off loosely adherent monocytes with a rubber police-
man under inverse microscopic control.
7. The monocyte-depleted fraction of peripheral blood lympho-
cytes (PBL) should contain less than 2 % monocytes, while the
monocyte-enriched fraction should contain 9095 % mono-
cytes by staining with CD14 monoclonal antibody.

3.1.3. Cell Culture, Human PBL undergo apoptosis in response to repetitive activation
Activation, and Viability through the T cell receptor, i.e., CD3/CD28 co-stimulation
Assays resulting in activation-induced cell death (AICD) (36, 37), cross-
linking of cell surface death receptors such as Fas/Apo-1/CD95
(15) or elevation of intracellular ROI levels after treatment with
H2O2 (24, 36). Monocytes/macrophages remove apoptotic bod-
ies via phagocytosis, therefore processing of cell death signals by
lymphocytes is best evaluated using PBL (see Note 2).

CD3/CD28 Co-stimulation 1. Precoat 10 cm diameter plastic Petri dishes with 100 mg/ml
of PBL goat anti-mouse IgG (diluted in PBS) for 2 h at 37 C.
2. Wash plates with PBS, add OKT3 monoclonal antibody (1 mg/
ml), and incubate for 1 h at 37 C.
3. Add PBL (106 cells/ml in complete RPMI medium).
4. For CD28 co-stimulation add 500 ng/ml mAb CD28.2 and
incubate cells at 37 C for the desired period of time.
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 71

Triggering Fas-Mediated 1. Sensitize peripheral blood T cells for Fas-mediated apoptosis

Cell Death of PBL by CD3/CD28 co-stimulation or treatment with 5 mg/ml
Con A for 57 days.
2. Monitor blast transformation by measurement of increased
side scatter (SSC) using flow cytometry (16) or 3HTdR incor-
poration via pulse-labeling with 0.4 mCi of 3HTdR of 105 cells
in 100 ml per well of a 96-well plate (54).
3. Determine cell viability by staining with 0.25 % trypan blue in
0.9 % NaCl. Viable cells should not stain with trypan blue.
4. Pellet and resuspend cells at 2 106 cells/ml in complete RPMI
medium.
5. Add an equal volume of complete RPMI medium without
(control) or with 2 mg/ml of CH11 IgM monoclonal
antibody.
6. Incubate cells at 37 C for the desired period of time.

Treatment with H2O2
1. Prepare fresh 10 mM H2O2 in PBS from 30 % stock solution.
2. Seed PBL at 2 106 cells/ml in complete RPMI medium.
3. Add an equal volume of complete RPMI medium without
(control) or with 100 mM H2O2.
4. Incubate cells at 37 C for the desired period of time.

Monitoring of Cell Death Apoptosis is monitored by observing cell shrinkage while counting
trypan blue-stained cells, DNA fragmentation using agarose gel
electrophoresis and quantified by flow cytometry after concurrent
staining with fluorescein-conjugated annexin V (annexin V-FITC,
R&D Systems, Minneapolis, MN; FL-1) and propidium iodide
(PI, FL-2) as earlier described (14, 15, 55, 56). Staining with phy-
coerythrin-conjugated annexin V (annexin V-PE, R&D Systems)
was used to monitor PS externalization (FL-2) in parallel with
measurement of ROI levels and ym (see below). Apoptosis rates
are expressed as percentage of annexin V-positive/PI-negative
cells. Necrosis is assessed by observing cellular and nuclear swell-
ing. Swollen nuclei of necrotic cells can be observed by staining
with propidium iodide (PI, 50 mg/ml). Necrotic cells are enumer-
ated by direct PI staining using flow cytometry and fluorescence
microscopy. Necrosis rates are expressed as percentage of PI-positive
population within annexin-positive cells (36) (see Note 4).
DNA Fragmentation Assay
1. Wash cells 3 in PBS using screw-capped 15 ml polypropylene
tubes.
2. Resuspend up to 5 107 cells in 100 mM NaCl, 10 mM
TrisHCL pH 8.0, and 1 mM EDTA.
3. Add 250 ml of 10 % SDS.
72 A. Perl et al.

4. Add 200 mg/ml proteinase K (keep 5 mg/ml aliquots

at 20 C).
5. Incubate overnight at 37 C.
6. Extract 2 with equal volume of phenol. Remove aqueous
phase only. Do not remove interface.
7. Extract 2 with equal volume of chloroform:isoamylalcohol
(24:1).
8. Precipitate DNA from aqueous phase with two volumes of
absolute ethanol for 10 min at room temperature.
9. Transfer DNA with Pasteur pipette to 500 ml of TE in
Eppendorf tube and let it dissolve for 30 min.
10. Add 1/10 volume of 3 M Na-acetate.
11. Add two volumes of absolute ethanol.
12. Precipitate DNA for 10 min at room temperature.
13. Repeat steps 912.
14. Dissolve DNA in 500 ml of 10 mM TrisHCL pH 8.0 and
1 mM EDTA.
15. Quantify DNA by reading optical density at 260 nm (OD260).
Read OD at 280 nm as well. OD260/280 should be 1.82.0.
1 OD corresponds to a DNA concentration of 50 mg/ml.
16. Store DNA at 4 C until analysis in a 2 % agarose gel (14).
Assessment of Apoptosis and Necrosis by Flow Cytometry
1. Resuspend 2 105 cells to be analyzed in 200 ml of annexin
binding buffer.
2. Add 5 ml of Annexin V-FITC (10 mg/ml) and 5 ml of PI
(50 mg/ml).
3. Incubate cells at room temperature in the dark.
4. Analyze by flow cytometry by electronic gating on live cells
based on forward scatter (FSC) and side scatter (SSC) and
measuring Annexin-FITC binding on the FL1 channel (emis-
sion at 530 nm) and PI staining on the FL-2 channel (emission
at 625 nm).

3.2. Flow Cytometric Mitochondrial transmembrane potential (ym) can be estimated by

Analysis of the cationic lipophilic dyes with high binding affinity to the negatively
Mitochondrial charged inner mitochondrial membrane (9, 57, 58). Since binding
Transmembrane characteristics do not completely overlap, parallel staining with sev-
Potential eral dyes is recommended (see Note 5).
1. Resuspend 2 105 cells in 200 ml of annexin binding buffer if
concurrently stained with Annexin V-FITC or Annexin V-PE
matching a potentiometric dye emitting FL-2 or FL-1
fluorescence, respectively. Alternatively, 2 105 cells can be
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 73

resuspended in 200 ml 5 mM HEPES-buffered saline (HBS

containing 0.9 % NaCl pH 7.4). This buffer, lacking Ca2+, does
not allow concurrent staining with Annexin V.
2. Aliquots of cell suspensions are stained with several potentio-
metric dyes in parallel.
(a) Add 200 ml of dye solution containing 20 nm DiOC6
(excitation: 488 nm, emission: 525 nm recorded in FL-1).
This dye can be added in combination with Annexin V-PE
emitting FL-2 fluorescence (see Note 3).
(b) Add 200 ml of dye solution containing 1 mM TMRM
(excitation: 549 nm, emission: 573 nm recorded in FL-2).
This dye can be added in combination with Annexin
V-FITC emitting FL-1 fluorescence.
(c) Add 200 ml of dye solution containing 0.5 mM JC-1. JC-1
selectively incorporates into mitochondria, where it forms
monomers (fluorescence in green, 527 nm) or aggregates,
at high transmembrane potentials (fluorescence in red,
590 nm) (59, 60).
3. Parallel cell suspensions should be treated 5 mM mClCCP.
Co-treatment with this protonophore for 15 min at 37 C
results in decreased DiOC6, TMRM, and JC-1 fluorescence
and serves as a positive control for disruption of mitochondrial
transmembrane potential (15).
4. Incubate cells in the dark for 15 min at 37 C before flow
cytometry.
5. For each sample, measurements are carried out on 10,000 cells.

3.3. Measurement Production of ROI can be assessed fluorometrically using

of ROI Production oxidation-sensitive fluorescent probes 5,6-carboxy-2,7-
dichlorofluorescein-diacetate (DCFH-DA), dihydrorhodamine
123 (DHR), and hydroethidine (HE, Molecular Probes, Eugene,
OR) as earlier described (14). Cells are stained in annexin binding
buffer or HBS (see Specific Protocol below) with 0.1 mM DHR for
2 min, 1 mM DCFH-DA for 15 min, or 1 mM HE for 15 min and
samples are analyzed using a Becton Dickinson FACStar Plus flow
cytometer equipped with an argon ion laser delivering 200 mW of
power at 488 nm. Fluorescence emission from 5,6-carboxy-2,7-
dichlorofluorescein (DCF; green) or DHR (green) is detected at a
wavelength of 530 30 nm. Fluorescence emission from oxidized
HE, ethidium (red), was detected at a wavelength of 605 nm.
Dead cells and debris are excluded from the analysis by electronic
gating on FSC and SSC measurements. While R123, the fluorescent
product of DHR oxidation, binds selectively to the inner mito-
chondrial membrane, ethidium and DCF remain in the cytosol of
living cells (61), thus, allowing measurement of ROI levels in dif-
ferent subcellular compartments.
74 A. Perl et al.

Specific Protocol
1. Following apoptosis assay, wash cells two times in 5 mM
HEPES-buffered saline (HBS, containing 0.9 % NaCl)
pH 7.4.
2. Resuspend 2 105 cells in 200 ml of annexin binding buffer if
concurrently stained with Annexin V-FITC or Annexin V-PE
matching oxidation-sensitive dyes emitting FL-2 (HE) or FL-1
fluorescence (DCF, R123), respectively. Alternatively, 2 105
cells can be resuspended in 200 ml of 5 mM HBS. This
buffer, lacking Ca2+, does not allow concurrent staining with
Annexin V.
3. Subsequently, aliquots of cell suspensions are stained with
several potentiometric dyes in parallel.
(a) Add 200 ml of dye solution containing 0.1 mM DHR (exci-
tation: 488 nm, emission: 530 nm recorded in FL-1). This
dye can be added in combination with Annexin V-PE
emitting FL-2 fluorescence. Staining is done in the dark at
room temperature for 2 min, timed with a stopwatch, fol-
lowed by running on the flow cytometer.
(b) Add 200 ml of dye solution containing 1 mM DCFH-DA
(excitation: 488 nm, emission: 525 nm recorded in FL-1).
This dye can be added in combination with Annexin V-PE
emitting FL-2 fluorescence. Staining is done in the dark at
room temperature for 15 min, timed with a stopwatch,
followed by running on the flow cytometer.
(c) Add 200 ml of dye solution containing 1 mM HE (excita-
tion: 488 nm, emission: 605 nm recorded in FL-2). This
dye can be added in combination with Annexin V-FITC
emitting FL-1 fluorescence. Staining is done in the dark at
room temperature for 15 min, timed with a stopwatch,
followed by running on the flow cytometer.
4. For each sample, measurements are carried out on 10,000
cells.

3.4. Intracellular Intracellular pH measurements are carried out with flow cytometry
pH Measurement using the pH-sensitive dye carboxy SNARF-1-acetoxymethyl ester
acetate (SNARF-1) as described by Wieder et al. (62). SNARF-1
enters cells passively as a nonpolar ester. It is then hydrolyzed by
intracellular esterases into a polar compound unable to leave mem-
brane-intact cells. The emission spectrum of SNARF-1 undergoes
a pH-dependent wavelength shift. The ratio of fluorescence inten-
sities emitted at two different wavelengths (FL2:580 nm and
FL3:650 nm) is used for determination of pH.
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients
3.5. Measurement
of Intracellular ATP
and ADP Levels
3.5.1. Collection of Cells
for ATP Assay
75
1. Make fresh 0.5 mg/ml stock solutions of SNARF-1 daily in
DMSO.
2. Resuspend 5 105 cells in 500 ml of PBS.
3. Add 5 mg/ml SNARF-1 to the cells and incubate samples
30 min at 37 C.
4. Wash cells once in 1 ml of PBS and resuspend in 500 ml of
PBS.
5. Analyze on a Becton Dickinson FACStar Plus flow cytometer.
The SNARF-1 dye is excited with 200 mW of the 488 nm
argon laser and fluorescence is collected in two wavelengths
(FL2:580 nm and FL3:650 nm) in the pulse processing mode.
6. Generate a standard calibration curve for each experiment by
staining the cells in high K+ buffers of varying pH values
(120 mM KCl, 30 mM NaCl, 0.5 mM MgSO4, 1 mM CaCl2,
1 mM NaHPO4, 5 mM glucose and 10 mM HEPES) in the
presence of 5 mg/ml nigericin to equilibrate the intracellular/
extracellular pH.
7. Calculate intracellular pH based on FL3/FL2 ratio.
T cell activation and apoptosis require the energy provided by
ATP (63). Intracellular ATP concentration is a key switch in the
cells decision to die via apoptosis or necrosis (64) and, therefore,
depletion of ATP may be responsible for defective apoptosis and a
predisposition to necrosis in patients with SLE (36). Intracellular
ATP levels can be determined with great sensitivity and specificity
using the luciferinluciferase method (65). Addition of luciferin
and firefly luciferase to ATP-containing biological sample results in
light emission. The luminometric ATP assay is based on the firefly
luciferase reaction:
ATP + D-Luciferin + O2 AMP + pyrophosphate
+ oxyluciferin + CO2 + light.
The quantum efficiency is very high resulting in almost one
photon per ATP molecule consumed in the reaction. The light is
measured in luminometer. Under assay conditions of constant
luciferase activity, intensity of the emitted light is proportional to
the ATP concentration. The assay is calibrated by the addition of a
known amount of ATP.
1. Collect 5 106 PBL by centrifugation at 300 g for 10 min and
wash once in PBS.
2. Resuspend cell pellet in 50 ml of PBS and mix with equal vol-
umes of 2.5 % trichloroacetic acid. Such extracts can be stored
at 20 C.
3. Measure the total protein content of each sample using the
Lowry assay (66).
76 A. Perl et al.

3.5.2. Lowry Assay Lowry A: 2 % Na2CO3 in 0.1 M NaOH.

Lowry B: 1 % CuSO4 in diH2O.
Stock Solutions
Lowry C: 2 % sodium potassium tartrate (NaKC4H4O64H2O).

Reagents Lowry Stock Reagent

49 ml Lowry A.
0.5 ml Lowry B and 0.5 ml Lowry C.
Folins Reagent: Phenol reagent2N (Folin-Ciocalteau reagent).
Dilute 1:1 in diH2O before use. BSA standard solution is prepared
as described in Table 2. The standard calibration solution is dis-
solved at a concentration of 1 mg/ml in a buffer similar to the
biological sample of unknown protein content, such as PBS.

Assay Procedure 1. Add 100 ml of sample (sample + buffer = 100 ml) per tube.
2. Add 1.0 ml of Lowry stock reagent to each tube.
3. Incubate 30 min at room temperature.
4. Add 100 ml of Folins reagent to each tube.
5. Incubate 30 min at room temperature.
6. Read in a spectrophotometer at 595 nm.

3.5.3. ATP Assay
The ATP contents of PBLs from patients with SLE and control
donors were assayed in parallel.
The bioluminescence assay was performed using an ATP deter-
mination kit (A22066, Invitrogen, Carslbad, CA) according to the
manufacturers instructions. Utilizing a Synergy 2 Multi-Mode
Microplate Reader (BioTek, Winooski, VT), equipped with auto-
matic reagent dispensers; in all-white, flat-bottom, 96-well plates
(Cat. No 7571, Thermo Scientific, Rochester, NY), ATP standard

Table 2
Preparation of protein standard solution for Lowry assay

Standard solution protein amount (mg) Standard solution volume(ml) Buffer volume (ml)
0 0 100
10 10 90
20 20 80
30 30 70
50 50 50
75 75 25
100 100 0
The standard calibration solution is dissolved at a concentration of 1 mg/ml in a buffer similar to the biological sample
of unknown protein content, such as PBS
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 77

curves are established in each experiment and should be linear in

the 5- to 5,000-nM range. In our laboratory, the sample volume
added to the reaction mixtures was less than 5 % of the total assay
volume (67). In our lab, the sample volume added to the reaction
mixtures was less than 2 % of the total assay volume.

Precautions Because of the high sensitivity of the luciferinluciferase reaction,

avoid contamination with ATP from exogenous biological sources,
such as bacteria or fingerprints. Therefore, latex or vynil gloves are
to be worn at all times.
Protect the D-luciferin and firefly luciferase reagents from light.
Mix solutions containing firefly luciferase gently, for example, by
inversion; vortex mixing may denature the enzyme.
Arsenate compounds may inhibit the reaction.
The temperature optimum for the reaction is 28 C. At higher
temperatures, the reaction is slower.

Reagents 1. D-Luciferin (MW 302) 3 mg of lyophilized powder per vial.

2. Luciferase, firefly recombinant,40 ml of a 5 mg/ml solution in
25 mM TrisHCL-acetate, pH 7.8, 0.2 M ammonium sulfate,
15 % (v/v) glycerol and 30 % (v/v) ethylene glycol.
3. Dithiothreitol (DTT) (MW 154) 25 mg.
4. Adenosine 5-triphosphate (ATP), 400 ml of a 5 mM solution
in TE buffer.
5. 20 Reaction Buffer, 10 ml of 500 mM Tricine buffer, pH 7.8,
100 mM MgSO4, 2 mM EDTA, and 2 mM sodium azide.
6. Standard reaction solution containing 0.5 mM D-luciferin,
1.25 mg/ml firefly luciferase, 25 mM Tricine buffer, pH 7.8,
5 mM MgSO4, 100 mM EDTA and 1 mM DTT.
Store reagent frozen at 80 C. Avoid repeated freezing and
thawing.

ATP Assay Protocol 1. Make 1.0 ml of 1 Reaction Buffer by adding 50 ml of 20

Reaction Buffer to 950 ml of deionized autoclaved H2O. This
volume will be sufficient to make 1 ml of 10 mM D-luciferin
stock solution.
2. Make 1 ml of a 10 mM D-luciferin stock solution by adding
1 ml of 1 Reaction Buffer (prepared in step 1 in ATP Assay
Protocol) to one vial of D-luciferin (3 mg of lyophilized pow-
der). Protect from light until use. The D-luciferin stock solu-
tion is reasonably stable for several weeks if stored at 20 C,
protected from light.
3. Prepare a 100 mM DTT stock solution by adding 1.62 ml of
H2O to 25 mg of DTT. Aliquot into ten 160 ml volumes and
78 A. Perl et al.

store frozen at 20 C. Stock solutions of DTT stored properly

are stable for 6 months to 1 year. Thawed aliquots should be
kept on ice until ready for use.
4. Prepare low-concentration ATP standard solutions from 5 nM
to 5 mM by diluting the 5 mM ATP stock solution in H2O.
These dilute solutions are stable for several weeks when stored
at 20 C.
5. Make 10 ml of a standard reaction solution by combining the
following:
8.9 ml dH2O.
0.5 ml 20 Reaction Buffer.
0.1 ml 0.1 M DTT.
0.5 ml of 10 mM D-luciferin.
2.5 ml of firefly luciferase 5 mg/ml stock solution.
6. Gently mix the tube by inverting THREE times. The firefly
luciferase enzyme is easily denatured by vortexing. Keep the
reaction solution protected from light until use.
7. Create standard curve.
(a) Measure luminescence of standard reaction solution (pre-
pared in step 5 in Subheading ATP Assay Protocol)
which is considered as background.
(b) Start the reaction by adding the desired amount of dilute
ATP standard solution (prepared in step 4 in Subheading
ATP Assay Protocol) and read the luminescence. The
volume of the dilute ATP standard solution that is added
to the standard assay solution should be no more than
10 %, preferably less than 2 %, of the total assay volume.
(c) Subtract the background luminescence.
(d) Generate a standard curve for a series of ATP concentra-
tions. Be sure to always add a constant sample volume of
the ATP-containing solution as internal standard.
8. Sample Analysis
(a) Add experimental sample to standard reaction solution.
The total volume of the experimental sample assays should
be equal to that of the ATP standard assays, with the
amount of experimental sample added no more than 10 %
of the total assay volume.
(b) Calculate the amount of ATP in the experimental samples
from the standard curve.
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients
Assessment of ATP/ADP
Ratio and ADP Levels
3.6. HPLC Assay
of Glutathione Levels
iodoacetic acid
temperature, in dark,
Fig. 2. The two-step derivatization of GSH.
79
ADP can be measured by its conversion to ATP, using the ApoGlow
kit (Lumitech, Nottingham, UK). The produced ATP is then
detected by the above luciferinluciferase method.
1. Dispense 100 ml of standard reaction solution with experimental
sample as described in step 8a in ATP Assay Protocol into 96
well plates.
2. Load plate into 96-well plate reader and record luminescence:
reading A.
3. After 10 min lag period, add 20 ml of ADP-converting reagent
to each well using multichannel pipettor or autodispenser built
into luminometer if available.
4. Take a 1 s integrated reading: reading B. If autoinjector is
unavailable, reading B should be taken before addition of
ADP-converting reagent.
5. After 5 min incubation allowing conversion of ADP to ATP
take a final 1 s integrated reading: reading C.
6. ADP:ATP ratio is calculated as follows: (C B)/A.
Reduced (GSH) and oxidized glutathione (GSSG) as well as other
intermediates of GSH metabolism can be concurrently measured
by reverse phase ion-exchange high-performance liquid chroma-
tography (HPLC) using UV detection at 365 nm (68).
We use a two-step derivatization procedure: (1) S-carbo-
xymethylation of the reduced SH groups with iodoacetic acid to
prevent their oxidation and (2) N-dinitrophenylation with
1-fluoro,2,4-dinitrobenzene to allow UV detection (Fig. 2).
1. Wash 2 107 PBL once in 5 ml of PBS and store cell pellet
at 80 C until assay.
2. Resuspend cell pellet in 250 ml of H2O. Use 10 ml of cell
suspension to measure protein content as described in
Subheading 3.5.2.
3. Add 50 ml of 70 % perchloric acid, 25 ml of 15 mM BPDS to
deproteinize sample as well as 25 ml of g-Glu-Glu as internal
standard.
1. 2.
1-fluoro-2,4-
dinitrobenze
10 min, room 4 C,
overnight, in
pH=8-9 dark
80 A. Perl et al.

4. Vortex, freeze, and thaw the sample in two cycles.

5. Pellet sample in microcentrifuge at 15,000 g for 5 min and
save supernatant.
6. Add 25 ml of 100 mM mono-iodo-acetic acid in 0.2 mM
m-cresol purple to 250 ml of supernatant.
7. Adjust pH of acidic solution (pink in color) to pH 89 (purple
in color) by addition of 240 ml of 2 M KOH and 2.4 M
KHCO3.
8. Incubate sample in the dark at room temperature for 10 min.
9. Add 1 ml of 1 % fluoro-dinitro-benzene, vortex, and incubate
the samples in the dark at 4 C overnight.
10. Centrifuge sample at 15,000 g for 10 min at 4 C.
11. Filter supernatant through 4 mm diameter 0.45 mm polypro-
pylene filter (Whatman, Maidstone, England).
12. Inject 50 ml of each sample into HPLC equipped with a Model
996 photodiode array detector (Waters Alliance System,
Milford, MA) and a Waters Spherisorb 3-NH2-propyl column
(4.6 250 mm; 10 mm). A UV detector set to a wavelength of
365 nm can also be utilized.
13. After sample injection, a mobile phase of HPLC comprised of
80 % of Mobile Phase Solution A (80 % methanol) and 20 % of
Mobile Phase Solution B (0.5 M sodium acetate dissolved in
64 % methanol) is maintained for 5 min, followed by a 10-min
linear gradient to 1 % Mobile Phase Solution A/99 % Mobile
Phase Solution B at a flow rate of 1.5 ml/min. Then, the
mobile phase is held at 99 % Mobile Phase Solution B until the
final compound, usually GSSG, has eluted (510 min).

3.7. Caspase Enzyme Activation of the caspase enzyme cascade is a hallmark of apoptosis.
Assays Caspase-3 is a key effector of all apoptosis pathways, amplifying the
signal from initiator caspases (such as caspase-8) and indicating a
final commitment to cellular disassembly. In addition to cleaving
other caspases in the enzyme cascade, caspase-3 has been shown to
cleave poly(ADP-ribose) polymerase (PARP), DNA-dependent
protein kinase, protein kinase C, the 70 kDa component of U1
snRNP, and actin (see Note 6).
1. Induce apoptosis in cells by desired method. Remember to
incubate a concurrent control culture without induction.
Include cells treated with caspase inhibitor DEVD-CHO as
negative control (15).
2. After washing cells once in PBS, pellet 106 cells per experimental
sample at 400 g for 5 min. Cell pellet can be stored at 80 C
until measurement.
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 81

3. Resuspend cell pellet in 25 ml of chilled cell lysis buffer

comprised of 10 mM HEPES/KOH (pH 7.4), 2 mM
EDTA, 0.1 % CHAPS, 5 mM dithiothreitol, 1 mM
phenylmethylsulphonylfluoride, 10 mg/ml pepstatin A, 20 mg/
ml leupeptin, 10 mg/ml aprotinin. Alternatively, cell lysis buf-
fer from Clontech can be used.
4. Incubate cell lysate on ice for 15 min.
5. Pellet cell lysates in a microcentrifuge at 15,000 g for 10 min
at 4 C.
Transfer the supernatants to new microcentrifuge tubes.
Samples may be assayed immediately or frozen at -80 C until
measurement.
6. Add 25 ml of 2 reaction buffer containing 80 mM zDEVD-
AFC substrate (derived from 1 mM stock in DMSO), 250 mM
sucrose, 20 mM HEPESKOH (pH 7.5), 50 mM KCl, 2.5 mM
MgCl, 1 mM freshly added DTT. Set up parallel samples lack-
ing zDEVD-AFC substrate and containing caspase 3 inhibitor
peptide zDEVD-AFC.
7. Incubate at 37 C for 1560 min (depending on caspase activ-
ity) and add ice-cold H2O to 1 ml. Reaction can be stored
frozen at 80 C for later measurement.
8. Prepare 12-point calibration curve with 0.0024 mM AFC.
9. Read fluorescence of experimental samples with 400 nm exci-
tation filter and 505 nm emission filter.
10. After reading the fluorescence take 0.4 ml aliquot from experi-
mental samples and add 0.4 ml Lowry reagent and 0.2 ml Folin
reagent (according to Lowry protocol described in
Subheading 3.5.2) to measure protein content. Add lysis and
reaction buffer to set up standard blank tubes.
11. Express caspase activity as pmoles AFC/min/mg protein.
Activities vary between 1,000 and 5,000 U/mg protein.
12. Western blot analysis of caspase activity.
Caspases are activated through proteolysis that can be
monitored by Western blot analysis.
(a) For visualization of poly(ADP-ribose) polymerase (PARP)
caspase 3 and caspase 8, lyse 2 106 cells in 100 ml of
62.5 mM TrisHCl pH 6.8, 6 M urea, 10 % glycerol, 2 %
SDS, 0.00125 % bromophenol blue, and 5 %
b-mercaptoethanol.
(b) Sonicate lysate for 15 s, boil for 5 min, and store at 80 C.
(53).
(c) Assess protein concentration by Lowry method (see
Subheading 3.5.2).
82 A. Perl et al.

(d) Separate 40 mg of total cell lysate in 10 ml per well by

SDS-PAGE and electroblot to nitrocellulose (69).
(e) Incubate nitrocellulose strips in 100 mM TrisHCL pH 7.5,
0.9 % NaCl, 0.1 % Tween 20, and 5 % skim milk with the
primary antibodies, anti-PARP monoclonal antibody C-2-
10 (53), anti-caspase 3 (GTS), anti-caspase-8 (Panvera),
or actin mAb C4 (Boehringer, Indianapolis, IN) at a
1,000-fold dilution at room temperature overnight.
(f) After washing, incubate blots with biotinylated secondary
antibodies, and, subsequently, with horseradish peroxi-
dase-conjugated avidin. In between incubations the strips
are vigorously washed in 0.1 % Tween-20, 100 mM TrisHCL
pH 7.5, and 0.9 % NaCl.
(g) The blots can be developed with a substrate comprised of
1 mg/ml 4-chloronaphthol and 0.003 % hydrogen peroxide
or using enhanced chemiluminescence (ECL) detection.
(h) Activation of the caspase cascade is monitored by cleavage
of caspase 3 (32 kDa precursor into 17 kDa active form),
caspase 8 (55 kDa precursor into 42 kDa active doublet),
and PARP (116 kDa precursor into 85 kDa fragment).

3.8. Measurement The method described here explains (a) how to permeabilize PBL
of Mitochondrial (70); (b) and measure the activity of the mitochondrial ETC
Electron Transport (71, 72).
Chain Activity 1. Mannitol (Sigma).
3.8.1. Reagents 2. KCl (Fisher).
3. MgCl (J.T. Baker, Phillipsburg, NJ).
4. K2PO4 (USB).
5. Digitonin (Sigma).
6. Bovine serum albumin fraction V, heat shock, fatty-acid free
(BSA; Roche).
7. ADP (Sigma).
8. Sodium pyruvate (Sigma).
9. Malic acid (Sigma).
10. Rotenone (Sigma).
11. Succinic acid (Fisher).
12. ATP (Sigma).
13. Antimycin A (Sigma).
14. L-Ascorbic acid (Sigma).
15. N,N,N ,N -Tetramethyl-p-phenylenediamine (TMPD; Sigma).
16. Oxygraph system (Hansatech Instruments, Norfolk,
England).
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients
3.8.2. Permeabilizing PBL
3.8.3. Testing ETC
Complexes IIV
Testing Complex I
Testing Complexes IIIV
(If Possible Done
in Separate Chamber)
3.8.4. Digitonin Purification
83
1. Wash PBL in PBS twice this is to ensure the cells are as clean as
possible if they are not truly clean it can affect how well the
cells are permeabilized.
2. Resuspend at a concentration of 5 106/ml in respiration
buffer (RB) containing 0.3 M mannitol, 10 mM KCl, 5 mM
MgCl, 10 mM K2PO4 pH adjusted to 7.4.
3. Set aside 300 ml of the cell suspension for whole cell respiration
to determine the baseline respiration rate of your cells.
4. Add digitonin to a final concentration of 60 mg/ml, as an
example, add 21 ml of a 2 mg/ml stock solution to 700 ml of
cell suspension; digitonin purification is described in 3.9.4.
5. Incubate for 1 min at room temperature.
6. Add 5 volumes (e.g., 5 21 ml = 105 ml, for 700 ml of cell
suspension) of RB containing 1 mg/ml of BSA (made fresh
that day) to stop the permeablization of the cell membranes.
7. Spin down cells at 500 g for 10 min.
8. Resuspend the pellet at 5 106 cells/ml in RB containing
1 mg/ml BSA and 0.5 mM ADP (made fresh that day).
9. Let the cells rest at room temperature for 5 min.
O2 consumption is measured in a Hansatech Instruments Oxygraph
system which uses of a Clark type polarographic sensor. A water
bath is used to maintain the temperature in the chamber at 37 C.
The electrode disc is stored in a desecrator under vacuum when
not in use, and is prepared prior to each use. Cleaning of the elec-
trode is done when deemed necessary (see Notes 710).
(a) Place 300 ml of cell suspension into the oxygraph chamber.
(b) Collect a baseline reading.
(c) Add pyruvate and malate to final concentrations of 8 mM and
0.2 mM, respectively, to measure complex I activity.
(a) Inhibit complex I by adding Rotenone to a final concentration
of 3 mM.
(b) Complex II is measured with the addition of 10 mM final
concentration succinate and 130 mM ATP.
(c) Inhibit complex II with a final concentration of 1 mM anti-
mycin A.
(d) Complexes III and IV are measured through the addition of
ascorbate and TMPD to final concentrations of 10 and 0.2 mM
respectively.
1. Weigh an empty Eppendorf tube.
2. Add 4050 mg of digitonin.
84 A. Perl et al.

3. Add 1 ml of pure ethanol; the digitonin will not dissolve;

however, this step will remove most impurities. Some batches
of digitonin may be only 70 % pure.
4. Spin at 14,000 g for 10 s discard the supernatant.
5. Repeat steps 3 and 4 two more times.
6. Air-dry pellet, weighing every 5 min until you get two consistent
weights.
7. Resuspend the digitonin in H2O to a final concentration of
2 mg/ml.
8. Spin at 14,000 g for 1 min to remove any final impurities.
9. Collect the supernatant, this is to be considered the pure
digitonin.

4. Notes

1. When assessing T cell activation and apoptosis in PBL of patients

with SLE, parallel processing of PBL from healthy and
inflammatory disease, such as rheumatoid arthritis, control
donors is essential. We typically establish values for normal
donors, and process cells from two controls in parallel with cells
from two patients. Whenever possible, we use freshly isolated
cells. Alternatively, PBL frozen in complete RMPI 1640
medium with 7.5 % DMSO/30 % FCS can be utilized when
viability of defrosted cells exceeds 98 % by trypan blue
staining.
2. Testing of apoptosis by PBMC vs. PBL may reflect influence of
phagocytic activity by monocytes/macrophages. Such com-
parative analysis should be supplemented by direct analysis of
macrophage phagocytosis. However, phagocytosis depends on
many factors, such as Fc receptor polymorphisms, release of
macrophage-activating factors by T and B cells. Therefore, we
routinely analyze PBL as effectors.
3. Fluorochromes are kept as highly concentrated (>10 mM)
stock solutions in DMSO at 80 C, unless indicated other-
wise. SNARF-1 is kept aliquoted in powder form and reconsti-
tuted in DMSO on the day of assay.
4. PI directly stains necrotic cells. As earlier described (14, 73), live
or apoptotic cells do not stain with PI and require permeabiliza-
tion with 0.1 % Triton X-100. When using hydroethidine (HE,
FL-2) for ROI measurement, cells are co-stained with fluorescein-
conjugated annexin V (annexin V-FITC, R&D Systems, FL-1).
Thus, annexin V-PE or annexin V-FITC are matched with emis-
sion spectra of potentiometric and oxidation-sensitive fluorescent
probes. Staining with annexin V alone or in combination with
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 85

DHR or DiOC6 was carried out in 10 mM HEPES pH 7.4,

140 mM NaCl, and 2.5 mM CaCl2. Using three-color
fluorescence, mitochondrial ROI levels, ym, and PS external-
ization within T cell subsets can be concurrently analyzed by
parallel staining with DHR or DiOC6 (FL1), annexin V-PE
(FL2), and Quantum Red/Cy5-conjugated monoclonal anti-
bodies directed to CD3, CD4, CD8 (Sigma, St. Louis, MO;
FL3), CD45RA, and CD45RO (Pharmingen). Quantum Red
contains two covalently linked fluorochromes, PE and Cy5. PE
absorbs light energy at 488 nm and emits at 670 nm, in the exci-
tation range of Cy5 which acts as an acceptor dye. For fluorescence
microscopy, cells were photographed using a Nikon Eclipse
E800 camera (Nikon Corporation, Tokyo, Japan). Green and
red fluorescent images were digitally superimposed using SPOT
software (Diagnostic Instruments, Sterling Heights, MI).
5. Mitochondrial transmembrane potential (ym) can be esti-
mated by cationic lipophilic dyes with high binding affinity to
the negatively charged inner mitochondrial membrane (9, 57,
58). Since binding characteristics do not completely overlap,
parallel staining with several dyes is recommended.
6. The AFC calibration curve is very stable when measured on
the same instrument and setting.
The caspase assay is time- and protein-dependent when
done with the suggested cell number and substrate concentra-
tion. Specificity of the enzymatic reaction was tested by
using caspase-3 inhibitor DEVD-CHO and caspase-1/ICE
inhibitor YVAD-CMK at a concentration range of 50300 mM
(74). Caspase 8 activity can be tested by using Z-IETD-AFC as
substrate. Caspase inhibitors Z-Val-Ala-Asp(Ome).fmk
(Z-VAD) and Boc-Asp.fmk (Boc-Asp) as well as non-caspase
cysteine protease inhibitor, Z-Phe-Ala.fmk (Z-FA) were tested
at concentrations of 20, 50, and 300 mM (75).
7. For permeabilization of cell membrane prior to mitochondrial
O2 consumption assay, add digitonin until 80 % of cells stain
with trypan blue. Permeabilized state is indicated by trypan
blue staining of the cytosol but not the nucleus of cells.
8. The Hansatech O2 electrode is cleaned whenever the silver ring
builds up as black oxidized film. This will cause spikes in the O2
in the rate of consumption. It is important to not overclean the
electrode as the cleaning solutions can damage the resin.
9. Keep substrates at 37 C when running O2 consumption
experiments. This will reduce fluctuations in O2 reading from
the addition of the substrates to 37 C chamber.
10. Rotenone can stick to the plastic of the Oxygraph chamber. To
prevent inadvertent inhibition of complex I, it is suggested
to use separate chambers or two separate Oxygraph systems to
measure complexes I and complexes IIIV, respectively.
86 A. Perl et al.
References
1. Elkon KB (1994) Apoptosis in SLEtoo little
or too much? [Review] [45 refs]. Clin Exp
Rheumatol 12:553559
2. Perl A, Banki K (1999) Molecular mimicry,
altered apoptosis, and immunomodulation as
mechanisms of viral pathogenesis in systemic
lupus erythematosus. In: Kammer GM, Tsokos
GC (eds) Lupus: molecular and cellular patho-
genesis, 1st edn. Humana, Totowa, NJ, pp
4364
3. Cohen JJ, Duke RC, Fadok VA, Sellins KS
(1992) Apoptosis and programmed cell death
in immunity. Annu Rev Immunol 10:267293
4. Thompson CB (1995) Apoptosis in the patho-
genesis and treatment of disease. Science
267:14561462
5. Emlen W, Niebur JA, Kadera R (1994)
Accelerated in vitro apoptosis of lymphocytes
from patients with systemic lupus erythemato-
sus. J Immunol 152:36853692
6. Casciola-Rosen LA, Anhalt G, Rosen A (1994)
Autoantigens targeted in systemic lupus ery-
thematosus are clustered in two populations of
surface structures on apoptotic keratinocytes. J
Exp Med 179:13171330
7. Kovacs B, Vassilopoulos D, Vogelgesang SA,
Tsokos GC (1996) Defective CD3-mediated
cell death in activated T cells from patients with
systemic lupus erythematosus: role of decreased
intracellular TNF-alpha. Clin Immunol
Immunopathol 81:293302
8. Skulachev VP (1999) Mitochondrial physiol-
ogy and pathology; concepts of programmed
death of organelles, cells and organisms. Mol
Aspects Med 20:139140
9. Xiang J, Chao DT, Korsmeyer SJ (1996) BAX-
induced cell death may not require interleukin
1b-converting enzyme-like proteases. Proc
Natl Acad Sci U S A 93:1455914563
10. Susin SA, Zamzami N, Castedo M, Daugas E,
Wang H-G, Geley S et al (1997) The central
executioner of apoptosis: multiple connections
between protease activation and mitochondria
in Fas/Apo-1/CD95- and ceramide-induced
apoptosis. J Exp Med 186:2537
11. Vander Heiden M, Chandel NS, Williamson
EK, Schumaker PT, Thompson CB (1997)
Bcl-XL regulates the membrane potential and
volume homeostasis of mitochondria. Cell
91:627637
12. Constantini P, Chernyak BV, Petronilli V,
Bernardi P (1996) Modulation of the mitochon-
drial permeability transition pore by pyridine
nucleotides and dithiol oxidation at two sep-
arate sites. J Biol Chem 271:67466751
13. Mayes PA (1993) The pentose phosphate path-
way & other pathways of hexose metabolism.
In: Murray RK, Granner DK, Mayes PA,
Rodwell VW (eds) Harpers biochemistry, 23rd
edn. Appleton & Lange, Norwalk, CT, pp
201211
14. Banki K, Hutter E, Colombo E, Gonchoroff
NJ, Perl A (1996) Glutathione levels and sensi-
tivity to apoptosis are regulated by changes in
transaldolase expression. J Biol Chem
271:3299433001
15. Banki K, Hutter E, Gonchoroff N, Perl A
(1999) Elevation of mitochondrial transmem-
brane potential and reactive oxygen intermedi-
ate levels are early events and occur
independently from activation of caspases in
Fas signaling. J Immunol 162:14661479
16. Perl A, Gergely P Jr, Puskas F, Banki K (2002)
Metabolic switches of T-cell activation and
apoptosis. Antiox Redox Signal 4(5):427443
17. Meier B, Radeke HH, Selle S, Younes M, Sies
H, Resch K et al (1989) Human fibroblasts
release reactive oxygen species in response to
interleukin-1 or tumor necrosis factor-a.
Biochem J 263:539545
18. Hennet T, Richter C, Peterhans E (1993)
Tumor necrosis factor-a induces superoxide
anion generation in mitochondria of L929
cells. Biochem J 289:587592
19. Schulze-Osthoff K, Krammer PH, Droge W
(1994) Divergent signaling via APO-1/Fas and
the TNFreceptor, two homologous molecules
involved in physiological cell death. EMBO J
13:45874596
20. Gergely P, Nekam K, Lang I, Kalmar L,
Gonzalez-Cabello R, Perl A (1984)
Ketoconazole in vitro inhibits mitogen-induced
blastogenesis, antibody-dependent cellular
cytotoxicity, natural killer activity and random
migration of human leukocytes.
Immunopharmacology 7:167170
21. Williams MS, Henkart PA (1996) Role of reac-
tive oxygen intermediates in TCR-induced
death of T cell blasts and hybridomas.
J Immunol 157:23952402
22. Gulbins E, Brenner B, Schlottmann K, Welsch
J, Heinle H, Koppenhoefer UL et al (1996)
Fas-induced programmed cell death is medi-
ated by a Ras-regulated O2-synthesis.
Immunology 89:205212
23. Um HD, Orenstein JM, Wahl SM (1996) Fas
mediates apoptosis in human monocytes by a
reactive oxygen intermediate dependent path-
way. J Immunol 156:34693477
24. Puskas F, Gergely P, Banki K, Perl A (2000)
Stimulation of the pentose phosphate pathway
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients
and glutathione levels by dehydroascorbate,
the oxidized form of vitamin C. FASEB J 14:
13521361
25. Li P-F, Dietz R, von Harsdorf R (1999) p53
regulates mitochondrial membrane potential
through reactive oxygen species and induces
cytochrome c-independent apoptosis blocked
by bcl-2. EMBO J 18:60276036
26. Gottlieb E, Vander Heiden MG (2000)
Thompson CB. Bcl-XL prevents the initial
decrease in mitochondrial membrane potential
and subsequent reactive oxygen species produc-
tion during tumor necrosis factor alpha-induced
apoptosis. Mol Cell Biol 20:56805689
27. Scarlett JL, Sheard PW, Hughes G, Ledgerwood
EC, Ku H-H, Murphy MP (2000) Changes in
mitochondrial membrane potential during
staurosporin-induced apoptosis in Jurkat cells.
FEBS Lett 475:267272
28. Sanchez-Alcazar JA, Ault JG, Khodjakov A,
Schneider E (2000) Increased mitochondrial
cytochrome c levels and mitochondrial hyper-
polarization precede camptothecin-induced
apoptosis in Jurkat cells. Cell Death Differ
7(11):10901100
29. Almeida A, Almeida J, Bolanos JP, Moncada S
(2001) Different responses of astrocytes and
neurons to nitric oxide: the role of glycolyti-
cally generated ATP in astrocyte protection.
Proc Natl Acad Sci U S A (2006) Rapamycin reduces disease activity and
98(26):1529415299
30. Tewari M, Dixit VM (1995) Fas- and tumor
necrosis factor-induced apoptosis is inhibited
by the Poxvirus crmA gene product. J Biol
Chem 270:32553260
31. Los M, Van de Craen M, Penning LS, Schenk
H, Westendorp M, Baeuerle PA et al (1995)
Requirement of an ICE/CED-3 protease for
Fas/Apo-1-mediated apoptosis. Nature
375:8183
32. Enari M, Hug H, Nagata S (1995) Involvement
of an ICE-like protease in Fas-mediated apop-
tosis. Nature 375:7881
33. Salvesen GS, Dixit VM (1997) Caspases:
Intracellular signaling by proteolysis. Cell
91:443446
34. Gross A, McDonnell JM, Korsmeyer SJ (1999)
BCL-2 family members and the mitochondria
in apoptosis [Review] [122 refs]. Genes Dev
13:18991911
35. Banki K, Hutter E, Gonchoroff NJ, Perl A
(1998) Molecular ordering in HIV-induced
apoptosis: oxidative stress, activation of caspases,
and cell survival are regulated by transaldolase.
J Biol Chem 273:1194411953
36. Gergely PJ, Grossman C, Niland B, Puskas F,
Neupane H, Allam F et al (2002) Mitochondrial
87
hyperpolarization and ATP depletion in patients
with systemic lupus erythematosus. Arthritis
Rheum 46:175190
37. Gergely PJ, Niland B, Gonchoroff N, Pullmann
R Jr, Phillips PE, Perl A (2002) Persistent mito-
chondrial hyperpolarization, increased reactive
oxygen intermediate production, and cytoplas-
mic alkalinization characterize altered IL-10
signaling in patients with systemic lupus ery-
thematosus. J Immunol 169:10921101
38. Stryer L (1988) Biochemistry, 3rd edn. W.H.
Freeman and Company, New York
39. Nagy G, Koncz A, Perl A (2003) T cell activa-
tion-induced mitochondrial hyperpolarization
is mediated by Ca2+- and redox-dependent pro-
duction of nitric oxide. J Immunol
171:51885197
40. Clementi E, Brown GC, Feelisch M, Moncada
S (1998) Persistent inhibition of cell respira-
tion by nitric oxide: crucial role of S-nitrosylation
of mitochondrial complex I and protective
action of glutathione. Proc Natl Acad Sci U S A
95(13):76317636
41. Desai BN, Myers BR, Schreiber SL (2002)
FKBP12-rapamycin-associated protein associ-
ates with mitochondria and senses osmotic
stress via mitochondrial dysfunction. Proc Natl
Acad Sci U S A 99:43194324
42. Fernandez D, Bonilla E, Mirza N, Perl A
normalizes T-cell activation-induced calcium
fluxing in patients with systemic lupus erythe-
matosus. Arthritis Rheum 54(9):29832988
43. Fernandez DR, Telarico T, Bonilla E, Li Q,
Banerjee S, Middleton FA et al (2009)
Activation of mTOR controls the loss of TCRz
in lupus T cells through HRES-1/Rab4-
regulated lysosomal degradation. J Immunol
182:20632073
44. Sen CK, Packer L (1996) Antioxidant and
redox regulation of gene transcription [see
comments]. [Review] [94 refs]. FASEB J
10:709720
45. Li N, Karin N (1999) Is NF-kB the sensor of
oxidative stress? FASEB J 13:11371143
46. Le Moine O, Louis H, Stordeur P, Collet JM,
Goldman M, Deviere J (1997) Role of reactive
oxygen intermediates in interleukin 10 release
after cold liver ischemia and reperfusion in
mice. Gastroenterology 113:17011706
47. Georgescu L, Vakkalanka RK, Elkon KB, Crow
MK (1997) Interleukin-10 promotes activa-
tion-induced cell death of SLE lymphocytes
mediated by Fas ligand. J Clin Invest
100:26222633
48. Suwannaroj S, Lagoo A, Keisler D, McMurray
RW (2001) Antioxidants suppress mortality in
88 A. Perl et al.
the female NZB NZW F1 mouse model of
systemic lupus erythematosus (SLE). Lupus
10(4):258265
49. Demedts M, Behr J, Buhl R, Costabel U,
Dekhuijzen R, Jansen HM et al (2005) High-
dose acetylcysteine in idiopathic pulmonary
fibrosis [see comment]. N Engl J Med
353:22292242
50. Nagy G, Barcza M, Gonchoroff N, Phillips PE,
Perl A (2004) Nitric oxide-dependent mito-
chondrial biogenesis generates Ca2+ signaling
profile of lupus T cells. J Immunol
173(6):36763683
51. Weinberg JB, Granger DL, Pisetsky DS, Seldin
MF, Misukonis MA, Mason SN et al (1994)
The role of nitric oxide in the pathogenesis of
spontaneous murine autoimmune disease:
Increased nitric oxide production and nitric
oxide synthase expression in MRL-lpr/lpr
mice, and reduction of spontaneous glomeru-
lonephritis and arthritis by orally administered
NG-monomethyl-L-arginine. J Exp Med
179:651660
52. Gilkeson GS, Mudgett JS, Seldin MF, Ruiz P,
Alexander AA, Misukonis MA et al (1997)
Clinical and serologic manifestations of auto-
immune disease in MRL-lpr/lpr mice lacking
nitric oxide synthase type 2. J Exp Med
186:365373
53. Lamarre D, Talbot B, De Murcia G, LaPlante
C, LeDuc Y, Mazen A et al (1988) Structural
and functional analysis of poly(ADP-ribose)
polymerase: an immunological study. Biochim
Biophys Acta 950:147160
54. Colombo E, Banki K, Tatum AH, Daucher J,
Ferrante P, Murray RS et al (1997) Comparative
analysis of antibody and cell-mediated autoim-
munity to transaldolase and myelin basic pro-
tein in patients with multiple sclerosis. J Clin
Invest 99:12381250
55. Vermes I, Haanen C, Steffens-Nakken H,
Reutelingsperger C (1995) A novel assay for
apoptosis. Flow cytometric detection of phos-
phatidylserine expression on early apoptotic
cells using fluorescein labelled Annexin V. J
Immunol Methods 184:3951
56. Martin SJ, Reutelingsperger CPM, McGahon
AJ, Rader JA, van Schie CAA, LaFace DM et al
(1995) Early redistribution of plasma mem-
brane phosphatidylserine is a general feature of
apoptosis regardless of the initiating stimulus:
inhibition by overexpression of bcl-2 and Abl. J
Exp Med 182:15451556
57. Petit PX, OConnor JE, Grunwald D, Brown
SC (1990) Analysis of the membrane potential
of rat- and mouse-liver mitochondria by flow
cytometry and possible applications. Eur J
Biochem 194:389397
58. Tanner MK, Wellhausen SR, Klein JB (1993)
Flow cytometric analysis of altered mononu-
clear cell transmembrane potential induced by
cyclosporin. Cytometry 14:5969
59. Smiley ST, Reers M, Mottola-Hartshorn C,
Lin M, Chen A, Smith TW et al (1991)
Intracellular heterogeneity in mitochondrial
membrane potentials revealed by a J-aggregate-
forming cation JC-1. Proc Natl Acad Sci U S A
88:36713675
60. Cossarizza A, Franceschi C, Monti D, Salvioli
S, Bellesia E, Rivabene R et al (1995) Protective
effect of N-acetylcysteine in tumor necrosis
factor-a-induced apoptosis in U937 cells: The
role of mitochondria. Exp Eye Res
220:232240
61. Royall JA, Ischiropoulos H (1993) Evaluation
of 2,7-dichlorofluorescein and dihydrorhod-
amine 123 as fluorescent probes for intracellu-
lar H2O2 in cultured endothelial cells. Arch
Biochem Biophys 302:348355
62. Wieder ED, Hang H, Fox MH (1993)
Measurement of intracellular pH using flow
cytometry with carboxy-SNARF-1. Cytometry
14:916921
63. Fiers W, Beyaert R, Declercq W, Vandenabeele
P (1999) More than one way to die: apoptosis,
necrosis and reactive oxygen damage. Oncogene
18:77197730
64. Leist M, Single B, Castoldi AF, Kuhnle S,
Nicotera P (1997) Intracellular adenosine
triphosphate (ATP) concentration: a switch in
the decision between apoptosis and necrosis. J
Exp Med 185:14811486
65. Lundin A (2000) Use of firefly luciferase in
ATP-related assays of biomass, enzymes, and
metabolites. Methods Enzymol 305:346370
66. Lowry OH, Rosebrough NJ, Farr AL, Randall
RJ (1951) Protein measurement with the Folin
phenol reagent. J Biol Chem 193:265275
67. Lundin A (1994) ATP extractants neutralized
by cyclodextrins. In: Campbell AK, Kricka LJ,
Stanley PE (eds) Biolumimescence and chemi-
luminescence. Wiley, Chichester, pp 399402
68. Fariss MW, Reed DJ (1987) High-performance
liquid chromatography of thiols and disulfides:
dinitrophenol derivatives. Methods Enzymol
143:101109
69. Towbin HH, Staehelin T, Gordon J (1979)
Electrophoretic transfer of proteins from poly-
acrylamide gels to nitrocellulose sheets: proce-
dure and some applications. Proc Natl Acad Sci
U S A 76:43504354
70. Rustin P, Parfait B, Chretien D, Bourgeron T,
Djouadi F, Bastin J et al (1996) Fluxes of nico-
tinamide adenine dinucleotides through mito-
chondrial membranes in human cultured cells.
J Biol Chem 271(25):1478514790
 4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients
71. Barrientos A (2002) In vivo and in organello
assessment of OXPHOS activities. Methods
26(4):307316
72. Rustin P, Chretien D, Bourgeron T, Gerard B,
Rotig A, Saudubray JM et al (1994) Biochemical
and molecular investigations in respiratory
chain deficiencies. [Review] [25 refs]. Clin
Chim Acta 228(1):3551
73. Nicoletti I, Migliorati G, Pagliacci MC,
Grignani F, Riccardi C (1991) A rapid and sim-
ple method for measuring thymocyte apoptosis
by propidium iodide staining and flow cytom-
etry. J Immunol Methods 139:271279
74. Nicholson DW, Ali A, Thornberry NA,
Vaillancourt JP, Ding CK, Gallant M et al
(1995) Identification and inhibition of ICE/
CED-3 protease necessary for mammalian
apoptosis. Nature 376:3743
75. Armstrong RC, Aja T, Xiang J, Gaur S, Krebs
JF, Hoang K et al (1996) Fas-induced activa-
tion of the cell death-related protease CPP32 is
inhibited by bcl-2 and by ICE family protease
inhibitors. J Biol Chem 271:1685016855
76. Lorenz HM, Grunke M, Hieronymus T,
Herrmann M, Kuhnel A, Manger B (1997) In
vitro apoptosis and expression of apoptosis-
related molecules in lymphocytes from patients
with systemic lupus erythematosus and other
autoimmune diseases. Arthritis Rheum
40:306317
77. Eisner MD, Amory J, Mullaney B, Tierney L
Jr, Browner WS (1996) Necrotizing lymph-
adenitis associated with systemic lupus erythe-
matosus. Semin Arthritis Rheum 26:477482
78. Llorente L, Zou W, Levy Y, Richaud-Patin Y,
Wijdenes J, Alcocer-Varela J et al (1995) Role
of interleukin 10 in the B lymphocyte hyperac-
tivity and autoantibody production of human
systemic lupus erythematosus. J Exp Med
181:839844
79. Green JM, Thompson CB (1994) Modulation
of T cell proliferative response by accessory cell
89
interactions [Review] [50 refs]. Immunol Res
13:234243
80. Los M, Schenk H, Hexel K, Baeuerle PA,
Droge W, Schulze-Osthoff K (1995) IL-2
gene expression and NF-kappa B activation
through CD28 requires reactive oxygen pro-
duction by 5-lipoxygenase. EMBO J
14:37313740
81. Tatla S, Woodhead V, Foreman JC, Chain BM
(1999) The role of reactive oxygen species in
triggering proliferation and IL-2 secretion in T
cells. Free Radic Biol Med 26:1424
82. Casciola-Rosen LA, Miller DK, Anhalt GJ,
Rosen A (1994) Specific cleavage of the 70 kDa
protein component of the U1 small nuclear
ribonucleoprotein is a characteristic biochemi-
cal feature of apoptotic cell death. J Biol Chem
269:3075730760
83. Perl A, Banki K (2000) Genetic and metabolic
control of the mitochondrial transmembrane
potential and reactive oxygen intermediate pro-
duction in HIV disease. Antioxid Redox Signal
2:551573
84. Puskas F, Gergely PJ, Niland B, Banki K, Perl A
(2002) Differential regulation of hydrogen
peroxide and Fas-dependent apoptosis path-
ways by dehydroascorbate, the oxidized form
of vitamin C. Antioxid Redox Signal
4(3):358369
85. Harada H, Becknell B, Wilm M, Mann M,
Huang LJ, Taylor SS et al (1999)
Phosphorylation and inactivation of BAD by
mitochondria-anchored protein kinase A. Mol
Cell 3:413422
86. Nisoli E, Carruba MO (2006) Nitric oxide and
mitochondrial biogenesis. J Cell Sci
119(14):28552862
87. Ibiza S, Victor VM, Bosca I, Ortega A,
Urzainqui A, OConnor JE et al (2006)
Endothelial nitric oxide synthase regulates T
cell receptor signaling at the immunological
synapse. Immunity 24(6):753765
 Chapter 5

The Role of Endocytic Recycling in Autoimmunity

Tiffany Telarico and Andras Perl

Abstract
Abnormalities in T cell signal transduction underlie pathology in systemic lupus erythematosus. Lupus T
cells are more sensitive to stimulation, yet have reduced expression of T cell antigen receptor (TCR) at the
surface. The amount of TCR expressed at the surface of a T cell directly determines the ability of a T cell
to become activated. The endocytic recycling machinery regulates transport of T cell receptors to the
plasma membrane, internalization of surface receptors, and recycling to the cell surface, which determines
the ability of a T cell to become activated. Increased recycling of CD3 and CD4 receptors occurs in lupus
T cells, and could represent a mechanism by which T cells are sensitized to stimulation. This chapter
explains methods used to investigate endocytic recycling of the TCR, CD4, and CD8 co-receptors in
peripheral blood lymphocytes, T cells, and in splenocytes from lupus-prone murine models. The assays
described will allow the study of surface receptor turnover in live untouched lymphocytes by flow
cytometry.

Key words: Receptor recycling, Early endosome, T cell antigen receptor, CD4, Mammalian target of
rapamycin, Systemic lupus erythematosus

1. Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease of

complex etiology characterized by autoantibody production and
dysregulation of T cell, B cell, and dendritic cell function (1, 2).
T cells contribute to SLE pathogenesis through secretion of pro-
inflammatory cytokines which provide help to autoreactive B cells.
SLE T cells have altered cell death pathway selection, with reduced
antigen-induced cell death (AICD) but increased spontaneous
apoptosis and necrosis (3, 4). Necrotic debris in the periphery
can be taken up and presented by dendritic cells and can activate
autoreactive B cells leading to production of autoantibodies (5).

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_5, Springer Science+Business Media New York 2012

91
92 T. Telarico and A. Perl

Upon stimulation through the T cell antigen receptor (TCR),

SLE T cells exhibit increased phosphorylation of cytosolic pro-
teins (6), have exaggerated production of IFN and other pro-
inflammatory cytokines (7), and enhanced intracytoplasmic
calcium (Ca2+) fluxing (8). Despite this hyperexcitable phenotype,
TCR surface expression and IL-2 production in SLE T cells are
reduced (9). Increased turnover of CD3 and CD4 receptors by
recycling on early endosomes can serve as a mechanism that sensi-
tizes SLE T cells to stimulation, despite reduced expression of
TCR on the surface.

1.1. Metabolic Control SLE T cells are metabolically active and have increased baseline
of the Endocytic nitric oxide (NO) production (10). NO production at the immu-
Pathway in SLE T Cells nological synapse (IS) is required to sustain the signal post
TCR ligation by MHCIIpeptide complex and maintain T cell
activation (11). NO overproduction leads to elevation of the
mitochondrial transmembrane potential (inducing mitochondrial
hyperpolarization, MHP) and increases mitochondrial biogenesis
(12). MHP increases production of reactive oxygen intermediates
(ROIs) and reduces ATP production, which predisposes chroni-
cally stimulated cells to undergo necrosis (4, 13).
The mammalian target of rapamycin (mTOR) is activated in
response to NO, and further enhances NO production through
activation of YY1 and PGC1, factors required for transcription of
nitric oxide synthases (14). Inhibition of mTORC1 activity by
rapamycin reduces NO production through inducing proteosomal
degradation of inducible nitric oxide synthase (iNOS) in mac-
rophages (15). mTOR senses MHP through association with the
voltage-dependent anion channel-1 (VDAC1). mTOR traffics to
mitochondria, through formation of a complex at the mitochon-
drial outer membrane with Bcl-xL and VDAC1 (16). mTOR activ-
ity has been directly implicated in promoting mitochondrial
function, as treatment with rapamycin reduces mitochondrial
transmembrane potential, decreases oxygen consumption, and
reduces ATP synthesis (17).
Target of rapamycin (TOR) proteins were identified as a
component of isolated endocytic patches, suggesting a role for
mTOR in endocytic trafficking (18). mTOR overexpression in
SLE T cells promotes expression of the small GTPase HRES-1/
Rab4, a regulator of endocytic recycling (19). Treatment with
rapamycin reduces HRES-1/Rab4 overexpression in SLE T cells
(19). mTOR and HRES-1/Rab4 colocalize on early endosomes
(19). The endocytic recycling machinery feeds back to regulate
mTOR activity through its intracellular location (20). Rab pro-
teins were found to be indispensible for signaling through mTOR
complex I (mTORC1) (20), and can serve as regulators of
mTORC1 activity (21).
 5 The Role of Endocytic Recycling in Autoimmunity 93

The early endocytic pathway is activated within SLE T cells

and is characterized by overexpression of Rab5A and HRES-1/
Rab4, which control internalization and recycling of surface recep-
tors on endosomes, respectively (19). HRES-1/Rab4 directly
interacts with CD3 and CD4 receptors (19). Expression of CD4
and CD3 inversely correlates with HRES-1/Rab4 in SLE CD4+
T cells. HRES-1/Rab4 expression is increased, while expression of
CD4 and CD3 is reduced in lupus CD4+ T cells (19). HRES-1/
Rab4 targets CD4 and CD3 for lysosomal degradation (19). Cbl
ubiquitinates CD3 (22) and degradation could be mediated via
an Rab4-CD2 adaptor proteinCbl complex targeted to the lyso-
some for degradation (23). Alternatively, degradation of CD3
and CD4 by HRES-1/Rab4 could be mediated through autophagy,
as early endosomes are required for formation of autophagosomes,
resulting in degradation of cargo in lysosomes (24) (Fig. 1).

1.2. Dysregulation Reduced expression of CD3 results in dysregulated signaling in

of TCR Signal SLE T cells. CD3 expression is reduced at both the mRNA and
Transduction Due protein level in SLE T cells (25). Reduced CD3 transcription
to Deficiency of CD3z occurs due to polymorphisms and mutations at the promoter
in SLE T Cells region (26), and oxidative stress-mediated increases in protein
phosphatase 2a activity that suppresses expression of the CD3
transcription factor, Elf-1 (27). Reduced protein occurs due to
reduction of transcript, as well as decreased protein stability due to
gene deletions in exon 7 (28), increased caspase-3 activity in SLE
T cells (29), and increased lysosomal degradation (30), which is
mediated by the endocytic pathway (19).
CD3 deficiency by mutation of a single ITAM is sufficient to
induce autoimmunity in mice (31). Reconstitution of CD3 chain
into SLE T lymphocytes normalizes Ca2+ responses, restores IL-2
production, and reduces phosphorylation of cellular proteins distal
to the TCR (32). CD3 is essential for normal T cell signaling, for
it regulates assembly and transport of the fully assembled TCR on
the cell surface and participates in TCR signal transduction by
phosphorylation at immunoreceptor tyrosine-based activation
motifs (ITAMs). CD3 is rate limiting in assembly of the TCR,
and thus reduced CD3 expression can result in reduced TCR at
the surface. Reduced CD3 expression in the T cell receptor com-
plex is functionally replaced by FcRI (33), and can form het-
erodimers with CD3 (34). FcRI contains a single ITAM in its
cytoplasmic domain and associates with Syk in place of ZAP-70
(34). Syk is a more potent kinase than ZAP-70, allowing increased
propagation of signaling distal of the TCR, even with reduced
TCR ligation (35). Reduced CD3 expression also renders SLE T
cells to be less responsive to CTLA-4-directed suppression, as
CTLA-4 requires association with phosphorylated CD3 for its
function (36).
94 T. Telarico and A. Perl

APC
MHC-II-peptide
complex

CD4 CD3
complex

eNOS

FcRI Syk
Lck
NO NOSIP P- PLC
Endocytic
recycling
HRES -1/Rab4
CD4,CD3,
Dy m NOSIP
EE
Lysosome

ROI NO Stim1 Orai1

Ca2+ Ca2+
PGC1

CRAC
NFAT
PP2a
IL-2
ER
Elf-1

CD3
Nucleus

T cell

Fig. 1. HRES-1/Rab4 overexpression in SLE T cells occurs downstream of NO-dependent mTOR activation (19). NO is
overproduced in SLE T cells, resulting in MHP, mitochondrial biogenesis, and transcription of mTOR via activation of PGC1
(12, 14). HRES-1/Rab4 feeds back to increase NO production via degradation of NOSIP, a negative regulator of eNOS
(unpublished data). HRES-1/Rab4 overexpression reorganizes the IS through promoting CD3 and CD4 receptor recycling
and lysosomal degradation, reducing expression of CD4 and CD3 (19). CD3 is functionally replaced with FcRI, which
interacts with Syk, resulting in activation of PLC and enhanced intracytoplasmic Ca2+ fluxing upon T cell activation (35).
APC, antigen-presenting cell; CRAC, Ca2+ release-activated calcium channel; EE, early endosome; ER, endoplasmic reticu-
lum; MHP, mitochondrial hyperpolarization; mTOR, mammalian target of rapamycin; NFAT, nuclear factor of activated
T cells; NOSIP, nitric oxide synthase interacting protein; eNOS, endothelial nitric oxide synthase; ROI, reactive oxygen inter-
mediates. Colorized lettering in red represents downregulation in SLE T cells; green lettering represents upregulation in
SLE T cells.
 5 The Role of Endocytic Recycling in Autoimmunity 95

1.3. Control T cell receptor surface expression on a cell surface depends on

of Proximal TCR synthesis and transport of newly assembled receptors, endocytosis
Signaling of TCR from the plasma membrane, and recycling of internalized
by the Endocytic receptors back to the cell surface (37). T cell receptors which have
Pathway undergone phosphorylation are constitutively recycled (38), and
recycling is required to maintain T cell responsiveness to allow for
serial activation of TCRs by a single peptideMHC complex (30).
Upon stimulation, TCR expression is downmodulated through
preventing recycling, while internalization in the presence or
absence of antigen remains the same (39). Downregulation of
TCRs prevents T cells from overstimulation and limits induction
of AICD. Reduced AICD in SLE is deleterious due to persistence
of autoreactive lymphocytes (40).
Rab GTPases of the endocytic pathway are recruited to the
immunological synapse and participate in T cell activation through
transporting and concentrating TCRs and costimulatory molecules
on the surface (41, 42). The recycling machinery compartmental-
izes the T cell receptor signaling machinery, recruits cargo to lipid
rafts at the IS (42), and regulates expression of the TCR and CD4
co-receptors (19). CD3 and CD4 recycling is increased in SLE T
lymphocytes (19). This may be a measure to compensate for
reduced surface receptor expression, as reduced CD3 expression
induces rapid internalization and recycling of the TCR, and the
half-life of TCR at the surface directly correlates with the expres-
sion of CD3 (37). The same motifs within ITAMs of the TCR,
including tyrosine residues and dileucine domains, participate in
both TCR signal transduction and recycling of the TCR receptor
complex; therefore endocytic recycling may be essential in T cell
signal transduction by allowing tyrosine residues within ITAMs to
be accessible for phosphorylation (39) (Fig. 2).

CD3 recycling: CD4 recycling: CD8 recycling:

130 140 130
Control Control Control
SLE SLE SLE
CD3 MFI, normalized

CD8 MFI, normalized

CD4 MFI, normalized

130
120 120

120
110 110
110
100 100
100

90 90 90
0 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150
Time (minutes) Time (minutes) Time (minutes)

Fig. 2. SLE T cells exhibit increased recycling of CD3 and CD4, but not CD8 receptors. T cells were negatively isolated from
seven SLE patients and seven healthy controls, then stained on ice with APC-Cy7-conjugated antibody to CD3, Alexa
647-conjugated antibody to CD4, and PE-conjugated antibody to CD8 for 30 min, and then washed three times in 4 C
RPMI 1640 medium. T cells were resuspended in pre-warmed 37 C medium and incubated at 37 C for 2.5 h, removing
samples every 30 min and placing them on ice. At the end of sample collection, the cells were restained with CD3, CD4,
and CD8 antibodies on ice for 30 min, then washed three times with ice-cold RPMI complete medium, and analyzed by flow
cytometry. Recycling was measured by the increase in mean fluorescence intensity (MFI) at each time point over the time
0 sample.
96 T. Telarico and A. Perl

2. Materials

1. Heparin (Sigma-Aldrich, St. Louis, MO).

2. 60 ml Luer-lock syringes (Becton-Dickinson, Franklin Lakes,
NJ).
3. 1 ml Tuberculin syringes (Becton-Dickinson, Franklin Lakes,
NJ).
4. Blood collection tubes containing no additives (Becton-
Dickinson, Franklin Lakes, NJ).
5. Ficoll-Paque PLUS solution (Amersham Pharmacia, Uppsala,
Sweden).
6. Phosphate-buffered saline (PBS) (8 g NaCl, 0.2 g KCl, 1.44 g
Na2HPO4, 0.24 g KH2PO4 in 1 L H2O), autoclaved and
adjusted to pH 7.4.
7. Beckman tissue culture centrifuge.
8. Microcentrifuge.
9. Light microscope.
10. Hemacytometer.
11. RPMI-1640 complete medium (RPMI base medium, 10 %
heat-inactivated fetal bovine serum (FBS), 1 % penicillin/
streptomycin/amphotericin B solution, 1 % L-glutamine solu-
tion). FBS from Hyclone (Thermo-Fisher Scientific, Pittsburgh,
PA). All other components from Invitrogen-Gibco (Carlsbad,
CA).
12. Dulbeccos modified Eagles medium (DMEM) complete
medium (DMEM base, 10 % heat-inactivated FBS, 1 % penicil-
lin/streptomycin/amphotericin B solution, 1 % L-glutamine
solution, 1 % sodium pyruvate solution, 1 % MEM nonessen-
tial amino acids, and 50 M beta-mercaptoethanol). FBS from
Hyclone (Thermo-Fisher Scientific, Pittsburgh, PA). All other
components from Invitrogen-Gibco (Carlsbad, CA).
13. ACK lysis buffer (8.29 g NH4Cl, 1 g KHCO3, 37.2 mg Na2-
EDTA in 1 L autoclaved H2O, filter-sterilized and pH adjusted
to 7.4).
14. 0.45 m Bottle top filter and syringe filters.
15. 70 m Cell strainer for 50 ml conical tubes (Becton-Dickinson,
Franklin Lakes, NJ).
16. 50 and 15 ml Polypropylene conical tubes (Becton-Dickinson,
Franklin Lakes, NJ).
 5 The Role of Endocytic Recycling in Autoimmunity 97

17. 10 cm Plastic petri dishes (Corning Life Sciences, Corning,

NY).
18. T cell negative isolation kit, containing magnetic beads conju-
gated to IgG antibodies against CD14, CD16, HLA class II
DR/DP, CD56, and CD235a (Invitrogen, Carlsbad, CA).
29. DynaMag-15 magnet (Invitrogen, Carlsbad, CA).
20. Multi-purpose rotator (Scientific Industries, Springfield, MA).
21. Cycloheximide (Sigma-Aldrich, St. Louis, MO).
22. Fluorescent conjugated anti-human antibodies: APC Cy7 con-
jugated CD3 antibody, Alexa 647 conjugated CD4 antibody,
PE Cy7 conjugated CD19 antibody, PE conjugated CD8 anti-
body (Becton-Dickinson, Franklin Lakes, NJ).
23. Fluorescent conjugated anti-mouse antibodies: PE Cy7 conju-
gated CD3 antibody, PerCP conjugated CD4 antibody, Alexa
Fluor 700-conjugated CD19 antibody, PE conjugated CD8
antibody (Biolegend, San Diego, CA).
24. 96-Well assay plates with 600 l wells (Corning Incorporated,
Corning, NY).
25. 5 ml Flow cytometry tubes (Becton-Dickinson, Franklin Lakes,
NJ).
26. Becton Dickinson LSR-II flow cytometer, equipped with FACS
Diva software (Becton-Dickinson, Franklin Lakes, NJ).
27. FlowJo cytometry analysis software (Treestar Incorporated,
Ashland, OR).
28. Statview statistical analysis software (SAS Institute, New York
City, NY).
29. Radio-immuno precipitation assay (RIPA) buffer with protease
inhibitors (150 mM NaCl, 2 % NP-40, 0.5 % sodium deoxy-
cholate, 0.1 % SDS, 50 mM Tris pH 8.0, 1 mM PMSF, 1 g/
ml aprotinin, 1 g/ml pepstatin, 1 g/ml leupeptin, 1 mM
NaF, 1 mM sodium orthovanadate, 0.1 mM sodium molyb-
date, 10 mM sodium pyrophosphate).
30. Laemmli protein sample buffer (2.4 ml 1 M Tris pH 6.8, 0.8 g
SDS, 4 ml glycerol, 0.02 % bromophenol blue, 1 ml beta-mer-
captoethanol, 2.8 ml H2O, pH 6.8).
31. Enhanced chemiluminescence detection kit (General Electric
Healthcare, Piscataway, NJ).
32. Kodak image station 440CF, equipped with Kodak 1D image
analysis software (Eastman Kodak Company, Rochester, NY).
33. Horseradish peroxidase conjugated goat anti-mouse IgG (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA).
34. Horseradish peroxidase conjugated donkey anti-rabbit IgG
(General Electric Healthcare, Piscataway, NJ).
98 T. Telarico and A. Perl

35. Mouse monoclonal antibody to CD4 (Leica Microsystems,

Buffalo Grove, IL).
36. Mouse monoclonal antibody to CD3 (Santa Cruz biotechnol-
ogy, Santa Cruz, CA).
37. Mouse monoclonal antibody to CD8 (Santa Cruz biotechnol-
ogy, Santa Cruz, CA).
38. Western blotting equipment, including 12 % SDS-PAGE gels,
Trans-Blot cell apparatus, prestained protein standards,
0.45 M nitrocellulose membrane.
39. TBS-T blocking solution (5 % nonfat dry milk, 100 mM Tris
HCl pH 7.5, 0.9 % NaCl, 0.1 % Tween-20).
40. TBS-T washing solution (100 mM TrisHCl pH 7.5, 0.9 %
NaCl, 0.1 % Tween-20).

3. Methods

The methods described here outline (a) preparation of human lym-

phocytes; (b) preparation of mouse splenocytes; (c) recycling assay;
and (d) western blot analysis for expression of surface receptors
(see Note 2).

3.1. Preparation 1. Collect 50 ml of peripheral blood into sterile heparinized blood

of Human collection tubes, or a 60 ml syringe containing 2,500 units of
Lymphocytes heparin. Collect 5 ml of native blood into a blood collection
tube with no additives for separation of autologous serum
3.1.1. Separation (see Note 3).
of Peripheral Blood
Mononuclear Cells from
2. Dilute whole blood 1:1 with PBS in 50 ml conical tubes.
Whole Blood 3. Layer PBS-diluted blood onto room-temperature Ficoll-Paque
solution in 50 ml conical tubes. Layer diluted blood onto Ficoll
at a 2:1 ratio.
4. Centrifuge cells at 550 g for 30 min with brake of centrifuge off.
5. Remove plasma layer from Ficoll gradient and discard. Remove
peripheral blood mononuclear cells (PBMCs) from the buffy
coat interface between the plasma and Ficoll layer and pipette
into a sterile conical tube.
6. Wash PBMC by adding three times the volume of PBS.
Centrifuge at 500 g for 10 min at 8 C with centrifuge brake
off.
7. Resuspend PBMC in RPMI 1640 complete medium at a
density of 12 106 cells/ml.
 5 The Role of Endocytic Recycling in Autoimmunity 99

3.1.2. Separation 1. Pre-coat 10 cm Petri dishes with 750 l autologous serum and
of Peripheral Blood incubate plates at 37 C CO2 tissue culture incubator for
Lymphocytes from PBMC 30 min.
2. Add 515 ml of PBMC (12 106 cells/ml) onto coated dishes
and incubate at 37 C for 1 h.
After incubation period, remove peripheral blood lym-
phocytes (PBLs) into a sterile conical tube. Wash plate gently
three times with 5 ml PBS and add these washes into tube (see
Note 4).
3. Centrifuge PBL at 1,300 rpm for 10 min at 8 C.
4. Resuspend PBL in RPMI 1640 complete medium at
5 106 cells/ml.
5. Monocyte-depleted PBL should contain <2 % monocytes. This
can be verified by flow cytometry after staining with a fluorescent
conjugated anti-human CD11b antibody.

3.1.3. Negative Isolation Adapted from Invitrogen Dynabeads untouched human T cell kit
of T Lymphocytes manufacturers protocol (catalog #113.44D).
1. Aliquot 20 106 PBMC or PBL into a 15 ml conical tube and
pellet cells by centrifugation at 1,300 rpm for 10 min at 8 C.
2. Resuspend cells in 10 ml T cell isolation buffer (0.5 g BSA,
2 mM EDTA, in autoclaved PBS). Wash cells by centrifugation
at 1,300 rpm at 8 C for 10 min.
3. Resuspend cell pellet in 200 l T cell isolation buffer. Add
40 l heat-inactivated and filter-sterilized FBS. Add 40 l of
antibody mix from Invitrogen Dynabeads untouched human T
cell kit and mix by inverting tubes. The antibody mix contains
IgG antibodies against human CD14, CD16, HLA class II
DR/DP, CD56, and CD235a antigens.
4. Incubate cells on ice for 20 min.
5. After incubation period, add 10 ml of T cell isolation buffer to
cells to wash off non-bound antibody. Centrifuge at 1,300 rpm
at 4 C for 10 min.
6. Aspirate off isolation buffer and resuspend pellet in 200 l T
cell isolation buffer.
7. Wash 1 ml Dynabeads per sample by placing in DynaMag-15
magnet for 2 min and resuspending in an equal volume of T
cell isolation buffer. Add 1 ml of negative isolation Dynabeads
per sample.
8. Incubate cells for 20 min at room temperature with low-speed
rotation.
9. After incubation period, place tubes in DynaMag-15 magnet
for 2 min.
100 T. Telarico and A. Perl

10. With tube remaining in magnet, remove supernatant into a

sterile 15 ml conical tube. This supernatant contains untouched
T cells.
11. Add 1 ml of T cell isolation buffer to beads, mix well, and place
in magnet for 2 min. Add this wash to cells in 15 ml conical
tubes.
12. Place tube containing supernatant in magnet for another 2 min
and remove supernatant into a new tube to remove residual
Dynabeads.
13. Pellet cells and resuspend in RPMI 1640 complete medium at
5 106 cells/ml.
14. Purity of untouched T cells should be >95 %. This can be
verified by flow cytometry after staining with a fluorescent con-
jugated anti-human CD3 antibody.

3.2. Separation 1. Sacrifice mice by cervical dislocation. Remove blood by cardiac

of Mouse Splenocytes puncture for detection of antinuclear autoantibodies, using a
25-gauge needle, and transfer into a sterile tube without anti-
coagulants. Centrifuge whole blood at 1,600 rpm for 20 min.
Remove serum and transfer to a sterile tube. Store serum at
80 C. Remove spleen retaining as little fat as possible, and
transfer into cold DMEM complete medium.
2. Place a 70 m cell strainer in a 50 ml conical tube, and transfer
spleen onto strainer. Use the flat part of the plunger to mechan-
ically disrupt the spleen, pushing it through the strainer,
attempting to break up any large fragments. Wash the strainer
with 10 ml of DMEM complete medium. Discard the strainer
and pellet cells by centrifugation at 450 g at 4 C for 10 min.
3. Aspirate off medium and resuspend cells in 5 ml of ACK buffer
for lysis of red blood cells. Allow 5 min for lysis to occur, and
then add 10 ml of DMEM complete medium. Centrifuge cells
at 1,200 rpm at 4 C for 10 min.
4. Resuspend splenocytes at a concentration of 5 106 cells/ml in
ice-cold DMEM complete medium.

3.3. Recycling Assay
3.3.1. Recycling Assays
on Human PBL
and Isolated T Cells
1. Aliquot 3 106 PBL or isolated T cells in a 600 l volume into
sterile 5 ml tubes. Aliquot five tubes containing 1 106 PBL in
a 200 l volume to use as single color compensation controls
(see Note 5).
2. Add 50 g/ml cycloheximide to samples to inhibit synthesis of
new surface receptors.
3. Stain cells on ice with APC Cy7-conjugated antibody to CD3,
Alexa Fluor 647-conjugated antibody to CD4, PE-conjugated
antibody to CD8, and PE Cy7 antibody conjugated to CD19
(for PBL only) using 2 g per sample. Add 0.5 g antibody per
 5 The Role of Endocytic Recycling in Autoimmunity 101

sample for single color compensation controls. Allow cells to

incubate on ice for 30 min.
4. Wash cells by adding 4 ml ice-cold RPMI 1640 complete
medium and centrifuging samples at 1,300 rpm at 4 C for
10 min.
5. Repeat step 4 and wash cells two more times.
6. After three wash cycles, resuspend cell pellet in 1.2 ml of 37 C
RPMI 1640 complete medium.
7. Remove a 200 l aliquot of cells to an assay plate, prechilled
with 100 l PBS added per well.
8. Incubate the remaining cells at 37 C for 3 h, removing a
200 l aliquot of cells every 30 min to the chilled assay plate.
9. At the conclusion, re-stain cells with antibodies for 30 min
on ice.
10. Wash assay plate by centrifugation at 550 g at 4 C for 10 min,
with centrifuge brake off.
11. Resuspend cells in ice-cold RPMI 1640 complete medium,
and wash samples two more times. Keep cells on ice in the dark
until flow cytometry analysis.
12. Analyze by flow cytometry on a flow cytometer containing 488
and 633 nm lasers by gating on live cells based on forward
scatter (FSC) and side scatter (SSC) profile and collect a mini-
mum of 10,000 events per sample. Export samples in FCS3.0
format for analysis on FlowJo software.
13. Electronically gate T and B cell populations and record com-
pensated CD3 and CD19 mean channel fluorescence intensi-
ties (MFIs). Separate the live T cell subpopulation by gating
out CD4+ single-positive, CD8+ single-positive, and CD4CD8
double-negative T cells. Record MFIs of CD3 on each popula-
tion, CD4 on CD3+CD4+ T cells, and CD8 on the CD3+CD8+
T cell population.
14. Calculate recycling by the increase in compensated mean
fluorescence intensity at each time point compared to the base-
line time 0 sample, normalizing each assay to a baseline value of
100.
15. To determine differences in baseline receptor expression between
SLE patients and healthy controls, compare the receptor MFI of
the patient to that of the healthy control at time 0.
16. Plot graphs of normalized MFI over time for each receptor.
17. Determine whether significant differences in recycling curves
exist using a pairwise repeated measures analysis of variance
(ANOVA) test on Statview software. Measure the differences
in receptor expression at each time interval by paired t-tests
and ANOVA post hoc analysis (see Note 6).
102 T. Telarico and A. Perl
3.3.2. Recycling Assays
on Mouse Splenocytes
10. Wash assay plate by centrifugation at 1,500 rpm at 4 C for
11. Resuspend cells in ice-cold DMEM complete medium, and
12. Analyze recycling results as described in Subheading 3.3.1.
3.4. Western Blotting
for Surface Receptor
Expression
1. Aliquot 3 106 splenocytes in a 600 l volume of DMEM
complete medium into a sterile 5 ml tube. Aliquot five tubes
containing 1 106 PBL in a 200 l volume to use as single
color compensation controls (see Note 7).
2. Add 50 g/ml cycloheximide to inhibit synthesis of new
surface receptors.
3. Add 2 g each PE Cy7-conjugated CD3, PerCP-conjugated
CD4, APC Cy7-conjugated CD8, and Alexa Fluor 700-conju-
gated CD19 antibodies to splenocytes and incubate on ice for
30 min. Add 0.5 g antibody to single color compensation
controls.
4. Wash unbound antibody by adding 4 ml of ice-cold DMEM
complete medium and centrifuge at 1,300 rpm at 4 C for
10 min.
5. Wash splenocytes two more times in ice-cold DMEM com-
plete medium.
6. After three washes, resuspend splenocytes in 1.2 ml of 37 C
DMEM complete medium.
7. Remove a 200 l aliquot of cells to an assay plate, prechilled
with 100 l PBS added per well.
8. Incubate the remaining cells at 37 C for 3 h, removing a
200 l aliquot of cells every 30 min to the chilled assay plate.
9. At the conclusion, re-stain cells with antibodies for 30 min on
ice.
10 min, with centrifuge brake off.
wash samples two more times. Keep cells on ice in the dark
until flow cytometry analysis.
1. To determine differences in total CD3, CD4, and CD8 surface
receptor expression, lyse 2 106 cells in 50 l RIPA lysis buffer
(150 mM sodium chloride, 1 % Non-Idet-40, 0.5 % sodium
deoxycholate, 0.1 % SDS, 50 mM Tris pH 8.0, 1 mM PMSF,
1 g/ml aprotinin, 1 g/ml pepstatin, 1 g/ml leupeptin,
1 mM NaF, 1 mM sodium orthovanadate, 0.1 mM sodium
molybdate, 10 mM sodium pyrophosphate) (see Noe 1).
2. Place cells on ice after lysis, and use 2 l lysate to measure pro-
tein concentration by Bradford assay (43). For Bradford assay,
prepare a set of standard concentrations of BSA from 0 to
12 g in Bradford reagent (0.15 M NaCl, Coomassie brilliant
blue G-250) and measure absorbance in spectrophotometer at
595 nanometers to create a standard curve (40). Add 2 l of
 5 The Role of Endocytic Recycling in Autoimmunity 103

protein sample diluted into 998 l Bradford reagent and

measure absorbance. After measuring the absorbance of pro-
tein samples, use the standard curve to determine the concen-
tration of protein in g/l. Dilute samples to a concentration
of 40 g/10 l. Heat samples at 95 C for 5 min before immu-
noblotting or storing at 20 to 60 C.
3. Separate 40 g of whole cell lysate in a 10 l volume per well
on 12 % SDS-PAGE gels and transfer onto 0.45 m nitrocel-
lulose membranes.
4. Incubate nitrocellulose membrane in TBS-T blocking solution
(100 mM TrisHCl pH 7.5, 0.9 % NaCl, 0.1 % Tween-20, 5 %
nonfat dry milk) for 1 h, and then incubate with primary anti-
bodies anti-CD3, anti-CD4, and anti-CD8 at a 500-fold dilu-
tion overnight.
5. After overnight incubation, wash blots six times in TBS-T solu-
tion (100 mM TrisHCl pH 7.5, 0.9 % NaCl, 0.1 % Tween-
20) and incubate membranes in TBS-T blocking solution with
horseradish peroxidase-conjugated goat anti-mouse antibody
at a 2,000-fold dilution.
6. Develop blots by enhanced chemiluminescence (ECL)
detection.
7. Expression of CD3, CD4, and CD8 surface receptors should
be compared to expression of total protein, quantitating
expression of receptor/-actin by automated densitometry.

4. Notes

1. When evaluating endocytic recycling in PBL or T cells from

patients with SLE, it is important to compare to PBL or T cells
from control blood donors who are healthy and matched for
gender, ethnicity, and age within 10 years. To control for the
effect of chronic inflammation, study PBL and T cells from
blood donors who have a chronic inflammatory disease but not
SLE, such as rheumatoid arthritis. Measure recycling from at
least two control and two SLE donors at the same time; this
will allow the determination of variation within the controls.
2. Lupus patients taking hydroxychloroquine should be excluded
from the study, as this compound inhibits endosome
acidification and measurement of recycling requires intact
endosomal function.
3. Use freshly isolated PBL and T cells. PBL frozen in liquid
nitrogen in RPMI 1640 medium with 7.5 % DMSO and 30 %
FBS can be used only if the viability of recovered cells is >98 %
by trypan blue exclusion staining.
104 T. Telarico and A. Perl

4. Lymphocytes should not be cultured at 37 C for more than

1 h of monocyte depletion. Prolonged incubation at 37 C
results in a rebound increase in CD3 expression in lupus T
cells.
5. Whenever possible, receptor recycling should be measured on
freshly isolated lymphocytes in the absence of stimulation. Pre-
stimulation with anti-CD3/CD28, interleukin-2, or conca-
navalin-A (Con-A) induces T cell receptor downmodulation.
If stimulation is required, do not utilize phorbol esters, such as
Con-A, as activators of protein kinase C induce rapid TCR
internalization without normal degradation of receptors.
Phorbol ester treatment leads to concentration of receptors in
the early endocytic requirement, which can rapidly be returned
to the surface upon removal of the stimulus (44). Concentration
of receptors in early endosomes reflects reduced TCR residency
time on the surface, but the size of the intracellular pool of
receptors is unaltered and represents ~15 % of total receptors
in resting T lymphocytes (45).
6. There are circumstances under which a period of resting or
stimulation is required, to allow for changes in gene expression
post transfection, infection, or after in vitro treatment. In these
circumstances, it is important to concurrently measure recep-
tor internalization to determine whether changes in surface
receptor expression are due to altered endocytic recycling, or
due to a change in receptor internalization.
Receptor internalization can be measured by biochemical
or fluorescence methods. The original method established by
Krangel et al. utilized neuraminidase treatment to compare
expression of internalized receptors, protected from neuramin-
idase, to expression of receptors on the surface, removed by
neuraminidase treatment (45). Alternatively, the amount of
internalized TCR can be measured by labeling T cells with
NHS-SS-biotin, incubating cells at 37 C to induce internal-
ization, and treating cells with MESNA, a reducing agent to
cleave off biotin on the cell surface. Subtracting the signal from
the MESNA-treated sample from the biotinylated time 0 sam-
ple will determine the amount of receptor internalized over a
given time frame (46). A flow cytometry-based method to
measure receptor internalization is to label cells with fluorescent
PE-conjugated antibodies, incubating cells at 37 C to induce
internalization, and acid-stripping cells at various time points.
The fraction of receptor internalized over time can be deter-
mined by comparing the amount of internalized fluorescence
subtracted from the amount of surface fluorescence on non-
stripped cells. Internalized receptors are protected from acid
stripping (37).
 5 The Role of Endocytic Recycling in Autoimmunity 105

7. Lupus-prone mice should be studied before the onset of disease

development, which varies according to genetic background
and strain used. To confirm that mice studied are free of auto-
immune disease, remove blood by cardiac puncture at the time
of sacrifice and analyze titer of antinuclear autoantibody from
serum by ELISA. Alternatively, proteinuria can be measured
before sacrifice by detection of urine albumin.

References
1. Perl A, Fernandez DR, Telarico T, Doherty E,
Francis L, Phillips PE (2009) T-cell and B-cell
signaling biomarkers and treatment targets in
lupus. Curr Opin Rheumatol 21(5):454464,
http://www.ncbi.nlm.nih.gov/pubmed/
19550330
2. Banchereau J, Pascual V, Palucka AK (2004)
Autoimmunity through cytokine-induced den-
dritic cell activation. Immunity 20(5):539550,
http://www.ncbi.nlm.nih.gov/pubmed/
15142523
3. Emlen W, Niebur J, Kadera R (1994)
Accelerated in vitro apoptosis of lymphocytes
from patients with systemic lupus erythemato-
sus. J Immunol 152(7):36853692, http://
www.ncbi.nlm.nih.gov/pubmed/8144943
4. Fernandez D, Perl A (2009) Metabolic control
of T cell activation and death in SLE.
Autoimmun Rev 8(3):184189, http://www.
ncbi.nlm.nih.gov/pubmed/18722557
5. Ma L, Chan KW, Trendell-Smith NJ, Wu A,
Tian L, Lam AC, Chan AK, Lo CK, Chik S, Ko
KH, To CK, Kam SK, Xs L, Yang CH, Leung
SY, Ng MH, Stott DI, MacPherson GG, Huang
FP (2005) Systemic autoimmune disease
induced by dendritic cells that have captured
necrotic but not apoptotic cells in susceptible
mouse strains. Eur J Immunol 35(11):
33643375, http://www.ncbi.nlm.nih.gov/
pubmed/16224814
6. Blasini AM, Alonzo E, Chacon R, Riera R,
Stekman IL, Rodriguez MA (1998) Abnormal
pattern of tyrosine phosphorylation in unstim-
ulated peripheral blood T lymphocytes from
patients with systemic lupus erythematosus.
Lupus 7(8):515523, http://www.ncbi.nlm.
nih.gov/pubmed/9863892
7. Karonitsch T, Feierl E, Steiner CW, Dalwigk K,
Korb A, Binder N, Rapp A, Steiner G,
Scheinecker C, Smolen J, Aringer M (2009)
Activation of the interferon-gamma signaling
pathway in systemic lupus erythematosus
peripheral blood mononuclear cells. Arthritis
Rheum 60(5):14631471, http://www.ncbi.
nlm.nih.gov/pubmed/19404947
8. Fernandez D, Bonilla E, Mirza N, Niland B,
Perl A (2006) Rapamycin reduces disease activ-
ity and normalizes activation-induced calcium
fluxing in patients with systemic lupus erythe-
matosus. Arthritis Rheum 54(9):29832988,
http://www.ncbi.nlm.nih.gov/pubmed/
16947529
9. Solomou EE, Juang YT, Gourley MF, Kammer
GM, Tsokos GC (2001) Molecular basis of
deficient IL-2 production in T cells from
patients with systemic lupus erythematosus.
J Immunol 166(6):42164222, http://www.
ncbi.nlm.nih.gov/pubmed/11238674
10. Nagy G, Barcza M, Gonchoroff N, Phillips PE,
Perl A (2004) Nitric oxide-dependent mito-
chondrial biogenesis generates Ca2+ signaling
profile of lupus T cells. J Immunol 173(6):
36763683, http://www.ncbi.nlm.nih.gov/
pubmed/15356113
11. Ibiza S, Victor VM, Bosca I, Ortega A,
Urzainqui A, OConnor JE, Sanchez-Madrid
F, Espluques JV, Serrador JM (2006)
Endothelial nitric oxide synthase regulates T
cell receptor signaling at the immunological
synapse. Immunity 24(6):753765, http://
www.ncbi.nlm.nih.gov/pubmed/16782031
12. Nagy G, Koncz A, Fernandez D, Perl A (2007)
Nitric oxide, mitochondrial hyperpolarization,
and T cell activation. Free Radic Biol Med
42(11):16251631, http://www.ncbi.nlm.nih.
gov/pubmed/17462531
13. Leist M, Single B, Castoldi AF, Kuhnle S,
Nicotera P (1997) Intracellular adenosine
triphosphate concentration: a switch in the
decision between apoptosis and necrosis. J Exp
Med 185(8):14811486, http://www.ncbi.
nlm.nih.gov/pubmed/9126928
14. Cunningham JT, Rodgers JT, Arlow DH,
Vazquez F, Mootha VK, Puiqserver P (2007)
mTOR controls mitochondrial oxidative func-
tion through a YY1-PGC1alpha transcriptional
complex. Nature 450(7170):736740, http://
www.ncbi.nlm.nih.gov/pubmed/18046414
15. Jin HK, Ahn SH, Yoon JW, Park JW, Lee EK,
Yoo JS, Lee JC, Choi WS, Han JW (2009)
106 T. Telarico and A. Perl
Rapamycin down-regulates inducible nitric
oxide synthase by inducing proteasomal degra-
dation. Biol Pharm Bull 32(6):988992,
http://www.ncbi.nlm.nih.gov/pubmed/
19483303
16. Ramanathan A, Schreiber SL (2009) Direct
control of mitochondrial function by mTOR.
Proc Natl Acad Sci 106(52):2222922232,
http://www.ncbi.nlm.nih.gov/pubmed/
20080789
17. Schieke SM, Phillips D, McCoy JP Jr, Aponte
AM, Shen RF, Balaban RS, Finkel T (2006)
The mammalian target of rapamycin (mTOR)
pathway regulates mitochondrial oxygen con-
sumption and oxidative capacity. J Biol Chem
281(37):2764327652, http://www.ncbi.nlm.
nih.gov/pubmed/16847060
18. Michelot A, Costanzo M, Sarkeshik A, Boone
C, Yates JR III, Drubin DG (2010)
Reconstitution and protein composition analy-
sis of endocytic actin patches. Curr Biol
20:18901899, http://www.ncbi.nlm.nih.
gov/pubmed/21035341
19. Fernandez DR, Telarico T, Bonilla E, Li Q,
Banerjee S, Middleton FA, Phillips PE, Crow
MK, Oess S, Muller-Esterl W, Perl A (2009)
Activation of mammalian target of rapamycin
controls the loss of TCR zeta in lupus T cells
through HRES-1/Rab4-regulated lysosomal
degradation. J Immunol 182(4):20632073,
http://www.ncbi.nlm.nih.gov/pubmed/
19201859
20. Flinn RJ, Yan Y, Goswami S, Parker PJ, Backer
JM (2010) The late endosome is essential for
mTORC1 signaling. Mol Biol Cell 21(5):
833841, http://www.ncbi.nlm.nih.gov/
pubmed/20053679
21. Li L, Kim E, Yuan H, Inoki K, Goraksha-Hicks
P, Schiesher RL, Neufeld TP, Guan KL (2010)
Regulation of mTORC1 by the Rab and Arf
GTPases. J Biol Chem 285(26):1970519711,
http://www.ncbi.nlm.nih.gov/pubmed/
20457610
22. Huang H, Jeon MS, Liao L, Yang C, Elly C,
Yates JR 3rd, Liu YC (2010) K33-linked polyu-
biquination of T cell receptor zeta regulates
proteolysis-independent T cell signaling.
Immunity 33(1):6070, http://www.ncbi.nlm.
nih.gov/pubmed/20637659
23. Cormont M, Meton I, Mari M, Monzo P,
Keslair F, Gaskin C, McGraw TE, Le Marchand-
Brustel Y (2003) CD2AP/CMS regulates
endosome morphology and traffic to the degra-
dative pathway through its interaction with
Rab4 and c-Cbl. Traffic 4(2):97112, http://
www.ncbi.nlm.nih.gov/pubmed/12559036
24. Razi M, Chan EY, Tooze SA (2009). Early
endosomes and endosomal coatomer are
required for autophagy. Journal of Cell Biology,
305-321. http://www.ncbi.nlm.nih.gov/
pubmed/19364919
25. Nambiar MP, Enyedy EJ, Fisher CU, Krishnan
S, Warke VG, Gilliland WR, Oglesby RJ, Tsokos
GC (2002) Abnormal expression of various
molecular forms and distribution of T cell
receptor zeta chain in patients with systemic
lupus erythematosus. Arthritis Rheum
46(1):163174, http://www.ncbi.nlm.nih.
gov/pubmed/11817588
26. Nambiar MP, Enyedy EJ, Warke VG, Krishnan
S, Dennis G, Kammer GM, Tsokos GC (2001)
Polymorphisms/mutations of TCR-zeta-chain
promoter and 3 untranslated region and selec-
tive expression of TCR zeta-chain with an
alternatively spliced 3 untranslated region in
patients with systemic lupus erythematosus.
J Autoimmun 16(2):133142, http://www.
ncbi.nlm.nih.gov/pubmed/11247639
27. Juang YT, Wang Y, Jiang G, Peng HB, Ergin S,
Finnell M, Magilavy A, Kyttaris VC, Tsokos
GC (2008) PP2A dephosphorylates Elf-1 and
determines the expression of CD3 zeta and
FcRgamma in human systemi lupus erythema-
tosus T cells. J Immunol 181(5):36583664,
http://www.ncbi.nlm.nih.gov/pubmed/
18714041
28. Takeuchi T, Tsuzaka K, Pang M, Amano K,
Koide J, Abe T (1998) TCR zeta chain lacking
exon 7 in two patients with systemic lupus ery-
thematosus. Int Immunol 10(7):911921,
http://www.ncbi.nlm.nih.gov/pubmed/
9701029
29. Krishnan S, Kiang JG, Fisher CU, Nambiar
MP, Nguyen HT, Kyttaris VC, Chowdhury B,
Rus V, Tsokos GC (2005) Increased caspase-3
expression and activity contribute to reduced
CD3zeta expression in systemic lupus erythe-
matosus T cells. J Immunol 175(5):
34173423, http://www.ncbi.nlm.nih.gov/
pubmed/16116236
30. Valitutti S, Muller S, Salio M, Lanzavecchia A
(1997) Degradation of T cell receptor (TCR)-
CD3zeta complexes after antigenic stimulation.
J Exp Med 185(10):18591864, http://www.
ncbi.nlm.nih.gov/pubmed/9151711
31. Holst J, Wang H, Eder KD, Workman CJ, Boyd
KL, Baguet Z, Singh H, Forbes SK, Chruscinski
A, Smeyne R, van Oers NS, Utz PJ, Vignali DA
(2008) Scalable signaling mediated by T cell
antigen receptor-CD3 ITAMs ensures effective
negative selection and precents autoimmunity.
Nat Immunol 9(6):658666, http://www.ncbi.
nlm.nih.gov/pubmed/18469818
32. Nambiar MP, Fisher CU, Warke VG, Krishnan
S, Mitchell JP, Delaney N, Tsokos GC (2003)
Reconstitution of deficient T cell receptor zeta
 5 The Role of Endocytic Recycling in Autoimmunity
chain restores T cell signaling and augments T
cell receptor/CD3-induced interleukin-2 pro-
duction in patients with systemic lupus erythe-
matosus. Arthritis Rheum 48(7):19481955,
http://www.ncbi.nlm.nih.gov/pubmed/
12847689
33. Howard FD, Rodewald HR, Kinet JP, Reinherz
EL (1990) CD3zeta subunit can substitute for
the gamma subunit of Fc epsilon receptor type
I in assembly and functional expression of the
high-affinity IgE receptor: evidence for inter-
receptor complementation. Proc Natl Acad Sci
87(18):70157019, http://www.ncbi.nlm.nih.
gov/pubmed/1698288
34. Enyedy EJ, Nambiar MP, Liossis SN, Dennis
G, Kammer GM, Tsokos GC (2001) Fc epsilon
receptor type I gamma chain replaces the
deficient T cell receptor zeta chain in T cells of
patients with systemic lupus erythematosus.
Arthritis Rheum 44(5):11141121, http://
www.ncbi.nlm.nih.gov/pubmed/11352243
35. Krishnan S, Farber DL, Tsokos GC (2003) T cell
rewiring in differentiation and disease. J Immunol
171(7):33253331, http://www.ncbi.nlm.nih.
gov/pubmed/14500623
36. Ueda H, Howson JM, Esposito L, Heward J,
Snook H, Chamberlain G, Rainbow DB,
Hunter KM, Smith AN, Di Genova G, Herr
MH, Dahlman I, Payne F, Smyth D, Lowe C,
Twells RC, Howlett S, Healy B, Nutland S,
Rance HE, Everett V, Smink LJ, Lam AC,
Cordell HJ, Walker NM, Bordin C, Hulme J,
Motzo C, Cucca F, Hess JF, Metzker ML,
Rogers J, Gregory S, Allahabadia A,
Nithiyananthan R, Tuomilehto-Wolf E,
Tuomilehto J, Bingley P, Gillespie KM, Undlien
DE, Rnningen KS, Guja C, Ionescu-Trgovi te
C, Savage DA, Maxwell AP, Carson DJ,
Patterson CC, Franklyn JA, Clayton DG,
Peterson LB, Wicker LS, Todd JA, Gough SC
(2003) Association of the T-cell regulatory
gene CTLA4 with susceptibility to autoim-
mune disease. Nature 423(6939):506511,
http://www.ncbi.nlm.nih.gov/pubmed/
12724780
37. D Oro U, Munitic I, Chacko G, Karpova T,
McNally J, Ashwell JD (2002) Regulation of
constitutive TCR internalization by the zeta
chain. J Immunol 169:62696278, http://
www.ncbi.nlm.nih.gov/pubmed/12444133
107
38. Krangel MS (1987) Endocytosis and recycling
of the T3 T-cell receptor complex. J Exp Med
165(4):11411159, http://www.ncbi.nlm.
nih.gov/pubmed/3104527
39. Liu H, Rhodes M, Wiest DL, Vignali DAA
(2000) On the dynamics of TCR:CD3 com-
plex cell surface expression and downmodula-
tion. Immunity 13:665675, http://www.
ncbi.nlm.nih.gov/pubmed/11114379
40. Kovacs B, Vassilopoulous D, Vogelgesang SA,
Tsokos GC (1996) Defective CD3-mediated
cell death in activated T cells from patients
with systemic lupus erythematosus: role of
decreased intracellular TNF-alpha. Clin
Immunol Immunopathol 81(3):293302,
http://www.ncbi.nlm.nih.gov/pubmed/
8938108
41. Patino-Lopez G, Dong X, Ben-Aissa K, Bernot
KM, Itoh T, Fukuda M, Kruhlak MJ, Samelson
LE, Shaw S (2008) Rab35 and its GAP EPI64C
in T cells regulate receptor recycling and immu-
nological synapse formation. J Biol Chem
283(26):1832318330, http://www.ncbi.
nlm.nih.gov/pubmed/18450757
42. Batista A, Millan J, Mittelbrunn-M Sanchez-
Madrid F, Alonso MA (2004) Recruitment of
transferrin receptor to immunological synapse
in response to TCR engagement. J Immunol
172(11):67096714, http://www.ncbi.nlm.
nih.gov/pubmed/15153487
43. Bradford MM (1976) Rapid and sensitive
method for the quantitation of microgram
quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:
248254, http://www.ncbi.nlm.nih.gov/
pubmed/942051
44. Alcover A, Alarcon B (2000) Internalization
and intracellular fate of TCR-CD3 complexes.
Crit Rev Immunol 20(4):325346, http://
www.ncbi.nlm.nih.gov/pubmed/11100805
45. Minami Y, Samelson LE, Klausner RD (1987)
Internalization and cycling of the T cell antigen
receptor. J Biol Chem 262(27):1334213347,
http://www.ncbi.nlm.nih.gov/pubmed/
3498715
46. Smythe E, Redelmeier TE, Schmid SL (1992)
Receptor-mediated endocytosis in semiintact
cells. Methods Enzymol 219:223234, http://
www.ncbi.nlm.nih.gov/pubmed/1487996
 Chapter 6

Multiparameter Flow Cytometry and Bioanalytics

for B Cell Profiling in Systemic Lupus Erythematosus
Denise A. Kaminski, Chungwen Wei, Alexander F. Rosenberg,
F. Eun-Hung Lee, and Ignacio Sanz

Abstract
B lymphocyte involvement in systemic lupus erythematosus has been recognized for several decades,
mainly in the context of autoantibody production. Both mouse and human studies reveal that different
types of antibody responses, as well as antibody-independent effector functions can be ascribed to distinct
subpopulations (subsets) of circulating B cells. Characterizing human B cell subsets can advance the field
of autoimmunity even further by establishing B cell signatures associated with disease severity, progression,
and response-to-treatment. For this purpose, we have developed specialized B cell reagent panels for mul-
tiparameter flow cytometry, and combine their use with advanced bioinformatics strategies that together
will likely be advantageous for improving the characterization, prognosis, and for possibly improving treat-
ment regimens of chronic inflammatory diseases such as lupus.

Key words: B lymphocyte, Autoantibody, 9G4, Transitional B cell, Mature-nave B cell, Memory B cell,
Antibody-secreting cell, B cell subset, Systemic lupus erythematosus, Principal component analysis

1. Introduction

Systemic lupus erythematosus (SLE) is a chronic inflammatory

autoimmune disorder whose clinical complications include skin
rashes, joint pain, and in more severe cases, kidney and nervous
system pathology (1, 2). Due to the enormous heterogeneity of
symptom types, severity, kinetics, and response-to-treatment
among patients (3), establishing reliable biological indicators asso-
ciated with different outcomes (biomarkers) is essential for improv-
ing diagnosis, for accuracy of prognosis, and for customizing
therapies. Both T and B lymphocyte abnormalities are observed in
SLE, including elevated levels of self-reactive antibodies that pre-
cede disease onset (4). The importance of B cells in SLE and the

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_6, Springer Science+Business Media New York 2012

109
110 D.A. Kaminski et al.

phenotypic diversity of human B cell subsets strongly suggest that

phenotypic profiles of B cells could be such a biomarker (5).
Herein, we review B cell phenotypic diversity in the context of SLE
and also describe reliable methods developed by our group to mea-
sure B cell changes in human peripheral blood using high-parame-
ter flow cytometry combined with advanced bioinformatics.

1.1. Importance Antibodies (immunoglobulins, Ig) are expressed on the surface of

of B Cells in SLE and secreted by B cells. Each B cell clone expresses an antibody [B
cell receptor (BCR), if surface membrane-bound] that is unique in
its variable (V) region, and whose constant region is one of nine
effector isotypes. Antibodies are designed by each B cell through
DNA recombination of Ig V region gene segments, by somatic
hypermutation of these assembled V region genes, and by isotype
(class) switching, which changes the Ig constant region to modify
its effector function.
During B cell development, Ig DNA rearrangements create a
diverse BCR repertoire encoding a wide range of antigen-binding
capabilities. Self-reactive B cells are usually deleted or inactivated at
developmental checkpoints, which are defective in SLE patients
(611). Common autoantibody reactivities include nuclear com-
ponents and plasma membrane inner leaflet phospholipids (12).
This reactivity may be attributed to poor clearance of apoptotic
cells and debris in SLE patients (13, 14), which can allow for expo-
sure to such internal antigens (15, 16). Thus, B cell tolerance
might be broken by extended exposure of these antigens stimulat-
ing self-reactive B cells, which in turn promote inflammation
through effector functions such as antigen presentation to T cells,
cytokine secretion, and/or pathogenic Ig secretion.
Despite significant technical advances in cell sorting, molecular
cloning, and re-expression of Ig-encoding cDNAs as recombinant
monoclonal antibodies, the use of anti-idiotype reagents recogniz-
ing self-reactive BCRs allows highly efficient characterization of
intact B cells by histology and by flow cytometry. One of these
reagents is a rat anti-human monoclonal antibody called 9G4. 9G4
binds to the V region of BCRs or of soluble antibodies (denoted
respectively, 9G4+ B cells or 9G4+ antibodies). Up to 10 % of
healthy-human B cells are 9G4+, but are largely confined to the
antigen-nave, undifferentiated B cell compartments, maintaining
very low-to-undetectable serum 9G4+ antibody levels (17).
However, SLE patients often have high titers of 9G4+ antibody
that correlates with disease activity (1820), with 9G4+ IgG levels
correlating more strongly with disease (especially nephritis) than
9G4+ IgM levels (20).
High-titer autoantibody in SLE has prompted interest in
developing biologicals that target B cells (3). Inhibiting the B cell
survival factor BAFF/Blys (B cell Activation Factor of the TNF
Family/B Lymphocyte Stimulator) or depleting B cells can alleviate
 6 Multiparameter Flow Cytometry and Bioanalytics 111

symptoms of lupus-associated disease in humans and mice (2128).

These outcomes strongly suggest that B cells actively contribute to
disease progression, although the exact mechanisms are not known.
Interestingly, both mouse and human data suggest that high-titer
autoantibody alone does not account for the B cell-mediated
effects (5, 22, 27, 29). In fact, B cells are important for memory
T cell accumulation in autoimmune mice, suggesting mechanisms
such as antigen presentation and/or cytokine production by the
B cells (29).
However, the B cell subsets responsible for each specific func-
tion are incompletely understood. Thus, in subsequent sections,
we discuss how B cell characterization could be further exploited
for understanding this disease and for optimizing clinical benefit
(5, 21, 30).

1.2. B Cell Subsets In adult bone marrow, nascent B cells begin expressing cell-surface
in SLE BCR. In a transitional stage, the BCR+ B cell enters the periph-
eral circulation and differentiates into a mature-nave cell that is
competent for responding to BCR engagement by antigen. In
addition to transitional and mature-nave B cells, human blood
contains 34 additional core B cell subsets with characteristics of
antigen exposure (Ig somatic hypermutation, Ig class switching,
and/or markers associated with Ig secretion). Such B cells that are
neither nave nor secreting antibody are generally regarded as
memory in most nomenclatures (31) (Fig. 1b, c), which are dis-
cussed below. All of these basic core CD19+ populations can be
identified using flow cytometry by various combinations of IgD
BCR (more nave) and CD27 (more differentiated) surface expres-
sion (Fig. 1c) (31). Further subsetting these core populations using
additional markers is a highly advantageous way to characterize
human B cells (31).

1.2.1. Transitional The CD10 marker on human BCR B cell progenitors continues to
and Mature-Nave B Cells be expressed on immature B cells that have acquired the first class
of BCR, IgM, as well as on IgM+ transitional B cells coexpressing
an IgD BCR with an identical V region. In healthy subjects, the
frequency of autoreactive B cells is reduced when the BCR reaches
the cell surface (6), and then again when CD10 is lost (7). At each
step, more of these B cells are self-reactive in SLE patients com-
pared with healthy subjects (7, 8). Despite breaching these toler-
ance checkpoints, SLE patients have decreased numbers of nave
B cells contributing to overall reduced numbers of total CD19+
B cells (13, 22, 32, 33). The cause and significance of this reduc-
tion is poorly understood.
Given the critical involvement of the transitional-to-mature
conversion in determining the SLE B cell repertoire, it is advanta-
geous to define such cells and their subsets in clearer detail. This
goal can be achieved using multiparameter flow cytometry that can
 a
112

250K 250K 250K 250K

105

200K 200K 200K 200K 4

99.3 10
150K 150K 150K 150K
3
10
98.2 98.1

SSC-A
SSC-A

SSC-W
100K 100K 100K 100K

FSC-W
2
10

CD3-Pacific Blue
50K 50K 50K 50K 10.3
42.2 0

0 0 0 0
0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 0
D.A. Kaminski et al.

102 103 104 105 102 103 104 105

FSC-A FSC-H SSC-H Live/Dead CD19-APC-Cy7

b
5 3.25
10 0.656
CD19+
populations in Bm3+4 Bm2
B-mature (Bm) 104
nomenclature
Bm2
103
74.2
0

CD38-PerCP-Cy5.5
Bm5 Bm1
14.2 7.72
0 102 103 104 105
IgD-FITC

c CD27+
SwMe NSM CD19+ gate CD19+ IgD- gate

105 12.2 105 12.1

CD19+ 1.69 105
populations in 1.06 PB
CD27 vs IgD 104 104 104
T1/2
nomenclature
3 3 103
10 10
CD27-Qdot605

0 0 102

CD27-Qdot605
CD24-PE-Alexa610

N
0
5.24 80.9
0 2 3 4 5 3 4 5 4
10 10 10 10 0 10 10 10 0 103 10 105
DN
IgD-FITC CD38-PerCP-Cy5.5 CD38-PerCP-Cy5.5
 IgD-CD27+ IgD-CD27- IgD+CD27-
d
100 100 100 100 100

80 80 80 80 80

60 60 60 60 60

% of Max
40 40 40 40 40

20 20 20 20 20

0 0 0 0 0
0 103 104 105 0102 103 104 105 0 103 104 105 0 102 103 104 105 0 102 103 104 105
B220-PE-Cy7 CD24-PE-Alexa610 CD21-PE-Cy5 CD95-APC CXCR3-PE
6

e CD19+ T1/2 N NSM CD27+SwMe DN

1500 25 60
15
105 4.78 200
20
104 1000 150 40
10 15
3 100
10 10
9.11 5.37 6.01 1.61

Cell number
5 500 20 2.67
5 50

CD27-Qdot605
102
0
0 0 0 0 0
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
9G4-Alexa680 9G4-Alexa680

Fig. 1. Healthy human PB B cell subsets and phenotypic characterization of memory B cells. (a) Sequential gating strategy for (left to right) lymphocytes, singlets based on FSC,
singlets based on SSC, viable cells that do not take up amine-reactive Aqua-fluorescent dye (Live/Dead-negative), CD3CD19+ B cells. (b) The Bm (mature B cell) nomenclature,
first developed to classify tonsil B cells by IgD and CD38 expression, is commonly used in PBL analysis. This approach fails to separate IgD+CD27+ nonswitched memory cells, which
fall into the Bm1 and Bm2 gates together with nave cells (Bm1 and Bm2). Transitional 1 and 2 (T1/2) are included with Bm2 (pre-GC). Furthermore, isotype-switched CD27+ cells
are not distinguished from CD27 memory cells within the Bm5 population (IgDCD38+/). (c) The more commonly used IgD/CD27 classification scheme identifies four core B cell
subsets in human peripheral blood: IgD+CD27 nave B cells (N), IgD+CD27+ nonswitched memory B cells (NSM), IgDCD27+ switched memory cells (CD27+ SwMe), and IgDCD27
double-negative B cells (DN). In this scheme, transitional T1/2 colocalize with nave cells in the CD27 vs IgD plot, but can be identified based on their CD24++CD38++ phenotype
(middle panel). Likewise, plasmablasts (PB) are embedded in the IgDCD27+ fraction and can be accurately identified by first gating on IgD fraction followed by gating on the
Multiparameter Flow Cytometry and Bioanalytics

CD27++CD38++ region as depicted in the third panel. (d) The differential expression patterns of the surface markers in the IgDCD27+ (blue histograms) and IgDCD27 (green histo-
grams) memory B cells were overlaid on the IgD+CD27 nave B cells (filled red histograms). (e) Most autoreactive 9G4+ B cells are substantially CD27 and thus underrepresented
within the CD27+ memory compartment as depicted in the bivariate plot of the total CD19+ B cells (right). Histograms further demonstrate that among the core subsets (gated as in
113

c), the transitional and nave B cells exhibit the highest fraction of 9G4+ cells.
114 D.A. Kaminski et al.

be efficiently executed in the laboratory with less than 10 cc of

blood, and also allows for isolating such subsets for functional
analyses. Transitional and mature-nave B cells are CD27. In addi-
tion to CD10, which typically has a poor staining index, the earli-
est transitional populations coexpress high levels of CD38 and
CD24. T1 transitional cells express slightly higher CD24 and
CD38 than T2 cells, their apparent progeny; however, visualiz-
ing this distinction is difficult without overlaying a profile of bone
marrow, which lacks T2 B cells. Compared with T1/T2 transi-
tional B cells, mature cells express lower levels of CD38, survive
longer in culture, and divide in response to BCR engagement (34).
Truly mature nave B cells can extrude dyes such as Rhodamine
123 using the mitochondrial ABCB1 transporter, which is absent
from both transitional and memory B cells (34). This characteristic
is especially useful in separating true-mature (mature-nave) B cells
from an additional transitional population (T3). T3 cells express
lower levels of CD24 and CD38 than T1/T2, but all three transi-
tional populations fail to express active ABCB1 (35). Thus, with-
out a dye-extrusion step, the T3 population is not distinguished
from true-mature B cells. It remains to be definitively determined
whether T3 is an obligate precursor to true-mature B cells.
Nonetheless, such phenotypic distinctions may be clinically infor-
mative, as preferential early reconstitution of transitional B cells
correlates with long-term remission in B cell-depleted SLE patients
(once treatment is stopped) (30). Thus, thorough characterization
of transitional and mature-nave B cell subsets may contribute to
defining more precise prognoses and perhaps predicting response
to treatment.

1.2.2. Germinal Center Antigen engagement of the BCR on a mature-nave B cell can
B Cells stimulate activation and differentiation along any of several path-
ways that, ideally, help eliminate an invading organism and protect
the host. One of these pathways is the germinal center (GC) reac-
tion in secondary lymphoid tissue follicles. Typically, GC reactions
require T cell help to promote B cell proliferation, BCR class
switching from IgM/IgD to IgG, IgA, or IgE, and also Ig V region
gene somatic hypermutation that changes the affinity of the
encoded BCR for its cognate antigen (36). Somatic mutations in
autoantibodies contribute to their self-reactivity (11, 37, 38).
Antigen-driven selection results in clonal expansion of mainly the
highest affinity B cells. In normal individuals with intact peripheral
tolerance, these B cells are ideally only reactive with antigens from
invading organisms. The surviving cells can differentiate into anti-
body-secreting plasma cells or into memory B cells (each discussed
below) that are ready for a rapid response to a second encounter
with an invading organism.
In healthy human tonsil, non-self-reactive B cells are evenly
distributed among the B cell populations, but self-reactive 9G4+ B
 6 Multiparameter Flow Cytometry and Bioanalytics 115

cells are confined to the nave compartment and excluded from

GCs (17, 39). Unlike in healthy humans, 9G4+ B cells in SLE
patients colocalize with proliferating CD38+ GC structures (39).
These observations strongly suggest that dysregulation of autore-
active B cells at GC differentiation stages contributes to SLE dis-
ease (40), and may explain the ability to detect IgG+ 9G4+ cells and
9G4+CD138+ (plasma) cells in SLE peripheral blood, each of which
are rare in healthy controls (39).

1.2.3. CD27+ Memory B cell memory (specific and rapid recall responses to previously
B Cells encountered antigens) is often inferred from cells restimulated
in vitro and from the ability to transfer such memory to nave
experimental animals (4143). In humans, most of this activity is
attributed to CD27+ B cells. Compared with CD27 (mostly
mature-nave and transitional) B cells, CD27+ B cells are slightly
larger (34, 44), are more proliferative in vitro (4547) and in vivo
(48), more efficiently stimulate allogeneic CD4 T cell proliferation
(46), and more readily differentiate into antibody-secreting cells
(47). GC, memory, and in vitro-activated nave B cells can all be
CD27+. However, as GC are generally confined to lymphoid tis-
sues, and few circulating CD27+ B cells show evidence of on-going
proliferation (34), GC and recently activated B cells unlikely
account for a significant portion of the CD27+ B cell pool, at least
in healthy humans. Additionally, CD27+ B cells are barely detect-
able in natal cord blood, but proportionally increase with age and
cumulative antigen exposure (44, 4850). The few CD27+ B cells
in the cord blood lack Ig V somatic hypermutations and have been
attributed to a newly described innate-like human B1 B cell
equivalent (51).
Adult CD27+ B cells either (1) express an IgD/IgM BCR
[IgM memory or nonswitched memory (NSM) B cells] or (2) have
lost expression of these markers after the irreversible process of
antibody class switching (switched memory B cells). CD27+ B cells
are ~1030 % each IgM+D+, IgG+, or IgA+ (34, 5254). It is
unknown whether these memory B cell subsets derive from com-
mon or from distinct differentiation pathways, or even if such
pathways are common to all cells in a given pool. Thus, the switched
memory B cell pool may contain cells that class-switched prior to
and also those that class-switched after acquiring CD27. Similarly,
the NSM pool may contain B cells that will remain NSM together
with intermediates that will eventually class-switch. Preliminary
analysis using high-parameter flow cytometry combined with auto-
mated analysis examining all parameters simultaneously (55) sug-
gests that most NSM cells have more in common with other
compartments than they have with each other (unpublished).
However, most studies to-date have compared all NSM with all
switched memory B cells using conventional manual gating meth-
ods. Briefly, both compartments have somatically hypermutated Ig
116 D.A. Kaminski et al.

V regions with characteristics of antigen selection (11, 36, 54, 56).

However, reports disagree on whether NSM have a unique (33,
57, 58) or similar (59) Vh repertoire compared with IgDCD27+
B cells. It is unknown whether sorting and/or sequencing strate-
gies account for these different observations.
The NSM pool is thought to consist of adult B1 B cells, regu-
latory B cells, and splenic marginal zone-like B cells that provide
carbohydrate-reactive antibody responses to encapsulated bacteria
(38, 50, 51, 60, 61). Recent studies in mice suggest that at least
some of the IgM memory pool functions to maintain very long-
term memory against protein antigen (62, 63).
Mice and humans with defective GC have a paucity of memory
B cells and profoundly decreased levels of class-switched serum
antibody (43). These observations led to the dogma that class-
switched humoral memory is GC-derived, whereas IgM memory is
GC-independent. Indeed, development of NSM B cells with
mutated Ig V regions does not require GC formation (64).
However, some NSM have mutations in a noncoding region of the
Bcl6 gene, which is characteristic of GC participation (59).
Although antibody class switching is also characteristic of GC reac-
tions, not all switched cells are necessarily GC-derived. A small
proportion of CD27+IgG+ cells express the IgG2+ subclass (34,
65), which can be induced independently of GC-type (i.e., T cell-
derived) stimuli in culture (66).
Some pediatric SLE patients have more IgG3-expressing and
fewer IgG2-expressing switched memory B cells compared with
healthy controls (65). Given the contribution of IgG2 to anti-
polysaccharide responses, this observation may suggest an over-
representation of GC-derived memory against protein-type
antigens. More notably, clinical relapse of B cell-depleted patients
correlates with rapid reconstitution of CD27+ memory B cells (30).
It is not clear if this effect is due to the functions of the memory
B cells or due to pathological circumstances favoring memory over
transitional B cell expansion. Interestingly still is the recurring
observation that NSM are proportionally reduced in active SLE
(32, 67, 68), correlating with higher autoantibody titers (67).
Together with the stronger clinical correlation between serum
9G4+ IgG compared with 9G4+ IgM (20), this result may suggest
that some of the IgM memory may play a protective role against
pathology otherwise exacerbated by IgG memory. Increasing
evidence supports such a dichotomy of protective and pathogenic
B cells in cardiovascular disease (6973).

1.2.4. Double-Negative A small proportion of class-switched B cells in healthy subjects do

B Cells not express CD27 (52, 53). These B cells make up most of the
CD19+IgDCD27 double-negative (DN) compartment, which
is typically less than 5 % of the CD19+ pool. Most DN B cells in
healthy subjects express either IgG or IgA (52). Some evidence
 6 Multiparameter Flow Cytometry and Bioanalytics 117

suggests that the DN compartment in unchallenged individuals

includes B cells that have further differentiated into memory B
cells without acquiring CD27. Both CD27+ and CD27 pools con-
tain B cells that can be readily induced to secrete Ig against tetanus
toxin and influenza virus (34), suggesting prior exposure to vac-
cines. Other than CD27 expression, the DN and CD27+ switched
memory B cell populations have similar surface phenotypes (34,
53) and have somatically mutated Ig V regions (52, 53). However,
Ig V region mutations in DN B cells are less frequent than in
switched CD27+ memory cells (52, 53). The DN compartment
may thus be a mixture of both effector and memory B cells.
To varying degrees, SLE patients have increased frequencies of
DN B cells (13, 22, 52) correlating with higher autoantibody titers
and with a greater incidence of nephritis (52). The higher fre-
quency of DN cells could result from B cells entering from other
compartments, such as the nave B cell pool becoming activated
and losing IgD expression, or from activated cells entering blood
from the tissues. Further investigation of the characteristics, ori-
gins, and functions of DN B cells in treated and untreated SLE
patients, and in newly vaccinated and acutely infected controls will
likely contribute to our understanding of B cells in this disease and
in B cell activation in general.

1.2.5. Antibody-Secreting Differentiation into an antibody-secreting cell (ASC) can occur

Cells downstream of the appropriate B cell-activating stimuli. ASC in
human peripheral blood are typically CD27highCD38high, and nearly
all express the intracellular Ki67 antigen, suggesting on-going pro-
liferation (34). ASCs detected by Ig secretion assays or by flow
cytometry are very rare in the blood of healthy, unchallenged indi-
viduals. However, a rapid and transient ASC response is readily
detectable as early as 4 days after booster vaccination (55, 7476).
During acute infection (especially viral), ASCs can be detected as
early as day 2 after symptom onset (77). Specificity of the circulating
ASCs is unique to antigens of recent exposure (94). Human and
mouse studies have identified at least two subsets of blood ASC
loosely referred to as plasmablasts and plasma cells (78, 79). True,
terminally differentiated plasma cells might be enriched in the
compartment expressing CD138, which is less than half of the ASC
in the blood, but nearly all of the ASC in the bone marrow (78,
79). Compared with CD38highCD138 ASC, considered to be plas-
mablasts, CD138+ plasma cells are larger with more cytoplasm,
lack surface Ig, and do not seem to be undergoing proliferation
(34, 80). As with the various kinds of memory B cells, it is not
entirely clear whether some or all plasmablasts and plasma cells
have precursorproduct relationships, even in elegant mouse
reporter systems (78, 79). However, given evidence that long-lived
specific antibody in the serum can be maintained without concur-
rently circulating memory B cells (42, 81), a model has arisen in
118 D.A. Kaminski et al.

which long-lived, CD138+ plasma cells in tissues (such as bone

marrow, where they are most readily detectable) provide a long-lived
Ig source, whereas plasmablasts in the circulation provide an imme-
diate, but transient boost to existing serum Ig levels. This model
does not favor nor exclude that plasmablasts and memory B cells
(from either tissues or circulation) could each progress to the
plasma cell stage upon migrating to the bone marrow.
ASC detected by functional secretion assays and by flow
cytometry are proportionally increased in SLE patients. However,
reports differ on whether this effect includes patients with quies-
cent and those with moderate disease (32, 67, 68, 82) or if it cor-
relates with disease activity (22, 33). The differences are not
accounted for by the choice of flow cytometry markers per se, and
thus may instead include disparities in patient groups biologically,
in clinical assessments, and even in technical strategies including
inconsistent resolution and/or event-gating of these rare popula-
tions. Biologically, the contribution of antibodies compared with
antibody-independent B cell functions might differ among patients
or patient groups. B cell depletion and CD40L blockade can each
reduce plasmablasts in SLE patients, correlating with symptom
reduction (13, 22). However, in only some of these patients do
serum autoantibody levels decrease. Thus, plasmablast expansion
in SLE could possibly be more of a consequence of systemic
inflammation, rather than a cause.

1.2.6. Evolving SLE B Cell We have thus far discussed the tendency of SLE patients to have
Signature proportional increases in CD19+IgDCD27 DN B cells (which
may become the largest switched memory subset) and in plas-
mablasts, but decreased proportions of NSM B cells. Previous
studies and our own observations suggest that including additional
cell-surface markers will provide additional advantages in further
characterizing these populations in lupus. As previously reported
(31), CD27+ resting memory cells in healthy subjects are predomi-
nantly B220 (Fig. 1d) as the expression of this marker is typically
lost during germinal center differentiation. By contrast, B220
expression predominates in IgDCD27 cells. This subset is char-
acterized in SLE by the downregulation of CD24, a marker typi-
cally expressed by most PBL B cells in general and memory B cells
in particular (see Fig. 1d for healthy subject example). Consequently,
expanded fractions of B220+CD24 cells within the IgDCD27
compartment are prominent in lupus patients. Loss of CD21 and
upregulation of CD95 have been independently associated with
memory B cell activation (83, 84). Accordingly, an indication of
memory B cell activation in SLE can be found in both SwMe and
DN cells in which the CD21 and CD95+ fractions are greatly
expanded in lupus compared to the healthy controls. By contrast,
CXCR3 expression (suggesting migratory potential of activated
cells to nonlymphoid, systemic inflamed tissues) is concentrated in
 6 Multiparameter Flow Cytometry and Bioanalytics 119

the IgDCD27+ memory subset. Therefore, the inclusion of CD21,

CD95, and CXCR3 in the same reagent panel determines the
coexpression of these informative functional markers. Consistent
with a resting phenotype, most IgDCD27+ memory cells are
CD21+CD95 in healthy controls (Fig. 1d). A significant fraction
of IgDCD27 cells, however, lack expression of CD21, a feature
consistent with activation. Yet, they lack CD95 expression, indicat-
ing that these two markers are not necessarily correlated. Combined,
CD95+ cells are relatively scarce in healthy memory cells. By con-
trast, CD95+ cells are greatly expanded in both memory subsets of
SLE patients. Interestingly, the vast majority of CD95+ IgDCD27
cells are CD21. CD95+ cells within the lupus IgDCD27+ memory
are almost equally split between CD21+ and CD21, illustrating
again that the expression of these markers is not necessarily recip-
rocal. Furthermore, the CXCR3+ fractions of both IgDCD27+
and IgDCD27 memory subsets exhibit CD21/CD95 expression
patterns that are similar to those of their respective total subsets
and are not therefore, particularly enriched in activated cells, at
least as defined by these markers.
Given the enormous heterogeneity in SLE disease presenta-
tion, and the contributions that B cells make to pathology, further
defining subpopulations of these core subsets using the markers
described above can establish signatures or patterns of changes
in B cell populations that may be associated with different clinical
outcomes. Such profiling to find B cell signatures could also help
prognosis and custom-designed treatments. Essentially, the appro-
priate modality would ideally eliminate B cells harboring patho-
genic properties, while it would enhance the numbers and/or
activity of B cells with regulatory properties (5, 21). However, cer-
tain signatures may also be predictive of whether more classical
therapies are or are not going to be useful in a given individual.

2. Materials

1. Human peripheral blood in a heparinized collection tube (e.g.,

green-top vacutainer; BD Biosciences, Franklin Lakes, NJ).
2. Phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA).
3. BSA (10 % solution; Miltenyi, Auburn, CA).
4. FACS buffer: 0.5 % BSA in PBS.
5. Ficoll-Paque Plus (GE/Amersham, Piscataway, NJ).
6. Freezing media: 10 % DMSO, 90 % fetal bovine serum.
7. Normal mouse serum (NMS; Jackson ImmunoResearch, West
Grove, PA).
120 D.A. Kaminski et al.

8. Normal rat serum (NRS; Jackson ImmunoResearch).

9. 5-mL FACS tubes (BD/Falcon, San Jose, CA).
10. 1.5-mL microcentrifuge tube (Costar, Austin, TX).
11. Formaldehyde (10 % UltraPure, methanol-free; Polysciences,
Warrington, PA).
12. Fixation buffer: 0.5 % formaldehyde in PBS.
13. Simply Cellular Compensation Standard (Bangs Laboratories,
Fishers, IN).
14. ArC Amine-Reactive Compensation Bead Kit (Invitrogen).
15. Rainbow Calibration Peak 6 particles (Spherotech, Lake Forest,
IL).
16. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Molecular
Probes/Invitrogen).
17. FITC-IgD (FITC-conjugated mouse anti-human IgD, IA6-2;
BD).
18. PE-CXCR3 [PE-conjugated mouse anti-human CXCR3
(CD183), 1C6/CXCR3; BD].
19. PE-A610-CD24 (PE-Alexa610-conjugated mouse anti-human
CD24, SN3; Caltag/Invitrogen).
20. PE-Cy5-CD21 (PE-Cy5-conjugated mouse anti-human
CD21, B-ly4; BD).
21. PerCP-Cy5.5-CD38 (PerCP-Cy5.5-conjugated mouse anti-
human CD38, HIT2; BD).
22. PE-Cy7-B220 (PE-Cy7-conjugated rat anti-mouse CD45/B220,
RA3/6B2; BD).
23. Pacific Blue-CD3 (Pacific Blue-conjugated mouse anti-human
CD3, SP34-2, BD).
24. Qdot 605-CD27 (Qdot 605-conjugated mouse anti-human
CD27, CLB-27/1; Invitrogen).
25. APC-CD95 (APC-conjugated mouse anti-human CD95,
DX2; BD).
26. APC-Cy7-CD19 (APC-Cy7-conjugated mouse anti-human
CD19, SJ25C1; BD).
27. Biotinylated 9G4 (rat anti-human Ig idiotype, Batch#3, 2008;
the 9G4 hybridoma was a gift from Dr. Freda S. Stevenson,
Southampton, UK).
28. SAv-A680 (streptavidin-Alexa680 conjugate; Invitrogen).
29. LSR II flow cytometer with Blue (488), Red (633), and violet
(405) lasers and desktop computer with Diva Software (both
from BD).
30. FlowJo (TreeStar, Ashland, OR) and MatLab (MathWorks,
Natick, MA) software.
 6 Multiparameter Flow Cytometry and Bioanalytics 121

Sample procurement &

preparation
Cryopreservation &
recovery
Cell staining

Acquire events with

cytometer

Primary analysis

Identify Profiling analysis

by clustering &
populations PCA

Calculate cell numbers, Seek B cell

graph signatures of
disease, or of
response-to-
treatment, etc

Draw
conclusions
Independently
validate with new
Develop sorting experiments patient samples
for functional analysis

Fig. 2. General work-flow for phenotypic B cell profiling.

3. Methods

Here, we describe procedures for analyzing B lymphocyte subsets

from human peripheral blood mononuclear cells (PBMC). We
include methods for preparing, staining, and analysis by flow
cytometry followed by bioinformatics analysis. As an example, we
focus on our recently developed 12-color memory B cell staining
panel (95) analyzed with a Becton-Dickson LSR II instrument.
The protocol assumes basic knowledge of operating the instru-
ment. Similar panels have been developed for transitional B cells
and plasma cells, and these methods can be amended for other
user-developed panels as well. For consistency in large studies, we
provide detail on critical steps such as freezing and thawing, with
recommended quality assurance/quality control to monitor and
regulate consistency (Fig. 2).
122 D.A. Kaminski et al.

3.1. Sample 1. Record the original volume of blood in the heparinized blood-
Preparation collection tube before proceeding. Centrifuge with no brake at
400 g for 15 min.
3.1.1. Isolation of PBMC
2. Remove plasma from the top of the sample, leaving ~0.2 mL
of plasma behind. If needed, reserve the plasma at 4 C.
3. Collect the white blood cells (WBC) from the interface (ini-
tially between the plasma and the middle layer of Ficoll; red
blood cells will be in the bottom layer) plus ~0.2 mL above
and below. Transfer to a clean 50-mL centrifuge tube.
4. Dilute the collected WBC up to the original blood-volume
with PBS.
5. Add 15 mL Ficoll-Paque into a separate, clean 50-mL conical
tube.
6. Hold the tube as close to horizontal as possible and slowly
layer the diluted WBC sample onto the Ficoll-Paque, being
careful not to mix layers.
7. Centrifuge at 400 g for 35 min at 20 C with brake off.
8. Using a Pasteur pipette and avoiding Ficoll-Paque, collect the
mononuclear cell layer at the interface (buffy coat), and trans-
fer to a clean 15-mL conical tube.
9. Fill tube to 15 mL with cold PBS or RPMI, cap the tube, and
mix by inverting. Centrifuge for 10 min at 400 g at 4 C with
high brake.
10. Discard supernatant and resuspend pellet in 10 mL sterile PBS
or RPMI. Centrifuge again.
11. Gently dislodge the pellet and repeat steps 9 and 10.
12. Remove supernatant and resuspend in FACS buffer.
13. Count the cells by Trypan blue exclusion.
14. For cells to be stained fresh, transfer 107 cells per sample for
each multicolor panel into separate FACS tubes. Remaining
cells can be frozen.

3.1.2. Freezing 1. Pellet cells reserved for freezing, discard supernatant, and
and Thawing resuspend in cold freezing medium at 107/mL. Densities less
than 5 106/mL will reduce cell recovery.
Freezing Cells
2. Immediately freeze at 80 C for 2448 h and then transfer to
permanent storage, such as a liquid N2 freezer (optimal
180 C).

Thawing Cells 1. Before retrieving cells from frozen storage, warm FACS buffer
to 37 C.
2. Remove the vial of cells from the freezer and hold in a 37 C
water bath while continuously shaking and monitoring the
 6 Multiparameter Flow Cytometry and Bioanalytics 123

thaw process. Do not submerge the vial and do not allow

incubation to proceed after thawing is complete.
3. Once thawed, immediately transfer the cells to a clean 15-mL
tube and wash in 10 mL warm FACS buffer.

3.1.3. Staining Cells with 1. Set up twelve, 1.5-mL microfuge tubes and dispense two drops
the 9G4 Memory B Cell of Simply Cellular Compensation Standard beads into each
Panel tube.
Stain Compensation 2. Reserve one tube as the unstained control. To each remaining
Controls tube, add 0.22 g of one of the other antibodies. Gently
vortex.
3. Incubate on ice for 30 min in the dark. [Note: for the Alexa680
channel, it is a 2-step staining: First with biotin-CD3 and then
with SAv-Alexa680. Stain 30 min for each step with a wash in
between.]
4. Wash the beads once with 1 mL FACS buffer. Pellet the beads
by centrifugation in a microcentrifuge at 900 g for 5 min.
Resuspend the beads in 200 L of 0.5 % formaldehyde, and
transfer to separate 5-mL FACS tubes.
5. For the Aqua compensation control, gently vortex ArC bead
components. Add one drop of Component A (reactive beads)
to a clean microfuge tube. Allow beads to sit at room tempera-
ture for at least 5 min. Add 1 L Aqua L/D stain directly to
the droplet of the reactive beads and incubate for 30 min.
6. Transfer to a FACS tube with the addition of 3 mL FACS buf-
fer. Centrifuge at 300 g for 5 min. Add 500 L FACS buffer
to the tube, plus one drop of ArC (negative beads) to the
tube.
Stain Blood-Cell Samples 1. Prepare antibody cocktails using FACS buffer in the presence
(3-Step Staining) of NMS and NRS (1:20 dilution each). Prepare a cocktail of
the fluorescent and biotinylated antibodies sufficient for stain-
ing the number of cell samples (100 L per sample). Prepare
also separate 1-sample mixtures of the same cocktail omitting
one reagent per cocktail for the fluorescence-minus-one
controls.
2. Pellet the cells reserved for staining at 300 g for 10 min at
4 C. Resuspend each pellet with 100 L of the appropriate
antibody cocktails. Incubate on ice for 30 min in the dark.
3. Wash the cells once with 2.5 mL FACS buffer.
4. Resuspend the cells with 100 L SAv-A680 (at 1:500 dilution)
on ice for 30 min in the dark.
5. Wash the cells once with 2.5 mL PBS (no BSA).
6. Incubate the cells in 1 mL PBS (no BSA) containing aqua-
fluorescent reactive dye (1:1,000) on ice for 30 min in the
dark.
124 D.A. Kaminski et al.

7. Wash the cells once with 2.5 mL FACS buffer.

8. Resuspend the cells in 0.5 mL 0.5 % formaldehyde.
9. Run stained cells and controls (single-color beads and FMOs)
on a flow cytometer.

3.2. Primary Analysis Another integral element needed for consistent and reproducible
flow results is a standardized strategy for flow data analysis. A ratio-
nalized gating strategy can significantly reduce user-to-user vari-
ability to ensure consistent interpretation of the data among all
operators (e.g., Fig. 1). The following protocol assumes basic
knowledge of FlowJo software. Tutorials on getting started with
FlowJo are available at the TreeStar Web site (http://www.flowjo.
com/home/basictutorial.html) and can also be arranged with
TreeStar personnel.

3.2.1. Gating Strategy 1. Gate on lymphocytes through the FSC-area vs SSC-area

for Memory B Cells (FSC-A/SSC-A) scatter plot (Fig. 1a).
2. Within the lymphoid gate, eliminate cell aggregates by display-
ing Height/Width for FSC. Gate a region only on events on
the optimal-ratio line. Within this gate, repeat the procedure
for SSC.
3. Within this region, gate-out dead cells that have taken up the
amine-reactive fluorescent dye (Aqua).
4. Gate on CD19+CD3 B cells for analysis.
5. Identify the core B cell subsets with either IgD/CD38
(Fig. 1b), IgD/CD27 (Fig. 1c, right), or CD24/CD38
(Fig. 1c, middle) plots, and also the 9G4+ B cell population,
depending on the gating strategy used.
6. Further subset the CD27+ and CD27 switched (IgD) mem-
ory cells by their differential expression of B220, CD24, CD21,
CD95, and CXCR3 (Fig. 1d).
7. Determine the percentages of 9G4+ B cells in each core subset
(Fig. 1e, histograms).
8. Numerical data can be exported from FlowJo by generating a
table (keyboard command + t or clicking the grid-shaped
icon), and exporting the data as a .txt file (click the grid icon in
the new table window, then save-as).

3.3. Secondary Once primary analysis (gating) has been performed and cross-
Analysis checked, the resulting data can be analyzed in numerous ways,
depending on the experimental question. Considerations include
whether to report the data as mean fluorescent intensity of a par-
ticular fluorescent parameter, the percentage of cells within a par-
ent population, as a percentage of all B cells, or as the absolute
cell number per volume of blood. For cell-number calculations, it
is necessary to acquire a clinical blood count (CBC) from an
 6 Multiparameter Flow Cytometry and Bioanalytics 125

aliquot of whole blood in which total white blood cells and lym-
phocytes are measured. These procedures can be requested from
clinical staff or performed by the analyst using conventional or
automated methods, such as a Sysmex XE-2100 instrument.
Multiparameter flow analysis invariably generates large quantities
of data that need to be managed efficiently in order to derive pro-
ductive output. For large-scale studies with associated clinical
information, it is recommended that the data be structured and
housed in a relational database to support flexible interrogation of
the data.
In some circumstances, many reagents in the panel are used for
narrowing down a particular subset of interest, which is then ana-
lyzed for various characteristics and by standard statistical tests with
conventional graphing techniques. In other circumstances, a more
global profiling approach can be used in which all of the data are
considered together to obtain a system-wide view of immune cell
populations. Profiling is useful for class-discovery in which natural
groupings of samples can be sought (85, 86). Furthermore, it may
be possible to relate those groupings to sets of samples with differ-
ent disease states, disease activities, or clinical manifestations.
Univariate approaches on individual subsets fail to reveal how col-
lections of subsets and their relative distributions might contribute
to such sample groupings.

3.3.1. Calculating Cell 1. Calculate B cell numbers with the following formula in which
Numbers both WBC/mL and % lymphocytes are taken from the CBC,
and % B cells among lymphocytes is taken from gating
analysis:
WBC %lymphocytes % CD19+ CD3 # B cells
= .
mL 100 100 mL blood
2. Calculate the core subset (e.g., switched memory) numbers:
# Bcells %IgD CD27 + # switchedmemoryB cells
= .
mL 100 mL blood
3. Calculate finer subpopulations (e.g., in which CD95+ was mea-
sured as a per cent of the switched memory parent):
# switched memoryB cells %CD95+

mL 100
#CD95+ switchedmemoryB cells
= .
mL blood
3.3.2. Clustering
The following represent a generic workflow that can be performed
and Principal-Component
by several software packages or by technical computing platforms.
Analysis
This protocol assumes basic knowledge of Matlab software
(Mathworks, Natick MA; http://www.mathworks.com/
products/matlab/).
126 D.A. Kaminski et al.

1. Construct a data matrix by arranging the cell-subset frequencies

(or numbers, MFIs, etc.) for each sample as a row in a sample-
by-feature matrix. Each row is then annotated by a sample ID
(which can be linked to a variety of clinical, demographic, or
other data) and each column is annotated by a cell subset name.
2. It may be beneficial to log-transform data, especially if some of
the cell subsets are rare. To determine whether transformation
is warranted, independently perform a QQ (QuantileQuantile)
plot on each data column (the values associated with a single
cell subset across all samples) as well as the log of a data col-
umn to see whether log transformation results in a distribution
that is closer to normal (Gaussian). It is common for log trans-
formation of a subset to result in a more Gaussian distribution.
The Matlab command is qqplot.
3. It is possible that there are both typically abundant and typi-
cally rare B cell subsets in the profile whose frequencies can be
orders of magnitude different. This situation can cause the
clustering to be dominated by abundant cell subsets (if
Euclidean distance measures are used). To standardize the scale
for cell subset expression, each column can be normalized by
subtracting the mean from each value, then dividing the result
by the standard deviation, so that the abundance (frequency)
measure for each subset will have a mean of 0 and a standard
deviation of 1.
4. Cluster the samples using the pdist command to find all the
pairwise distances (specifying a distance measure such as
Euclidean distance, Pearson correlation, or Spearman correla-
tion) followed by linkage to construct the dendrogram
(specifying a hierarchical clustering linkage method). To ren-
der, use the dendrogram command. Alternatively, see the
clusterdata command.
5. Render the heat map (with the reordered samples and/or sub-
sets) using the imagesc command. To align the sample and
subset dendrogram to the heat map, see the Matlab documen-
tation on multiple axes on a single figure. If showing normal-
ized data in a heat map, it is preferable to use a black-centered
color-map (e.g., either redblackgreen or cyanblackyellow)
so that black corresponds to the mean (0). Alternatively, if
showing nonnormalized data, a non-black-centered color-map
(e.g., jet in Matlab) is useful.
6. To perform PCA, use the princomp command on the trans-
formed sample-by-feature matrix. This command will display a
matrix of the same dimensionality, but where each column is a
principal component (typically the first two or three are con-
sidered). It will also return a square matrix from which the
principal-component loadings can be extracted.
 6 Multiparameter Flow Cytometry and Bioanalytics 127

4. Notes

1. It is essential to minimize the time between blood collection in

the clinic and running stained samples on the flow cytometer.
The Ficoll-Paque should be at room temperature prior to and
during the centrifugation. It is important not to overload the
Ficoll, keeping it one part Ficoll-Paque per two parts blood/
PBS. Apart from the Ficoll and the cell thawing steps, samples
should be kept cold (4 C or on wet ice), especially if they
need to be shipped to another facility. In this case, it is impor-
tant to invest in a carrier service with a minimal delivery time.
Depending on the reagent panel used and the cells of interest,
time constraints can be partially circumvented by cryopreserv-
ing the cells. Cryopreservation also allows excess cells not
needed for fresh staining to be reserved for future staining with
other panels. Additionally, frozen cells can be shipped on dry
ice and may thus provide an option for improving consistency
if samples are collected at multiple clinical sites, but stained and
analyzed at only one central facility. Even with the most careful
freezing and thawing techniques, however, cryopreservation
will always result in cell loss and may affect certain populations
more than others. The plasmablasts/plasma cells are one such
population. When cryopreservation is used, it is important to
minimize the time between blood sample collection in the
clinic and the freezing step. When freezing and thawing, it is
critical to minimize the time that nonfrozen cells are exposed
to the freezing media containing DMSO, which is toxic.
2. Fluorescent reagents should be kept cold and out of light. These
conditions are especially critical for tandem dyes, in which two
fluorochromes are linked (e.g., PE-Cy7). This linkage is highly
sensitive to oxidation, which can be catalyzed by both light and
increased temperature. The result of a degraded tandem dye is
that the reagent now fluoresces similarly as one of the parent
dyes, and not of the conjugate. Thus, completely erroneous
results can be acquired without careful control of this parame-
ter, which can be monitored with the single-color control sam-
ples. It is also important to run fluorescence-minus-one (FMO)
controls (panel lacking one reagent per FMO control sample)
on a regular basis. FMO controls are used to determine the
lower-limits of a positive stain when gating. Another critical
component of a flow cytometry experiment is to include a con-
sistency control, which is a single biological sample that has
been aliquotted and frozen for staining with each run. For
human B cell analysis, it is highly advantageous to establish such
a control from patients with hemachromatosis, as their blood is
highly enriched for B lymphocytes, typically without excessive
128 D.A. Kaminski et al.

perturbances to known B cell subsets. This control allows for

careful monitoring of changes in instrument performance that
are not readily detected with the bead preparations.
3. Some 9G4+ antibodies can bind to B220 (87). Thus, patients
with high titers of 9G4+ antibodies in the circulation can have
a painting effect in which B220+ (especially nave) B cells are
bound by the rat anti-idiotype 9G4 reagent, even if that B
cells BCR is not truly 9G4+ (i.e., usually encoded by the Vh4-
34 gene segment). This effect is apparent when an unusually
large percentage (<50 %) of total or of nave B cells stain with
the 9G4 reagent. To circumvent this issue, incubate the cells in
serum-containing media at 37 C for 1 h, then wash prior to
staining with the 9G4 memory B cell panel (87).
4. The quality and consistency of multiparameter flow data is
affected by each step in the operational process. Artifacts can
be avoided by addressing several technical challenges associ-
ated with these processes. Foremost is proper and reproducible
flow cytometer setup. Users and/or core facilities should fol-
low manufacturers (e.g., BD Biosciences) guidelines to estab-
lish the Baseline PMT Gains for each LSRII or other
cytometer with its particular configuration of lasers and filters
using Rainbow Calibration Peak 2 Particles (Spherotech, RCP-
30-5A-2). These baseline voltages are then used as starting-
points to fine-tune the voltage settings to achieve optimal
detection and resolution of each fluorescent reagent used in
each staining panel. The resulting settings are then saved as
application-specific settings, and used in all subsequent
experiments with the same reagent panel.
5. Once the application-specific settings are established for the
flow cytometer, the mid-range Rainbow Calibration Peak 6
Particles (RCP-30-5A-6) are used to determine target channel
values for each detector. The Peak 6 Particles are also run
before each subsequent experiment to check instrument per-
formance and to ensure that the particles fall into the target
channel values for the same voltage settings. These target chan-
nel values can also be used to convert the application-specific
settings determined on one instrument to voltage settings on
another instrument with the same laser and filter configuration.
With a properly set-up and regularly calibrated instrument,
experimental results can be meaningfully compared and com-
piled even if they were collected during a lengthy study, by
different operators, and from different instruments. Such an
instrument standardization process can also facilitate collabo-
rations among different laboratories and institutions.
6. Depending on the quality and distribution of each fluorescent
parameter in various two-dimensional plots, the axis scales may
need to be transformed (88) so that events at extremely low or
 6 Multiparameter Flow Cytometry and Bioanalytics 129

negative fluorescent intensities are clearly visible, and thus

accurately gated (e.g., note the events less than 0 on the CD38
scale in Fig. 1b, c). We recommend bi-exponential transforma-
tion. In Flowjo v9.2, Platform in the pull-down menu has an
option for bi-exponential transformation. Start with cus-
tom transformation and the default settings (0 for negative
region, 4.5 for log scale) in the IgD/CD27 plot (Fig. 1c). If
the results are not satisfactory (e.g., CD27), then manual
transformation can be used to modify the settings on the
CD27 channel. After that, visually inspect whether you need to
transform any other channels. These settings can be adjusted
for the optimal display of each parameter. After the transforma-
tion process, it is important to go back to check the parental
gates, such as live/dead and CD19 gates, which may shift upon
transformation. FSC and SSC will not be affected by
transformation.
7. Profiling-based visualization is enabled by structuring data into
a two-dimensional matrix, where each row is a sample and each
column represents a feature (or variable; in this case, cell
subsets). Data are then displayed as a heat-map by assigning
the numeric values (e.g., % of CD19+) to color in this matrix.
The samples, and independently, the features can then be clus-
tered, resulting in the rows and columns being rearranged so
that those most like each other are grouped closely.
Dendrograms can be displayed adjacent to the rows and col-
umns to indicate the nature of the clustering, namely, whether
the samples (or features) are grouped into distinct clusters or
whether they represent more of a continuum of diversity. This
approach has been widely used for representing gene expres-
sion data (89).
8. Principal-component analysis (PCA), can be used to map the
original variables (cell subsets) into a set of transformed vari-
ables that are uncorrelated with each other. Because many cell
subsets in a profile may covary across samples (this is especially
true in the extreme case where subsets are arithmetically
related, such as 9G4+ and 9G4 frequencies within the nave
parent population), those subsets do not provide additional
information regarding class separation of the samples. Most of
the variation in the entire data set is typically accounted for
by the first few components (Fig. 3a, b). Thus, PCA is a
dimension-reduction technique where the variation in the data
is explained by fewer informative variables (9092). By per-
forming PCA on the original sample-by-feature matrix, one
can plot the samples in two- or three-dimensional space, which
may be revealing if some attribute of the sample is coded
by color or symbol (Fig. 3c) (93). Note that the first few
principal-components highlight aspects of the data that
130 D.A. Kaminski et al.

a
c
% Variance Explained

100
HC
80 8 SLE
60
40 6
20
0 4
1 2 3 4 5 6 7 8 9 10
Principal component

Principal Component 2
b 2
40 B cell Subsets Ordered by PC1 Loadings

-2

-4

-6

-8

-0.3 0 0.3 -8 -6 -4 -2 0 2 4 6 8
Loading
Principal Component 1

Fig. 3. PCA applied to summarized human B cell phenotyping data. Twenty PBMC samples were selected from our lupus
cross-sectional study (in preparation) as follows: ten healthy controls were selected at random. Ten flaring lupus patients
were chosen, including two severely flaring patients, three moderately flaring patients and five additional mildly flaring
patients that were selected randomly from a larger pool of available samples. Primary analysis (gating as per Fig. 1) of these
samples resulted in 40 cell subsets (the original variables). (a) Pareto plot showing the contribution to the overall variance
by the first ten (out of 40) principal components (PCs). The line indicates the cumulative contribution of the PCs to the overall
variance of the data from the samples. Most of the variance is captured by the first few components. (b) Contributions
(loadings) of each original variable to the first principal component. Each horizontal line corresponds to one of the original
cell subsets. Similar plots can be generated for other principal components as well. The loading (x axis) is the coefficient of
an original variable for the principal component (with values from 1 to 1); larger coefficients indicate a larger contribution
of that subset to the principal component in question. (c) Score plot of 40 B cell subsets reduced to the first two principal
components, representing an informative summary of the phenotypic profiles. Frequency data were first log-transformed
and then normalized. Open circles are healthy controls (HC) and asterisks are flaring lupus patients (SLE). Note how the two
groups of samples segregate based on their phenotypic profiles (mainly along Principal Component 2, y axis).

maximally contribute to the overall variance, which is not

necessarily the consequence of some biological or clinically rel-
evant factor, but could instead be influenced by process noise
or experimental artifact. Clustering and PCA are both unsu-
pervised in that they are solely based on the data readout, not
the class-membership of the samples. They can be useful for
visualizing trends among many subsets in many subjects. The
underlying data structure is well-suited for a variety of data-
mining approaches, which, despite the exploratory nature, are
ideally utilized based on established experimental questions.
 6 Multiparameter Flow Cytometry and Bioanalytics
Acknowledgments
We thank John Jung and Ravi Misra for reading the manuscript
and members of the Sanz lab for help and advice. Supported by
NIH R01 AI049660-01A1 and U19 Autoimmunity Center of
Excellence AI56390 to I.S.
References
1. Pons-Estel GJ et al (2008) Understanding the
epidemiology and progression of systemic lupus
erythematosis. Semin Arthritis Rheum 39:
257268
2. Borchers AT et al (2010) The geoepidemiol-
ogy of systemic lupus erythematosus.
Autoimmun Rev 9:A277A287
3. Bertsias GK, Salmon JE, Boumpas DT (2010)
Therapeutic opportunities in systemic lupus ery-
thematosus: state of the art and prospects for the
new decade. Ann Rheum Dis 69:16031611
4. Arbuckle MR et al (2003) Development of
autoantibodies before the clinical onset of sys-
temic lupus erythematosus. N Engl J Med 349:
15261533
5. Anolik JH et al (2009) Insights into the het-
erogeneity of human B cells: diverse functions,
roles in autoimmunity, and use as therapeutic
targets. Immunol Res (2009) 45:144-158
6. Wardemann H et al (2003) Predominant
autoantibody production by early human B cell
precursors. Science 301:13741377
7. Yurasov S et al (2005) Defective B cell tolerance
checkpoints in systemic lupus erythematosus.
J Exp Med 201:703711
8. Yurasov S et al (2006) Persistent expression of
autoantibodies in SLE patients in remission.
J Exp Med 203:22552261
9. Yurasov S et al (2005) B-cell tolerance check-
points in healthy humans and patients with sys-
temic lupus erythematosus. Ann N Y Acad Sci
1062:165174
10. Yurasov S, Nussenzweig MC (2007) Regulation
of autoreactive antibodies. Curr Opin
Rheumatol 19:421426
11. Tiller T et al (2007) Autoreactivity in human
IgG+ memory B cells. Immunity 26:205213
12. Sherer Y et al (2004) Autoantibody explosion
in systemic lupus erythematosus: more than
100 different antibodies found in SLE patients.
Semin Arthritis Rheum 34:501537
13. Huang W et al (2002) The effect of anti-CD40
ligand antibody on B cells in human systemic
lupus erythematosus. Arthritis Rheum 46:
15541562
131
14. Erdei A et al (2009) Expression and role of
CR1 and CR2 on B and T lymphocytes under
physiological and autoimmune conditions. Mol
Immunol 46:27672773
15. Casciola-Rosen LA, Anhalt G, Rosen A (1994)
Autoantigens targeted in systemic lupus ery-
thematosus are clustered in two populations of
surface structures on apoptotic keratinocytes.
J Exp Med 179:13171330
16. Schutters K, Reutelingsperger C (2010)
Phosphatidylserine targeting for diagnosis and
treatment of human diseases. Apoptosis 15:
10721082
17. Pugh-Bernard AE et al (2001) Regulation of
inherently autoreactive VH4-34 B cells in the
maintenance of human B cell tolerance. J Clin
Invest 108:10611070
18. Isenberg D et al (1993) Identification of the
9G4 idiotope in systemic lupus erythematosus.
Br J Rheumatol 32:876882
19. van Vollenhoven RF et al (1999) VH4-34
encoded antibodies in systemic lupus erythe-
matosus: a specific diagnostic marker that
correlates with clinical disease characteristics.
J Rheumatol 26:17271733
20. Bhat NM et al (2002) VH4-34 encoded anti-
body in systemic lupus erythematosus: effect of
isotype. J Rheumatol 29:21142121
21. Sanz I, Lee FE (2010) B cells as therapeutic tar-
gets in SLE. Nat Rev Rheumatol 6:326337
22. Anolik JH et al (2004) Rituximab improves
peripheral B cell abnormalities in human sys-
temic lupus erythematosus. Arthritis Rheum
50:35803590
23. Calero I, Sanz I (2010) Targeting B cells for
the treatment of SLE: the beginning of the end
or the end of the beginning? Discov Med 10:
416424
24. Shlomchik MJ et al (1994) The role of B cells
in lpr/lpr-induced autoimmunity. J Exp Med
180:12951306
25. Chan OT et al (1999) A novel mouse with B
cells but lacking serum antibody reveals an
antibody-independent role for B cells in murine
lupus. J Exp Med 189:16391648
132 D.A. Kaminski et al.
26. Ahuja A et al (2007) Depletion of B cells in
murine lupus: efficacy and resistance. J Immunol
179:33513361
27. Looney RJ et al (2004) B cell depletion as a
novel treatment for systemic lupus erythemato-
sus: a phase I/II dose-escalation trial of ritux-
imab. Arthritis Rheum 50:25802589
28. Navarra SV et al (2001) Efficacy and safety of
belimumab in patients with active systemic
lupus erythematosus: a randomised, placebo-
controlled, phase 3 trial. Lancet 377:721731
29. Chan O, Shlomchik MJ (1998) A new role for
B cells in systemic autoimmunity: B cells pro-
mote spontaneous T cell activation in MRL-
lpr/lpr mice. J Immunol 160:5159
30. Anolik JH et al (2007) Delayed memory B cell
recovery in peripheral blood and lymphoid tissue
in systemic lupus erythematosus after B cell deple-
tion therapy. Arthritis Rheum 56:30443056
31. Sanz I et al (2008) Phenotypic and functional
heterogeneity of human memory B cells. Semin
Immunol 20:6782
32. Wehr C et al (2004) A new CD21low B cell
population in the peripheral blood of patients
with SLE. Clin Immunol 113:161171
33. Odendahl M et al (2000) Disturbed peripheral
B lymphocyte homeostasis in systemic lupus
erythematosus. J Immunol 165:59705979
34. Wirths S, Lanzavecchia A (2005) ABCB1
transporter discriminates human resting naive
B cells from cycling transitional and memory B
cells. Eur J Immunol 35:34333441
35. Palanichamy A et al (2009) Novel human tran-
sitional B cell populations revealed by B cell
depletion therapy. J Immunol 182:59825993
36. Pascual V et al (1994) Analysis of somatic
mutation in five B cell subsets of human tonsil.
J Exp Med 180:329339
37. Wellmann U et al (2005) The evolution of
human anti-double-stranded DNA autoantibod-
ies. Proc Natl Acad Sci USA 102:92589263
38. Tsuiji M et al (2006) A checkpoint for autore-
activity in human IgM+ memory B cell devel-
opment. J Exp Med 203:393400
39. Cappione A 3rd et al (2005) Germinal center
exclusion of autoreactive B cells is defective in
human systemic lupus erythematosus. J Clin
Invest 115:32053216
40. Vinuesa CG, Sanz I, Cook MC (2009)
Dysregulation of germinal centres in autoim-
mune disease. Nat Rev Immunol 9:845857
41. Crotty S, Ahmed R (2004) Immunological mem-
ory in humans. Semin Immunol 16:197203
42. Mamani-Matsuda M et al (2008) The human
spleen is a major reservoir for long-lived vaccinia
virus-specific memory B cells. Blood 111:
46534659
43. Anderson SM, Tomayko MM, Shlomchik MJ
(2006) Intrinsic properties of human and
murine memory B cells. Immunol Rev 211:
280294
44. Agematsu K et al (1997) B cell subpopulations
separated by CD27 and crucial collaboration of
CD27+ B cells and helper T cells in immuno-
globulin production. Eur J Immunol 27:
20732079
45. Tangye SG et al (2003) Intrinsic differences in
the proliferation of naive and memory human
B cells as a mechanism for enhanced secondary
immune responses. J Immunol 170:686694
46. Good KL, Avery DT, Tangye SG (2009)
Resting human memory B cells are intrinsically
programmed for enhanced survival and respon-
siveness to diverse stimuli compared to naive
B cells. J Immunol 182:890901
47. Tangye SG, Avery DT, Hodgkin PD (2003)
A division-linked mechanism for the rapid gen-
eration of Ig-secreting cells from human mem-
ory B cells. J Immunol 170:261269
48. Macallan DC et al (2005) B-cell kinetics in
humans: rapid turnover of peripheral blood
memory cells. Blood 105:36333640
49. van Gent R et al (2009) Refined characterization
and reference values of the pediatric T- and B-cell
compartments. Clin Immunol 133:95107
50. Kruetzmann S et al (2003) Human immuno-
globulin M memory B cells controlling
Streptococcus pneumoniae infections are gener-
ated in the spleen. J Exp Med 197:939945
51. Griffin DO, Holodick NE, Rothstein TL (2011)
Human B1 cells in umbilical cord and adult
peripheral blood express the novel phenotype
CD20+CD27+CD43+CD70RDDDDD.
J Exp Med 208:6780
52. Wei C et al (2007) A new population of cells
lacking expression of CD27 represents a nota-
ble component of the B cell memory compart-
ment in systemic lupus erythematosus.
J Immunol 178:66246633
53. Fecteau JF, Cote G, Neron S (2006) A new
memory CD27-IgG+ B cell population in
peripheral blood expressing VH genes with low
frequency of somatic mutation. J Immunol
177:37283736
54. Klein U, Rajewsky K, Kuppers R (1998)
Human immunoglobulin (Ig)M+ IgD+ periph-
eral blood B cells expressing the CD27 cell sur-
face antigen carry somatically mutated variable
region genes: CD27 as a general marker for
somatically mutated (memory) B cells. J Exp
Med 188:16791689
55. Qian Y et al (2010) Elucidation of seventeen
human peripheral blood B-cell subsets and
quantification of the tetanus response using a
density-based method for the automated
 6 Multiparameter Flow Cytometry and Bioanalytics
identification of cell populations in multidi-
mensional flow cytometry data. Cytometry B
Clin Cytom 78(Suppl 1):S69S82
56. Dunn-Walters DK, Isaacson PG, Spencer J
(1995) Analysis of mutations in immunoglobu-
lin heavy chain variable region genes of micro-
dissected marginal zone (MGZ) B cells suggests
that the MGZ of human spleen is a reservoir of
memory B cells. J Exp Med 182:559566
57. Weller S et al (2008) Somatic diversification in
the absence of antigen-driven responses is the
hallmark of the IgM+ IgD+ CD27+ B cell rep-
ertoire in infants. J Exp Med 205:13311342
58. Wu YC et al (2010) High-throughput immu-
noglobulin repertoire analysis distinguishes
between human IgM memory and switched
memory B-cell populations. Blood
116:10701078
59. Seifert M, Kuppers R (2009) Molecular foot-
prints of a germinal center derivation of human
IgM+(IgD+)CD27+ B cells and the dynamics
of memory B cell generation. J Exp Med 206:
26592669
60. Wasserstrom H et al (2008) Memory B cells
and pneumococcal antibody after splenectomy.
J Immunol 181:36843689
61. Iwata Y et al (2011) Characterization of a rare
IL-10-competent B-cell subset in humans that
parallels mouse regulatory B10 cells. Blood
117:530541
62. Dogan I et al (2009) Multiple layers of B cell
memory with different effector functions. Nat
Immunol 10:12921299
63. Pape KA et al (2011) Different B cell popula-
tions mediate early and late memory during an
endogenous immune response. Science 331:
12031207
64. Weller S et al (2001) CD40-CD40L indepen-
dent Ig gene hypermutation suggests a second
B cell diversification pathway in humans. Proc
Natl Acad Sci USA 98:11661170
65. Mietzner B et al (2008) Autoreactive IgG
memory antibodies in patients with systemic
lupus erythematosus arise from nonreactive
and polyreactive precursors. Proc Natl Acad Sci
USA 105:97279732
66. Snapper CM, Mond JJ (1993) Towards a com-
prehensive view of immunoglobulin class
switching. Immunol Today 14:1517
67. Rodriguez-Bayona B et al (2010) Decreased
frequency and activated phenotype of blood
CD27 IgD IgM B lymphocytes is a permanent
abnormality in systemic lupus erythematosus
patients. Arthritis Res Ther 12:R108
68. Korganow AS et al (2010) Peripheral B cell
abnormalities in patients with systemic lupus
133
erythematosus in quiescent phase: decreased
memory B cells and membrane CD19 expres-
sion. J Autoimmun 34:426434
69. Houtkamp MA et al (2001) Adventitial
infiltrates associated with advanced atheroscle-
rotic plaques: structural organization suggests
generation of local humoral immune responses.
J Pathol 193:263269
70. Kyaw T et al (2010) Conventional B2 B cell
depletion ameliorates whereas its adoptive
transfer aggravates atherosclerosis. J Immunol
185:44104419
71. Sjoberg BG et al (2009) Low levels of IgM
antibodies against phosphorylcholine-A poten-
tial risk marker for ischemic stroke in men.
Atherosclerosis 203:528532
72. Zhou X, Hansson GK (1999) Detection of B
cells and proinflammatory cytokines in athero-
sclerotic plaques of hypercholesterolaemic apo-
lipoprotein E knockout mice. Scand J Immunol
50:2530
73. Ait-Oufella H et al (2010) B cell depletion
reduces the development of atherosclerosis in
mice. J Exp Med 207:15791587
74. Halliley JL et al (2010) Peak frequencies of cir-
culating human influenza-specific antibody
secreting cells correlate with serum antibody
response after immunization. Vaccine 28:
35823587
75. Gonzalez-Garcia I et al (2006) Immunization-
induced perturbation of human blood plasma
cell pool: progressive maturation, IL-6 respon-
siveness, and high PRDI-BF1/BLIMP1 expres-
sion are critical distinctions between
antigen-specific and nonspecific plasma cells.
J Immunol 176:40424050
76. Odendahl M et al (2005) Generation of migra-
tory antigen-specific plasma blasts and mobili-
zation of resident plasma cells in a secondary
immune response. Blood 105:16141621
77. Lee FE et al (2010) Circulating antibody-
secreting cells during acute respiratory syncytial
virus infection in adults. J Infect Dis 202:
16591666
78. Fairfax KA et al (2008) Plasma cell develop-
ment: from B-cell subsets to long-term survival
niches. Semin Immunol 20:4958
79. Oracki SA et al (2010) Plasma cell development
and survival. Immunol Rev 237:140159
80. Caraux A et al (2010) Circulating human B
and plasma cells. Age-associated changes in
counts and detailed characterization of circulat-
ing normal CD138 and CD138+ plasma cells.
Haematologica 95:10161020
81. Bernasconi NL, Traggiai E, Lanzavecchia A
(2002) Maintenance of serological memory by
134 D.A. Kaminski et al.
polyclonal activation of human memory B cells.
Science 298:21992202
82. Jacobi AM et al (2010) Effect of long-term
belimumab treatment on B cells in systemic
lupus erythematosus: extension of a phase II,
double-blind, placebo-controlled, dose-ranging
study. Arthritis Rheum 62:201210
83. Moir S et al (2008) Normalization of B cell
counts and subpopulations after antiretroviral
therapy in chronic HIV disease. J Infect Dis
197:572579
84. Jacobi AM et al (2008) Activated memory B
cell subsets correlate with disease activity in sys-
temic lupus erythematosus: delineation by
expression of CD27, IgD, and CD95. Arthritis
Rheum 58:17621773
85. Habib LK, Finn WG (2006) Unsupervised
immunophenotypic profiling of chronic lym-
phocytic leukemia. Cytometry B Clin Cytom
70:124135
86. Diaz-Romero J et al (2010) Hierarchical clus-
tering of flow cytometry data for the study of
conventional central chondrosarcoma. J Cell
Physiol 225:601611
87. Cappione AJ et al (2004) Lupus IgG VH4.34
antibodies bind to a 220-kDa glycoform of
CD45/B220 on the surface of human B
lymphocytes. J Immunol 172:42984307
88. Herzenberg LA et al (2006) Interpreting flow
cytometry data: a guide for the perplexed.
Nat Immunol 7:681685
89. Eisen MB et al (1998) Cluster analysis and
display of genome-wide expression patterns.
Proc Natl Acad Sci USA 95:1486314868
90. Dhaeseleer (2005) How does gene expression
clustering work? Nat Biotechnol 23:
14991501
91. Quackenbush J (2001) Computational analy-
sis of microarray data. Nat Rev Genet 2:
418427
92. Misra J et al (2002) Interactive exploration of
microarray gene expression patterns in a
reduced dimensional space. Genome Res 12:
11121120
93. Lugli E et al (2007) Subject classification
obtained by cluster analysis and principal com-
ponent analysis applied to flow cytometric data.
Cytometry A 71:334344
94. Lee FE et al (2011) Circulating human anti-
body-secreting cells during vaccinations and
respiratory viral infections are characterized by
high specificity and lack of bystander effect.
J Immunol 186:55145521
95. Wei et al (2011) OMIP-003: Phenotypic analy-
sis of human memory B cells. Cytometry A
79:894896
 Chapter 7

Experimental Use of Mouse Models of Systemic Lupus

Erythematosus
Stanford L. Peng

Abstract
Mouse models of lupus have for many years provided accessible and reliable research systems for the
pathogenesis and therapy of systemic autoimmune disease, spanning a spectrum of inbred strains that
develop spontaneous disease to experimentally induced, sometimes genetically manipulated animals.
Nearly all the models share in common the development of glomerulonephritis and autoantibodies, includ-
ing antinuclear and DNA specificities, the most common endpoints examined in experimental studies, but
exhibit specific differences in the incidence of other end-organ manifestations such as hemolytic anemia,
arthritis, dermatitis, and vasculitis. This chapter contrasts the clinical characteristics of these various mod-
els, providing an outline for their use and analysis.

Key words: Lupus, Mice, inbred, Mice, mutant, Mice, knockout, Mice, transgenic, Autoimmune
diseases, Autoantibodies, Glomerulonephritis, Arthritis, Hemolytic anemia

1. Introduction

Systemic lupus erythematosus (SLE) remains a prototypical systemic

autoimmune disease, capable of affecting virtually any organ, and
is hallmarked by the presence of autoantibodies against a wide
spectrum of ubiquitous autoantigens, including DNA and ribonu-
cleoproteins (1). Because of such myriad manifestations, studies of
disease pathogenesis and clinical therapeutics have often turned
toward animal models, primarily mice, whose disease often resem-
bles the serologies and pathologies of human SLE (25). Lupus or
lupus-like manifestations have been described in several other spe-
cies, including cats, dogs, guinea pigs, hamsters, horses, minks,
pigs, rabbits, rats, and primatesbut the mouse system has

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_7, Springer Science+Business Media New York 2012

135
136 S.L. Peng

remained a focus of scientific investigations, in large part because

of the availability of genetically homogeneous yet susceptible
strains, as well as immunological and genetic reagents (5). Herein
is provided an overview of these various mouse models, focusing
primarily upon their experimental utility and the assessment of
their most commonly studied disease manifestations, autoantibod-
ies, and renal disease (Table 1): a complete discussion of pathology,
immunology, and pathogenesis of such models is beyond the scope
of this chapter and can be found in several other reviews (26).

1.1. Lupus Models The vast majority of mouse lupus models develop autoantibodies
in Mice such as antinuclear antibodies (ANA) and anti-DNA antibodies, as
well as renal disease, particularly glomerulonephritis, and this com-
bination of manifestations is often used as the primary outcome
measure in experimental studies. Several models, however, develop
additional lupus-like features such as dermatitis, arthritis, cytope-
nias, vasculitis, and neurological manifestations; the pathogenesis
of such manifestations has been less well studied, perhaps in part
because of a less consistent incidence of these manifestations in the
models (Table 1). Because the specific details of these different
models have been well detailed in several prior reviews (26), the
listings here only outline key similarities and differences by which
to guide experimental model selection.

1.1.1. Inbred and The most well studied of the mouse lupus models include poly-
Spontaneous Models genic, inbred mouse strains which spontaneously develop lupus-
like syndromes, particularly the New Zealand (NZ) group of
strains, in addition to Murphys Recombinant Large (MRL) and
BSXB.

New Zealand Group The NZ group of mouse strains includes a number of different
strains that alone or in combination produce spontaneous lupus-
like disease models. In addition, other otherwise normal strains
which have been hybridized with NZ mice to generate other mouse
lupus models are included here since their clinical disease and
pathogenesis resembles the NZ strains. These include the
following:
NZB NZW F1. NZ Bielschowsky black (NZB/Bl) female NZ
White (NZW) male F1 (BW) mice, perhaps the most com-
monly studied hybrid, are considered by many researchers to
resemble the human disease most closely (2, 79). NZB mice
themselves are characterized by B cell hyperactivity and thymic
loss, and spontaneously develop a fatal autoimmune hemolytic
anemia due to anti-erythrocyte antibodies, sometimes in asso-
ciation with a typically mild glomerulonephritis (10). NZW
mice are clinically healthy in isolation but confer lupus in hybrid
with several other lupus-prone strains; this strain therefore
 Table 1
Differentiating characteristics of commonly used mouse lupus models

Other key Other key Key issues for

Model Nephritis Dermatitis Arthritis Vascular clinical findings Anti-DNA autoantibodies experimental use
7

NZB/NZW Proliferative Cerebritis + Cardiolipin, Most commonly used

F1 Choroiditis erythrocyte
Oophoritis
MRL/Mp-+/+ Mild, delayed Mild, Pannus Vasculitis Cerebritis + Rheumatoid factor; Most diverse lupus-like
delayed Choroiditis Ribonucleoproteins manifestations
MRL/Mp-lpr/ Diffuse, Ulcerative, Oophoritis including Sm; Lymphoproliferation
lpr proliferative; chronic Sialoadenitis ribosomal P may confound
interstitial clinical disease
(lpr confers CD95
deficiency)
BXSB Diffuse, + Erythrocyte Male lupus
proliferative (Yaa confers Tlr7
overexpression)
Hydrocarbon Focal, + Hepatitis + Rheumatoid factor; Inducible lupus-like
oil (pristane)- proliferative Ribonucleoproteins disease in all genetic
induced including Sm; backgrounds tested
ribosomal P
Chronic Diffuse, Scleroderm- Perivasculitis Enteritis + T cell, erythrocyte Most cumbersome to
graft-versus- membranous atous Hepatitis implement; possible
host disease Pancreatitis sclerodermalupus
Pneumonitis overlap
Sialoadenitis
BXD2 Diffuse, Erosive Splenomegaly + Resembles rheumatoid
Experimental Use of Mouse Models of Systemic Lupus Erythematosus

proliferative arthritislupus
overlap
NZW BXSB Atherosclerosis Coronary infarcts Cardiolipin Resembles antiphos-
137

F1 pholipid syndrome
Adapted from ref. 2
138 S.L. Peng

possesses several pathogenic genetic determinants, but at the

same time possesses repressor or epistatic genes that allow
the true-bred strain to otherwise appear normal. BW hybrid
mice develop autoantibodies including ANA and anti-DNA
which correlate with the development of proliferative glomer-
ulonephritis, associated with polyclonal B cell activation and
T cell-dependent autoantibody production. Inbred strains with
more specific or exaggerated autoimmune characteristics have
been established by recombinant inbreeding of BW progeny,
such as the NZ Mixed (NZM) NZM.2410 strain, which devel-
ops more rapid and severe glomerulonephritis than BW (11).
NZB SWR F1. SWR mice are clinically normal, but NZB SWR
F1 (SNF1) hybrid progeny develop clinical disease similar to
BW animals, including ANA, anti-DNA, and glomerulone-
phritis, B cell hyperactivity and T cell-dependent autoantibod-
ies (12).
NZB SJL F1. SJL mice are also generally clinically normal, but
NZB SJL F1 (NS) hybrid progeny develop clinical disease
resembling but perhaps milder than BW animals, including
glomerular and cutaneous immune deposits in association with
ANA and anti-DNA antibodies (13).
SWR SJL F1. These hybrid progeny also develops several
autoantibodies and glomerulonephritis (14).

MRL and CD95 Mutants This group of lupus models includes a handful of strains which are
characterized by diffuse autoimmunity, more extensive than the
NZ group, including, in some cases, lymphoproliferation:
MRL/Mp-+/+. These mice, derived from LG/J, C3H,
C57BL/6, and AKR strains, develop late-life lupus including
glomerulonephritis, ANA, and anti-DNA (69). In contrast to
NZ strains, they develop a broader spectrum of autoantibod-
ies, including anti-Sm and rheumatoid factor, as well as more
diverse organ involvement, including arthritis, sialoadenitis,
and ocular disease; many researchers therefore consider this
model a more severe or widespread lupus than NZ. B cell
hyperactivity is characteristic, but T cell dependence has been
demonstrated for only some of the autoimmune manifesta-
tions, such as nephritis and autoantibody production.
Faslpr (lpr). This mutation in the apoptosis-related CD95 gene
arose spontaneously during inbreeding of the MRL/Mp strain
(79, 15). In the MRL/Mp background, it confers dramatic
lymphadenopathy due to the expansion of CD4+ and
B220+CD4CD8 T cells which are unable to undergo normal
apoptosis, correlating with a significant acceleration of the
underlying lupus autoimmunity and resulting in early death
from glomerulonephritis. Lymphoproliferation is also seen
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 139

when lpr is bred on to nonautoimmune strains, such as

C57BL/6, but the development of true end-organ autoim-
munity, such as lethal glomerulonephritis, is only observed in
some backgrounds like MRL/Mp (1618). Other CD95
mutant strains have been described, both spontaneous (e.g.,
Faslpr-cg) and targeted (1921).
FasLgld (gld). This mutation in CD95 ligand arose spontane-
ously among C3H/HeJ mice and confers an analogous lym-
phoproliferative and accelerated autoimmune phenotype as
Faslpr (2224).

Other Inbred Mouse Strains Several additional inbred or spontaneously arising mice develop
lupus-like syndromes:
BXSB. These mice, derived from SB/Le and C57BL/6 strains,
develop lupus similar to NZ animals, consisting also of glom-
erulonephritis, ANA, and anti-DNA, associated with B cell
hyperactivity and T cell-dependent autoantibody production,
and are the most well characterized of this group of strains
(79). However, in these animals, males develop early, acceler-
ated disease, while females develop late disease, due to the
presence of the Y chromosome-linked autoimmune accelerator
(Yaa), a duplication in and overexpression of the toll-like
receptor Tlr7 gene (25, 26).
NZW SB/Le F1. NZW female SB/Le male F1 hybrid ani-
mals also develop lupus-like disease, virtually identical to BXSB
(27).
NZW BSXB F1. These hybrid mice develop accelerated coro-
nary artery disease in addition to anti-DNA and anti-phospho-
lipid antibodies, sometimes associated with glomerulonephritis,
and may therefore be considered a model for antiphospholipid
antibody syndrome (28).
BDX2. This recombinant inbred strain derived from a
C57BL/6 DBA2/J hybrid cross develops spontaneous ero-
sive arthritis in addition to generalized autoimmune disease
consisting of glomerulonephritis, rheumatoid factor, and anti-
DNA autoantibodies. Such features of both rheumatoid arthri-
tis and lupus have suggested this strain as a model for
rheumatoid arthritislupus overlap (rhupus) (29).
SCG/Kj. The spontaneous crescentic glomerulonephritis-
forming mouse/Kinjoh (SCG/Kj), derived from a BXSB/
Mp MRL/Mp-lpr/lpr F1 hybrid, interestingly develops anti-
neutrophil cytoplasmic antibodies which cross-react with
DNA, in addition to glomerulonephritis (30, 31).
Palmerston North (PN). This mouse strain develops necrotiz-
ing vasculitis of the small and medium arteries, in addition to
proliferative glomerulonephritis in association with autoanti-
bodies (32).
140 S.L. Peng

Flaky skin (fsn). These mice develop a psoriasiform dermatitis

and anemia in association with glomerulonephritis and anti-
DNA antibodies (33, 34), due to an endogenous retroviral
insertion in the tetratricopeptide repeat domain 7 (Ttc7) gene,
which currently is of unknown immunological function (35).

1.1.2. Experimentally A number of different experimental manipulations in mice have

Induced Models successfully (or inadvertently) produced lupus-like phenotypes.
These include the following:
Hydrocarbon oil. A single intraperitoneal injection of
2,6,10,14-tetramethylpentadecane (pristane) induces lupus-
like autoimmunity in several mouse strains, consisting of
autoantibody production and an immune-complex glomerulo-
nephritis, as well as a late-onset arthritis, and is sometimes con-
sidered a model of environmentally induced lupus (36).
Because of its simplicity and reproducibility, this model can be
useful to assess the role of novel therapeutic targets, e.g., by
allowing control of timing of a therapeutic intervention rela-
tive to disease induction, or by facilitating the assessment of a
knockout or transgenic strain without requiring tedious back-
crosses to one of the inbred strains.
Chronic GVHD. Although acute graft-versus-host disease
(GVHD) does not resemble lupus, chronic GVHD, such as
that induced in (C57BL/6 DBA/2) F1 (BDF1) mice by the
injection of DBA/2 mouse spleen cells, results in the forma-
tion of several autoantibodies including anti-DNA in associa-
tion with glomerulonephritis in a T cell-dependent manner
(3739).
16/6 Idiotype. Immunization of C3H mice and other suscep-
tible strains with the common human anti-DNA idiotype 16/6
results in an anti-idiotype response leading to a syndrome
highly reminiscent of lupus after a booster, including anti-
DNA and anti-ribonucleoprotein autoantibodies, leukopenia,
proteinuria, and renal immune deposits and sclerosis, as well as
anticardiolipin antibodies and antiphospholipid antibody syn-
drome (40, 41).
Bacillus Calmette-Guerin (BCG) injection in nonobese dia-
betic (NOD) mice induces hemolytic anemia, autoantibodies,
worsened sialadenitis, and immune complex glomerulonephri-
tis (42).
Genetically targeted animals. An extensive and growing num-
ber of genetically manipulated mouse strains have been shown
to develop features of lupus and/or systemic autoimmunity,
including transgenic animals such as those overexpressing
bcl-2, IEX-1, LIGHT, or IFN-, and a number of knockout
animals such as those de fi cient in ER , TACI, or Aiolos
(an incomplete list is shown in Table 2). Collectively, these findings
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 141

Table 2
Targeted genetic mutations which produce lupus-like phenotypes in mice

Functional group/gene product Mutation References

Apoptosis and cell cycle
B-cell leukemia/lymphoma (Bcl)-2 Transgenic (65)
Bcl2-like 11 (Bcl2l11; Bim) Knockout (66, 67)
Fas (CD95, TNFR6) Dominant-interfering (68)
transgenic
E2F transcription factor 2 (E2F2) Knockout (69)
Growth arrest and DNA-damage-inducible 45 (GADD45) Knockout (70)
Immediate early response 3 (Ier3; IEX-1) Transgenic (71)
MC159 (viral FLIP) Transgenic (72)
Cdkn1a (p21/Waf1/Cip1) Knockout (73, 74)
Phosphatase and tensin homolog (Pten) Heterozygote (75)
T cell-specific knockout (76)
Clearance of immune complexes, apoptotic cells, and nucleic acids
CD21/CD35 (Complement receptor 2) Knockout (77)
c-mer proto-oncogene tyrosine kinase (Mertk, Mer) Knockout (78)
Complement component 1, q subcomponent (C1q) Knockout (79, 80)
Complement C4 Knockout (77)
Deoxyribonuclease I (Dnase1) Knockout (81)
Fc receptor, IgG, low affinity IIb (FcRIIb) Knockout (82)
IgM (secretory) Knockout (83)
Nuclear receptor Nr1h3 (Liver receptor, LXR) Knockout (84)
Milk fat globule-EGF factor 8 (MFG-E8) Knockout (85)
Ro60 (Trove2) Knockout (86)
Serum amyloid P (SAP, Apcs) Knockout (87)
Tyro3 family (Tyro3, Axl, Mer) Knockout (88)
Cellular receptors
CD22 Knockout (89)
CD40-LMP1 Transgenic (90)
CD40 Ligand (CD154, Tnfsf5) Transgenic (91, 92)
CD45 (Protein tyrosine phosphatase Ptprc) Phosphatase-deficient (93)
CD72 Knockout (94)
CD152 (Cytotoxic T-lymphocyte protein 4, CTLA4) Knockout (95, 96)
Decoy receptor 3 (DcR3, TNFRSF6B) Transgenic (97)
G protein-coupled receptor Gpr132 (G2A) Knockout (98)
Inducible T-cell co-stimulator ligand (ICOSL, B7RP-1) Transgenic (99)
Programmed cell death 1 (PD-1, Pdcd1) Knockout (100)
Platelet/endothelial cell adhesion molecule PECAM-1 (CD31) Knockout (101)
TACI (Tnfrsf13b) Knockout (102)
T cell receptor TCR Knockout (103)
(continued)
142 S.L. Peng

Table 2
(continued)

Functional group/gene product Mutation References

Cellular signaling or regulation

Aiolos (Znfn1a3) Knockout (104)
Bright (Arid3a) Transgenic (105)
Cbl-b/Vav-1 protooncogenes Knockout (106)
CD19 Trasngenic (107)
Deltex1 (Dtx1) Knockout (108)
Early growth response 2 (Egr2) Knockout (109)
Estrogen receptor (ER) Knockout (110)
Friend leukemia integration 1 (Fli-1) Transgenic (111)
Fyn Knockout (112)
Id3 (Inhibitor of DNA binding 3) Knockout (113)
IRF-2 (Interferon regulatory factor-2) Knockout (114)
JunB transcription factor Conditional knockout (115)
LAT (Linker for activation of T cells) Point mutant (116)
Lyn Knockout (117119)
MAP/microtubule affinity-regulating kinase 2 (Mark2, Par-1) Knockout (120)
NF-E2 related factor 2 (Nrf2, Nfe2l2) Knockout (121)
Protein kinase C (PKC)- (Prkcd) Knockout (122)
Rasgrp1 (RAS guanyl releasing protein 1) Spontaneous mutant (123)
RbAp48 (retinoblastoma binding protein 4, Rbbp4) Transgenic (124)
Ro52 (Trim21) Knockout (125)
Roquin (RING C3H domains 1, Rc3h1) Induced mutant (126)
p66ShcA (Shc1) Knockout (127)
ShcC (Rai, Shc3) Knockout (128)
SHP-1 (Nr0b2) Spontaneous mutant (129, 130)
Supressor of cytokine signaling 1 (SOCS-1) Partial knockout (131)
Stra13 Knockout (132)
T cell-specific adaptor protein (TSAd, Sh2d2a) Knockout (133)
Toll-IL-1R8 (Tir8, Sigirr) Knockout (134)
Cytokines and related genes
BLys/BAFF/TALL-1 (TNFSF13b) Transgenic (135137)
Interferon (IFN)- Transgenic (138)
Interleukin (IL)-2 Knockout (139)
IL-2R Knockout (140)
IL-2R Knockout (141)
IL-4 Transgenic (142)
IL-12 Transgenic (143)
IL-14 Transgenic (144)
LIGHT (TNFSF14) Transgenic (145)
Tristetraprolin (TTP) Knockout (146, 147)
Transforming growth factor (TGF)- Knockout (148150)
Tumor necrosis factor (TNF)- Transgenic (151)
Miscellaneous
-Mannosidase II Knockout (152)
1,6-N-acetylglucosaminyltransferase V (Mgat5) Knockout (153)
miR-17-92 Transgenic (154)
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 143

have suggested that lupus-like disease may arise from multiple

alterations in B- or T cell hyperactivity or clearance of apoptotic
or necrotic cells or other self-antigens such as nucleosomes.

1.2. Selection The diverse array of inbred and genetically altered mice provides
of Mouse Lupus many models for systemic lupus erythematosus, each with particu-
Models lar clinical manifestations and unique pathogeneses (Table 1).
Investigators should therefore choose models based upon areas or
phenomena of interest within the lupus autoimmune spectrum.
Some key issues for consideration include the following:
End-organ disease manifestations. Whereas almost all the mod-
els develop anti-DNA antibodies and glomerulonephritis and
therefore may be used to model the effect of a therapeutic
intervention of interest on lupus nephritis, only some of the
models (e.g., MRL, BDX2, pristane) develop arthritis and so
may be preferable over the NZ strains when arthritis is of
experimental interest. Similarly, only some of the models
develop significant cutaneous or vascular involvement, or non-
DNA autoantibodies such as ribonucleoprotein or phospho-
lipid specificities.
Non-lupus-like traits. Several mouse lupus models exhibit
characteristics that do not resemble typical human lupus: e.g.,
the male predominance of BXSB, or the extreme lymphade-
nopathy conferred by lpr or gld. Depending on the specific
context, these issues may detract from the scientific impact of a
given study.
Timeframe. Although many of the lupus models require sev-
eral months for maturation of disease, some models, such as
MRL/Mp-lpr/lpr, develop rapid disease, allowing earlier
assessment of interventions (e.g., 23 versus 69 months of
age). At the same time, the overall timing of experimental
setup and execution should be considered: for instance, the
study of knockouts or other genetic manipulations in the
inbred lupus-prone backgrounds requires extensive backcross-
ing, preferably with congenic genotyping to confirm strain
homozygosity at target strain chromosomes, and this typically
requires at least 1518 months or more simply to generate the
desired derivative strain. In contrast, the pristane/hydrocar-
bon model, although requiring several months for disease phe-
notypes to manifest, may require less total experimental set up
and execution time since it does not require a backcrossed
genetic locus.
Existing reagents or knowledge. The most well-studied mouse
lupus systems include the NZ, MRL, and BXSB strains: many
studies have already explored the impact of several therapeutic
interventions (e.g., T cell depletion or other costimulatory
144 S.L. Peng

pathway blockade), established congenic lines with targeted

genetic mutations, and identified multiple pathogenic genetic
loci in these models. The availability of such reagents and/or
knowledge may be advantageous for many studies, e.g., by
allowing more robust interpretation of the role of a novel
gene.
Hereafter is described a typical necropsy of the lupus-prone
mouse with particular focus upon serological and renal assessment,
which together remain the hallmark manifestations of mouse
lupus.

2. Materials

2.1. Implementation Mice of desired strain(s) and age(s):

of Mouse Lupus Inbred models: e.g.
Models
BXSB/MpJ (Jackson Laboratories 000740).
MRL/MpJ (Jackson Laboratories 000486).
MRL/MpJ-Faslpr/J (Jackson Laboratories 000485).
NZBWF1/J (Jackson Laboratories 100008).
NZM2410/J (Jackson Laboratories 002676).
Inducible models:
Chronic GVHD: adult parental and F1 mice, e.g.
DBA/2J (Jackson Laboratories 000671).
B6D2F1/J (Jackson Laboratories 100006).
Hydrocarbon oil-induced: adult mice of a susceptible strain, e.g.
BALB/cJ (Jackson Laboratories 000651).
129X1/SvJ (Jackson Laboratories 000691).
Chronic GVHD models:
Hanks Balanced Salt Solution (HBSS).
Microscope slides, frosted (e.g., Fisher 12-550-33).
Needles, 2730 G.
Red blood cell lysis solution (e.g., Sigma R7757).
Syringes, 1 mL.
Hydrocarbon oil-induced models:
2,6,10,14-tetramethylpentadecane (pristane; e.g., Sigma
P2870).
Needles, 2225 G.
Syringes, 1 mL.
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 145

2.2. Lupus-Oriented Blood collecting tubes, heparin-coated, Natelson or Caraway (e.g.,

Mouse Necropsy Fisher 02-668-10 or 02-668-25).
Dissecting tools: scissors, forceps.
Dissection boarde.g., a styrofoam box lid.
Dry ice.
Formalin, 10 % buffered.
Ethanol, 70 % and absolute.
Medium for tissue culture (depending upon follow-up
applications).
Needles, 2227 G.
OCT Tissue-Tek medium (e.g., Andwin scientific #4583).
Pasteur pipettes.
Phosphate-buffered saline (PBS).
Syringes, 13 and 10 mL.
Tubes, microcentrifuge.

2.3. Analysis of Serum Alkaline phosphatase substrate (e.g., Sigma 104105 tablets).
2.3.1. Immunoglobulin Antibodies:
Isotype Titers by ELISA Capture (e.g., from Southern Biotechnology Associates, SBA):
Goat F(ab)2 anti-mouse Ig (e.g., SBA 1012-01)for
IgM, IgA, and IgGs.
Rat anti-mouse IgE (e.g., 23G3SBA 1130-01).
Detection:
Goat anti-mouse IgM-Alkaline phosphatase (AP) (e.g., SBA
1020-04).
Goat anti-mouse IgA-AP (e.g., SBA 1040-04).
Goat anti-mouse IgG1-AP (e.g., SBA 1070-04).
Goat anti-mouse IgG2a-AP (e.g., SBA 1080-04).
Goat anti-mouse IgG2b-AP (e.g., SBA 1090-04).
Goat anti-mouse IgG3-AP (e.g., SBA 1100-04).
Rat anti-mouse IgE-AP (e.g., SBA 1130-04).
Standards:
Purified mouse IgM, IgA, IgG1, IgG2a, IgG2b, IgG3, IgE
(e.g., myeloma proteins).
Buffers:
2 % Bovine serum albumin in PBS.
Carbonate buffer (325 mL 0.1 M NaHCO3, 50 mL 0.1 M
Na2CO3).
DEA buffer: mix 49 mg MgCl26H2O and 96 mL dietha-
nolamine in 800 mL H2O.
146 S.L. Peng

Titrate to pH 9.8 by HCl, raise to 1 L.

Store at 4 C in foil, protected from light.
PBS: PBS-Tween (PBS with 0.05 % Tween-20).
Microtiter plates, 96-well, high absorbency (e.g., Nunc Maxisorp
430341).
Microplate reader, capable of OD405.
Helpful, but not required:
Multichannel pipettes, 20200 L
Microplate washer.

2.3.2. Fluorescent Coverslips for microscope slides.

Antinuclear Antibody Test HEp-2 cell substrate, mounted on microscope slide (e.g., INOVA
Diagnostics, Zeus Scientific, etc.).
FITC-anti-mouse IgG (e.g., Pierce 31541).
Microscope, fluorescence-ready.
Mounting medium.
PBS.

2.3.3. Anti-dsDNA: Coverslips for microscope slides.

Crithidia Crithidia cell substrate, mounted on microscope slide (e.g.,
Antibodies Incorporated, Inc.).
FITC-anti-mouse IgG (e.g., Pierce 31541).
Microscope, fluorescence-ready.
Mounting medium.
PBS.

2.3.4. Total Rheumatoid Alkaline phosphatase substrate (e.g., Sigma 104105 tablets).
Factor ELISA Antibodies:
Purified murine IgG1, ; IgG2a, ; IgG2b, ; and IgG3,
(e.g., myeloma proteins).
Anti-mouse Ig-AP (e.g., SBA 1050-04).
Buffers:
2 % Bovine serum albumin in phosphate-buffered saline (PBS).
Carbonate buffer (325 mL 0.1 M NaHCO3, 50 mL 0.1 M
Na2CO3).
DEA buffer: mix 49 mg MgCl26H2O and 96 mL dietha-
nolamine in 800 mL H2O, titrate to pH 9.8 by HCl, raise
to 1 L store at 4 C in foil, protected from light.
PBS: PBS-Tween (PBS with 0.05 % Tween-20).
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus
2.4. Renal Disease
Assessment
2.4.1. Light Microscopy
2.4.2. Immunofluorescence
for Immune Deposits
3. Methods
3.1. Implementation
of Mouse Lupus
Models
147
Microtiter plates, 96-well, high absorbency (e.g., Nunc Maxisorp
430341).
Microplate reader, capable of OD405.
Helpful, but not required:
Multichannel pipettes, 20200 L.
Microplate washer.
Acid/alcohol differentiator: 10 % acetic acid in 95 % ethanol.
Coverslips for microscope slides.
Eosin Y Alcoholic (e.g., EMD Chemicals 588X-75).
Ethanol: 100, 95, 90, and 70 %.
Harris Hematoxylin, mercury free (e.g., EMD Chemicals 638A-71).
Microscope slides, poly-L-lysine coated.
Mounting media.
Paraffin.
Water: distilled or deionized, tap.
Xylene.
Acetone (at 20 C).
Coverslips for microscope slides.
Cryostat.
FITC-anti-mouse IgG (e.g., Pierce 31541).
Microscope, fluorescence-ready.
Microscope slides, poly-L-lysine coated.
Mounting media.
PBS.
1. For spontaneous models, lupus-prone mice may simply be
allowed to age (see Note 1).
2. Two inducible models are described here:
(a) Hydrocarbon oil:
Use adult mice, aged 68 weeks, e.g., BALB/cJ.
Administer one single intraperitoneal injection of
pristanebased in part on established protocols (43).
Prepare a syringe and needle containing 0.5 mL
pristane.
148 S.L. Peng

Restrain the mouse and tilt so head is facing

downward and abdomen exposed. This may be
achieved by grasping the mouse by the scruff of its
neck close to the ears.
Disinfect the injection site, such as with 70 % ethanol.
Insert the needle cranially into the abdomen at a
3045 angle, caudal to the umbilicus and lateral
to the midline.
Attempt to aspirate the needle: greenish-brown or
yellow aspirate indicates penetration into the
intestine or bladder, respectivelyin which case
the syringe should be discarded and the proce-
dure repeated with a new syringe and needle.
Inject the pristane.
(b) Chronic GVHD (see Note 2).
Isolate red blood cell-cleared splenocytes from
DBA/2J mice.
Harvest spleens.
Euthanize mouse by CO2 inhalation.
Fix the mouse on a flat surface (e.g., styrofoam
box cover or dissecting tray) with 22 G and/or
24 G needles.
Spray fur with 70 % ethanol. The ethanol prevents
fur from loosening and interfering with the
dissection.
Cut the skin, including the peritoneum, along the
abdomen.
Harvest the spleen just inferior to the stomach,
placing in HBSS without serum.
Isolate splenocytes.
In a tissue culture hood, dissociate splenocytes
from whole spleen by gently grinding between
the prewetted frosted ends of two microscope
slides over the collection medium.
When completely disaggregated, transfer the cell
suspension to a sterile conical tube, e.g., 50 mL
(Fisher 14-432-22).
Centrifuge at 1,800 g, 5 min.
Resuspend the cell pellet in ~3 mL red blood cell
lysis solution per spleen.
Incubate at room temperature, ~1 min.
Dilute the suspension with HBSS (e.g., fill the
remainder of the tube completely).
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus
3.2. Lupus-Oriented
Mouse Necropsy
3.2.1. Tissue
HarvestBased in Part
on Established Protocols
(44, 45)
149
Centrifuge at 1,800 g, 5 min.
Resuspend the cell pellet in ~1 mL HBSS per
spleen.
Determine cell concentration with a hemocytom-
eter and adjust to approximately 180 106 cells/
mL in HBSS.
Prepare individual syringes and needles with
90 106 splenocytes per <0.6 mL aliquot.
Intravenous injection of cells into B6D2F1/J recipi-
entsbased in part on established protocols (43).
Restrain mouse with physical or chemical
restraint.
Rotate the tail slightly to visualize the veins.
Disinfect injection site, e.g., with 70 % ethanol.
Insert needle into the vein at a slight angle.
Inject slowlyintravenous location is confirmed
by clearing of the vein lumen. Incorrect position-
ing will result in a visible bulge in the tail, in which
case the needle should be removed and the pro-
cess repeated proximal to the previous site.
When injection complete, remove needle and
apply brief pressure to the injection site.
3. Tissues are collected at appropriate time points, often
612 months for most models such as the NZ strains, or earlier
such as 34 months for more aggressive models such as MRL/
Mp-lpr/lpr (see below).
1. Collect urine for analysis, if desired (see Note 3)based on
established protocols (46).
(a) Cover the bottom of an empty mouse cage (i.e., with no
bedding) with plastic wrap.
(b) Place mouse on plastic wrap.
(c) After 1015 s, remove the mouse.
(d) Collect urine with a micropipette or Pasteur pipette.
2. Collect blood by retro-orbital approach (see Note 4)based
in part on established protocols (47).
(a) Anesthetize the mouse.
(b) Apply pressure to the external jugular vein caudal to the
mandible with thumb, and gently elevate the upper eyelid
with the index finger of the same hand.
(c) Insert a hematocrit tube into the medial canthus of the eye.
(d) Gently direct the hematocrit tube in a ventrolateral direc-
tion until blood is obtained.
150 S.L. Peng

(e) Once the desired amount of blood is obtained, discontinue

the external jugular pressure and remove the hematocrit
tube.
(f) Gentle pressure on the globe may be used to provide
hemostasis.
(g) Empty the collected blood into a 1.5 mL Eppendorf tube
or equivalent.
3. Euthanize mouse by CO2 inhalation.
4. Collect peritoneal cells, if desired (see Note 5).
(a) Nick the abdominal skin below the sternum, taking care
not to nick the peritoneum.
(b) Grab the two sides of the cut and gently pull apart the
abdominal skin with a firm but gentle movement, expos-
ing the sternum and the pelvis.
(c) Fit a 10 mL syringe with a 2627 G 1/2 needle, fill with
57 mL of medium and 23 mL of air. Air is necessary to
allow for intraperitoneal movement of fluid.
(d) Holding the sternum, fill the abdominal cavity with the
medium and air. Remove the needle; the peritoneum self-
seals.
(e) Vigorously shake the mouse in a supine position, caudad
cephalad about 1015 times.
(f) Prepare a Pasteur pipette with a rubber pipettor.
(g) Holding the sternum and peritoneum with forceps, insert
the air-filled Pasteur pipette into the peritoneal cavity with
a swirling movement.
(h) Express the air from the pipette into the peritoneum as
much as possible.
(i) Collect as much fluid as possible into the pipette.
(j) Longitudinally cut the peritoneum from sternum to pel-
vis, and collect the remaining fluid.
5. Fix the mouse on a flat surface (e.g., styrofoam box cover or
dissecting tray) with 22 and/or 24 G needles.
6. Spray fur with 70 % ethanol. The ethanol prevents fur from
loosening and interfering with the dissection.
7. Cut the skin (not the peritoneum) from leg to leg, arm to arm,
and perineum to chin.
8. Pull apart the fur, exposing the neck, thorax, and abdomen.
Pin the anterior skin flaps to the dissecting board with needles,
exposing the mammary glands.
9. If the lung is to be harvested for histopathology, use a 13 mL
syringe and 2527 G needle to cannulate the trachea and infuse
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 151

approximately 1 mL of fixative. This step expands the alveolar

spaces prior to final fixation.
10. Superficial lymph nodes are easily recognizable in the mouse as
small, grayish nodules resembling peas or beans.
(a) Harvest the inguinal lymph nodes at the crossing of the
mammary arteries and superficial epigastric vein by grasp-
ing with forceps the fat pad in which it is contained.
(b) Harvest the submandibular lymph nodes located just dis-
tal to the darker submandibular glands which lie inferior
to the mandible. Use forceps to grasp the nodes, and scis-
sors to dissect them away from the glands (see Note 6).
(c) Harvest axillary lymph nodes in the axillary fossae.
(d) Harvest brachial lymph nodes just posterolateral to the
axillae and near the scapular angle.
11. Harvest the submandibular glands, if desired.
12. Open the peritoneum (if not already done) and harvest the
spleen just inferior to the stomach.
13. Cut away a piece of liver approximately the size of a spleen.
14. Remove kidneys.
15. Open the thorax by cutting the ribs on either side and cutting
along the diaphragm in between. Gently lift the sternum to
reveal the intrathoracic contents.
16. Harvest the thymus by holding it at the isthmus and dissecting
the mediastinal tissue with scissors.
17. Remove the heart and lungs, if desired.
18. Harvest bones for bone marrow, if desired.
(a) Long bone dissection.
Insert the tips of a closed sharp pair of scissors in the
thigh muscles just inferior to the femoral artery, meet-
ing the femoral shaft.
Slide the closed scissors anterior to the femur, and open
them, detaching the anterior muscles from the femur.
Repeat a and b on the posterior side of the femur.
Gently dislocate the femoral head from the acetabulum.
Gently dislocate the distal femoral diaphysis from the
knee.
Tibiae and humeri can be isolated in fashions similar to
15.
(b) Vertebrae dissection.
Remove all the visceral organs and most of the retro-
peritoneal tissue.
152 S.L. Peng

Deeply incise the left and right psoas muscles, as close

to the spinous processes as possible.
Cut the spine at the base of the thoracic vertebrae
(at the level of the ribs).
Cut the spine at the base of the lumbar vertebrae (at
the level of the pelvis).
Remove the lumbar spine en bloc.

3.2.2. Tissue Processing 1. Serum isolation from whole blood (see Note 7).
(a) Allow the blood to clot at 4 C for 2 h to overnight.
(b) Microcentrifuge the sample at maximum speed (e.g.,
20,800 g) for 5 min.
(c) Collect the serumthe beige-colored, clear supernatant
with a micropipette.
2. Routine histopathology-buffered formalin (see Note 8).
(a) Fix tissues of interest in 30 volume equivalents of 10 %
buffered formalin for 810 h at room temperature, or
24 h at 4 C.
(b) Wash once with PBS (224 h) and transfer to 70 % etha-
nol. Store at room temperature or 4 C until ready for
processing.
3. Frozen tissue specimens (see Note 9).
(a) Prepare a dry iceethanol bath.
(b) Cut tissue into pea-sized specimen, but still containing the
anatomy of interest: e.g., for kidney, this usually equals
about one-half of a hemisphere.
(c) Cut off the cap from a microcentrifuge tube, to be used as
a mounting board. Place tissue on the cap (or other
mounting apparatus as desired). Embed with OCT
medium, using just enough as necessary to cover the
tissue.
(d) Immediately place the cap-tissue complex into the super-
chilled ethanol. Store at 70 C or below until
sectioning.
(e) Section and stain per protocol for specific antigens (e.g.,
mouse IgG for kidneys).
4. Specimens to be analyzed in tissue culture or by flow cytome-
try can be rinsed in PBS and/or placed directly in complete
cell culture medium.
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus
3.3. Analysis of Serum
(See Note 10)
3.3.1. Immunoglobulin
Isotype Titers by ELISA
10. Wash each well 46 times with 200 L PBS-Tween.
11. Dissolve phosphatase substrate in DEA buffer to 1 mg/mL
12. Read OD405 in a spectrophotometer. Interpolate and extrapo-
3.3.2. Fluorescent
Antinuclear Antibody Test
(FANA) (See Note 16)
153
1. Coat 96-well plates with 50 L/well of 2 g/mL capture
antibody in carbonate buffer, 4 C overnight (see Note 11).
2. Remove coating solution by tapping.
3. Block with 200 L/well 2 % Bovine serum albumin in PBS,
37 C, 2 h (see Note 12).
4. Remove blocking solution by tapping.
5. Make serial dilutions of each serum sample, such as 1:100,
1:200, 1:400, etc. up to 1:102,400 in PBS, and add 50 L per
well in duplicate (see Note 13).
6. Make serial dilutions of isotype standard control (e.g., 4,000,
2,000, 1,000, 500, 250, 125 ng/mL) and add 50 L per well
in duplicate. Use a separate standard curve for each plate (see
Note 14).
7. Incubate at room temperature for 12 h.
8. Wash each well 46 times with 200 L PBS-Tween.
9. Dilute detection antibody 1:1,0001:2,000 in PBS (according
to manufacturers instructions) and add 50 L per well.
Incubate at room temperature for 4560 min.
(e.g., one 5 mg tablet in 5 mL), and add 50 L per well. Watch
yellow color development in standard curve.
late Ig concentration in each sample using the standard curve
and dilutions, averaging the duplicate samples. Serial OD read-
ings may be necessary to obtain interpretable results for every
sample. Discard sample readings that are outside the range of
the standard curve. Note that OD and concentration is a semi-
log relationship (see Note 15).
1. For screening, dilute test serum 1:40 or 1:50 in PBS, and add
50 L to a slide well of fixed, permeabilized HEp-2 cells.
Incubate for 3045 min, room temperature.
2. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
the excess fluid away after the last wash.
3. Dilute FITC-anti-mouse IgG 1:500 in PBS or as directed by
manufacturer, and add 50 L to each well. Incubate for
3045 min, room temperature, protected from light.
4. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
the excess fluid away after the last wash.
5. Add 35 drops of mounting medium and coverslip to the slide.
Observe slide immediately by fluorescence microscopy.
6. Titers less than 1:40 are generally considered negative. Positive
samples, identified by the presence of any type of apple-green
154 S.L. Peng

nuclear staining, can be fully titered by making serial dilutions

(1:40, 1:80, 1:160, etc.), and repeating steps 15 until an endpoint
is reached.

3.3.3. Anti-dsDNA 1. Dilute test serum 1:10 in PBS, add 25 L to a slide well of
Assessment: Crithidia fixed, permeabilized Crithidia. Incubate for 3045 min, room
luciliae temperature.
Immunofluorescence (See 2. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
Note 17) the excess fluid away after the last wash.
3. Dilute FITC-anti-mouse IgG 1:500 in PBS or as directed by
manufacturer, and add 25 L to each well. Incubate for
3045 min, room temperature, protected from light.
4. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
the excess fluid away after the last wash.
5. Add 35 drops of mounting media and coverslip to the slide.
Observe slide immediately by fluorescence microscopy.
6. Titers of less than 1:10 are generally considered negative.
Positive samples are identified by the presence of apple-green
fluorescence of both the nucleus (indicating positive antinu-
clear antibodies) and kinetoplast, the dsDNA-containing
organelle near the base of the flagellum.

3.3.4. Total Rheumatoid 1. Coat 96-well plates with 50 L/well of 0.5 g/mL each of
Factor Assessment (ELISA) IgG1, ; IgG2a, ; IgG2b, ; and IgG3, in carbonate buffer,
(See Note 18) 4 C overnight.
2. Remove coating solution by tapping.
3. Block with 200 L/well 2 % bovine serum albumin in PBS,
37 C, 2 h.
4. Remove blocking solution by tapping.
5. Make serial dilutions of each serum sample, such as 1:100,
1:200, 1:400, etc. up to 1:102,400 in PBS, and add 50 L per
well in duplicate.
6. Make serial dilutions of isotype standard control (e.g., 4,000,
2,000, 1,000, 500, 250, 125 ng/mL) and add 50 L per well
in duplicate. Use a separate standard curve for each plate.
7. Incubate at room temperature for 12 h.
8. Wash each well 46 times with 200 L PBS-Tween.
9. Dilute alkaline phosphatase-conjugated anti-mouse Ig anti-
body 1:1,0001:2,000 in PBS (according to manufacturers
instructions) and add 50 L per well. Incubate at room tem-
perature for 4560 min.
10. Wash each well 46 times with 200 L PBS-Tween.
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 155

11. Dissolve phosphatase substrate in DEA buffer to 1 mg/mL

(e.g., one 5 mg tablet in 5 mL), and add 50 L per well. Watch
yellow color development in standard curve.
12. Read OD405 in a spectrophotometer. Total RF activity is usually
expressed as the absolute OD reading, but can be titrated against
a positive control, e.g., a diseased MRL/Mp-lpr/lpr mouse.

3.4. Renal Disease 1. Embedding of tissue.

Assessment (a) Dehydrate tissue in 70 % ethanol, room temperature, 1 h.
3.4.1. Light Microscopic (b) Dehydrate tissue in 90 % ethanol, room temperature, 1 h.
(c) Dehydrate tissue in 100 % ethanol, room temperature,
1 h.
(d) Clear the tissue with three changes of xylene, room tem-
perature, 1 h.
(e) Infiltrate the tissue with two changes of paraffin wax at
65 C.
(f) Reinfiltrate the tissue with paraffin wax at 65 C under
vacuum.
(g) Embed the tissue and allow to set.
2. Cut 57 m sections of kidney, placing them on a poly-L-lysine-
coated slide.
3. Dewax slides at room temperature.
(a) Change twice in xylene, 3 min each.
(b) Change twice in absolute ethanol, 2 min each.
(c) Change twice in 95 % ethanol, 1 min each.
(d) Change twice in 70 % ethanol, 2 min each.
(e) Place in distilled water, 5 min.
4. Stain with hematoxylin and eosin.
(a) Stain with hematoxylin, 68 min.
(b) Rinse in tap water, 45 s.
(c) Rinse in acid/alcohol differentiator, 3045 s.
(d) Rinse with distilled water, 1 min.
(e) Rinse with tap water, 1 min.
(f) Stain in eosin Y alcoholic, 2040 s.
(g) Place in 95 % ethanol, 30 s.
(h) Change twice in absolute ethanol, 1 min each.
(i) Change three times in xylene, 1 min each.
5. Add 35 drops of mounting media and coverslip to the slide.
Allow to set overnight in fume hood before observing under
light microscopy.
156 S.L. Peng

6. Disease can be assessed in three locations (see Note 19).

1. Glomerular, for the presence of glomerulonephritis, typi-
cally diffusely proliferative and necrotizing, often with
crescents. Focal, segmental, membranous and/or sclerotic
lesions are common as well.
2. Tubular, for the presence of tubulointerstitial inflammation,
typically composed of dense infiltrates of granulocytes and
mononuclear cells, most common in the MRL strains.
3. Perivascular, for the presence of perivascular inflammation,
typically composed of dense infiltrates of granulocytes,
lymphocytes, monocytes and plasmacytoid cells, most
common in MRL/Mp-lpr/lpr and related strains.

3.4.2. Immunofluorescence 1. Place the frozen kidney in OCT in the cryostat chamber for
for Immune Deposits 1020 min to allow the block to equilibrate in temperature.
This is critical for quality of sections.
2. Place the tissue block on the cryostat specimen disk. Face the
block until desired tissue is exposed.
3. Cut 57 m sections of frozen kidney in OCT, placing them
on a poly-L-lysine-coated slide (see Note 20).
4. Fix sections in cold acetone, 20 C, 2 min.
5. Wash slide 3 in PBS, room temperature.
6. Add FITC-anti-mouse IgG, diluted 1:500 in PBS, just enough
to cover the tissue (typically 2550 L). Incubate in a
humidified chamber, room temperature, protected from light
(see Note 21).
7. Wash slide three times in PBS, room temperature.
8. Add 35 drops of mounting media and coverslip to the slide.
Observe slide immediately by fluorescence microscopy.
9. Positive samples are identified by apple-green fluorescence at
the glomeruli (see Note 22).

3.4.3. Surrogate Assays 1. Serum analysis can be performed using commercially available
for Renal Function kits for creatinine and/or urea nitrogen (e.g., Sigma), if
desired.
2. Urine protein excretion can be quantified by various protein
assays (e.g., Pierce), if desired.

3.5. Diagnosis At present there is no consensus as to the diagnosis of lupus in

of Murine Lupus murine models, but in general, most studies judge experimental
interventions based on the presence and/or titers of antinuclear anti-
bodies including anti-dsDNA, and immune-deposit related
glomerulonephritis. Thus, minimal analysis generally includes assess-
ment of FANA and anti-dsDNA antibodies, and histopathological
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 157

analysis of kidneys, often including immunofluorescence for IgG.

The assessment of other organs, such as salivary glands or joints,
have not been broadly studied or codified, at least in the study of
mouse lupus. In some investigations, serial analysis has been per-
formed for antinuclear antibodies and/or specific autoantibodies,
serum creatinine and urea nitrogen, urine proteinuria, and even kid-
ney biopsies (48)but unfortunately such use remains nonstan-
dardized and the predictive value of any of these tests is incompletely
known (49). Consequently, most experiments are performed as
cross-sectional, necropsy studies in animals at a specified age (since
birth or immunization), based principally on serology and histopa-
thology, as described above.

4. Notes

1. Although not formally documented, environmental factors

may affect the penetrance or severity of the lupus phenotype,
resulting in variability between and within individual colonies.
For instance, exposure of BXSB mice to ultraviolet B light pro-
motes autoantibody production and early death (50), but this
has not been seen in other strains such as BW (51). Similarly,
the 16/6 Id model was not reproducible in a British labora-
tory, suggesting that differences in ancillary reagents or hous-
ing environments may influence disease expression (52). In
some cases, reduced environmental antigen exposure such as
germ-free environments appears to improve or exacerbate
autoimmunity (53, 54). Consistency in environments and
reagents is therefore advised to ensure reproducibility of
findings.
2. The protocol indicated includes the technique used for chronic
graft-versus-host disease induced by transfer of DBA/2J sple-
nocytes into B6D2F1/J recipients. Other strain combinations
may optimally require different numbers of splenocytes, with
or without T cell-depleted bone marrow cells, using irradiated
or nonirradiated recipients (39).
3. Mice usually urinate without provocation within 510 s or so
by this method. Alternatively, multiple animals can be sampled
simultaneously by using a multicompartment tray. Wire mesh-
bottom cages, such as used in metabolic assays, can be used,
but are somewhat cumbersome for the present purposes.
4. Blood collection by cardiac puncture is not preferred since
spilled blood may contaminate organs to be collected. The ret-
roorbital (RO) approach provides a rapid means to obtain
blood; many investigators have found that anesthesia and
158 S.L. Peng

postprocedure global pressure are unnecessary. However,

proper training is required since ocular trauma may result. In
addition, it is not amenable to multiple serial samples in a short
period of time, although switching eyes allows for at least two
relatively closely timed samples is possible. Several alternatives
exist for mouse blood collection (55); below are three alterna-
tives often used in immunological studies because they are
nonterminal:
(a) Alternative 1: lateral saphenous vein technique. This
approach is often suggested because of its amenability to
multiple serial sampling and low potential for animal harm
and distress. However, like RO collection, it requires
proper training; and unlike RO, it generally requires spe-
cialized restraints or additional personnel, and often
requires 12 min for collection due to reduced blood flow
(56).
Restrain the mouse; this may require two people or a
restraining device (57).
Shave the hair covering the lateral saphenous vein with
a scalpel blade (the vein is located caudal and lateral to
the fibula and tibia).
Clean the shaved area.
Apply pressure around the leg above the stifle (knee)
this will help improve venous filling.
Puncture the saphenous vein with a 25 G needle.
Collect the blood accumulating over the incision using
a hematocrit tube.
Apply direct pressure to the incision for 13 min to
facilitate hemostasis.
Repeated blood samples may be obtained by removing
the scab.
(b) Alternative 2: tail sectioning. This approach is a rapid, sim-
ple procedure that does not require significant training.
Serial blood samples can be performed, but must be lim-
ited because of the length of the tail and the increasing risk
of inducing pain and distress. In general, a restraining
device is required, but anesthesia is not (58).
Restrain the mouse (57).
Clean the tail with an appropriate antiseptic solution.
With a sterile scalpel blade, make a transverse section
through the long axis of the tail 2 mm from the tip.
Use a hematocrit tube or blood-collecting tube to col-
lect blood dripping from the sectioned tail.
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 159

Massage the tail by passing the thumb and index finger

from the base to the tip of the tail if blood flow is
inadequate.
Apply direct pressure to the incision for 13 min to
facilitate hemostasis.
Repeated blood sampling may be obtained by remov-
ing the clot, or following the above protocol with a
new cut 1 mm from tip.
(c) Alternative 3: tail vein aspiration. This technique can
potentially allow for multiple serial samples with little harm
to the animal, but requires significant proficiency to avoid
permanent damage to the vein, which would disallow
future aspirations. Restraint is required, and blood collec-
tion is often slow.
Anesthetize the mouse.
Clean the area over the tail vein 3 cm from the base of
the tail; use an acceptable antiseptic scrub.
Make a small transverse incision with a sterile scalpel
blade, partially through the lateral tail vein.
Use a hematocrit tube or blood-collection tube to col-
lect the blood dripping from the incision.
Apply direct pressure to the incision for 13 min to
facilitate hemostasis.
Subsequent blood samplings may be obtained in awake
animals by removing the scab; if a new incision is
required, the animal must be anesthetized.
5. Peritoneal cell collection is particularly used for B1 cell studies
in NZ mice. During harvesting, too much force will cause lac-
eration of the large vessels and intraperitoneal bleeding. Care
should be taken to keep the needle hole as small as possible.
Best results are generally obtained if a firm hold on the ster-
num is maintained until the end of the peritoneal fluid
harvest.
6. Here, investigators often also take several nodes from the
nearby cervical chain, located just superior to the submandibu-
lar glands.
7. The stated protocol is a routine serum collection method,
which allows the blood to fully clot before serum collection.
Alternatively, the serum can be collected immediately after
blood collection (skipping step 2), as long as care is taken to
avoid aspirating unclotted blood. Regardless, this type of serum
collection will miss cold-precipitable cryoglobulins, which
often comprise a significant part of the autoantibody repertoire
of lupus-prone animals (9, 59, 60).
160 S.L. Peng

(a) Alternative: Cryoglobulin serum collection.

Collect blood at 37 C, with prewarmed instruments
and without anticoagulants (e.g., non-heparin-coated
hematocrit tubes).
Allow the blood to clot at 37 C for 12 h.
Collect the serum by centrifugation at 37 C, e.g., by
microcentrifuge at maximum speed for 5 min.
Incubate the serum at 04 C for 24120 h and observe
the development of a precipitatethe packed (centri-
fuged) volume of the precipitate is expressed as a per-
centage of the original serum volume, the cryocrit.
Often, further confidence in the cryocrit is obtained
by washing the precipitate 36 times in normal saline
solution to reduce the possibility of precipitated salts
or other proteins. In addition, the precipitate is often
redissolved in saline at 37 C to confirm the warm
solubility of the CGs; at this time, CG concentration
can be determined by spectrophotometry; and further
characterization can be accomplished by immunoelec-
trophoresis, ELISA, or other specific immunological
assay (61).
8. Prolonged fixation does not affect morphology, but will affect
immunodetection. Larger specimens will obviously need lon-
ger fixation times, but care should be taken since the outer
regions of a large specimen will be fixed longer than the center,
resulting in differential antigen availability during immunohis-
tochemistry. For bone or bone marrow specimens,
decalcification (e.g., via Decal; Decal Chemical Corporation)
should be performed according to manufacturer protocol prior
to ethanol dehydration.
9. Here, larger specimens are sometimes technically difficult to
section. Also, a mount is helpful to allow the histopathologist
to section through as much of the specimen as possible. Excess
OCT will prolong the sectioning time. During sectioning, care
should be taken that the cap does not fall away from the tissue
with manipulation. Once frozen, however, this is usually not a
problem. Alternatively, many investigators simply place their
tissue in a tissue cassette, add OCT or other embedding
medium compatible with frozen specimens, and douse the
sample directly into the dry iceethanol bath. The tissue qual-
ity is no different than the protocol described above, but the
tissue will need to be processed without a mount.
10. For other specificities than those mentioned, many commercial
sources produce ELISA systems with recombinant human
autoantigens, which may be used for the assessment of murine
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 161

autoantibodies, such as specificities against snRNPs (including

Sm and RNP), Ro, La, and antiphospholipid. Some investigators
have successfully purified their own autoantigens, such as chro-
matin or snRNPs, for their own autoantibody ELISA systems;
but the sensitivity and specificity of such protocols can be vari-
ably dependent upon the quality of the antigen preparation.
Immunoblot and immunoprecipitation techniques are also
available, but also generally require the purification of
autoantigen(s) and/or lengthy assays, and so have generally
not been widely used (62).
11. Several alternative capturedetection antibody combinations
are available (e.g., BD Pharmingen); the SBA reagents are
those routinely used by the author. Note that most IgG2a
reagents, including those listed in Section 2.3.1, capture and
detect only IgG2aa, not IgG2ab, which is found in strains like
C57BL/6 or SJLsuch strains require the use of allotype-
specific reagents (e.g., BD Pharmingen). In addition, coating
can be performed for shorter periods, like 4 h at 37 C or 8 h
at room temperature, but the efficiency of bonding seems to
be diminished.
12. Longer periods, like overnight at 4 C, tend to overblock,
leading to diminished OD signals.
13. High-titer dilutions are necessary to allow for the detection of
the extreme hypergammaglobulinemia seen in many lupus
strains.
14. Higher concentrations than as stated tend to exceed the bind-
ing capacity of most assay plates, leading to non-linear signals.
15. The OD of a sample should lie within the linear range of the
standard curve (on semi-log paper) for proper interpretation.
16. Although HEp-2 cells are of human epithelial origin, they
remain the most popular substrate due to their widespread
commercial availability as part of human diagnostic kits, and
the ability of murine autoantibodies to cross-react with human
antigens. Some investigators have elected to grow and/or pre-
pare their own murine targets, such as murine spleen or liver,
but the convenient, commercial HEp-2 substrates have largely
supplanted such assays.
17. For most investigators, anti-dsDNA assessment by the Crithidia
immunofluorescence technique affords the best compromise
between reliability, reproducibility, and convenience (62). In
contrast, the Farr radioimmunoassay is often considered the
gold standard assay, but requires radioactivity and is somewhat
unwieldy. Many ELISA systems are available, including many
commercial systems, but significantly vary in terms of their
specificity and convenience, most commonly because dsDNA
162 S.L. Peng

often denatures to ssDNA during the coating process. In addition,

DNA binds poorly to many microtiter plate materials, prompting
some investigators to use various conjugation methods to
enhance substrate binding: e.g., precoating the microtiter plate
with avidin, and then adding biotinylated dsDNAbut such a
protocol also risks the development of ssDNA during the con-
jugation process. As such, many anti-dsDNA ELISAs are
reported simply as anti-DNA to indicate the likely assessment
of total anti-DNA, rather than specific anti-dsDNA, activity.
18. This rheumatoid factor ELISA generally provides the best
compromise between reliability, reproducibility and conve-
nience. Limitations include the use of denatured, rather than
native, autoantigen (IgG) and the detection of only -chain
containing Igs. In addition, this technique does not discrimi-
nate among rheumatoid factor isotypes, so activity is generally
reported in this assay as total RF activity. IgM, IgA, and IgE
rheumatoid factors could be assayed using appropriate second-
ary antibodies, but IgG RFs could not since the secondary
antibodies would cross-react with the coated substrates. On
the other hand, RF activity by this assay correlates strongly
with disease activity in many murine models (e.g., ref. 63).
Investigators should note that the IgG2a component of the
target will need to be changed to IgG2c/IgG2ab during the
assessment of IgHb allotype strains (e.g., C57BL/6).
Otherwise, classical rheumatoid factor assays include solid-
phase assays like radioimmunoassays, which are somewhat
tedious but nonetheless can discriminate specific RF isotypes.
Finally, general comments for ELISAs as described for
Section 3.3.1 also apply here.
19. Many studies evaluate only the glomerular lesions, which is the
only area of consistent involvement across lupus-prone strains.
These lesions have not been codified into pathological groups,
so descriptions remain largely investigator-dependent.
20. Some investigators use uncoated slides, but the sections have a
tendency to detatch from the slide in steps 5 and 7.
21. Blocking, e.g., with BSA or serum, is not necessary in this pro-
tocol. Other isotype-specific antibodies, such as for IgG sub-
types or IgA, can be used here as well (64).
22. Often, nuclear staining is present in the tubular epithelial cells
which probably reflects serum antinuclear antibodies, not
renal-specific immune deposits. Sometimes, staining is present
in the tubular epithelium or glomerular capsule, but is of
unclear significance.
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus
References
1. Rahman A, Isenberg DA (2008) Systemic
lupus erythematosus. New Engl J Med
358:929939
2. Hahn BH, Singh RR (2007) Animal models
of systemic lupus erythematosus. In: Wallace
DJ, Hahn BH (eds) Dubois lupus erythema-
tosus. Lippincott Williams & Wilkins,
Philadelphia, PA
3. Davidson A, Aranow C (2009) Lupus nephri-
tis: lessons from murine models. Nat Rev
Rheumatol 6:1320
4. Ghoreishi M, Dutz JP (2009) Murine models
of cutaneous involvement in lupus erythema-
tosus. Autoimmun Rev 8:484487
5. Rottman JB, Willis CR (2010) Mouse models
of systemic lupus erythematosus reveal a com-
plex pathogenesis. Vet Pathol 47:664676
6. Peng SL (2004) Experimental use of murine
lupus models. Methods Mol Med 102:227272
7. Izui S, McConahey PJ, Dixon FJ (1978)
Increased spontaneous polyclonal activation of
B lymphocytes in mice with spontaneous auto-
immune disease. J Immunol 121:22132219
8. Andrews BS, Eisenberg RA, Theofilopoulos
AN et al (1978) Spontaneous murine lupus-
like syndromes. Clinical and immunopatho-
logical manifestations in several strains. J Exp
Med 148:11981215
9. Dixon FJ, Andrews BS, Eisenberg RA et al
(1978) Etiology and pathogenesis of a spon-
taneous lupus-like syndrome in mice. Arthritis
Rheum 21:S64S67
10. Bielschowsky M, Helyer BJ, Howie JB (1959)
Spontaneous haemolytic anemia in mice of
the NZB/BL strain. Proc Univ Otago Med
Sch 37:9
11. Morel L, Rudofsky UH, Longmate JA et al
(1994) Polygenic control of susceptibility to
murine systemic lupus erythematosus.
Immunity 1:219229
12. Kaliyaperumal A, Mohan C, Wu W et al
(1996) Nucleosomal peptide epitopes for
nephritis-inducing T helper cells of murine
lupus. J Exp Med 183:24592469
13. Dumont F, Monier JC (1983) Sex-dependent
systemic lupus erythematosus-like syndrome
in (NZB SJL)F1 mice. Clin Immunol
Immunopathol 29:306317
14. Vidal S, Gelpi C, Rodriguez-Sanchez JL
(1994) (SWR SJL)F1 mice: a new model
of lupus-like disease. J Exp Med 179:
14291435
15. Watanabe-Fukunaga R, Brannan CI, Copeland
NG et al (1992) Lymphoproliferation disorder
163
in mice explained by defects in Fas antigen that
mediates apoptosis. Nature 356:314317
16. Kelley VE, Roths JB (1985) Interaction of
mutant lpr gene with background strain
influences renal disease. Clin Immunol
Immunopathol 37:220229
17. Eisenberg RA, Craven SY, Fisher CL et al
(1989) The genetics of autoantibody produc-
tion in MRL/lpr lupus mice. Clin Exp
Rheumatol 7:S35S40
18. Peng SL, Craft J (1999) Lessons from knock-
out and transgenic lupus-prone mice (chapter
10). In: Kammer GM, Tsokos GC (eds) Lupus:
molecular and cellular pathogenesis. Humana
Press Inc., Totowa, NJ, pp 152166
19. Matsuzawa A, Moriyama T, Kaneko T et al
(1990) A new allele of the lpr locus, lprcg, that
complements the gld gene in induction of
lymphadenopathy in the mouse. J Exp Med
171:519531
20. Kimura M, Mohri H, Shimada K et al (1990)
Serological and histological characterization
of the new mutant strain of lpr mice, CBA/
KlJms-lprcg/lprcg. Clin Exp Immunol
79:123129
21. Adachi M, Suematsu S, Kondo T et al (1995)
Targeted mutation in the Fas gene causes
hyperplasia in peripheral lymphoid organs and
liver. Nat Genet 11:294300
22. Roths JB, Murphy ED, Eicher EM (1984) A
new mutation, gld, that produces lymphopro-
liferation and autoimmunity in C3H/HeJ
mice. J Exp Med 159:120
23. Lynch DH, Watson ML, Alderson MR et al
(1994) The mouse Fas-ligand gene is mutated
in gld mice and is part of a TNF family gene
cluster. Immunity 1:131136
24. Takahashi T, Tanaka M, Brannan CI et al
(1994) Generalized lymphoproliferative dis-
ease in mice, caused by a point mutation in
the Fas ligand. Cell 76:969976
25. Pisitkun P, Deane JA, Difilippantonio MJ et al
(2006) Autoreactive B cell responses to RNA-
related antigens due to TLR7 gene duplica-
tion. Science 312:16691672
26. Subramanian S, Tus K, Li QZ et al (2006) A
Tlr7 translocation accelerates systemic auto-
immunity in murine lupus. Proc Natl Acad Sci
USA 103:99709975
27. Izui S, Masuda K, Yoshida H (1984) Acute
SLE in F1 hybrids between SB/Le and NZW
mice; prominently enhanced formation of
gp70 immune complexes by a Y chromosome-
associated factor from SB/Le mice. J Immunol
132:701704
164 S.L. Peng
28. Hashimoto Y, Kawamura M, Ichikawa K et al
(1992) Anticardiolipin antibodies in
NZW BXSB F1 mice. A model of antiphospho-
lipid syndrome. J Immunol 149:10631068
29. Mountz JD, Yang P, Wu Q et al (2005)
Genetic segregation of spontaneous erosive
arthritis and generalized autoimmune disease
in the BXD2 recombinant inbred strain of
mice. Scand J Immunol 61:128138
30. Kinjoh K, Kyogoku M, Good RA (1993)
Genetic selection for crescent formation yields
mouse strain with rapidly progressive glomer-
ulonephritis and small vessel vasculitis. Proc
Natl Acad Sci USA 90:34133417
31. Jethwa HS, Nachman PH, Falk RJ et al (2000)
False-positive myeloperoxidase binding activity
due to DNA/anti-DNA antibody complexes: a
source for analytical error in serologic evalua-
tion of anti-neutrophil cytoplasmic autoanti-
bodies. Clin Exp Immunol 121:544550
32. Walker SE, Gray RH, Fulton M et al (1978)
Palmerston North mice, a new animal model
of systemic lupus erythematosus. J Lab Clin
Med 92:932945
33. Sundberg JP, France M, Boggess D et al
(1997) Development and progression of pso-
riasiform dermatitis and systemic lesions in the
flaky skin (fsn) mouse mutant. Pathobiology
65:271286
34. Pelsue SC, Schweitzer PA, Schweitzer IB et al
(1998) Lymphadenopathy, elevated serum
IgE levels, autoimmunity, and mast cell accu-
mulation in flaky skin mutant mice. Eur J
Immunol 28:13791388
35. White RA, McNulty SG, Nsumu NN et al
(2005) Positional cloning of the Ttc7 gene
required for normal iron homeostasis and
mutated in hea and fsn anemia mice. Genomics
85:330337
36. Satoh M, Reeves WH (1994) Induction of
lupus-associated autoantibodies in BALB/c
mice by intraperitoneal injection of pristane.
J Exp Med 180:23412346
37. Gleichmann H, Gleichmann E, Andre-
Schwartz J et al (1972) Chronic allogeneic
disease. 3. Genetic requirements for the induc-
tion of glomerulonephritis. J Exp Med
135:516532
38. Van Elven EH, van der Veen FM, Rolink AG
et al (1981) Diseases caused by reactions of T
lymphocytes to incompatible structures of the
major histocompatibility complex. V. High
titers of IgG autoantibodies to double-
stranded DNA. J Immunol 127:24352438
39. Chu Y-W, Gress RE (2008) Murine models of
chronic graft-versus-host disease: insights and
unresolved issues. Biol Blood Marrow
Transplant 14:365378
40. Mendlovic S, Brocke S, Shoenfeld Y et al
(1988) Induction of a systemic lupus erythe-
matosus-like disease in mice by a common
human anti-DNA idiotype. Proc Natl Acad
Sci USA 85:22602264
41. Blank M, Cohen J, Toder V et al (1991)
Induction of anti-phospholipid syndrome in
naive mice with mouse lupus monoclonal and
human polyclonal anti-cardiolipin antibodies.
Proc Natl Acad Sci USA 88:30693073
42. Silveira PA, Baxter AG (2001) The NOD
mouse as a model of SLE. Autoimmunity
34:5364
43. Duke Animal Care and Use Program (2011)
Guidelines for techniques with rodents.
http://vetmed.duhs.duke.edu/guidelines_
for_techniques_in_rodents.htm. Accessed 1
Mar 2011
44. Cattoretti G (2002) Pathology and autopsy of
a mouse. http://icg.cpmc.columbia.edu/cat-
toretti/Protocol/MousePathology/
mouseTissue.html. Accessed 1 Mar 2011
45. Covelli V (1972) Guide to the necropsy of the
mouse. http://eulep.pdn.cam.ac.uk/
Necropsy_of_the_Mouse/. Accessed 1 Mar
2011
46. Kurien BT, Scofield RH (1999) Mouse urine
collection using clear plastic wrap. Lab Anim
33:8386
47. Duke Animal Care and Use Program (2011)
Guidelines for retro-orbital blood collection.
In: Mice. http://vetmed.duhs.duke.edu/
guidelines_for_retroorbital_blood_coll_mice.
htm. Accessed 1 Mar 2011
48. Kimura M, Nagase M, Hishida A et al (1987)
Intramesangial passage of mononuclear
phagocytes in murine lupus glomerulonephri-
tis. Am J Pathol 127:149156
49. Hoffsten PE, Hill CL, Klahr S (1975) Studies
of abluminuria and proteinuria in normal mice
and mice with immune complex glomerulo-
nephritis. J Lab Clin Med 86:920930
50. Ansel JC, Mountz J, Steinberg AD et al
(1985) Effects of UV radiation on autoim-
mune strains of mice: increased mortality and
accelerated autoimmunity in BXSB male mice.
J Invest Dermatol 85:181186
51. Davis P, Percy JS (1978) Effect of ultraviolet
light on disease characteristics of NZB/W
mice. J Rheumatol 5:125128
52. Isenberg DA, Katz D, Le Page S et al (1991)
Independent analysis of the 16/6 idiotype
lupus model. A role for an environmental fac-
tor? J Immunol 147:41724177
53. Maldonado MA, Kakkanaiah V, MacDonald
GC et al (1999) The role of environmental
antigens in the spontaneous development of
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus
autoimmunity in MRL-lpr mice. J Immunol
162:63226330
54. Mizutani A, Shaheen VM, Yoshida H et al
(2005) Pristane-induced autoimmunity in
germ-free mice. Clin Immunol 114:110118
55. Hoff J (2000) Methods of blood collection in
the mouse. Lab Anim 29:4753
56. Hem A, Smith AJ, Solberg P (1998) Saphenous
vein puncture for blood sampling of the
mouse, rat, hamster, gerbil, guinea pig, ferret
and mink. Lab Anim 32:364368
57. Furner RL, Mellett LB (1975) Mouse restrain-
ing chamber for tail-vein injection. Lab Animal
Sci 25:648
58. Durschlag M, Wurbel H, Stauffacher M et al
(1996) Repeated blood collection in the labo-
ratory mouse by tail incisionmodification of
an old technique. Phys Behavior
60:15651568
59. Hijmans W, Radema H, van Es L et al (1969)
Cryoglobulins in New Zealand Black mice.
Clin Exp Immunol 4:227239
60. Reininger L, Berney T, Shibata T et al (1990)
Cryoglobulinemia induced by a murine IgG3
rheumatoid factor: skin vasculitis and glom-
erulonephritis arise from distinct pathogenic
mechanisms. Proc Natl Acad Sci USA
87:1003810042
61. Dammacco F, Sansonno D, Piccoli C et al
(2001) The cryoglobulins: an overview. Eur J
Clin Invest 31:628638
62. Peng SL, Craft J (2008) Antinuclear antibod-
ies (chapter 50). In: Firestein GS, Budd RC,
Harris ED et al (eds) Kellys textbook of rheu-
matology, 8th edn. Elsevier, Inc., Philadelphia,
PA
63. Shlomchik MJ, Madaio MP, Ni D et al (1994)
The role of B cells in lpr/lpr-induced autoim-
munity. J Exp Med 180:12951306
64. Peng SL, Cappadona J, McNiff JM et al
(1998) Pathogenesis of autoimmunity in T
cell-deficient lupus-prone mice. Clin Exp
Immunol 111:107116
65. Strasser A, Whittingham S, Vaux DL et al
(1991) Enforced BCL2 expression in
B-lymphoid cells prolongs antibody responses
and elicits autoimmune disease. Proc Natl
Acad Sci USA 88:86618665
66. Bouillet P, Metcalf D, Huang DC et al (1999)
Proapoptotic Bcl-2 relative Bim required for
certain apoptotic responses, leukocyte homeo-
stasis, and to preclude autoimmunity. Science
286:17351738
67. Bouillet P, Purton JF, Godfrey DI et al (2002)
BH3-only Bcl-2 family member Bim is
required for apoptosis of autoreactive thymo-
cytes. Nature 415:922926
165
68. Choi Y, Ramnath VR, Eaton AS et al (1999)
Expression in transgenic mice of dominant
interfering Fas mutations: a model for human
autoimmune lymphoproliferative syndrome.
Clin Immunol 93:3445
69. Murga M, Fernandez-Capetillo O, Field SJ
et al (2001) Mutation of E2F2 in mice causes
enhanced T lymphocyte proliferation, leading
to the development of autoimmunity.
Immunity 15:959970
70. Salvador JM, Hollander MC, Nguyen AT et al
(2002) Mice lacking the p53-effector gene
Gadd45a develop a lupus-like syndrome.
Immunity 16:499508
71. Zhang Y, Schlossman SF, Edwards RA et al
(2002) Impaired apoptosis, extended dura-
tion of immune responses, and a lupus-like
autoimmune disease in IEX-1-transgenic
mice. Proc Natl Acad Sci USA 99:878883
72. Woelfel M, Bixby J, Brehm MA et al (2006)
Transgenic expression of the viral FLIP
MC159 causes lpr/gld-like lymphoprolifera-
tion and autoimmunity. J Immunol
177:38143820
73. Balomenos D, Martin-Caballero J, Garcia MI
et al (2000) The cell cycle inhibitor p21 con-
trols T-cell proliferation and sex-linked lupus
development. Nat Med 6:171176
74. Lawson BR, Kono DH, Theofilopoulos AN
(2002) Deletion of p21 (WAF-1/Cip1) does
not induce systemic autoimmunity in female
BXSB mice. J Immunol 168:59285932
75. Di Cristofano A, Kotsi P, Peng YF et al (1999)
Impaired Fas response and autoimmunity in
Pten+/ mice. Science 285:21222125
76. Suzuki A, Yamaguchi MT, Ohteki T et al
(2001) T cell-specific loss of Pten leads to
defects in central and peripheral tolerance.
Immunity 14:523534
77. Prodeus AP, Goerg S, Shen LM et al (1998) A
critical role for complement in maintenance of
self-tolerance. Immunity 9:721731
78. Scott RS, McMahon EJ, Pop SM et al (2001)
Phagocytosis and clearance of apoptotic cells
is mediated by MER. Nature 411:207211
79. Nash JT, Taylor PR, Botto M et al (2001)
Immune complex processing in C1q-deficient
mice. Clin Exp Immunol 123:196202
80. Botto M, DellAgnola C, Bygrave AE et al
(1998) Homozygous C1q deficiency causes
glomerulonephritis associated with multiple
apoptotic bodies. Nat Genet 19:5659
81. Napirei M, Karsunky H, Zevnik B et al (2000)
Features of systemic lupus erythematosus in
Dnase1-deficient mice. Nat Genet 25:177181
82. Bolland S, Ravetch JV (2000) Spontaneous
autoimmune disease in FcRIIB-deficient
166 S.L. Peng
mice results from strain-specific epistasis.
Immunity 13:277285
83. Boes M, Schmidt T, Linkemann K et al (2000)
Accelerated development of IgG autoantibod-
ies and autoimmune disease in the absence of
secreted IgM. Proc Natl Acad Sci USA
97:11841189
84. A-Gonzalez N, Bensinger SJ, Hong C et al
(2009) Apoptotic cells promote their own
clearance and immune tolerance through acti-
vation of the nuclear receptor LXR. Immunity
31:245258
85. Hanayama R, Tanaka M, Miyasaka K et al
(2004) Autoimmune disease and impaired
uptake of apoptotic cells in MFG-E8-deficient
mice. Science 304:11471150
86. Xue D, Shi H, Smith JD et al (2003) A lupus-
like syndrome develops in mice lacking the Ro
60-kDa protein, a major lupus autoantigen.
Proc Natl Acad Sci USA 100:75037508
87. Bickerstaff MC, Botto M, Hutchinson WL
et al (1999) Serum amyloid P component con-
trols chromatin degradation and prevents anti-
nuclear autoimmunity. Nat Med 5:694697
88. Lu Q, Lemke G (2001) Homeostatic regula-
tion of the immune system by receptor
tyrosine kinases of the Tyro 3 family. Science
293:306311
89. OKeefe TL, Williams GT, Davies SL et al
(1996) Hyperresponsive B cells in CD22-
deficient mice. Science 274:798801
90. Stunz LL, Busch LK, Munroe ME et al (2004)
Expression of the cytoplasmic tail of LMP1 in
mice induces hyperactivation of B lympho-
cytes and disordered lymphoid architecture.
Immunity 21:255266
91. Mehling A, Loser K, Varga G et al (2001)
Overexpression of CD40 ligand in murine
epidermis results in chronic skin inflammation
and systemic autoimmunity. J Exp Med
194:615628
92. Higuchi T, Aiba Y, Nomura T et al (2002)
Cutting edge: ectopic expression of CD40
ligand on B cells induces lupus-like autoim-
mune disease. J Immunol 168:912
93. Majeti R, Xu Z, Parslow TG et al (2000) An
inactivating point mutation in the inhibitory
wedge of CD45 causes lymphoproliferation
and autoimmunity. Cell 103:10591070
94. Li DH, Winslow MM, Cao TM et al (2008)
Modulation of peripheral B cell tolerance by
CD72 in a murine model. Arthritis Rheum
58:31923204
95. Tivol EA, Borriello F, Schweitzer AN et al
(1995) Loss of CTLA-4 leads to massive lym-
phoproliferation and fatal multiorgan tissue
destruction, revealing a critical negative regu-
latory role of CTLA-4. Immunity 3:541547
96. Waterhouse P, Penninger JM, Timms E et al
(1995) Lymphoproliferative disorders with
early lethality in mice deficient in Ctla-4.
Science 270:985988
97. Han B, Moore PA, Wu J et al (2007)
Overexpression of human decoy receptor 3 in
mice results in a systemic lupus erythemato-
sus-like syndrome. Arthritis Rheum
56:37483758
98. Le LQ, Kabarowski JH, Weng Z et al (2001)
Mice lacking the orphan G protein-coupled
receptor G2A develop a late-onset autoim-
mune syndrome. Immunity 14:561571
99. Yoshinaga SK, Zhang M, Pistillo J et al (2000)
Characterization of a new human B7-related
protein: B7RP-1 is the ligand to the co-stim-
ulatory protein ICOS. Int Immunol 12:
14391447
100. Nishimura H, Nose M, Hiai H et al (1999)
Development of lupus-like autoimmune dis-
eases by disruption of the PD-1 gene encod-
ing an ITIM motif-carrying immunoreceptor.
Immunity 11:141151
101. Wilkinson R, Lyons AB, Roberts D et al
(2002) Platelet endothelial cell adhesion mol-
ecule-1 (PECAM-1/CD31) acts as a regula-
tor of B-cell development, B-cell antigen
receptor (BCR)-mediated activation, and
autoimmune disease. Blood 100:184193
102. Seshasayee D, Valdez P, Yan M et al (2003)
Loss of TACI causes fatal lymphoproliferation
and autoimmunity, establishing TACI as an
inhibitory BLyS receptor. Immunity
18:279288
103. Wen L, Roberts SJ, Viney JL et al (1994)
Immunoglobulin synthesis and generalized
autoimmunity in mice congenitally deficient
in (+) T cells. Nature 369:654658
104. Wang JH, Avitahl N, Cariappa A et al (1998)
Aiolos regulates B cell activation and matura-
tion to effector state. Immunity 9:543553
105. Shankar M, Nixon JC, Maier S et al (2007)
Anti-nuclear antibody production and auto-
immunity in transgenic mice that overexpress
the transcription factor Bright. J Immunol
178:29963006
106. Krawczyk C, Bachmaier K, Sasaki T et al
(2000) Cbl-b is a negative regulator of recep-
tor clustering and raft aggregation in T cells.
Immunity 13:463473
107. Stohl W, Xu D, Kim KS et al (2005) Humoral
autoimmunity in mice overexpressing B cell
surface CD19: vital role for MHC class II.
Clin Immunol 116:257264
108. Hsiao HW, Liu WH, Wang CJ et al (2009)
Deltex1 is a target of the transcription factor
NFAT that promotes T cell anergy. Immunity
31:7283
 7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus
109. Zhu B, Symonds AL, Martin JE et al (2008)
Early growth response gene 2 (Egr-2) con-
trols the self-tolerance of T cells and prevents
the development of lupuslike autoimmune
disease. J Exp Med 205:22952307
110. Shim GJ, Kis LL, Warner M et al (2004)
Autoimmune glomerulonephritis with spon-
taneous formation of splenic germinal centers
in mice lacking the estrogen receptor gene.
Proc Natl Acad Sci USA 101:17201724
111. Zhang L, Eddy A, Teng YT et al (1995) An
immunological renal disease in transgenic
mice that overexpress Fli-1, a member of the
ets family of transcription factor genes. Mol
Cell Biol 15:69616970
112. Yu CC, Yen TS, Lowell CA et al (2001)
Lupus-like kidney disease in mice deficient in
the Src family tyrosine kinases Lyn and Fyn.
Curr Biol 11:3438
113. Li H, Dai M, Zhuang Y (2004) A T cell intrin-
sic role of Id3 in a mouse model for primary
Sjgrens syndrome. Immunity 21:551560
114. Hida S, Ogasawara K, Sato K et al (2000)
CD8(+) T cell-mediated skin disease in mice
lacking IRF-2, the transcriptional attenuator
of interferon-/ signaling. Immunity 13:
643655
115. Pflegerl P, Vesely P, Hantusch B et al (2009)
Epidermal loss of JunB leads to a SLE pheno-
type due to hyper IL-6 signaling. Proc Natl
Acad Sci USA 106:2042320428
116. Sommers CL, Park CS, Lee J et al (2002) A
LAT mutation that inhibits T cell develop-
ment yet induces lymphoproliferation. Science
296:20402043
117. Hibbs ML, Tarlinton DM, Armes J et al
(1995) Multiple defects in the immune sys-
tem of Lyn-deficient mice, culminating in
autoimmune disease. Cell 83:301311
118. Nishizumi H, Taniuchi I, Yamanashi Y et al
(1995) Impaired proliferation of peripheral B
cells and indication of autoimmune disease in
lyn-deficient mice. Immunity 3:549560
119. Cornall RJ, Cyster JG, Hibbs ML et al (1998)
Polygenic autoimmune traits: Lyn, CD22,
and SHP-1 are limiting elements of a bio-
chemical pathway regulating BCR signaling
and selection. Immunity 8:497508
120. Hurov JB, Stappenbeck TS, Zmasek CM et al
(2001) Immune system dysfunction and auto-
immune disease in mice lacking Emk (Par-1)
protein kinase. Mol Cell Biol 21:32063219
121. Yoh K, Itoh K, Enomoto A et al (2001) Nrf2-
deficient female mice develop lupus-like auto-
immune nephritis. Kidney Int 60:13431353
122. Miyamoto A, Nakayama K, Imaki H et al
(2002) Increased proliferation of B cells and
167
auto-immunity in mice lacking protein kinase
C. Nature 416:865869
123. Layer K, Lin G, Nencioni A et al (2003)
Autoimmunity as the consequence of a spon-
taneous mutation in Rasgrp1. Immunity
19:243255
124. Ishimaru N, Arakaki R, Yoshida S et al (2008)
Expression of the retinoblastoma protein
RbAp48 in exocrine glands leads to Sjgrens
syndrome-like autoimmune exocrinopathy.
J Exp Med 205:29152927
125. Espinosa A, Dardalhon V, Brauner S et al (2009)
Loss of the lupus autoantigen Ro52/Trim21
induces tissue inflammation and systemic auto-
immunity by disregulating the IL-23-Th17
pathway. J Exp Med 206:166171
126. Yu D, Tan AH-M, Hu X et al (2007) Roquin
represses autoimmunity by limiting inducible
T-cell co-stimulator messenger RNA. Nature
450:299303
127. Finetti F, Pellegrini M, Ulivieri C et al (2008)
The proapoptotic and antimitogenic protein
p66SHC acts as a negative regulator of lym-
phocyte activation and autoimmunity. Blood
111:50175027
128. Savino MT, Ortensi B, Ferro M et al (2009) Rai
acts as a negative regulator of autoimmunity by
inhibiting antigen receptor signaling and lym-
phocyte activation. J Immunol 182:301308
129. Shultz LD, Schweitzer PA, Rajan TV et al
(1993) Mutations at the murine motheaten
locus are within the hematopoietic cell pro-
tein-tyrosine phosphatase (Hcph) gene. Cell
73:14451454
130. Tsui HW, Siminovitch KA, de Souza L et al
(1993) Motheaten and viable motheaten mice
have mutations in the haematopoietic cell
phosphatase gene. Nat Genet 4:124129
131. Fujimoto M, Tsutsui H, Xinshou O et al
(2004) Inadequate induction of suppressor of
cytokine signaling-1 causes systemic autoim-
mune diseases. Int Immunol 16:303314
132. Sun H, Lu B, Li RQ et al (2001) Defective T cell
activation and autoimmune disorder in Stra13-
deficient mice. Nat Immunol 2:10401047
133. Drappa J, Kamen LA, Chan E et al (2003)
Impaired T cell death and lupus-like autoim-
munity in T cell-specific adapter protein-
deficient mice. J Exp Med 198:809821
134. Lech M, Kulkarni OP, Pfeiffer S et al (2008)
Tir8/Sigirr prevents murine lupus by suppress-
ing the immunostimulatory effects of lupus
autoantigens. J Exp Med 205:18791888
135. Mackay F, Woodcock SA, Lawton P et al
(1999) Mice transgenic for BAFF develop
lymphocytic disorders along with autoimmune
manifestations. J Exp Med 190:16971710
168 S.L. Peng
136. Gross JA, Johnston J, Mudri S et al (2000)
TACI and BCMA are receptors for a TNF
homologue implicated in B-cell autoimmune
disease. Nature 404:995999
137. Khare SD, Sarosi I, Xia XZ et al (2000) Severe
B cell hyperplasia and autoimmune disease in
TALL-1 transgenic mice. Proc Natl Acad Sci
USA 97:33703375
138. Seery JP (2000) IFN- transgenic mice: clues
to the pathogenesis of systemic lupus erythe-
matosus? Arthritis Res 2:437440
139. Sadlack B, Lohler J, Schorle H et al (1995)
Generalized autoimmune disease in
interleukin-2-deficient mice is triggered by an
uncontrolled activation and proliferation of
CD4+ T cells. Eur J Immunol 25:30533059
140. Willerford DM, Chen J, Ferry JA et al (1995)
Interleukin-2 receptor chain regulates the
size and content of the peripheral lymphoid
compartment. Immunity 3:521530
141. Suzuki H, Kundig TM, Furlonger C et al
(1995) Deregulated T cell activation and
autoimmunity in mice lacking interleukin-2
receptor . Science 268:14721476
142. Erb KJ, Ruger B, von Brevern M et al (1997)
Constitutive expression of interleukin (IL)-4
in vivo causes autoimmune-type disorders in
mice. J Exp Med 185:329339
143. Vosters JL, Landek-Salgado MA, Yin H et al
(2009) Interleukin-12 induces salivary gland
dysfunction in transgenic mice, providing a
new model of Sjgrens syndrome. Arthritis
Rheum 60:36333641
144. Shen L, Zhang C, Wang T et al (2006)
Development of autoimmunity in IL-14-
transgenic mice. J Immunol 177:56765686
145. Wang J, Lo JC, Foster A et al (2001) The
regulation of T cell homeostasis and autoim-
munity by T cell-derived LIGHT. J Clin Invest
108:17711780
146. Carballo E, Lai WS, Blackshear PJ (1998)
Feedback inhibition of macrophage tumor
necrosis factor- production by tristetrapro-
lin. Science 281:10011005
147. Taylor GA, Carballo E, Lee DM et al (1996)
A pathogenetic role for TNF in the syn-
drome of cachexia, arthritis, and autoimmu-
nity resulting from tristetraprolin (TTP)
deficiency. Immunity 4:445454
148. Dang H, Geiser AG, Letterio JJ et al (1995)
SLE-like autoantibodies and Sjgrens syn-
drome-like lymphoproliferation in TGF-
knockout mice. J Immunol 155:32053212
149. Yaswen L, Kulkarni AB, Fredrickson T et al
(1996) Autoimmune manifestations in the
transforming growth factor-1 knockout
mouse. Blood 87:14391445
150. Gorelik L, Flavell RA (2000) Abrogation of
TGF signaling in T cells leads to spontane-
ous T cell differentiation and autoimmune
disease. Immunity 12:171181
151. Cheng J, Turksen K, Yu QC et al (1992)
Cachexia and graft-vs.-host-disease-type
skin changes in keratin promoter-driven
TNF transgenic mice. Genes Dev
6:14441456
152. Green RS, Stone EL, Tenno M et al (2007)
Mammalian N-glycan branching protects
against innate immune self-recognition and
inflammation in autoimmune disease patho-
genesis. Immunity 27:308320
153. Demetriou M, Granovsky M, Quaggin S et al
(2001) Negative regulation of T-cell activa-
tion and autoimmunity by Mgat5
N-glycosylation. Nature 409:733739
154. Xiao C, Srinivasan L, Calado DP et al (2008)
Lymphoproliferative disease and autoimmu-
nity in mice with increased miR-17-92 expres-
sion in lymphocytes. Nat Immunol
9:405414
 Chapter 8

Murine Models of Lupus Induced by Hypomethylated T Cells

(DNA Hypomethylation and Lupus)
Bruce Richardson, Amr H. Sawalha, Donna Ray, and Raymond Yung

Abstract
CD4+ T cell DNA hypomethylation may contribute to the development of drug induced and idiopathic
human lupus. Inhibiting DNA methylation in mature CD4+ T cells causes MHC-specific autoreactivity
in vitro. The lupus-inducing drugs hydralazine and procainamide also inhibit T cell DNA methylation and
induce autoreactivity, and T cells from patients with active lupus have hypomethylated DNA and a similarly
autoreactive T cell subset. Further, T cells treated with DNA methylation inhibitors demethylate the same
sequences that demethylate in T cells from patients with active lupus. The pathologic significance of the
autoreactivity induced by inhibiting T cell DNA methylation has been tested by treating murine T cells
in vitro with drugs which modify DNA methylation, then injecting the cells into syngeneic female mice.
Mice receiving CD4+ T cells demethylated by a variety of agents including procainamide and hydralazine
develop a lupus-like disease. Further, transgenic mice with an inducible T cell DNA methylation defect also
develop lupus-like autoimmunity. This chapter describes the protocols for inducing autoreactivity in
murine T cells in vitro and for inducing autoimmunity in vivo using an adoptive transfer approach or trans-
genic animal models.

Key words: Lupus, Drug-induced lupus, DNA methylation, Animal models, Autoimmunity

1. Introduction

1.1. Background DNA methylation is an essential determinant of chromatin structure

and gene expression. DNA methylation refers to the postsynthetic
methylation of deoxycytosine (dC) bases at the 5th position to
form deoxymethylcytosine (dmC). Nearly all dmC is found in CpG
dinucleotides, although only 7080 % of the CpG pairs are methy-
lated. In general, the methylation of CG pairs in regulatory
sequences results in transcriptional suppression, while demethyla-
tion permits active transcription. Methylation patterns are estab-
lished during differentiation, and serve to suppress expression of

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_8, Springer Science+Business Media New York 2012

169
170 B. Richardson et al.

genes not necessary for the function of fully differentiated cells.

The enzymes responsible for establishing methylation patterns
include Dnmt3a and Dnmt3b, referred to as de novo methyltrans-
ferases. The methylation patterns are then maintained during mito-
sis by maintenance methyltransferases including Dnmt1 (1).
The importance of DNA methylation is evidenced by its role in
differentiation, genomic imprinting, and X chromosome inactiva-
tion (1). Disrupting both DNMT1 alleles in embryonal stem cells
results in embryonic death, indicating that DNA methylation is
important in ontogeny (2). Dnmt3a and 3b are also essential for
mammalian development: homozygous Dnmt3a deficiency causes
runting and death at 4 weeks of age, while Dnmt3b deficiency is
embryonic lethal (3). Inhibiting DNA methylation in differenti-
ated cells can have profound effects on cells. For example, treating
the mouse fibroblast cell line 10T1/2 with the irreversible DNA
methyltransferase inhibitor 5-azacytidine (5-azaC) causes the cells
to differentiate into myocytes, adipocytes, and chondrocytes (4).

1.2. DNA Methylation DNA methylation is also important in regulating T lymphocyte

and T Cell Function gene expression. Methylation patterns change during thymic mat-
uration (5), similar to the changes that occur during differentiation
of other cell types. DNA methylation is implicated in the differen-
tiation of Th0 cells into Th1 and Th2 phenotypes as well: the
interferon-g (IFN-g) gene is methylated in nonexpressing Th2
cells, but demethylated in Th1 cells, while the IL-4 gene methy-
lated in Th1 but not Th2 cells (6, 7). Demethylation of the Foxp3
locus is also important in the differentiation and function of regu-
latory T cells (8). 5-azaC can also modify T cell gene expression.
Examples include effects on IFN-g and perforin expression in
CD4+ T cells (9, 10).
Demethylating T cell DNA with 5-azaC can change T cell
reactivity and function. Treating CD4+ T cell clones, as well as
polyclonal CD4+ T cells, with DNA methylation inhibitors causes
autoreactivity. The treated cells lose restriction for nominal antigen
and respond to self class II MHC molecules without added antigen
(11, 12). The autoreactivity is due at least in part to overexpression
of the adhesion molecule LFA-1 (CD11a/CD18), and causing
LFA-1 overexpression by transfection induces a similar MHC-
restricted autoreactivity (1315). The autoreactivity may reflect
overstabilization of the normally low affinity interaction between
the TCR and class II MHC molecules presenting inappropriate
antigen (16). 5-azaC increases steady state levels of CD11a but not
CD18 mRNA, and the increase in CD11a mRNA appears to be
due to demethylation of repetitive elements 5 to the CD11a pro-
moter (14, 17). In contrast, CD8+ T cells do not become autore-
active following 5-azaC treatment (12), and the reason is
unexplored.
 8 Murine Models of Lupus Induced by Hypomethylated T Cells 171

1.3. T Cell DNA The pathologic significance of 5-azaC induced autoreactivity has
Hypomethylation been tested in animal models. The approach is to treat stimulated
and Autoimmunity CD4+ T cells with 5-azaC in vitro, culture for at least 12 cell
cycles, and then inject the treated cells into syngeneic recipients.
5-azaC and other DNA methylation inhibitors prevent the methy-
lation of newly synthesized DNA during S phase, referred to as
passive demethylation. Thus, these agents are only effective when
added to dividing cells. Further, 12 rounds of cell division are
often required before changes in gene expression are observed
(18). Adoptive transfer of murine CD4+ T cells, made autoreactive
either by treatment with 5-azaC or by transfection with CD18,
causes a lupus-like disease in syngeneic recipients (15). The disease
induced closely resembles chronic graft-vs-host disease in mice, in
which features of lupus-like autoimmunity are also induced by
CD4+ T cells responding to host class II MHC molecules (19).
The DNA hypomethylation model has been used successfully
with polyclonal CD4+ T cells in DBA/2 mice (20), cloned Th2
cells in AKR mice (21), and cloned Th1 cells in B10.A mice (22).
We have also used a panel of DNA methylation inhibitors, includ-
ing 5-azaC, procainamide, hydralazine, and the ERK pathway
inhibitor U0126 to induce autoimmunity (23, 24). 5-azaC and
procainamide are DNA methyltransferase inhibitors (18, 25), while
hydralazine and the ERK pathway inhibitors prevent the upregula-
tion of Dnmt1 and Dnmt3a during T cell stimulation (24). All the
DNA hypomethylation models develop anti-DNA antibodies but
vary to some degree with respect to the histologic changes induced,
either due to the different repertoire of effector functions displayed
by the treated cells, or due to host-specific genetic influences. The
mechanism common to all models is promiscuous killing of host
macrophages (M). This may contribute to the development of
anti-DNA antibodies by increasing the total amount of potentially
antigenic apoptotic material (26), and/or by removing the cells
responsible for removing apoptotic debris, analogous to knock-
out mice with defective clearance of apoptotic material, which
develop anti-DNA antibodies (27).
Next, we designed experiments to provide further evidence
that the T cell ERK signaling defect and DNA hypomethylation
described in lupus patients plays a pathogenic role rather than
merely being a result of the disease process. We generated trans-
genic mice that express a dominant negative MEK (dnMEK) under
the control of a promoter sequence that contains multiple tetracy-
cline-responsive elements. By breeding these mice onto another
transgenic mouse strain that expresses a reverse tetracycline trans-
activator (rtTA) under the control of a CD2 promoter, we can
induce T cell-specific expression of the dominant negative MEK by
adding doxycycline to the drinking water of the double transgenic
mice (B6.dnMEK/CD2-rtTA). This allowed us to study the effects
172 B. Richardson et al.

of an induced T cell ERK signaling defect, caused by overexpressing

the dominant negative MEK, in these mice (28).
T cell specific expression of the dominant negative MEK and
the resulting reduction of phosphorylated ERK only in the pres-
ence of doxycycline were confirmed. As expected, inducing a T cell
ERK signaling defect resulted in reduced expression of Dnmt1, the
main maintenance DNA methylation enzyme that is known to be
regulated by ERK signaling. The expression of the methylation
sensitive genes Itgal (CD11a) and Tnfsf7 (CD70) were increased
in the spleen, similar to that observed in T cells from lupus patients.
Further, B6.dnMEK/CD2-rtTA mice given doxycycline devel-
oped anti-dsDNA antibodies, and gene expression profiling in the
spleen revealed an interferon-signature consistent with that
described in PBMCs from lupus patients (28). No clinical pheno-
type was observed in the B6 autoimmune-resistant genetic back-
ground. This emphasizes the importance of genetic susceptibility
in the pathogenesis of lupus and indicates that DNA demethyla-
tion is not sufficient to induce clinical autoimmunity in the absence
of a genetic susceptibility background. Indeed, this is similar to
what we observe in patients who develop drug-induced lupus as a
result of treatment with procainamide or hydralazine (both are
DNA methylation inhibitors). The majority of patients treated
with these medications develop autoantibodies, but only a small
fraction develops clinical drug-induced lupus. Those who go on to
develop clinical disease are arguably those who also carry appropri-
ate genetic susceptibility loci.
We therefore studied the effect of the dnMEK/CD2-rtTA on
mice with a more autoimmune susceptible genetic background by
crossing B6.dnMEK/CD2-rtTA with SJL mice and examining the
development of autoimmunity in B6.SJLdnMEK/CD2rtTA F1
mice (29). Female but not male mice developed glomerular IgG
deposition suggestive of immune-complex glomerulonephritis.
Platelet thrombi were also observed in the small vessels of the lungs
of female but not male mice. To determine if the development of
autoimmunity in female B6.SJLdnMEK/CD2rtTA F1 mice is due
to a gene dosage effect from the X chromosome (presumably due
to demethylation and reactivation of the inactive X chromosome)
or due to hormonal factors, both male and female B6.SJLdnMEK/
CD2rtTA F1 mice were neutered and implanted with time-release
pellets delivering placebo or a supra-estrus dose of estrogen.
Doxycycline induced anti-dsDNA antibodies in intact and neu-
tered, placebo-treated female transgenic mice. No anti-DNA anti-
bodies were found in doxycycline-treated intact or neutered,
placebo-treated male transgenic mice. Doxycycline induced even
greater amounts of anti-dsDNA antibodies in neutered females
given high dose estrogen, but none were detected in neutered
 8 Murine Models of Lupus Induced by Hypomethylated T Cells 173

males given high dose estrogen. No anti-DNA antibodies developed

in the absence of doxycycline treatment or without the transgenes.
These experiments confirm the X-chromosome gene-dose effect in
lupus as an important factor for the female predominance of the
disease. Indeed, doxycycline induced overexpression of the X-linked
methylation-sensitive gene Cd40lg on T cells in female but not
male mice, consistent with demethylation and reactivation of the
second X chromosome in the females.

1.4. Relevance At least six lines of evidence support the contention that the DNA
to Human Lupus hypomethylation model has relevance to human lupus. First, the
two drugs that most clearly cause a lupus-like disease in people,
procainamide and hydralazine, are T cell DNA methylation inhibi-
tors (30). Cloned murine Th2 cells treated with these drugs induce
a lupus-like disease identical to that caused by 5-azaC (23), sug-
gesting a mechanism by which they might cause lupus in humans.
Second, T cells from patients with active lupus have decreased lev-
els of total genomic dmC, similar to 5-azaC treated cells (31).
Third, T cells from patients with active lupus overexpress LFA-1
on an autoreactive T cell subset (14), and the overexpression is
associated with hypomethylation of the same sequences flanking
the CD11a promoter that demethylate following 5-azaC treatment
(17). Fourth, T cells from patients with active lupus have a selec-
tive defect in ERK pathway signaling, the pathway inhibited by
hydralazine (32), and inhibiting this pathway with hydralazine or
U0126 causes a lupus-like disease in the adoptive transfer model
and in the dnMEK/CD2-rtTA mouse model (2428). Fifth,
LFA-1 overexpressing T cells isolated from patients with active
lupus spontaneously kill autologous monocytes with a specificity
identical to experimentally hypomethylated T cells (14), and by the
same mechanisms (FasL, TRAIL, and TWEAK) as experimentally
hypomethylated T cells (33, 34). Finally, the X-linked methylation
sensitive gene CD40LG is overexpressed in CD4+ T cells from
female but not male lupus patients (35). The overexpression of
CD40LG in female patients with active lupus is associated with
hypomethylation of the CD40LG promoter sequence in both
alleles, allowing for transcriptional activity of the CD40LG
gene from the normally heavily methylated and inactive X chro-
mosome (35). These findings are consistent with a gene-dose effect
on the X-chromosome as demonstrated by the dnMEK/CD2-rtTA
mice experiments and suggest that chromosomal sex is perhaps
more important than hormonal sex in explaining the female pre-
dominance of lupus. Together, these studies strongly suggest that
similar mechanisms contribute to the development of autoimmu-
nity in the DNA hypomethylation models and in drug induced and
idiopathic human lupus.
174 B. Richardson et al.

2. Materials

2.1. Mice Young (68 weeks) female AKR and B10.A mice are obtained from
Jackson Labs, and DBA/2 mice from Charles River. The dnMEK
transgenic mice were generated by subcloning a dominant negative
MEK1 into the pTRE2 construct (Clontech), containing multiple
tetracycline response elements and a minimal CMV promoter.
These mice were then bred with CD2-rtTA mice, obtained from
Dr. Rose Zamoyska, to produce dnMEK/CD2-rtTA double trans-
genic mice. Doxycycline, added to the drinking water at a concen-
tration of 2 mg/ml, was used to induce expression of the dominant
negative MEK in vivo.

2.2. Cell Lines D10.G4.1 (D10) cells are obtained from the American Type
Culture Collection (ATCC). AE7 cells were obtained from Dr.
Ronald Schwartz.

2.3. 5-Azacytidine 5-Azacytidine (Aldrich) is dissolved in tissue culture media, typi-

cally at 0.258.0 mM, and is made fresh just before use (see Note 1).
The solution is filter sterilized before adding to culture. 5-aza-2-
deoxycytidine may also be used, and is more potent (see Note 1).
5-Azacytidine is potentially mutagenic and should be handled with
appropriate precautions.

2.4. IL-2 The IL-2 secreting T cell line MLA-144, obtained from the ATCC,
was cultured in RPMI 1640 supplemented with 3 % fetal calf serum
(FCS). Three times a week the cells were sedimented by centrifu-
gation, the conditioned media filtered to remove any remaining
cells, and then stored frozen at 20 C.

2.5. Media RPMI-1640 supplemented with 10 % heat-inactivated fetal calf

serum (FCS), 40 % IL-2 containing conditioned media, 2 mM glu-
2.5.1. Polyclonal T Cells
tamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and
5 105 M 2-mercaptoethanol.

2.5.2. D10 Cells Clicks medium supplemented with 10 % FCS, 40 % IL-2 contain-
ing conditioned media, 2 mM glutamine, 100 IU/ml penicillin,
100 mg/ml streptomycin, and 5 105 M 2-mercaptoethanol.

2.5.3. AE7 Cells 50 % Clicks medium/50 % RPMI 1640 supplemented with 10 %

FCS, 40 % IL-2 containing conditioned media, 2 mM glutamine,
100 IU/ml penicillin, 100 mg/ml streptomycin, and 5 105 M
2-mercaptoethanol.
 8 Murine Models of Lupus Induced by Hypomethylated T Cells 175

3. Methods

3.1. Cells Spleens are removed from young (6- to 8-week old) female
and 5-Azacytidine DBA/2 (H-2d) mice and dissociated with forceps followed by
Treatment forcing through a sterile disposable plastic screen with a syringe
piston. Splenocytes are then isolated by density gradient centrifu-
3.1.1. Polyclonal CD4+ T
gation through Lympholyte-M (Cedarlane). CD8+ cells are
Cell Lines
depleted with magnetic beads (Miltenyi) according to the manu-
facturers instructions, then 106 CD4+ cells are cultured in 2 ml of
IL-2 containing media (see Subheading 2) and stimulated with
either 106 irradiated (3000R) allogeneic splenocytes (e.g.,
C57BL/6, H-2b) or 5 mg/ml Concanavalin A (Pharmacia) using
flat-bottom 24-well plates. The cultures are maintained at 37 C
in 5 % CO2 and humidified atmosphere, and rocked on a rocker
platform (Bellco) at 56 cycles/min (see Note 2). The lines are
maintained by the addition of fresh IL-2 containing media every
23 days, and restimulating every 710 days, using ~106 cells/
well and equal numbers of irradiated allogeneic splenocytes or
0.51.0 106 irradiated syngeneic splenocytes + 1 mg/ml
Concanavalin A, as appropriate. One day after restimulation the
cells are treated with 5-azaC and 6 days later the cells tested for
autoreactivity and/or used for adoptive transfer. The cells should
also be tested for CD4 and CD8 expression by flow cytometry at
this point to exclude overgrowth by CD8+ cells.

3.1.2. D10 Cells
D10.G4.1 (D10) cells (American Type Culture Collection) are
maintained in IL-2 containing media (see Subheading 2) using flat-
bottomed 24-well plates and a rocker platform as for polyclonal cells.
The line is maintained by challenging 0.11.0 106 D10 cells with
5 105 irradiated (3000R) AKR splenocytes and 100 mg/ml conal-
bumin (Sigma) every 710 days. The D10 line contains an autoreac-
tive subset (see Note 3) and must be subcloned by limiting dilution
and a nonautoreactive subclone selected prior to use. The cells are
treated with 5-azaC and used for functional characterization or given
in adoptive transfer at least 6 days after treatment.

3.1.3. AE7 Cells
AE7 cells are maintained in IL-2 containing media (see
Subheading 2) and stimulated weekly with irradiated syngeneic
(B10.A) splenocytes and antigen (100 mg/ml pigeon cytochrome
C) on a rocker platform as described for polyclonal CD4+ cells and
D10 cells. To induce autoreactivity the cells are treated with 5-aza-
2-deoxycytidine (see Note 4) and used 6 days later.
176 B. Richardson et al.
3.1.4. Variations
3.2. Autoreactivity
Assays
3.2.1. Proliferation Assays
3.2.2. Cytotoxicity Assays
3.3. Adoptive Transfer
of Autoreactive Cells
3.4. IgG, IgM, and
Anti-DNA Antibody
Assays
See Note 5.
For D10 cells, 2 104 treated or untreated cells are cultured with
graded numbers (210 104) of irradiated syngeneic (AKR) sple-
nocytes in 200 ml of the same media but without IL-2, with or
without 100 mg/ml conalbumin, using round-bottom 96-well
plates. Proliferation is tested 45 days later by adding 1 mCi triti-
ated thymidine/well and 6 h later determining 3H incorporation
into DNA. Polyclonal CD4+ T cells are similarly tested, using
5 104 T cells and ~105 irradiated syngeneic splenocytes/well
(range 5 1045 105), using allogeneic splenocytes or Conca-
navalin A as appropriate for the positive control. In all cases deter-
minations are performed in triplicate or quadruplicate.
For D10 cells, thioglycollate elicited syngeneic (AKR) M are labeled
with 100 mCi 51Cr in 1 ml of RPMI/10 % FCS for 1 h 37 C in a
round-bottom culture tube. The cells are washed, then 5,000 labeled
M cultured with 125,000 D10 cells with or without 100 mg/ml
conalbumin in a total volume of 200 ml of sterile media lacking IL-2,
using round-bottom microtiter plates. 18 h later chromium release
is measured using a scintillation spectrometer (33). AE7 killing
assays are performed similarly, except that an effectortarget ratio of
10:1 is used, and the antigen (positive control) is 100 mg/ml pigeon
cytochrome C. Splenocyte killing assays are similarly performed,
using an effectortarget ratio of 25:1. Percent cytotoxicity is calcu-
lated as [(experimental background release)/(total incorpora-
tion background release)] 100 (20) (see Note 6).
All adoptive transfer models are performed similarly. The treated
cells are washed, dead cells removed by centrifugation through
Lympholyte M, then 5 106 viable cells are suspended in 0.2 ml
sterile PBS and injected into the tail vein of young (<12 weeks)
syngeneic female mice, using a 26-gauge needle. A total of six
injections are performed, spaced 2 weeks apart. The rationale for
repeated adoptive transfers derives from the observation that
5-azaC induced autoreactivity is self-limited (12). Four weeks after
the last injection the mice are euthanized and studied for the devel-
opment of serologic and histologic evidence of autoimmunity. The
development of proteinuria and hematuria may be monitored by
holding Chemstrips (Boehringer Mannheim) under the mouse
while picking it up (see Note 7).
Total serum IgG and IgM concentrations are measured using
Immulon 4 plates (Dynatech Laboratories) coated with 2.5 mg
anti-mouse IgG or IgM (Sigma) in 100 ml 0.01 M PBS, pH 7.4 for
18 h at 4 C. The plates are washed 3 with PBS containing 0.05 %
Tween 20, then 200 ml of PBS supplemented with 3 % BSA, 0.1 %
gelatin and 0.05 % Tween 20 are added and incubated 2 h 23 C.
 8 Murine Models of Lupus Induced by Hypomethylated T Cells 177

Serum samples or purified standards (murine IgG or IgM, from

Sigma) are diluted to the desired concentrations in PBS containing
3 % BSA and 0.1 % gelatin, added to the wells and incubated 18 h
at 4 C, then washed 3. 100 ml horseradish peroxidase (HRP)-
conjugated goat anti-mouse IgG (heavy and light chains) or IgM
(m chain specific) are added at a final dilution of 1:2,500 in PBS
containing 0.05 % Tween 20 and incubated for 2 h at room tem-
perature. The plates are developed using Sigma Fast tablets (Sigma)
according to the manufacturers instructions.
Anti-ssDNA and anti-dsDNA titers are determined by coating
Immulon 4 plates with 2.5 mg purified ssDNA (Sigma) or dsDNA
(cesium chloride-purified KS+-SV2CAT plasmid) in 100 ml 0.01 M
PBS, pH 7.4 for 18 h at 4 C. The protocols used are the same as
for the immunoglobulin ELISAs described above, except for coat-
ing the wells. HRP-conjugated goat anti-mouse polyvalent (IgG,
IgM, IgA) antibody (Sigma) is again used as the secondary anti-
body before developing with Sigma Fast tablets. Positive controls
should include pooled serum from >6 month old female NZB/W
mice or >5 month old MRL/lpr mice (see Note 8).
For the experiments performed in the dnMEK/CD2-rtTA
mice, anti-dsDNA antibody was detected using an ELISA kit
(Alpha Diagnostic Intl. Inc.).

4. Notes

1. 5-Azacytidine: 5-Azacytidine and 5-aza-2-deoxycytidine are

unstable in aqueous media (18), and must be prepared just
before use. The purchased chemicals have some variability in
potency, and each lot should be tested. Typical concentrations
for 5-azacytidine are 0.258.0 mM, with 1 mM most often
being effective. 5-aza-2-deoxycytidine is more specific for
DNA methylation inhibition and is also more potent (18), so
lower concentrations may be used. Both compounds inhibit
both DNA methylation and DNA synthesis, and concentra-
tions inhibiting DNA methylation are only slightly lower than
concentrations inhibiting DNA synthesis (18). Further, treated
cells must undergo 12 cycles of cell division for the changes in
gene expression to occur (18), highlighting the importance of
establishing optimal concentrations. Significant cell death also
occurs during treatment. Changes in T cell gene expression are
typically seen 36 days after treatment, and kinetic analysis
should be performed in order to determine the optimal time.
2. Polyclonal Cell Lines: Cellcell contact is maximized in flat-
bottom culture plates by using a rocker platform, and the cul-
tures should be rocked for at least 24 h following stimulation.
178 B. Richardson et al.

3. D10 Cells: With prolonged culture, D10 cells tend to lose the
restriction for antigen and proliferate to syngeneic APC with-
out added antigen. The mechanism is unknown, but adoptive
transfer of these cells does not induce autoimmunity (Bruce
Richardson, MD, PhD, University of Michigan). The cells also
tend to lose the requirement for IL-2 for sustained growth
over time, causing high backgrounds in proliferation assays.
Consequently, it is necessary to repeatedly subclone this line
and select antigen-specific cells. Alternatively, multiple aliquots
of quality tested subcloned cells may be stored in liquid nitro-
gen, and thawed as needed.
4. AE7 Cells: AE7 cells are more refractory to the induction of
autoreactivity than normal T cells or D10 cells. Treatment with
5-aza-2-deoxycytidine is required, and concentrations up to
8 mM are sometimes needed (22). Also, in our hands, prolif-
eration assays are less reliable than cytotoxicity assays for both
antigen reactivity and autoreactivity.
5. Variations: Activated T cells can be modified with other DNA
methylation inhibitors, or by transfection as needed. Our
group has found D10 cells to be the best suited for these stud-
ies, and have successfully compared procainamide with
N-acetylprocainamide, and hydralazine with phthalazine in this
model (23). Similarly, we have used the ERK pathway inhibitor
U0126 (24) and used D10 cells transfected with CD18 (15).
Other modifications of the cells may be similarly tested.
6. If desired, MHC specificity of the autoreactivity assays may be
tested using monoclonal antibodies to the relevant class II
MHC molecules, or congenic mouse strains.
7. Following injection of 5-azaC treated polyclonal CD4+ T cells
from DBA/2 mice, hematuria is first seen between weeks 1
and 3, and usually lasts 714 days then resolves. Proteinuria,
defined as >30 mg/dl, was more persistent. Immunofluorescent
evidence of renal Ig deposition correlates with active hematu-
ria and resolves at later time points.
8. Sometimes the control sera give a relatively high background
in the anti-DNA ELISAs. The specificity of the ELISAs may be
tested by adding 5 mg/ml of soluble ss-DNA or ds-DNA as
appropriate to replicate wells. Lack of inhibition is indicative of
nonspecificity, while inhibition is indicative of autoantibodies.

References
1. Attwood JT, Yung RL, Richardson BC (2002)
DNA methylation and the regulation of gene
transcription. Cell Mol Life Sci 59:241257
2. Li E, Bestor TH, Jaenisch R (1992) Targeted
mutation of the DNA methyltransferase gene
results in embryonic lethality. Cell 69:
915926
3. Okano M, Bell DW, Haber DA, Li E (1999)
DNA methyltransferases Dnmt3a and Dnmt3b
are essential for de novo methylation and mam-
malian development. Cell 99:247257
4. Taylor SM, Jones PA (1979) Multiple new
phenotypes induced in 10T1/2 and 3T3 cells
treated with 5-azacytidine. Cell 17:771779
 8 Murine Models of Lupus Induced by Hypomethylated T Cells
5. Golbus J, Palella TD, Richardson BC (1990)
Quantitative changes in T cell DNA methyla-
tion occur during differentiation and ageing.
Eur J Immunol 20:18691872
6. Young HA (1996) Regulation of interferon-
gamma gene expression. J Interferon Cytokine
Res 16:563568
7. Santangelo S, Cousins DJ, Winkelmann NE,
Staynov DZ (2002) DNA methylation changes
at human Th2 cytokine genes coincide with
DNase I hypersensitive site formation during
CD4(+) T cell differentiation. J Immunol
169:18931903
8. Floess S, Freyer J, Siewert C et al (2007)
Epigenetic control of the foxp3 locus in regula-
tory T cells. PLoS Biol 5:e38
9. Young HA, Ghosh P, Ye J et al (1994)
Differentiation of the T helper phenotypes by
analysis of the methylation state of the IFN-
gamma gene. J Immunol 153:36033610
10. Lu Q, Wu A, Ray D, Deng C, Attwood J,
Hanash S, Pipkin M, Lichtenheld M,
Richardson B (2003) DNA methylation and
chromatin structure regulate T cell perforin
gene expression. J Immunol 170:51245132
11. Richardson BC, Liebling MR, Hudson JL (1990)
CD4+ cells treated with DNA methylation inhib-
itors induce autologous B cell differentiation.
Clin Immunol Immunopathol 55:368381
12. Richardson B (1986) Effect of an inhibitor of
DNA methylation on T cells. II. 5-Azacytidine
induces self-reactivity in antigen-specific T4+
cells. Hum Immunol 17:456470
13. Richardson B, Powers D, Hooper F, Yung RL,
ORourke K (1994) Lymphocyte function-
associated antigen 1 overexpression and T cell
autoreactivity. Arthritis Rheum 37:13631372
14. Richardson BC, Strahler JR, Pivirotto TS et al
(1992) Phenotypic and functional similarities
between 5-azacytidine-treated T cells and a T
cell subset in patients with active systemic lupus
erythematosus. Arthritis Rheum 35:647662
15. Yung R, Powers D, Johnson K et al (1996)
Mechanisms of drug-induced lupus. II. T cells
overexpressing lymphocyte function-associated
antigen 1 become autoreactive and cause a
lupuslike disease in syngeneic mice. J Clin
Invest 97:28662871
16. Kaplan MJ, Beretta L, Yung RL, Richardson
BC (2000) LFA-1 overexpression and T cell
autoreactivity: mechanisms. Immunol Invest
29:427442
17. Lu Q, Kaplan M, Ray D, Zacharek S, Gutsch
D, Richardson B (2002) Demethylation of
ITGAL (CD11a) regulatory sequences in
systemic lupus erythematosus. Arthritis Rheum
46:12821291
179
18. Jones PA (1984) Gene activation by 5-azacyti-
dine. In: Razin A, Cedar H, Riggs A (eds)
DNA methylation biochemistry and biological
significance. Springer-Verlag, New York,
pp 165187
19. van der Veen FM, Rolink AG, Gleichmann E
(1982) Autoimmune disease strongly resem-
bling systemic lupus erythematosus (SLE) in
F1 mice undergoing graft-versus-host reaction
(GVHR). Adv Exp Med Biol 149:669677
20. Quddus J, Johnson KJ, Gavalchin J et al
(1993) Treating activated CD4+ T cells with
either of two distinct DNA methyltransferase
inhibitors, 5-azacytidine or procainamide, is
sufficient to cause a lupus-like disease in syn-
geneic mice. J Clin Invest 92:3853
21. Yung RL, Quddus J, Chrisp CE, Johnson KJ,
Richardson BC (1995) Mechanism of drug-
induced lupus. I. Cloned Th2 cells modified with
DNA methylation inhibitors in vitro cause auto-
immunity in vivo. J Immunol 154:30253035
22. Yung R, Kaplan M, Ray D et al (2001)
Autoreactive murine Th1 and Th2 cells kill
syngeneic macrophages and induce autoanti-
bodies. Lupus 10:539546
23. Yung R, Chang S, Hemati N, Johnson K,
Richardson B (1997) Mechanisms of drug-
induced lupus. IV. Comparison of procain-
amide and hydralazine with analogs in vitro
and in vivo. Arthritis Rheum 40:14361443
24. Deng C, Lu Q, Zhang Z et al (2003) Hydralazine
may induce autoimmunity by inhibiting extra-
cellular signal-regulated kinase pathway signal-
ing. Arthritis Rheum 48:746756
25. Scheinbart LS, Johnson MA, Gross LA, Edelstein
SR, Richardson BC (1991) Procainamide inhibits
DNA methyltransferase in a human T cell line.
J Rheumatol 18:530534
26. Mevorach D, Zhou JL, Song X, Elkon KB
(1998) Systemic exposure to irradiated apop-
totic cells induces autoantibody production.
J Exp Med 188:387392
27. Walport MJ (2000) Lupus, DNase and defec-
tive disposal of cellular debris. Nat Genet
25:135136
28. Sawalha AH, Jeffries M, Webb R et al (2008)
Defective T-cell ERK signaling induces inter-
feron-regulated gene expression and overex-
pression of methylation-sensitive genes similar
to lupus patients. Genes Immun 9:368378
29. Strickland FM, Hewagama R, Lu Q, Johnson
K, Webb R, Sawalha A, Richardson BC (2010)
Both Estrogen and female sex contribute to
disease development in an epigenetic model of
lupus. Lupus 19:91
30. Cornacchia E, Golbus J, Maybaum J, Strahler
J, Hanash S, Richardson B (1988) Hydralazine
180 B. Richardson et al.
and procainamide inhibit T cell DNA methyla-
tion and induce autoreactivity. J Immunol
140:21972200
31. Richardson B, Scheinbart L, Strahler J, Gross
L, Hanash S, Johnson M (1990) Evidence for
impaired T cell DNA methylation in systemic
lupus erythematosus and rheumatoid arthritis.
Arthritis Rheum 33:16651673
32. Deng C, Kaplan MJ, Yang J et al (2001)
Decreased Ras-mitogen-activated protein
kinase signaling may cause DNA hypomethyla-
tion in T lymphocytes from lupus patients.
Arthritis Rheum 44:397407
33. Kaplan MJ, Lewis EE, Shelden EA et al (2002)
The apoptotic ligands TRAIL, TWEAK, and Fas
ligand mediate monocyte death induced by autol-
ogous lupus T cells. J Immunol 169:60206029
34. Kaplan MJ, Ray D, Mo RR, Yung RL,
Richardson BC (2000) TRAIL (Apo2 ligand)
and TWEAK (Apo3 ligand) mediate CD4+ T
cell killing of antigen-presenting macrophages.
J Immunol 164:28972904
35. Lu Q, Wu A, Tesmer L, Ray D, Yousif N,
Richardson B (2007) Demethylation of CD40LG
on the inactive X in T cells from women with
lupus. J Immunol 179:63526358
 Chapter 9

Aspects of CNS Lupus: Mouse Models of Anti-NMDA

Receptor Antibody Mediated Reactivity
Czeslawa Kowal and Betty Diamond

Abstract
This chapter describes methods utilized in establishing a mouse model of neuropsychiatric lupus encompassing
both cognitive and emotional dysfunction, and a model of the influence of maternal antibody on the devel-
oping brain. The antibody of interest binds the N-methyl-D-aspartate receptor (NMDAR), a receptor for
glutamate that is a major excitatory neurotransmitter in the brain involved in synaptic plasticity, in memory
and learning, and in emotional responses.
We introduce basic concepts of these models and provide protocols for the following: (1) the induction
of anti-dsDNA, anti-NMDAR antibodies, (2) testing serum antibody titer by ELISA, (3) breaching blood
brain barrier (BBB) integrity with LPS and epinephrine, (4) passive transfer of pathology by injecting
human and mouse brain-reactive antibodies into adult mouse as well as injecting the antibody into gestat-
ing mice and transfer of antibody from dam to fetus, (5) blocking NMDAR-mediated pathogenicity
in vivo, (6) evacuation of blood from the brain by cardiac perfusion to preserve the brain for histology,
(7) evaluating injured/apoptotic neurons in brain histology, (8) preparing membrane-enriched brain
fractions for NMDAR analysis.

Key words: CNS lupus, Neuropsychiatric lupus, Anti-NMDA receptor antibodies, Blood brain barrier,
Pathogenic maternal autoantibody

1. Introduction

The past decade has witnessed a rapid grow of the information on

autoantibody-mediated pathogenicity in the central nervous sys-
tem (CNS). The putative role of brain-reactive antibodies in CNS
diseases has long intrigued scientists, but only recently the molecu-
lar aspects of this pathogenicity have begun to emerge. Experimental
animal models of neuronal pathology mediated by autoantibodies
which arise spontaneously or by immunization or are delivered by
passive transfer have contributed significantly to an understanding
of the molecular mechanisms leading to CNS pathology (1, 2).

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_9, Springer Science+Business Media New York 2012

181
182 C. Kowal and B. Diamond

Passive transfer studies in particular have provided direct proof of

the role of antibody in pathology. In lupus, research focusing on
brain-reactive antibodies is of particular significance as approxi-
mately 80 % of lupus patients experience neuropsychiatric symp-
toms. Among these, cognitive and emotional disturbances are most
common (3, 4). We have developed a mouse model of CNS lupus
that allows for study of both these symptoms (5, 6). First, we
identified a peptide mimetope of dsDNA, the pentameric consen-
sus sequence D/EWE/DYS/G (7). This sequence is present
within both the NR2A and NR2B subunits of the NMDAR. We
immunize mice with a multimerized form of the DWEYS sequence,
a decapeptide octamerized on a poly-L-lysine backbone (MAP-
DWEYSVWLSN) and MAP-core, polylysine backbone without
peptide, as control. MAP-DWEYSVWLSN immunized mice
develop elevated serum titers of cross-reactive, anti-dsDNA, anti-
NMDAR antibodies, while MAP-core immunized mice do not
(8). The brain of the adult animal is protected from harmful effects
of the anti-NMDAR antibodies present in the circulation by the
BBB, a complex multifunctional, multicellular system of brain cap-
illaries that shields the brain from harmful plasma contents and
that allows nutrient transport to the brain and removal of the
metabolites from the brain (9). The BBB consists of three major
components: endothelial cells, astrocytes, and pericytes (10). In
contrast to endothelial cells in the periphery, endothelial cells in
the brain are connected by a web of proteins that comprise the
tight junction that prevents cells and large molecules from entering
freely into brain parenchyma. This physical barrier of high electri-
cal resistance is additionally sealed by astrocytic foot processes. The
fact that the pericyte is involved in the BBB has been known for a
long time, but its crucial role in controlling the expression of pro-
teins that are critical in BBB function, both in endothelial cells and
in astrocytes, has been demonstrated only recently (11). The BBB
is essential for brain homeostasis and normal brain function.
However, the permeability of the BBB is increased in certain con-
ditions (12), among them infection or stress. We tested both con-
ditions in our animal model, as both are relevant in lupus patients.
We utilized bacterial lipopolysaccharide (LPS) as a surrogate for
infection, and epinephrine, the stress hormone, to evaluate stress
effects. Though we detected antibody extravasation in animals
injected with either LPS or epinephrine, we discovered a surprising
regional specificity of the impact of these two agents. LPS injection
leads to the extravasation of the antibody in the hippocampus caus-
ing a pronounced neuronal loss in that area of the brain and, con-
sequently, cognitive (memory) deficits (6). In contrast, epinephrine
injection affects mainly the amygdala, and neuronal loss in that
region leads to emotional disturbances that can be assessed in fear
conditioning experiments in animals (5). Mice given intravenous
transfer of lupus serum containing anti-dsDNA, anti-NMDAR
 9 Aspects of CNS Lupus 183

antibodies or purified IgG antibodies from those sera and subsequently

treated with LPS revealed cognitive abnormalities, while mice
given serum depleted of DWEYS reactivity did not (13).
The clinical literature reports an increased incidence of learn-
ing disabilities among children of lupus patients, specifically among
children of lupus mothers but not in the small number of lupus
fathers that were studied (1418). We hypothesized that this could
occur due to the permeability of the immature BBB to pathogenic
maternal antibodies during gestation. We tested this hypothesis in
a mouse model. We immunized female BALB/c mice with MAP-
DWEYSVWLSN peptide and Map-core and two additional MAP-
peptides (WSDYEVWLSN and AAAAAVWLSN) as control
antigens and tested the offspring of mice harboring anti-dsDNA,
anti-NMDAR antibodies during pregnancy. We discovered brain
abnormalities with a thinned cortical plate during fetal develop-
ment that resulted in an impairment in reflexes in newborn pups
and in cognitive tasks dependent on cortical function in young
animals in MAP-DWEYSVWLSN immunized mice but not in
MAP-core immunized mice or mice immunized with control
MAP-peptides. We concluded that maternal antibody can alter
brain development during gestation (19).
Antibodies with this cross-reactive specificity are also present in
serum and CSF of lupus patients and can be extracted from brain tissue.
Antibody titers in CSF correlate with symptomatology (2022).
The following are commonly used immunological and bio-
chemical assays used in these studies. We hope that experimental
details of the animal models of CNS lupus provided here that are
not published due to the limited space in most of the scientific
journals would be helpful not only in lupus studies but more gen-
erally. Most of the equipment described here can be replaced by
equipment routinely used in any laboratory. However, finding the
proper reagents and protocols are time-consuming, so anticipating
the usefulness of this information we specify the reagents we use,
with the exception of truly common ones.

2. Materials

2.1. Induction Immunization. Inducing anti-dsDNA, anti-NMDAR antibodies

of Anti-dsDNA, for both the adult CNS lupus model and the model of maternal
Anti-NMDA Receptor pathogenic antibody transfer, the embryonal model.
Antibodies
by Immunization

2.1.1. Immunization Animals. 6 8 week old BALB/c female mice (see Note 1).
Equipment. Eppedorf centrifuge, two pairs of glass syringes and
3-way Luer-lock stopcock for emulsifying the antigens, glass
184 C. Kowal and B. Diamond

capillaries for bleeding animals, eppendorf tubes, 1 ml Luer-lock

plastic syringes for immunization, 26 G 3/8 needle.
Reagents and buffers. MAP-DWEYSVWLSN and MAP-core, the
MAP-WSDYEVWLSN or MAP-AAAAAVWLSN control peptides
(see Note 2), Complete Freunds Adjuvant, Incomplete Freunds
Adjuvant, saline, Dulbeccos phosphate buffered saline (PBS),
dimethyl sulfoxide (DMSO), saturated (hydroxymethyl)amin-
omethane (Tris) base (see Note 3).

2.2. Testing Serum ELISA assays. The following protocols adapted from Scripps
Antibody Titer Research Laboratory Manual (La Jolla, CA) for phage combinato-
by ELISA rial libraries are used in our laboratory for the analysis of serum
titers from immunized mice as well as for the analysis of patients
sera for the presence of anti-dsDNA, anti-NMDAR antibodies. We
use these protocols with slight modifications for other antigens, for
quantification of immunoglobulin in the serum human and in
other media and for an inhibition ELISA.

2.2.1. ELISA Assays Equipment. Costar 3690 plates (see Note 4), polyvinyl chloride
plates (see Note 5), ELISA plate reader (see Note 6), ELISA plate
washer (see Note 7), multichannel pipettes and plastic tips, any
kind of plastic reagent reservoir.
Reagents and buffers. Antigens used in immunization (see
Subheading 2.1.1), DWEYS peptide, DWEYSVWLSN peptide,
extracellular domain of the NMDARrecombinant protein (see
Note 8), calf thymus double stranded DNA (dsDNA) (see Note
9), 0.1 M NaHCO3, pH 8.6 (see Note 10), PBS-Tween 20, 0.05 %
(PBS-T), PBS, 1 % bovine serum albumin (BSA) in PBS (see Note
11), alkaline phosphatase (AP)-labeled secondary antibody (see
Note 12), AP substrate, AP substrate buffer: 0.001 M magnesium
chloride (MgCl2) plus 0.05 M sodium carbonate (Na2Co3) (see
Note 13).

2.3. Opening the BBB We based our experiments of the opening of the BBB on the
by LPS and by groundbreaking work done by Bank and colleagues (23, 24).
Epinephrine in NPSLE There is a significant strain and age dependent sensitivity to LPS
Mouse Model toxicity so the conditions described here are for young BALB/c
mice. This is still work in progress so here we present effective pro-
cedures but by no means the final ones.

2.3.1. Opening the BBB Animals. BALB/c female mice immunized with MAP-
DWEYSVWLSN peptide and with MAP-core.
Reagents and buffers. Bacterial LPS (see Note 14), epinephrine
(see Note 15), saline.
Equipment. Water bath sonicator, freezing vials, eppendorf tubes,
1 ml plastic syringes for injections.
 9 Aspects of CNS Lupus 185

2.4. Passive Transfer Passive transfer studies are an integral part of any animal model of
of Pathology antibody-mediated disease. We started by injecting of R4A, a mono-
by Intravenous clonal antibody that binds dsDNA and NMDAR, and the isotype
Injection of Human control antibody directly into mouse cortex and hippocampus using
and Mouse Brain- stereotaxic coordinates. Mice exposed to NMDAR-reactive anti-
Reactive Antibodies body but not control mice analyzed 1 and 2 days post injection
into Adult Mouse showed neuronal loss (25). Similar results were obtained by direct
and into the Embryo injection of lupus IgG affinity purified on DWEYSVWLSN peptide
via Maternal column, with normal IgG purified on a protein G column as con-
Circulation at Different trol. We also tested and confirmed NMDAR-mediated pathogenic-
Gestational Stages
ity by direct injection to the brain of undiluted, NMDAR-reactive
CSF from a lupus patient and normal CSF as control as well as anti-
body purified from the frozen brain tissue of a lupus patient with
neuropsychiatric symptoms and normal IgG subjected to the same
procedures (13, 25). We then established a more physiological
model and intravenously injected NMDAR-reactive antibodies
from lupus patient purified on protein G and antibodies from simi-
larly treated normal serum. In the same model, we intravenously
injected the same lupus serum depleted of NMDAR-reactivity on a
DWEYSVWLSN peptide column as control. The animals were
treated subsequently with LPS to compromise the BBB. Mice that
received pathogenic, NMDAR-specific antibody showed neuronal
deficits primarily in the hippocampus and, consequently, deficits in
hippocampus-dependent cognitive tasks (13). For the embryonal
model, we injected timed-pregnant mice with anti-dsDNA, anti-
NMDAR-reactive antibody, R4A, and isotype control on day E12
and analyzed the embryonal brain by histology at E15, just before
birth and postnatally. We observed cortical abnormalities and behav-
ioral deficits in the offspring of mice exposed to the anti-NMDAR
antibody in utero but not in control animals. Similar results were
obtained in fetuses exposed to human monoclonal anti-dsDNA,
anti-NMDAR reactive antibody, G11, using monoclonal B1 anti-
body that did not bind these antigens as a control (19).

2.4.1. Passive Transfer Animals. Adult male C57BL/6 (direct intraparenchymal

injection), adult female BALB/c (intravenous injection).
Equipment. Hollow Fiber Bioreactor and glucose level monitoring
system (see Note 16), or any currently used system in the laboratory
for growing monoclonal antibody, 70 % alcohol spray, alcohol wipes,
20 ml syringes, glass columns, centrifuge tubes, sterile pipettes, ste-
reotaxic frame, centrifuge, ultracentrifuge, plastic tubes, small syringes
with 2730 G 1/2 needle for intravenous injection and 25 G 3/8
for intraperitoneal injection, thin glass capillaries, precision peristaltic
pump, all equipment for ELISA (see Subheading 2.2.1).
Reagents and buffers. Low IgG fetal bovine serum (FBS), serum-
free buffer and serum replacement (see Note 17), PBS, Protein G
sepharose or Protein G spin columns (see Note 18), 0.1 M glycine
pH 2.5 with and without 0.15 M sodium chloride, Ringers solution,
186 C. Kowal and B. Diamond

saline, 0.3 mg/ml LPS (see above), protein G slurry or Nunc spin
column, Amicon centrifugal units, purified antibodies, sera, CSF,
all ELISA reagents (see Subheading 2.2.1).

2.5. Blocking NMDAR- In parallel to establishing the animal model of CNS lupus, we tested
Mediated the blocking of antibody-mediated damage as a potential future
Pathogenicity In Vivo therapy. We have successfully tested two blocking reagents, the
NMDARs specific blocker memantine (see Note 19) and a D-isoform
of DWEYS peptide (see Note 20) in a MAP-DWEYSVWLSN
immunized mouse (5). We are currently developing new blocking
reagents (26). We describe here a simple dose and injection sched-
ule that resulted in successful inhibition of pathology.

2.5.1. Blocking NMDAR- Animals. Immunized 68-week-old female BALB/c mice (as
Mediated Pathogenicity described in paragraph 3.1 in this chapter) (see Note 21 and
In Vivo Note 65).
Equipment. Insulin syringes (see Note 22), isoflurane anesthesia
machine (see Note 23), small volume, 0.250.5 ml, with a needle
gauge 2729.
Reagents. Ringers solution, memantine hydrochloride, D-DWEYS
peptide.

2.6. Evacuation Cardiac perfusion. In order to perform brain histology the blood
of Blood from the needs to be evacuated from the animals brain. We use transcardiac
Brain by Cardiac perfusion that is effective and relatively easy to learn.
Perfusion and Fixing
the Brain for Histology

2.6.1. Cardiac Perfusion Equipment. Anesthesia machine for isoflurane with oxygen tank
(see Note 23), peristaltic pump (see Note 24), proper tubing (see
Note 25) with the needle adaptor on one side, stainless steel con-
tainer for collecting the fluids during procedure, stainless steel
mesh to cover the container, 27 G 3/8 needle, rongeur for remov-
ing the skull (see Note 26), surgical scissors (see Note 27), spatula,
50 ml conical tubes, eppendorf tubes, glass capillaries, small slotted
spoon (see Note 28).
Reagents and buffers. Pre-perfusion buffer: 0.9 % sodium chloride
(NaCl), 0.5 % sodium nitrate, 1,000 U Heparin (see Note 29);
perfusion buffer: 4 % paraformaldehyde, 0.1 M sodium phosphate
buffer (PB) pH 7.4 (see Note 30); 30 % sucrose (see Note 31),
PBS, isoflurane.

2.7. Evaluating In histology, we focus on basic procedures that are essential in

Injured/Apoptotic detecting antibody-mediated brain pathogenicity. Variations of
Neurons and these methods are available in the literature and we use some of
Immunoglobulin them with only slight modifications. However, details of these pro-
Deposition by Brains cedures such as antibody source and concentration used in binding,
Histology type of blocking buffer and time of reaction are of value and may
 9 Aspects of CNS Lupus 187

be crucial to a successful outcome. We describe here methods for

detecting immunoglobulin deposition in brain tissue and methods
detecting stressed/dying neurons by FluoroJade, and apoptotic
neurons by activated Capase 3. Though we also use Nissl-cresyl
violet staining of nucleic acids to detect picnotic structure of dying
neurons, the staining protocol is as commonly used.

2.7.1. Histology Equipment. Cryostat or microtome for frozen tissue or paraffin

embedded tissue, vibratome for fresh fixed tissue (see Note 32);
microscope (see Note 33), microscope slides (see Note 34), glass
chambers and slide racks for washing the slides, humidified, light-
proof, flat chamber for binding assays using slides, ceramic dish or
tissue culture dish for free floating sections binding (see Note 35),
camel hair brushes, tissue culture dishes with different number of
wells for collecting sections, hydrophobic slide marker, vacuum
suction system, coverslip for microscope slides.
Buffers and reagents. PBS, PBS-T (see Subheading 2.2.1 buffers),
PBS-T 0.2 % Triton X-100, 1 % BSA/PBS, 1 % BSA/PBS 0.2 %
Triton X-100, phosphate buffer in saline (PB-Sal) pH 7.4
(see Note 36), mounting media and M-1 embedding matrix for fro-
zen sectioning (see Note 37), nail polish, 0.06 % potassium perman-
ganate (KMnO4) (see Note 38), ethanol (EtOH): 100 and 70 %,
0.0010.0001 % of FluoroJade (see Note 39), ddH2O, xylene.

2.8. Preparing Membrane-enriched brain fractions. We searched the literature and

Membrane-Enriched compiled the information to have the protocol that gives good
Fractions of Brain yield, is relatively easy, and is very reproducible.
Lysate and Analysis
of NMDAR by Western
Blot

2.8.1. Membrane-Enriched Animals. BALB/c mice (see Note 40).

Brain Fractions Equipment. Brain extraction surgical tools (see equipment in
Subheading 2.6.1), small (2550 ml) metal beaker, cryogenic Dewar
for liquid nitrogen, small slotted spoon (see Subheading 2.6.1),
porcelain mortar and pestle, snap-cap or eppendorf tubes, glass tis-
sue homogenizing tube with Teflon pestle (see Note 41), portable
centrifuge (see Note 42), ultracentrifuge (see Note 43).
Reagents and buffers. Homogenizing buffer: 0.32 M sucrose,
10 mM TrisCl pH 7.4, 1 mM NaHCO3 pH 7.4, 1 mM MgCl2,
1 mM Na orthovanadate (see Note 44), 1 % protease inhibitors (see
Note 45); lysis buffer: 10 mM TrisCl pH 9, 150 mM NaCl, 0.5 %
TritonX, 1 % Na deoxycholate, 0.5 % SDS, 2 mM EDTA, 1 mM Na
orthovanadate, 1 % protease inhibitors (as above), 0.1 mM PMSF.

2.8.2. Western Blot For western blot (WB) we use Invitrogens (Carlsbad, CA) system
Analysis of NMDARs for electrophoresis and protein transfer and their gels and buffers.
188 C. Kowal and B. Diamond

The system is well designed, user friendly, and essentially error-proof.

We follow the manufacturers instructions, so we minimize details
of preparing the membrane for WB.
For detection we use LICOR (Lincoln, NE) infrared Odyssey
machine and their infrared-labeled secondary antibodies and block-
ing buffer. The infrared detection is sensitive and the signal is very
stable. It eliminates film usage and guessing of exposure time.
We provide information on the source of antibodies that work in
our hands and conditions for binding as not all the anti-NMDAR
specific antibodies work in WB.
Reagents and buffers. NuPAGE reagents: MES SDS running buf-
fer, transfer buffer and 412 % Bis-Tris gel, all from Invitrogen (see
Note 46). PBS and PBS-T buffer (see Subheading 2.2.1 above),
LICORs blocking buffer (see Note 47). Antibodies for western
blot analysis: mouse-anti-NR1 antibody from Molecular Probes
(Invitrogen, Carlsbad, CA), rabbit-anti-NR2A antibody from
Chemicon (Millipore, Billerica, MA), and rabbit-anti-NR2B anti-
body from Upstate (Millipore, Billerica, MA). Secondary antibod-
ies: anti-mouse and anti-rabbit infrared labeled antibodies from
LICOR. Nitrocellulose membrane (Stratagene, Santa Clara, CA).
Equipment. Novex Mini Cell electrophoresis chamber, XCell II Blot
Module for protein transfer, rotor/shaker and plastic containers for
incubating membrane and Odyssey Infrared Imager (LICOR).

3. Methods

3.1. Immunization 1. Pre-bleed the animals before immunization. Obtain about

50100 l of blood using glass capillary or any method of your
choice. Separate the serum by centrifuging at 10,000 g for
15 min at 4 C after deactivating serum complement at 37 C for
30 min and coagulating blood at 4 C. Store at 20 C until use.
2. Prepare antigens at 10 mg/ml in saline for immediate use or in
PBS for short storage at 4 C (see Note 48). A dose per mouse
is 100 g in 100 l of 50 % Complete Freunds Adjuvant in
saline for the first immunization and 50 % Incomplete Freunds
Adjuvant for boosts; add 1020 % for losses.
3. Preparing Complete Freunds Adjuvant/Incomplete Freunds
Adjuvant emulsion. Calculate the volume depending on the
number of mice to be immunized. The volume of the glass
syringe should be more than double (see Note 49). Connect
two syringes with a 3-way stopper, remove the plunger from one
syringe, secure the leakage by putting the stopper at the right
position, and put Complete Freunds Adjuvant or Incomplete
Freunds Adjuvant into the syringe while pressing the plunger in
 9 Aspects of CNS Lupus 189

the other syringe. Add 100 l extra for lubrication (you may
need more for bigger syringes). Remove the air bubbles using
the plunger and add the antigen in saline through the open
syringe. Secure the open syringe with the plunger and carefully
remove the air from the syringe by opening the stopper. Close
the stopper and start emulsification by pushing the solution
from one syringe to the other until emulsion is homogenous
and you start feeling the resistance (see Note 50). Test the emul-
sion by placing a drop on water in a small container. The prop-
erly prepared emulsion is thick and does not disperse on water.
4. Transfer emulsion for immunization by removing one glass
syringe and replacing it by Leur-lock 1 ml plastic syringe. Push
the proper amount of emulsion. Place 26 G 3/8 needle on the
top. Remove air by delicately tapping the syringe on the hard
surface. Check the air bubble in a syringe against good light
and press the air out through the needle.
5. First immunization. Inject BALB/c female mice intraperitone-
ally (see Note 51), with 100 l of emulsion/mouse. Wait for
2 weeks.
6. First boost. Repeat intraperitoneal injection of the same
amount of antigen in Incomplete Freunds Adjuvant. Wait for
2 weeks.
7. Second boost. Repeat intraperitoneal injection of the same
amount of antigen in Incomplete Freunds Adjuvant.
8. Bleeding. Obtain about 50100 l of blood at desired time
point. Test the serum titer (see Note 52).

3.2. ELISA Assays 1. Prepare 0.1 M NaHCO3, pH 8.6 buffer in ddH2O.

2. Prepare the antigen in 0.1 M NaHCO3 buffer for coating the
plate. Concentration of antigens: 15 g/ml of all peptides,
10 g/ml of recombinant protein, and 100 g/ml of dsDNA
in 0.1 M NaHCO3, pH 8.6 buffer.
3. Coat the Costar 3690 plate with any of the above antigens by
adding 25 l per well using a multichannel pipette and plastic
reagent reservoir.
4. Incubate protein coated plates overnight at 4 C or 1 h at
37 C; dry coat dsDNA (see Note 53).
5. Dump the contents of the plate into the sink. Pat dry on the
paper towels.
6. Block plate with 50 l/well of 1 % BSA/PBS for 1 h at
37 C.
7. Dilute serum/primary antibody in 0.2 % BSA/PBS using poly-
vinyl chloride plate (see Note 54).
8. Dump the blocking contents of the plate into the sink. Pat dry
on the paper towels.
190 C. Kowal and B. Diamond

9. Load serum/primary antibody sample diluted in 0.2 % BSA/

PBS, 25 l /well. Include positive and negative controls on
each plate; leave some wells with 0.2% BSA/PBS only.
10. Incubate at 37 C for 1 h.
11. Wash six times in PBS-T.
12. Load secondary antibody, usually 1:1,000 dilution, 25 l/well
in 0.2 % BSA/PBS.
13. Incubate at 37 C for 1 h.
14. Wash six times in PBS-T.
15. Add the substrate (see Note 55)50 l/well.
16. Incubate at room temperature or 37 C. The temperature is
your choice, but be consistent.
17. For AP read OD at 405 nm, use the proper filters for other
detection system.

3.3. Opening
of the BBB by LPS
and by Epinephrine
3.3.1. Opening of the Blood
Brain Barrier Using LPS
1. Prepare LPS solution at 0.3 mg/ml in saline (see Note 14).
2. Inject BALB/c mice intraperitoneally using 3 mg/kg (see
Note 56). Observe the animals for septic effects. Use saline for
control animals. Wait for 48 h.
3. Repeat injection using the same dose (see Note 57). Wait for
the proper time for neuronal loss analysis or for behavioral
experiments. We have observed neuronal loss at day 7 and did
not see any increase in neuronal loss past 30 days after LPS
injection. Behavioral experiments were performed 48 weeks
after opening of the BBB.
4. Perform cardiac perfusion (described below) for histological
analysis of the brain at the conclusion of the experiment.

3.3.2. Opening of the Blood
Brain Barrier Using
Epinephrine
1. Prepare epinephrine solution in saline at the dose of
200 nM/100 l per mouse (see Note 58).
2. Inject BALB/c mice intraperitoneally using 200 nM/100 l
per mouse (see Note 59). Wait for 48 h.
3. Repeat injection using the same dose. Wait for the proper time
for neuronal loss analysis or for behavioral experiments. We
performed the same 7 and 30 day analysis as with LPS and see
similar neuronal loss but in different brain areas. Behavioral
studies were done 48 weeks after opening of the BBB.
4. Perform cardiac perfusion (described below) for histological
analysis of the brain at the conclusion of the experiment.

3.4. Passive Transfer
3.4.1. Production of
Monoclonal Antibodies
Using Hollow Fiber
Bioreactor
1. Grow monoclonal antibody in 10 % ultra low FBS in DMEM
medium using Petri dish until you obtain 1 108 hybridoma
cells.
2. Equilibrate the Hollow Fiber Bioreactor with PBS and DMEM
medium according to manufacturers instruction.
 9 Aspects of CNS Lupus 191

3. Pellet the cells, resuspend in the above medium, and inoculate

1 108 cells into the Bioreactor. Monitor glucose consumption
and change the medium when the glucose level reaches 2 g/l.
4. Start replacing the FBS for Chemically Defined Medium for
High Density Cell Culture (CDM HD) serum replacement in
serum free medium when the glucose consumption reaches
approximately 1 g/day. Exchange it gradually, first to 5 % FBS,
then to 2.5 %, and then to serum-free medium monitoring
glucose consumption, ensuring cell growth.
5. Harvest the supernatant when the medium is completely serum-
free. Check the concentration of the antibody by any available
method. Proceed to purify the antibody using protein G.

3.4.2. Purification 1. Prepare the column using Protein G sepharose for gravity
of Antibodies from Serum purification or use commercially available Protein G spin col-
and Culture Supernatant umns following manufacturers instruction. Wash the columns
and from Brain Tissue with Protein G binding buffer or PBS.
2. Filter (0.2 m) serum/supernatant and load the column. Do
not exceed column capacity.
3. Let the antibody flow through the column by gravity or spin
the column if using the Nunc system.
4. Wash the column with neutral buffer (PBS) until no protein is
detectable in the flowthrough.
5. Elute antibody using 0.1 M glycine pH 2.5 with or without
0.15 M sodium chloride (see Note 60) and immediately neu-
tralize each fraction using 2 M TrisCl pH 9.0 (see Note 61).
6. Dialyze or exchange buffer using Amicon filtration system to
experimental buffer or PBS.
7. Check the concentration of purified antibody using any avail-
able method (see Note 62). If the antibody concentration is
high, dilute it in PBS before use (see Note 63).
8. Test antibody binding by ELISA, remove the aggregates by
centrifugation at 1,000 x g for 10 min and filter-sterilize it.
For extraction of antibody from frozen brain tissue we followed
the excellent method of Owens et al. (27) and then purified IgG
using Protein G as above.

3.4.3. Passive Transfer Use sterile solutions for all of the injections.
of Pathogenicity in Mouse
1. For direct injection to the brain parenchyma use 15 g in
Model of CNS Lupus
12 l volume. Inject slowly (0.25 l/min) using stereotaxic
frame and coordinates. Use precision peristaltic pump and glass
capillaries.
2. For intravenous injection of timed-pregnant mice use 200
400 g of purified monoclonal antibody in 100 l (see
Note 64). For adult model we injected 2 mg of antibodies
192 C. Kowal and B. Diamond

purified from lupus NMDAR-reactive serum achieving a level of

the antibody similar to that in immunized mice. Both mice
were injected by retroorbital injection.
3. For breaching of the BBB wait for 5 min after antibody injec-
tion and inject LPS or epinephrine (see above). Wait for proper
time for animal analysis. Embryos from pregnant mice injected
at E12 were analyzed on E15, E18, and postnatally. Adult mice
were analyzed as described above (see Subheading 3.3).

3.5. Blocking NMDAR- 1. Prepare blocking reagents: memantine hydrochloride at 5 mg/

Mediated ml and D-DWEYS peptide at 2 mg/ml in Ringers solution.
Pathogenicity In Vivo 2. Bring the animals, one cage at a time, to reduce stress. Return
the cage to the previous location after treatment.
3. Anesthetize animal using low level isoflurane (Subheadings 3.6,
step 4 and 5). Inject intravenously 100 l of blocking reagent.
Wait for 5 min.
4. Inject the proper amount of the BBB breaching agent, 5 mg/
kg of memantine or 200 micrograms/mouse of D-DWEYS
peptide.
5. Repeat intravenous administration of blocking agent every
24 h till the end of the 96 h experiment.
6. Repeat the BBB breach 48 h after the first injection.
7. Analyze the result by histology 48 h and 7 days after the sec-
ond insult to the BBB integrity and behavior and neuronal loss
at selected time points as in the immunized model (see
Subheading 3.3, above).

3.6. Cardiac Perfusion 1. Prepare the buffers, 50 ml each, pre-perfusion and 4 % para-
formaldehyde perfusion buffer/mouse.
2. Prepare all of the equipment: anesthesia machine, peristaltic
pump and surgical instruments.
3. Put 27 G 3/8 needle at the end of the Tygon tubing of the
peristaltic pump and the other end in 50 ml conical tube con-
taining pre-perfusion buffer. Flush small volume of pre-perfu-
sion buffer to remove the air from the tubing.
4. Preset the oxygen level at 1.51.7 ml/min and the isoflurane
at the higher level (44.5 is that higher level of isoflurane on
the anesthesia machine, no subheadings 44.5) at the start so
that the animal is anesthetized quickly (see Note 65).
5. Anesthetize the mouse (see Note 66).
6. Place the mouse nose cone (see Note 67) on the animal and
turn the switch to allow anesthetic delivery and continuing
anesthesia until the animal expires.
7. Open abdominal cavity at the edge of the rib cage. Make a
wide cut from one side to the other using sharp surgical scis-
sors avoiding any organ injury.
 9 Aspects of CNS Lupus 193

8. Carefully open thoracic diaphragm without cutting any vessel

or organ and get an access to the heart.
9. Clip or punch a tiny whole (see Note 68) in the right (darker)
ventricle, place the needle with pre-perfusion buffer in the left
ventricle and slowly start pumping the buffer. Set the pump at
level 2.
10. Pump 4045 ml of the buffer and switch the tubing to the 50 ml
conical tube containing fixative paraformaldehyde buffer.
11. Pump 4045 ml of paraformaldehyde buffer, reverse the pump,
and evacuate the paraformaldehyde back to 50 ml conical
tube.
12. Wash the tubing with water to remove remaining paraformal-
dehyde, let then some air in, and flush pre-perfusion buffer if
you are perfusing more than one animal.
13. Decapitate the animal, remove skin and muscles from the top
and around the scull. Cut open the scull and remove it using
rongeur or scissors and extract the brain by placing the spatula
underneath and releasing all connections. Put the brain in the
remaining 4 % paraformaldehyde and continue fixation for at
least 2 h (see Note 69).
14. Using slotted spoon remove the brain from fixative, rinse it
with PBS and for cryo-preserve equilibrate in 30 % sucrose
buffer. Wait until the brain sinks in, or equilibrate it for at least
24 h.
15. Freeze the brain in embedding medium (see Note 70). Place a
layer of embedding medium at the bottom of the plastic freez-
ing mold, place the mold on flat dry ice and cool it down; do
not let it freeze. Place the brain on the layer using tweezers,
cover it with O.C.T. and freeze it. Mark the tube for the orien-
tation for the cut in cryostat. Wrap it in aluminum foil and
freeze it at 80 C until use. Do not keep it for too long to
have the best quality of the tissue.

3.7. Histology Preparing brain sections. Select the proper thickness for the sec-
tions of the brain and the method of staining.
(a) For thin, 1020 m sections use cryostat or microtome and
Super Frost microscope slides. Equilibrate frozen tissue to cry-
ostat temperature before starting sectioning. You may brush
the slide with water for better adherence, but you will need to
dry these sections longer (see Note 71). Place thin sections on
microscope slides and let it adhere for at least 1 h. You can
freeze the slides in a slide cassette at 80 C until use.
(b) For thicker sections, use microtome or vibratome and free-
floating binding. Use M-1 embedding matrix for microtome
to freeze the brain and to adhere the brain to the platform for
194 C. Kowal and B. Diamond

sectioning. Use glue (we use Crazy Glue) to immobilize the

tissue to the vibratome platform. Select the proper thickness
and collect the sections in wells containing 0.1 M PB buffer.
The following are two general protocols for staining brain sec-
tions, using microscope slides and floating sections. Details for
particular staining are described bellowed that.

3.7.1. Staining Brain 1. Equilibrate the frozen slides to room temperature for at least
Sections Using Microscope 20 min if frozen.
Slides 2. Rehydrate the slide in PBS in a histology jar for 5 min.
3. Remove an excess of PBS by tilting the slide on the edge and
using Kimwipes or paper towel.
4. Make a circle around the section using hydrophobic slide
marker (see Note 72).
5. Place the slide in humidified, light-proof, flat chamber and put
the proper volume of blocking buffer (see Note 73) on a sec-
tion; usually 100 l is sufficient for mouse brain section.
6. Block for 30 min to 1 h at room temperature.
7. Remove blocking solution by using low vacuum suction or by
Kimwipe.
8. Apply primary antibody in the blocking buffer or in PBS-T,
same volume as above (see Note 74).
9. Incubate for 12 h at room temperature or overnight at 4 C
in a light-proof chamber (see Note 75).
10. Wash the slides in a battery histology jars filled with PBS.
Typically, wash 35 times for 10 min each time. Remove an
excess of washing buffer after final wash.
11. Apply secondary antibody in the blocking buffer or in PBS-T
(see Note 74).
12. Incubate for 0.51 h at room temperature.
13. Wash 35 times for 10 min each time. Remove an excess of the
washing buffer. For staining without DAPI move on directly
to Subheading 3.7.1 step 15.
14. For staining with DAPI, cover the tissue with 1 g/ml of
DAPI in PBS in 100150 l PBS, wait for 12 min, remove
DAPI solution, and move on to Subheading 3.7.1 step 15.
15. Apply the mounting medium near the tissue, place the glass
coverslip carefully on the top avoiding air bubbles and press
delicately to remove the excess of the mounting medium.
Drain an excess of the mounting medium by tilting the slide
on the edge and using Kimwipe or Whatman paper (see
Note 76). Seal the coverslip using nail polish.
16. Analyze sections under the microscope.
 9 Aspects of CNS Lupus 195

3.7.2. Free-Floating 1. Collect the sections in the tissue culture dish containing
Section Staining Used 0.1 M PB buffer, pH 7.4.
for Thicker Sections
2. Prepare the blocking solution (see Note 73).
for Detecting Antibody
Deposition in the Brain 3. Place the proper volume of blocking solution in the wells of
ceramic dish or in the tissue culture dish (see Note 35). Do not
fill up, as the binding is done with slow rotating shaker.
4. Using camel hair brush place the sections in the blocking solu-
tion containing wells.
5. Incubate at room temperature for 1 h with delicate rotating
shaking.
6. Transfer the sections to new wells containing primary antibody
(see Note 74).
7. Incubate at room temperature for few hours or overnight at
4 C on a slow rotating shaker.
8. Wash the sections by transferring them to the well containing
PBS washing buffer and incubate with rotating for 510 min.
9. Repeat three times.
10. Transfer the sections to the wells containing secondary anti-
body (see Note 74).
11. Incubate for 2 h at room temperature or overnight at 4 C.
12. Wash three times for 510 min as in step 8.
13. Mount on a glass or gelatinized slide as in 3.7.1 step 15. For
DAPI staining see step 14 in 3.7.1.
14. Analyze in the microscope.

3.7.3. Detecting Antibody Use floating method and 40100 m sections.

Deposition in the Brain
1. Block in PB-Sal buffer with two drops of Vectors blocking
reagent for 30 min.
2. Incubate with primary antibodies: biotinylated horse anti-
mouse or horse anti-human antibody (Vector Laboratories,
Burlingame, CA) at 1:200 dilution in PB-Sal buffer with two
drops of Vectors blocking reagent for 1 h.
3. Incubate with avidin-peroxidase complex at 1:100 dilution in
blocking solution (see above) for 1 h. Alternatively, use
AlexaFluor 488-labeled streptavidin (Invitrogen) at 1:500
dilution in blocking solution (see above) as a fluorescent detec-
tion system. In this case skip step 4 and proceed directly to
washing. Wash three times 5 min each.
4. Develop in diaminobenzidine (0.5 mg/ml) in the presence of
0.03 % hydrogen peroxide (H2O2) (see Note 77). Watch for
the proper intensity of the staining, 210 min.
5. Wash 5 min in ddH2O and mount.
196 C. Kowal and B. Diamond

3.7.4. Staining with 1. Prepare buffers: 0.06 % potassium permanganate (KMnO4)

FluoroJade [Done (see Note 38), ethanol (EtOH): 100 and 70 %, 0.0010.0001 %
Essentially as Described of FluoroJade (see Note 39), ddH2O, xylene.
(28, 29) with Slight
2. Allow tissue sections to dry on a slide at room temperature.
Modifications in time and
Concentration of Reagents] 3. Place the slide on staining rack and immerse in 100 % EtOH
for 3 min.
4. Immerse in 70 % EtOH for 2 min.
5. Wash in ddH2O for 2 min.
6. Immerse in 0.06 % KMnO4 solution for 1015 min with gentle
shaking.
7. Rinse in ddH2O for 2 min. Protect from the light from this
step on.
8. Incubate in 0.0010.0001 % of FluoroJade staining solution
for 30 min with gentle shaking in dark.
9. Wash in ddH2O for 2 min, three times.
10. Remove the slide from the rack and let dry flat on a paper
towel at room temperature. The best method in our experi-
ence is placing the slide under fan for about 20 min or until
completely dry.
You may leave it overnight at room temperature in darkness.
11. Immerse in xylene for 1 min.
12. Repeat three times.
13. Coverslip as above and analyze.

3.7.5. Detecting 1. Rinse tissue sections in PB-Sal buffer for 10 min.

Preapoptotic Neurons
Using Activated Caspase-3
2. Permeabilize in 0.2 % Triton X-100 in PB-S buffer containing
2 % BSA for 15 min.
3. Wash in PB-S buffer two times for 15 min.
4. Block in 2 % BSA in PB-S buffer for 1 h.
5. Incubate with rabbit anti-activated Caspase-3 antibody
(Pharmingen, San Diego, CA) in 2 % BSA in PB-S buffer over-
night at 4 C.
6. Wash two times in PBS buffer, 1 in PB-S buffer, 15 min
each.
7. Incubate with donkey anti-rabbit IgG-Texas red (Jackson
Immunochemicals, West Grove, PA) at 1:200 or with goat
anti-rabbit IgG-AlexaFluor 594 1:500 in 2 % BSA in PB-S buf-
fer for 45 min.
8. Wash three times in PB-S buffer 15 min each.
9. Mount on a slide, air dry and coverslip.
 9 Aspects of CNS Lupus 197

3.8. Membrane- 1. Snap-freeze freshly harvested brains using liquid nitrogen (see
Enriched Brain Note 78). For the whole brain use metal beaker or 50 ml coni-
Fractions and WB cal tube, for a fraction of a brain you may use slotted spoon or
Analysis of NMDARs eppendorf tube. Use cold reagents and tools through the
preparation.
3.8.1. Membrane-Enriched
Fractions 2. Make a powder of the frozen brain tissue in porcelain mortar
and pestle. Small fraction of a brain can be homogenized
directly.
3. Transfer the brain powder or small piece of the brain to the
glass homogenizing tube using ten times brain volume of
homogenizing buffer. For mouse brain, the volume will be
45 ml.
4. Homogenize tissue using Teflon pestle: 1015 strokes.
5. Transfer homogenate to spinning tube and spin at 1,000 g for
15 min. Save the supernatant.
6. Transfer the supernatant to high speed ultracentrifuge tube
and spin in 60 Ti rotor at 50,000 for 30 min or an equivalent
(see Note 43). Save the pellet.
7. Resuspend the pellet in 2 ml of homogenizing buffer and
repeat the spin (see Note 79). Save the pellet.
8. Resuspend the pellet in the lysis buffer (see Note 80) or freeze
the pellet at 80 C until use.
9. Check the protein concentration using any method that is
compatible with the buffer components or dialyze the product
against PBS (see Note 81).
10. Freeze the aliquots of the preparation at 80 C until use.

3.8.2. Western Blot 1. Prepare 1025 g of brain proteins in 1015 l total volume
Analysis of NMDARs Using in 1 loading buffer.
Membrane Enriched Brain 2. Heat 10 min at 70 C. Place on ice.
Fractions
3. Load the gel using protein standard and run the gel at 180
200 V (see Note 46) until the blue line reaches the bottom of
the gel.
4. Prepare the transfer system and transfer proteins at 30 V for
12 h. You may check the progress by checking the transfer of
the protein marker without disassembling the system.
5. Rinse the membrane with PBS and block it for 1 h at room
temperature in a blocking buffer (see Note 47).
6. Incubate membrane in primary antibody in a blocking buffer;
typically, overnight at 4 C with shaking. We use anti-NR1
antibody at 1:500 dilution, anti-NR2A at 1:200 dilution, and
anti-NR2B antibody at 1:500 dilution.
7. Wash the membrane 34 times 10 min each, in PBS-T with
shaking.
198 C. Kowal and B. Diamond

8. Incubate with infrared secondary antibody (see Note 82) at

1:5,0001:25,000 dilution for 0.51 h at room temperature.
9. Wash 34 times 10 min each, in PBS-T.
10. Develop using any sensitive system or check the binding on
Odyssey (see Note 83).

4. Notes

1. We have tested several mouse strains: A/J, AKR/J. DBA/2,

C57bl/6, C57bl/6 H2d and BALB/c, but only BALB/c and
C57bl/6 H2d haplotype gave the appropriate response (8).
The response is Ed restricted.
2. All peptides are custom made by AnaSpec (Fremont, CA).
MAP-core is commercially available from AnaSpec.
3. The solubility of Tris base in water is ~50 g/100 ml (25 C).
Simply put ddH2O in any plastic tube, add an excess of Tris
base (more than 0.5 g/ml), vortex it and leave it overnight.
There should be some Tris crystals on the bottom. Add more
Tris base if needed. Keep at room temperature.
4. Any plastic plate can be used for this assay; Costar 3690 plates
(Corning, NY) give an advantage of using only 25 l of reac-
tion volume thus saving serum and reagents without compro-
mising the sensitivity of the assay. The background is usually
lower on this plate. Assays are slightly more sensitive in 0.1 M
NaHCO3 than in PBS.
5. These are 96-well, u-bottom, non-protein-binding plates (BD
Falcon; Durham, NC, REF 353911) that we use for diluting the
primary antibodies for ELISA. The dilution can be done in this
plate ahead of time without losing antibody activity. It allows
using multichannel pipettes for loading the ELISA plates and
better control of time and volume in each well. Never dilute
serum/antibody directly on the ELISA plate. Binding can be
immediate and you will lose the sensitivity of the binding curve.
6. We have a PerkinElmer Multilabel Counter VICTOR3 and
appreciate its versatility but any ELISA reader can be used.
7. We use BioTek EL405 that has a built-in self-maintenance pro-
gram and is very reliable. However, any ELISA washer is fine,
as is manual washing.
8. Both NR2A and NR2B recombinant proteins were developed
in our laboratory. They were expressed in Escherichia coli and
purified using a French press and nickel columns. They give
excellent results in ELISA, but preparation and purification is
laborious and DWEYS peptides are a good replacement.
 9 Aspects of CNS Lupus 199

9. Prepare calf thymus dsDNA solution. Cut lyophilized calf

thymus (SIGMA, St. Louis, MO) to smaller pieces and dissolve
it in ddH2O overnight at 4 C with magnetic stirring. Next day
sonicate DNA to pieces less than 1 kb, aliquot and store at
20 C for longer time. You may keep the currently used solu-
tion at 4 C.
10. We prepare 0.1 M NaHCO3 in ddH2O and adjust pH to 8.6 if
necessary. The pH of 0.1 M NaHCO3 is very close to 8.6. We
filter-sterilize the solution using 0.2 m filter and keep it at
room temperature for a long time.
11. BSA comes in many forms and the effectiveness of blocking
may depend on the particular fraction. We use BSA Fraction V
heat shock from Roche Diagnostic (Indianapolis, IN) that is
good block in ELISAs and in histology. You may prepare 5 %
BSA/PBS or higher concentration, filter-sterilize, keep it at
4 C and dilute in PBS as desired.
12. We use anti-IgG AP-labeled secondary antibodies of different
kind and AP phosphatase substrate from SIGMA. AP gives a
lower background, but is relatively slow in developing. Horse
radish peroxidase (HRP) is faster but the background is higher.
You may try any system you like.
13. We prepare ten times (10) concentrated solution of each,
filter-sterilize, and keep them at room temperature indefinitely,
mixing the desired amount just before use. You will need 5 ml
of the buffer/substrate tablet which is enough for one Costar
plate (see below). For any other plate you will need 10 ml.
14. We use bacterial LPS from E. coli serotype 055:B5 from SIGMA
catalog number L 4524. We dissolve lyophilized LPS in ddH2O
at 1 mg/ml and sonicate it twice for 13 min at medium level
set in water bath sonicator, then aliquot it and store in freezing
vials at 20 C until use. Sonication increases the activity of
LPS (30). Never boil the LPS, as boiling destroys its
proinflammatory activity (31).
15. We use () Epinephrine (+) Bitartrate salt (SIGMA E4375).
16. We routinely produce monoclonal antibody using FiberCell
bioreactors C2011 (FiberCell Systems, Frederick, MD). This
system allows continuous production of high titer of antibod-
ies in serum-free medium. It is the most efficient system we
have tested. We have produced the antibody using the same
cartridge for over 1 year. It requires some space in the incuba-
tor. For monitoring glucose level we use GlucoCell [CESCO
Bioengineering (Hsinchu, Taiwan)].
17. We use Ultra Low IgG FBS from Gibco (Invitrogen, Carlsbsd,
CA), HyClone DMEM/High Glucose serum-free buffer
(Thermo Scientific, Waltham, MA), and CDM HD Serum
Replacement (Fiber Cell System, Frederick, MD).
200 C. Kowal and B. Diamond

18. We use Protein G Sepharose 4 Fast Flow (GE Healthcare

Bio-Sciences, Piscataway, NJ) and Protein G spin columns
(Nunc, Denmark).
19. We use memantine chloride for its water solubility (SIGMA,
St. Louis, MO). Memantine is an approved treatment for
Alzheimers patients. It is safe and does not have any visible
adverse effects in contrast to another NMDAR-specific blocker,
dizocilpine (MK801).
20. Both L- and D-isoforms are effective as inhibitors of NMDAR
antibodies in vitro, while D-isoform is much more stable in vivo.
21. Take care to minimize the stress; do not transfer animals to
different location and treat them as described in the
Subheading 3.3 of this chapter.
22. These syringes guarantee better control of a volume and thus,
the concentration of the inhibitor.
23. You can use any anesthesia machine; we use the portable and
the mobile ones with CO2 absorber from VetEquip (Pleasanton,
CA).
24. We use MasterFlex Model 7553-70 from Cole-Parmer
Instruments (Vernon Hills, Illinois) that is durable and
reliable.
25. We use MasterFlex Tygon 06409-14 from the same company.
Check from time to time the flexibility of the tubing and
replace it if needed.
26. We use Friedman-Pearson Rongeur1.0 mm Cup Curved
(FST-16021-14). You can use a good surgical scissors too (see
Note 27).
27. It is important to have a good quality surgical scissors. We use
Germany Stainless (F.S.T 14586-12).
28. We use slotted spoon from Moria (Doylestown, PA).
29. This buffer can be prepared ahead of time with the exception
of Heparin that is added just before use. Filter-sterilize or auto-
clave sodium chloride/sodium nitrate solution and keep it at
room temperature or at 4 C.
30. You can use any method of preparing 0.2 M sodium phosphate
buffer. We prepare 0.2 M monobasic and dibasic solutions and
mix them at proportion 774 ml of dibasic and 226 ml of
monobasic and adjust pH using one or the other. We then
autoclave or filter-sterilize and keep it at room temperature.
For preparing 4 % paraformaldehyde PB buffer we prepare 8 %
paraformaldehyde in ddH2O and then mix 1:1 with 0.2 M PB
pH 7.4. Make it fresh, filter-sterilize, and use it, or freeze it at
20 C in aliquots until use. To make 8 % paraformaldehyde
use hot ddH2O, almost at the boiling point, place it on a
 9 Aspects of CNS Lupus 201

magnetic stirrer in a chemical hood, put paraformaldehyde in

the solution and pH it to 7.4 using 0.1 N sodium hydroxide.
You will need 810 ml/100 ml. Paraformaldehyde dissolves in
hot water after adding sodium hydroxide.
31. We prepare 30 % sucrose buffer in 0.1 M PB pH 7.4, filter-
sterilize it and keep it at 4 C.
32. You will need at least one of the following instruments. For
preparing thin, 520 m sections from frozen tissue we use
Leica CM 3050 S (Leica, Wetzlar, Germany) cryostat that is
particularly recommended for soft tissues such as the brain.
Alternatively, fixed fresh tissue can be cut by the microtome;
we use Microm HM 450 (Thermo Scientific, Waltham, MA)
microtome. Both instruments have an automatic and manual
feed function.
For thicker sections, more than 100 m, we use vibratome
from Leica, VT 1000 S, with included cooling system.
33. Any microscope with z-stack function can be used. To mini-
mize bleaching of fluorochromes use confocal or multiphoton
microscope.
34. Any microscope slides can be used. We use Superfrost Plus
from Fisher (Pittsburgh, PA).
35. We use 30- or 24-well ceramic dishes, but they can be replaced
by 24-well tissue culture plate, or bigger well for bigger
sections.
36. Mix 0.2 M phosphate buffer pH 7.4 with equal volume of 2
saline (1.8 g NaCl in 100 ml of ddH2O).
37. A variety of mounting anti-fade media are available. We use
fluorescent mounting medium from Dako (Carpinteria, CA)
for cryostat sectioning and M-1 embedding matrix for frozen
sectioning (Thermo Scientific) on a microtome.
38. You will need 60 mg for 100 ml. It is good for 23 days if kept
at 4 C.
39. Make 0.01 % FluoroJade stock solution by using 25 mg in
250 ml of ddH2O, keep at 4 C for up to 3 months. Make fresh
staining solution for the day of staining: use 10 ml of the
FluoroJade stock solution and mix with 90 ml of 0.1 % acetic
acid (0.1 % acetic acid is 100 l of glacial acetic acid in 100 ml
of ddH2O). For 0.0001 % FluoroJade use 1 and 99 ml, respec-
tively. Do not use after 4 h.
40. This protocol should be easy to scale up for any brain prepara-
tion, but we did not test it in any scale larger than the mouse
brain.
41. We have different size tubes from Kontes (Vineland, NJ). You
can use any homogenizing tube, but you will need sizes ten
times volume of the brain tissue.
202 C. Kowal and B. Diamond

42. Any centrifuge that provides the proper volume and speed is
fine. The volume depends on the size of the brain; mouse
whole brain is 0.40.5 g, so the volume is 45 ml; the speed
is 1,000 g. For a fraction of a brain you may use
microcentrifuge.
43. You will need an ultracentrifuge. We currently use Beckman
TLA120.2 at 75 K rpm (gmax 245000), for 15 min. For bigger
volumes, use bigger rotors. Use Beckman speed converter to
calculate the speed in your rotor.
44. Prepare higher concentration stock solutions of each compo-
nent and mix them accordingly. This is particularly important
for low solubility products such as sodium vanadate. Do not
add it as a powder; it is difficult to solubilize when the other
components are already present in the buffer.
45. We use Roches Complete Mini (Roche Applied Science,
Indianapolis, IN).
46. There are a variety of gels and buffer systems available. We use
one of the earlier systems, NuPAGE Bis-Tris gel and NuPAGE
MES SDS running buffer. The voltage and running time
depend on the gel-buffer system and so are the bands positions
of the markers. Invitrogen provides the templates for their
markers in different buffers. Watch the temperature of the gel.
Avoid overheating. You may run the gel in an ice-water bath.
47. Blocking time may vary and is quite flexible. We use LICORs
buffer diluted 1:1 with PBS, but 5 % milk or 1 % BSA in PBS
are good alternatives.
48. For MAP-peptides use few microliters of saturated Tris-base,
do not exceed 5 l/ml, as this slows down emulsification.
MAP-core dissolves easily without Tris. Map-AAAAAVWLSN
peptide is hardly soluble in water solution; you may use DMSO
or add the powder to CFA/IFA solution. Solubility in water-
based solution may depend on the purity of the product.
49. Do not use small syringes. They are leaky and you will lose too
much of the emulsion.
50. MAP-core emulsifies very easily; the other antigens require
more time. Remember not to use too much Tris.
51. Originally, we published a different schedule for immuniza-
tion, but we now routinely use the version described here.
52. Initially, we bled the animals 2 weeks after each immunization
to establish the time course of immunization and later reduced
the bleeding to pre-bleed and a final bleed at the conclusion of
the experiment.
53. Dry-coating for dsDNA is important as otherwise dsDNA may
not sufficiently bind to the plastic plate. An insufficient amount
of antigen may give too low a reading.
 9 Aspects of CNS Lupus 203

54. For dilution use BD Falcon polyvinyl chloride plates (see

Note 5). Start with slightly more than double the volume of
diluted serum/primary antibody in an eppendorf tube. Mix by
vortexing and place half the volume in a well of the polyvinyl
plate. Add the same volume of diluents to the tube, mix, and
do serial twofold dilutions. For testing serum of immunized
mice for dsDNA and linear peptides start with 1:100 or 1:250,
for immunizing antigen 1:500 or 1:1,000. Prepare dilutions
during blocking creating a replica of your experimental plate.
You can use the same dilution on multiple antigens.
55. Prepare substrate few minutes before loading and mix it well
by vortexing to allow the substrate tablet to dissolve.
Undissolved substrate particles may give false positives.
56. Preparing solution at 0.3 mg/kg eliminates volume calculation
for a dose of 3 mg/kg and makes a local concentration of LPS
in the peritoneum less toxic. Sensitivity to LPS is strain depen-
dent and this dose is at the highest level for BALB/c; C57bl/6
strain can take a dose at 10 mg/ml. This is particularly impor-
tant for the immunized and the older mice. Do not use LPS
just after immunization. Wait for at least 23 weeks as you may
lose too many animals to the septic shock. All the young and
nonimmunized BALB/c mice in passive antibody transfer sur-
vived the 3 mg/ml LPS.
57. The second injection has a diminished effect according to
Nagano et al. (32), and we observe it as well.
58. We use () Epinephrine (+) Bitartrate salt (see Note 15) for its
water solubility. Prepare 100 solution and keep it at 4 C until
use. Protect it from light. Epinephrine is light sensitive, so do
not use solutions that change the color.
59. We started using 100 nM/mouse and doubled the dose to
increase the effectiveness of the opening of the BBB.
Epinephrine is milder than LPS and 200 nM per mouse is a
safe dose.
60. Use sodium chloride if the yield of elution is low and more
stringent condition of elution buffer is needed.
61. Immediate neutralization is important; low pH may denature
antibody. Pretest the amount of TrisCl before purification on
the chosen fraction volume.
62. The method of verifying the concentration of antibody depends
on the buffer system, the range of the anticipated concentra-
tion and the subsequent utilization. Using ELISA for assess-
ment will eliminate the buffer compatibility problems and is
good for a wide range of concentration. We also use NanoDrop
spectrophotometer ND-1000 (Thermo Scientific, Wilmington,
DE) that requires only 12 l of sample and is fast and
accurate.
204 C. Kowal and B. Diamond

63. Antibodies are stable at concentrations above 1 mg/ml. You

may freeze the aliquots, but some isotypes are not cryogeni-
cally stable, so do not subject to repeated freezing and
thawing.
64. Do not exceed 5 % of the total blood volume otherwise you
may cause high blood pressure.
65. For the BBB experiments it is important to minimize the expo-
sure of the animal to stress as this may affect your results. This
is also why we do not recommend the injectable method of
anesthesia.
66. Wait for slow breathing, wait for 30 s to 1 min more, and then
perform the toe test; if the animal does not react to the toe
pinch, it is ready. Lower the level of anesthesia to 2.53 if you
need more time and for further procedure.
67. There are anatomical differences between animal strains, so
you have to buy the proper nose cone for your strain.
68. We use a small animal eye clipper or a needle; the latter is
sufficient and easier to use.
69. Brain is fixed immediately during perfusion and that preserves
the tissue but we postfix it for 24 h or overnight.
70. We use Optimal Cutting Temperature (O.C.T.), from Sakura
Finetek (Torrance, CA).
71. Dry the slide longer, 6 h to overnight. Alternatively, use gela-
tin-coated slides.
72. We use marker from Vector Laboratories (Burlingame, CA).
73. The choice of blocking buffer is critical in controlling the back-
ground. Typically, this is diluted serum from a host of the anti-
body used in binding assay or a blocking buffer recommended
by a company that produced the antibody. We also use 1 %
BSA/PBS with or without 0.10.2 % Triton X-100 that is very
effective.
74. The antibody dilution depends on the type of the antibody and
the source. In the detailed protocols that follow we provide
that information.
75. For detecting antibody deposition in the brain we use 12 h of
staining.
76. It will be difficult to seal the slide if the excess of mounting
medium is not removed. Whatman paper (1 mm) should do
the job.
77. H2O2 can be used from Vector and used as recommended or
can be purchased from SIGMA as 3 or 30 % solution which is
100 and 1,000 solution, respectively.
78. The tissue can be kept at 80 C until used.
 9 Aspects of CNS Lupus 205

79. For smaller size of tissue you may skip the second spin. The
quality of this preparation is good for most of the applications
after single spin.
80. The volume depends on the size of the brain. We resuspend
the whole brain preparation in 1 ml and dilute or concentrate
if necessary.
81. You may use the Amicon filtration system (Millipore, Billerica,
MA) for exchanging buffer or any dialysis system. Amicon filter
units come with different cut-offs and sizes and are very con-
venient. Protein loss in this system is minimal, as long as you
do not exceed three spins per unit.
82. The Odyssey secondary antibody can be used at two wave-
lengths 700 and 800 nm, red and green, respectively, that
allows simultaneous detection of two proteins. The recom-
mended concentration of the secondary antibody by manufac-
turer is effective. We use infrared labeled rabbit anti-mouse
(700) and goat anti-mouse 800 nm.
83. You may continue washing and checking if the background is
too high.

References

1. Alexander JJ, Quigg RJ (2007) Systemic lupus
erythematosus and the brain: what mice are
telling us. Neurochem Int 50:511
2. Banks WA (2010) Mouse models of neurologi-
cal disorders: a view from the bloodbrain bar-
rier. Biochim Biophys Acta 1802:881888
3. Denburg SD, Larocque L, Denburg JA (1999)
Cognitive dysfunction in systemic lupus ery-
thematosus. In: Systemic Lupus Erythematosus,
Fourth Edition, Lahita RG (ed.). Academic
Press, San Diego, pp 611629
4. Hanly JG (2007) New insights into central
nervous system lupus: a clinical perspective.
Curr Rheumatol Rep 9:116124
5. Huerta PT, Kowal C, DeGiorgio LA, Volpe
BT, Diamond B (2006) Immunity and behav-
ior: antibodies alter emotion. Proc Natl Acad
Sci USA 103:678683
6. Kowal C, DeGiorgio LA, Nakaoka T,
Hetherington H, Huerta PT, Diamond B, Volpe
BT (2004) Cognition and immunity; antibody
impairs memory. Immunity 21:179188
7. Gaynor B, Putterman C, Valadon P, Spatz L,
Scharff MD, Diamond B (1997) Peptide inhi-
bition of glomerular deposition of an anti-DNA
antibody. Proc Natl Acad Sci USA 94:
19551960
8. Putterman C, Diamond B (1998)
Immunization with a peptide surrogate for
double-stranded DNA (dsDNA) induces
autoantibody production and renal immuno-
globulin deposition. J Exp Med 188:2938
9. Hawkins BT, Davis TP (2005) The blood
brain barrier/neurovascular unit in health and
disease. Pharmacol Rev 57:173185
10. Paolinelli R, Corada M, Orsenigo F, Dejana E
(2011) The molecular basis of the blood brain
barrier differentiation and maintenance. Is it
still a mystery? Pharmacol Res 63:165171
11. Daneman R, Zhou L, Kebede AA, Barres BA
(2010) Pericytes are required for bloodbrain
barrier integrity during embryogenesis. Nature
468:562566
12. Banks WA, Erickson MA (2010) The blood
brain barrier and immune function and dys-
function. Neurobiol Dis 37:2632
13. Kowal C, Degiorgio LA, Lee JY, Edgar MA,
Huerta PT, Volpe BT, Diamond B (2006)
Human lupus autoantibodies against NMDA
receptors mediate cognitive impairment. Proc
Natl Acad Sci USA 103:1985419859
14. Lahita RG (1988) Systemic lupus erythemato-
sus: learning disability in the male offspring of
female patients and relationship to laterality.
Psychoneuroendocrinology 13:385396
15. McAllister DL, Kaplan BJ, Edworthy SM,
Martin L, Crawford SG, Ramsey-Goldman R,
Manzi S, Fries JF, Sibley J (1997) The influence
206 C. Kowal and B. Diamond
of systemic lupus erythematosus on fetal
development: cognitive, behavioral, and health
trends. J Int Neuropsychol Soc 3:370376
16. Neri F, Chimini L, Bonomi F, Filippini E,
Motta M, Faden D, Lojacono A, Rebaioli CB,
Frassi M, Danieli E, Tincani A (2004)
Neuropsychological development of children
born to patients with systemic lupus erythema-
tosus. Lupus 13:805811
17. Ross G, Sammaritano L, Nass R, Lockshin M
(2003) Effects of mothers autoimmune dis-
ease during pregnancy on learning disabilities
and hand preference in their children. Arch
Pediatr Adolesc Med 157:397402
18. Tincani A, Danieli E, Nuzzo M, Scarsil M,
Motta M, Cimaz R, Lojacono A, Nacinovich
R, Taddei F, Doria A, Brucato A, Meroni P
(2006) Impact of in utero environment on the
offspring of lupus patients. Lupus 15:801807
19. Lee JY, Huerta PT, Zhang J, Kowal C, Bertini
E, Volpe BT, Diamond B (2009) Neurotoxic
autoantibodies mediate congenital cortical
impairment of offspring in maternal lupus. Nat
Med 15:9196
20. Arinuma Y, Yanagida T, Hirohata S (2008)
Association of cerebrospinal fluid anti-NR2
glutamate receptor antibodies with diffuse neu-
ropsychiatric systemic lupus erythematosus.
Arthritis Rheum 58:11301135
21. Fragoso-Loyo H, Cabiedes J, Orozco-Narvaez
A, Davila-Maldonado L, Atisha-Fregoso Y,
Diamond B, Llorente L, Sanchez-Guerrero J
(2008) Serum and cerebrospinal fluid autoanti-
bodies in patients with neuropsychiatric lupus
erythematosus. Implications for diagnosis and
pathogenesis. PLoS One 3:e3347
22. Yoshio T, Onda K, Nara H, Minota S (2006)
Association of IgG anti-NR2 glutamate recep-
tor antibodies in cerebrospinal fluid with neu-
ropsychiatric systemic lupus erythematosus.
Arthritis Rheum 54:675678
23. Banks WA (2001) Enhanced leptin transport
across the blood-brain barrier by alpha
1-adrenergic agents. Brain Res 899:209217
24. Xaio H, Banks WA, Niehoff ML, Morley JE
(2001) Effect of LPS on the permeability of the
bloodbrain barrier to insulin. Brain Res
896:3642
25. DeGiorgio LA, Konstantinov KN, Lee SC,
Hardin JA, Volpe BT, Diamond B (2001) A
subset of lupus anti-DNA antibodies cross-
reacts with the NR2 glutamate receptor in sys-
temic lupus erythematosus. Nat Med 7:
11891193
26. Diamond B, Bloom O, Al Abed Y, Kowal C,
Huerta PT, Volpe BT (2011) Moving towards
a cure: blocking pathogenic antibodies in sys-
temic lupus erythematosus. J Intern Med 269:
3644
27. Owens GP, Burgoon MP, Devlin ME, Gilden
DH (1997) Extraction and purification of
active IgG from SSPE and MS brain. J Virol
Methods 68:119125
28. Fernandes AM, Maurer-Morelli CV, Campos
CB, Mello ML, Castilho RF, Langone F (2004)
Fluoro-Jade, but not Fluoro-Jade B, stains
non-degenerating cells in brain and retina of
embryonic and neonatal rats. Brain Res
1029:2433
29. Schmued LC, Albertson C, Slikker W Jr (1997)
Fluoro-Jade: a novel fluorochrome for the sen-
sitive and reliable histochemical localization of
neuronal degeneration. Brain Res 751:3746
30. Ogawa Y, Kanoh S (1984) Enhancement of
endotoxicity and reactivity with carbocyanine
dye by sonication of lipopolysaccharide.
Microbiol Immunol 28:13131323
31. Gao B, Wang Y, Tsan MF (2006) The heat sen-
sitivity of cytokine-inducing effect of lipopoly-
saccharide. J Leukoc Biol 80:359366
32. Nagano I, Takao T, Nanamiya W, Takemura T,
Nishiyama M, Asaba K, Makino S, De Souza
EB, Hashimoto K (1999) Differential effects
of one and repeated endotoxin treatment on
pituitaryadrenocortical hormones in the
mouse: role of interleukin-1 and tumor necro-
sis factor-alpha. Neuroimmunomodulation 6:
284292
 Chapter 10

Analysis of Renal Mononuclear Phagocytes

in Murine Models of SLE
Ramalingam Bethunaickan, Ranjit Sahu, and Anne Davidson

Abstract
In this chapter we present methods for the isolation and characterization of mononuclear phagocytes from
the kidneys of mice with SLE. Activation of these cells is associated with the onset of clinical disease in mice
and infiltration with these cells is associated with poor prognosis in humans. Using magnetic beads fol-
lowed by flow cytometric sorting, pure populations of cells are obtained that are functional in a variety of
assays. Sufficient numbers of cells are obtained for genomic characterization. An analysis of the function of
these cells should lead to a better understanding of the inflammatory processes that cause renal impairment
in SLE and other renal inflammatory diseases.

Key words: Lupus, Nephritis, Macrophages, Dendritic cells, Cell activation, Murine models

1. Introduction

Lupus nephritis affects between 30 and 60 % of adult and up to

70 % of pediatric patients with SLE and is responsible for significant
morbidity and mortality. The treatment of lupus nephritis remains
unsatisfactory with complete remission rates of <50 % and relapse
rates of up to 30 % over a 2-year period (1). Remission can be
maintained with long-term immunosuppressive treatment (24),
but chronic renal insufficiency still ensues in up to 30 % of patients
and the rate of end stage renal disease in SLE patients appears to be
increasing in the USA, especially in African-American patients (5).
This clinical picture mandates that we improve our understanding
of the pathogenesis of SLE nephritis including a definition of the
phenotype of this disease based not only on histologic appearance
but also on immunologic, genomic, and proteomic approaches. As
human clinical trials proceed, these phenotypes can then be linked
prospectively to responses to therapeutic intervention.

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_10, Springer Science+Business Media New York 2012

207
208 R. Bethunaickan et al.

Although hypertension and poor renal function at presentation

correlate with poorer prognosis in SLE nephritis, most clinical and
histologic features are not good predictors of disease response or
relapse (6). One predictor of poor renal outcome in lupus patients
is infiltration with inflammatory cells, particularly CD68+ mono-
nuclear cells (7, 8). In this review we present methods for analyz-
ing cellular infiltrates in the kidneys of mice with SLE with a focus
on the analysis of subsets of mononuclear phagocytes that contrib-
ute to renal damage.

1.1. Pathogenesis SLE nephritis is most often initiated by the glomerular deposition
of SLE Nephritis of immune complexes directed at a variety of antigens including
DNA, glomerular extracts, alpha actinin and laminin (911) and is
characterized by varying degrees of glomerular and tubulointersti-
tial inflammation. Although in most cases autoantibody deposition
in the kidneys is required to initiate a cascade of inflammatory
events, signals from the innate immune system and cellular immu-
nity are also crucial contributors to renal disease (Fig. 1). These
signals include complement activation (reviewed in ref. (12),
engagement of activating Fc receptors on mononuclear cells (13),
activation of resident renal cells through Toll-like receptors (14)
and recruitment of inflammatory cells to the kidney. Inflammatory
cytokines and other mediators induce endothelial cells to express
adhesion molecules, increasing the probability that they will recruit
inflammatory cells after contact with immune complexes (15). In
MRL/lpr mice T-cell mediated interstitial renal disease and vascu-
litis together with mild glomerular changes occur even in the com-
plete absence of immunoglobulins (16). Similarly, in human SLE
some pauci-immune forms of nephritis have been observed (6).
Microvascular damage and thromboses also occur in the setting of
SLE nephritis and may be more common in patients with anti-
phospholipid antibodies (17).
In both murine and human SLE, B-cells, T-cells, macrophages
and dendritic cells are recruited into the inflamed kidney (1820).
We have shown in NZB/W mice, that treatment of established
nephritis with a combination of Cytoxan and the costimulatory
antagonist CTLA4Ig does not alter renal immune complex deposi-
tion but does induce remission (21, 22). The mechanism for remis-
sion induction includes downregulation of local chemokine
expression in conjunction with a decrease in migration of lympho-
cytes and dendritic cells to the kidney, a decrease in renal endothe-
lial cell activation and a decrease in the activation state of resident
renal macrophages/DCs (21). Thus, the initiation of nephritis by
immune complexes can be delayed if downstream effector mecha-
nisms such as cell activation, migration or cell death are altered.
This concept offers new therapeutic approaches.
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 209

Fig. 1. Mechanisms for renal damage in SLE: renal damage is initiated by deposition of
immune complexes and complement in the glomeruli and may be amplified by high serum
levels of soluble cytokines and other inflammatory mediators. This is followed by endothe-
lial activation and recruitment of lymphoid cells. Activation of intrinsic renal cells such as
mesangial cells and intrinsic renal macrophages contribute to loss of integrity of the
glomerulus with damage to podocytes and development of proteinuria. Interstital
inflammation results in tubular damage and fibrosis.

1.2. Macrophage Macrophages and dendritic cells are known to be key players in
and Dendritic Cell acute renal inflammation (19, 2326). Macrophages have a high
Subtypes degree of plasticity and have complex responses to inflammatory
stimuli with distinct activation patterns and functions depending
on the stimuli to which they are exposed (2730). Inflammatory
or classically activated (M1) macrophages are induced during cell
mediated immune responses in response to IFN (or IFN) and
TNF and produce large amounts of proinflammatory cytokines
including IL-1, IL-6, IL-12, and IL-23 and inflammatory media-
tors including iNOS and ROS. These inflammatory macrophages
derive from peripheral Gr1+/CCR2+ monocytes that have a very
short half-life and egress from the blood during acute inflammation
or infection (3133). M1 macrophages help recruit neutrophils to
sites of inflammation and induce the differentiation of TH1 and
TH17 cells. Alternatively activated (M2) macrophages that are
induced by IL-4 secrete protective cytokines and promote wound
healing. They derive from Gr1/CD62L/CX3CR1+ monocytes
that have a longer half-life and patrol the endothelium (34). These
cells extravasate very rapidly upon tissue damage and take on an
inflammatory phenotype for a short time but then appear to switch
to the alternative M2 macrophage differentiation program with a
210 R. Bethunaickan et al.

reparative role (30). Finally, regulatory macrophages that are

induced by immune complexes or by corticosteroids produce
high levels of IL-10 and are anti-inflammatory (30, 35). Other
macrophage activation patterns represent a mixed phenotype likely
resulting from simultaneous exposure to inflammatory and homeo-
static/suppressive factors in vivo during chronic disease states (29,
36). Of relevance to SLE, macrophages exposed to immune com-
plexes and TLR agonists are characterized by an IL-10hi/IL-12lo
phenotype termed M2b (37). These cells have antigen presenta-
tion capabilities and may continue to produce inflammatory cytok-
ines but they are not involved in tissue repair.
Dendritic cells (DCs) belong to three main subgroups: plasma-
cytoid DCs that produce large amounts of Type I interferons,
conventional DCs that have a major antigen presentation role, and
migratory DCs that reside in peripheral tissue and capture antigen
that they then deliver to T cells in lymphoid organs (35, 38).
Conventional DCs derive from both myeloid and lymphoid lin-
eages and are very heterogeneous; within tissues, the microenviron-
ment can greatly influence their function. Tissue DCs are continually
replaced by short-lived blood borne precursors and undergo several
rounds of division in the periphery (39). Their life span in second-
ary lymphoid organs is 1014 days but may be significantly longer
in peripheral organs such as skin and brain (40, 41).

1.3. Renal Normal mouse kidneys have a heterogeneous resident population

Macrophages of cells of monocytic origin. The dominant population is high for
and Dendritic Cells F4/80, CX3CR1 and MHC Class II and intermediate for CD86,
CD11b and CD11c. This cell population forms a network that is
dense in the renal interstitium but less frequent in the renal cortex
(42, 43). Studies in human kidneys from normal and diseased indi-
viduals have similarly found a network of CD68 positive cells
throughout the interstitium (44). Functional studies in mice have
confirmed that these cells have antigen presenting function, that
they use dendrites to sample their local environment, and are poor
NO producers, suggesting that they are dendritic cells; their phago-
cytic capability, while lower than that of macrophages, is higher
than that of splenic dendritic cells (42, 43). Because these cells
express F4/80 they have also often been referred to as a resident
macrophage population. Several recent reviews debate the utility
of the macrophage vs. DC nomenclature for tissue derived mono-
nuclear phagocytes that can have diverse functions (25, 35).
A minor population of monocytic cells in normal kidneys is
high for CD11b, CD62L, and Gr-1, lo/intermediate for F4/80,
intermediate or negative for CD11c and low for MHC Class II and
CD86; this population expresses CCR2, secretes inflammatory
cytokines and most resembles classic inflammatory macrophages
(32). During acute ischemic injury this F4/80lo/CD11bhi/Gr1hi
inflammatory macrophage population increases markedly in the
kidneys, whereas the F4/80hi cell population does not change (32).
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 211

Two other small populations have been described but have not
been well characterized. One is high for CD11c and is also CD11b
positive. The other includes cells that are CD11c+/CD11b (45).
Finally a small number of plasmacytoid dendritic cells can be found
in the kidneys (46).

1.4. Murine Models Given the heterogeneity of disease course, outcome and response to
of SLE Nephritis therapy in SLE patients, even in those patients with comparable
renal histology, and the lack of sequential biopsy material from
humans, it remains essential to study informative animal models of
disease. Animal models of SLE nephritis offer the advantage that
disease pathogenesis can be studied in a timed manner and new
therapies tested at various disease stages (47). Two main categories
of mouse models exist, namely spontaneous models and induced
models. Of these, the spontaneous models have been extensively
studied and well characterized (48, 49). The classical models of
spontaneous lupus include the F1 hybrid between New Zealand
Black (NZB) and New Zealand White (NZW), MRL/lpr mice,
BXSB.Yaa mice, and NZM 2410. Not surprisingly, striking differ-
ences in responses to immunologic interventions have been observed
in the different mouse models (13, 5052) suggesting that multiple
animal models will be needed to study responses to new therapies
and to dissect pathogenetic mechanisms of SLE nephritis.
Despite the clear association of mononuclear phagocyte
infiltration in the kidneys with poor disease outcome in SLE, little
is known about the function of these cells in the kidneys in lupus
nephritis. Our preliminary studies in NZB/W mice show that renal
macrophages and dendritic cells may behave differently in mice
with lupus nephritis than in normal mice with acute renal
inflammation and that there is some variability between models. In
contrast to the findings in models of acute renal inflammation, the
inflammatory macrophage population (32) does not increase in
the nephritic kidneys of the three mouse models that we have stud-
ied in detail (NZB/W, NZW/BXSB.yaa, and NZM2410), nor is
there infiltration with neutrophils. These findings suggest that the
short-term inflammatory model of nephritis induced by immune
complexes (53) is not an adequate model for chronic SLE nephritis.
Chronic models of SLE nephritis include the following:
(a) Diffuse proliferative glomerulonephritis with anti-DNA anti-
bodies: NZB/W F1 is the oldest classical model with a pheno-
type comparable to lupus patients. Female mice develop
lymphadenopathy, splenomegaly, elevated antinuclear autoanti-
bodies including anti-dsDNA and immune complex mediated
glomerulonephritis (54). Nephritic kidneys are characterized
by extensive infiltration of activated renal macrophages
(F4/80hi) in the tubulointerstitial region and accumulation of
T and B cells along with dendritic cells (CD11chi) in perivascular
and periglomerular aggregates (21, 46). Complete remission
212 R. Bethunaickan et al.

of established nephritis (reversal of >300 mg/dl proteinuria to

<30 mg/dl) is achieved in >80 % of mice by combination
therapy either with a short course of Cytoxan together with
costimulatory blockade (21) or with a short course of CTLA4Ig
together with BAFF blockade (55).
(b) Sclerotic glomerulonephritis with anti-nucleosome and anti-
DNA antibodies: The NZM2410 strain was obtained as a result
of a backcross between NZB/W F1 and NZW followed by
brother sister mating. Twenty-seven different recombinant
inbred strains of New Zealand Mixed (NZM) mice were
obtained; among them NZM2410 and NZM2328 have been
used most commonly as lupus models (56). NZM2328 are
similar to NZB/W with the advantage that an F1 is not
required. NZM2410 mice are dominated by an IL-4 response
and they produce IgG1 and IgE anti-chromatin antibodies.
They develop sclerotic glomerulonephritis with the accumula-
tion of activated interstitial F4/80hi cells that have upregulated
expression of CD11b but they have little lymphocytic or
CD11chi dendritic cell infiltration (48). Disease is somewhat
accelerated in females but also occurs in males. As in humans
with little renal inflammatory disease, these mice are very
responsive to treatment and BAFF blockade alone induces
long-term survival (57).
(c) Proliferative disease associated with anti-RNA and anti-phos-
pholipid antibodies: BXSB.Yaa mice exhibit monocytosis and
lymphoid hyperplasia, immune complex mediated glomerulo-
nephritis and hypergammaglobulinemia as a part of their SLE
phenotype. Although the genetic background confers SLE
susceptibility in both sexes, males carry the Yaa (Y chromo-
some-linked autoimmunity accelerator) locus consisting of a
reduplication of at least 17 genes from the X chromosome,
including toll-like receptor 7 (TLR 7) resulting in dysregula-
tion of the innate immune response (5860). NZW/BXSB
(W/B) mice produce anti-Sm/RNP and anti-phospholipid
antibodies (6163) that cause clots within the myocardial small
vessels. These mice have severe proliferative glomerulonephri-
tis with sheets of F4/80hi cells in the interstitium and peri-
glomerular regions and the accumulation of CD11chi dendritic
cells within the glomeruli. The kidneys of these mice also have
TH17 cells scattered throughout the interstitium. W/B mice
are resistant to therapy with Cytoxan and costimulatory block-
ade once autoantibodies appear in the serum but have a partial
response to treatment with BAFF blockade (62).
(d) Proliferative and interstitial disease associated with anti-DNA,
anti-RNA and rheumatoid factor autoantibodies: MRL/lpr
mice are deficient in Fas resulting in massive lymphoprolifera-
tion due to accumulation of double negative (CD4 CD8) T
cells and B220+ cells (64). These mice display an accelerated
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 213

mortality, with both males and females significantly affected.

In addition they have elevated levels of multiple autoantibodies
including ANA, anti-ssDNA, anti-Sm and rheumatoid factors,
resulting in large amounts of immune complexes (65).
Macrophage infiltration is an essential feature of nephritis in
this model (66, 67). The infiltrating cells have not as yet been
well characterized in this model although recruitment of
F4/80lo/Ly6Chi inflammatory macrophages into the kidneys
is observed 12 days after adoptive transfer of bone marrow
derived monocytes (66).

1.5. Induction Remission induction studies were carried out in NZB/W mice
of Remission in with new onset proteinuria >300 mg/dl using a single dose of
NZB/W Mice cyclophosphamide with or without a 2-week course of murine
is Associated CTLA4Ig in combination with or without anti-CD154 (CD40L).
with Alterations Sixty to eighty percent of mice treated with the triple combination
in Renal Mononuclear entered remission, and this remission could be reinduced following
Phagocytes relapse. Triple therapy treatment induced remission for a period of
up to 20 weeks but did not alter anti-DNA antibody titers or ongo-
ing renal immune complex deposition. Flow cytometry and
ELISpot studies revealed that there was an initial decrease in the
frequency of anti-DNA producing B cells and activated T cells but
this persisted for only 36 weeks after treatment. Light microscopy
studies of kidneys from the mice which entered into remission
revealed a complete reversal of renal pathology, with complete
absence of glomerular proliferation, interstitial infiltrates and casts;
there was a gradual return of pathologic changes over time. These
data suggested that remission was associated with a decrease in the
renal inflammatory response to immune complex deposition (21).
To further investigate the reasons for the decrease in renal
inflammation, real-time PCR studies were carried out for 61
inflammatory molecules on RNA obtained from perfused kidneys
of treated and untreated mice at progressive disease stages. Only a
limited set of inflammatory markers were upregulated in the kid-
neys of prenephritic NZB/W mice. These markers include the
chemokine CXCL13 and its receptor CXCR5, CCL20, CCR6 and
CCR8. When age matched mice with and without proteinuria were
compared, both macrophage/DC markers (IL-1, TNF-, IL-6,
BAFF, IL-10 along with CCL3, CCL9 and CCR2) and endothe-
lial activation markers (VCAM-1, E-selectin, P-selectin) were
found to be upregulated. Flow cytometry experiments confirmed
both an increase in the number of renal mononuclear phagocytes
in the kidneys and an increase in CD11b expression on these cells.
When mice entered remission, a significant decrease was noted in
the expression of IL-1, IL-10, TNF-, BAFF, Ox40L, CCL5, and
CCL2. Reductions of these markers were associated with a sig-
nificant decrease in the renal macrophage-DC population and a
decrease in CD11b expression (68).
214 R. Bethunaickan et al.

Fig. 2. Flow cytometric analysis of renal mononuclear phagocytes: gating strategy to identify three major subpopulations
of CD11b positive renal cells in a young (16W) and nephritic (3640W) NZB/W mice. After exclusion of clumps and dead
cells, cells are gated on CD11b and then on CD11c and F4/80. The three populations include F4/80hi (right), F4/80lo (lower
left) and CD11chi (upper left). Note the differences in staining for MHCII and VLA4 between the F4/80hi and CD11chi popu-
lations and the appearance of the CD11chi, VLA4hi, MHCIIlo population in nephritic mice.

In order to better understand how macrophages/DCs

contribute to lupus nephritis, isolated cells from nephritic kidneys
of NZB/W mice were extensively characterized by flow cytometry.
Three populations of mononuclear phagocytes that increased in
frequency during nephritis were identified by the expression of a
panel of surface markers and were designated F4/80hi, F4/80lo,
and CD11chi (Fig. 2). F4/80hi cells were the major population in
normal NZB/W kidneys and expressed a phenotype of CD11bhi/
CD11cint/Gr1lo/Ly6Clo/VLA4lo/MHCIIhi/CD43lo/CD62Llo.
Morphologically, they were large cells with small dendrites.
Peripheral blood Gr1lo monocytes were identified as the precursors
of this F4/80hi population. The F4/80lo cells had macrophage like
morphology and a phenotype of CD11bhi/CD11cint/F4/80lo/
Gr1lo. The CD11chi cells appeared only in nephritic kidneys. They
had a veiled dendritic cell morphology and were CD11bhi/
D11cint/F4/80lo/Gr1lo/MHCIIhi/CD43hi/CD62Llo. A fourth
population comprising less than 2 % of all the cells in the lympho-
cyte monocyte gate was CD11blo/CD11chi/F4/80lo/Gr1lo. Only
few plasmacytoid DCs were found in the kidneys. Upon nephritis
induction, the F4/80hi cells became activated, upregulated cathepsin
and matrix metalloproteinase activity, and acquired autophagocytic
vacuoles. These characteristics reversed upon disease remission.
Cellular turnover studies carried out with BrdU revealed that the
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 215

F4/80hi population has a half-life of 16 days in both normal and

nephritic NZB/W mice whereas the CD11chi DCs has a shorter
half-life of 6.5 days.
To investigate the function of the major F4/80hi population
we performed microarray analysis of sorted cells from young and
nephritic NZB/W mice and from mice in which remission had
been induced with triple therapy. We found 694 genes were dif-
ferentially expressed in cells from nephritic compared with young
kidneys and 794 genes were differentially expressed in remission
mice compared with nephritic mice. A total of 378 genes over-
lapped between two comparisons and were considered to be mark-
ers for the nephritis activity. Analysis of this subset of genes
identified a nephritis-associated profile of pro- and anti-
inflammatory genes and genes involved in tissue repair that was
regulated upon remission. Our findings suggest that mononuclear
phagocytes with an aberrant activation profile contribute to tissue
damage in lupus nephritis by mediating both local inflammation
and excessive tissue remodeling (46). By analyzing isolated cells
from the kidneys using techniques that are well established for
other monocytic cell populations we expect to discover at least
some of the relevant activation pathways. A deeper understanding
of this population of renal mononuclear phagocytes may be useful
for the therapeutic targeting of the mononuclear cell infiltration
that is associated with poor prognosis in human lupus.
Herein we present a detailed methodology for isolation and
characterization of renal mononuclear phagocytes. These methods
require perfusion of the kidneys to remove blood, magnetic bead
isolation followed by cell sorting based on the markers we have
described and multiple methods for analysis of cell function.

2. Materials

2.1. Equipment Metabolic cages.

BD LSRII or similar flow cytometer.
FACS ARIA IIu or similar cell sorter.
LightCycler 480 (Roche).

2.2. Kits QuantiChrom Urea Assay Kit: Bio assay systems (Cat #DUIR-500).
Diff-Quick stain Kit: IMEB Inc. (Cat #K7128).
BRDU FITC flow kit: BD Pharmingen (Cat #559619).
PicoPure RNA isolation kit: Arcturus (Cat #KIT0202/KIT0204).
RNeasy micro Kit: Qiagen (Cat #74004).
Superscript III: Invitrogen (Cat #18080-051).
216 R. Bethunaickan et al.

WrightGiemsa stain kit: Volu-Sol, Inc. (Cat #VWG-300).

Mouse Albumin ELISA Kit: Bethyl Laboratories (Cat
#NC9285735).

2.3. Buffers Sterile PBS.

0.17 M ammonium chloride.
PEB: 0.5 % BSA, 2mm EDTA in PBS(pH 7.4).
Lysis buffer: TrisHCl 150 mM, NaCl 150 mM, NP40 1 % with pro-
tease inhibitors (Cat No. 11873580001, Roche Diagnostics).
FACS Staining Buffer: PBS, 3 % fetal bovine serum.

2.4. Chemical Multistix 10SG: Reagent Strips for Urinalysis (Cat #2161).
Reagents Collagenase Type I, CLS I: Worthington (Cat #4197, specific
activity 230 U/mg).
DMEM, High glucose: GIBCO (Cat #10313).
BRDU: Sigma (Cat #B5002-5G).
2 % Paraformaldehyde: Tousimis (Cat #1108A).
Prosense 680: VISEN (Part #10003).
MMPsense 680: VISEN (Part #10126).
Dako Glycergel Mounting medium (Ref #C0563).
Superscript III, Invitrogen company (Cat #18080-051).
LightCycler480 SYBR Green I master: Roche (Cat #04707516001).
Trizol Reagent: Invitrogen (Cat #15596-018).
Griess Reagent Kit: Invitrogen (Cat #G-7921).
IL4: R&D SYSTEMS INC (Cat #404-ML-010).
FN: R&D SYSTEMS INC (Cat #485-MI-100).
M-CSF: Invitrogen (Cat #PMC2044).
GM-CSF: Invitrogen (Cat #PMC2015).
L-Arginine: SIGMA-ALDRICH (Cat #A8094).
Manganese chloride: SIGMA-ALDRICH (Cat #244589).
Urea: SIGMA-ALDRICH (Cat #U5378).
a-Isonitrosopropiophenone: SIGMA-ALDRICH (Cat #I3502).

2.5. Plasticware BD cell strainer (40 nm): BD Falcon (Ref #352340).

30 ml syringe BD.
1 ml syringe.
20 G needles.
26 G needles.
V bottom 96-well Assay plate: Costar (Cat #3897).
LightCycler480 multiwell plate 384: Roche (Cat #04729749001).
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 217

Double cytology Funnel: Fisherbrand (Cat #10-356).

Glass slides.
5 ml Polypropylene Round-Bottom tube (12 mm 75 mm): BD
Falcon (Cat #352063).
5 ml Polystyrene Round-Bottom tube (12 mm 75 mm): BD
Falcon (Cat #352054).
5 ml polystyrene Round-Bottom tube with cell-strainer cap: BD
Falcon (Cat #352235).

3. Methods

The methods described in this chapter are outlined in the following

sections:
3.1. Assessment of Kidney damage.
3.2. Harvesting Kidneys for characterization of renal infiltrates.
3.3. Flow Cytometric characterization and Cell Sorting.
3.4. Enrichment of renal macrophages and dendritic cells by
magnetic beads.
3.5. Morphological studies.
3.6. Cellular turn over studies.
3.7. Functional studies of renal macrophages/dendritic cells.
3.8. Gene expression studies.
3.9. Immunohistologic studies.
3.10. Western blot studies for proteins.

3.1. Kidney Damage 1. Collect overnight urine from each individual mouse in a meta-
Assessment bolic cage.
2. Test the protein level in the urine with Multistix 10SG (Reagent
Strips for Urinalysis). Grade protein levels in the urine as 1+,
2+, 3+, or 4+ equivalent to 30, 100, 300 and 2,000 or more
mg/dl respectively. Note: all normal mice have 1+ proteinuria
by dipstick.
3. Test the mice twice a week for proteinuria.
4. Alternately, when metabolic cages are not available, bladder
massage can be used to obtain urine. The mouse is held in the
upright position. Use the thumb of the other hand to gently
milk the abdomen from top to the bottom. The urine bead is
then directly placed on the strip. Make sure the protein marker
square is completely covered with urine (see Note 1).
218 R. Bethunaickan et al.

3.1.1. Albumin Estimation Mouse albumin can also be measured in 12 or 24 h urine collections
in Urine by ELISA (Kit from Bethyl Laboratories Catalog #NC9285735).
In our experience, values obtained by ELISA fall within the range
indicated by the Multistix.

3.1.2. Blood Urea Nitrogen Blood urea nitrogen (BUN) levels in the blood are measured with
the Quantichrom Urea assay Kit (see Note 2).
1. Transfer 5 l of serum (fresh or stored) to a 96-well flat bot-
tom plate.
2. Prepare serial dilutions of the standards ranging from 100 to
0 mg/dl from the kits master stock. Transfer 5 l of the each
of the standards to the 96-well plate. Use 5 l of deionized
water as blank.
3. Mix equal volumes of Reagent A and Reagent B prior to assay.
From this mix dispense 200 l to the samples, standards and
the blank wells on the plate.
4. Gently tap the plate on the sides to mix the solutions and incu-
bate the plate at RT for exactly 30 min.
5. Read the plate at 470550 nm optical density.
6. The urea concentration is calculated by using the formula
shown below.
OD sample OD blank
N [STD]
OD standard OD blank
OD sample, OD blank, and OD standard are the OD values of
the sample, water and the standard respectively, N is the dilu-
tion factor and [STD] = 50 Urea std concentrations (mg/dl).
7. To obtain the BUN values the following conversion is made.
Urea (mg/dl) = BUN (mg/dl) 2.14.

3.2. Kidney Harvest
1. Anesthetize the mouse and perfuse with 60 ml of cold PBS
over 35 min through the left ventricle after snipping the right
atrium and observe for pale white color change in liver and
kidney. If needed, repeat perfusion with another 60 ml of cold
PBS (see Note 3a).
2. Carefully remove and cut the kidneys into 12 mm3 pieces,
excluding any adjoining renal fat.
3. Incubate the slices in DMEM containing 2 mg/ml Collagenase
Type I (Worthington) for 30 min at 37 C (use 10 ml per two
kidneys).
4. Gently disrupt the tissue by pipetting up and down sequen-
tially through 25 ml, 10 ml, 5 ml pipettes to obtain a fine cell
suspension.
5. Filter the cell suspension through a BD cell strainer (40 m)
into a conical tube.
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 219

6. Gently rub the remaining material between two glass slides,

resuspend in 2 ml DMEM, filter and add to the suspension.
7. Allow the suspension to settle briefly (35 min) during which
most of the larger fragments settle to the bottom. Harvest the
suspension excluding the bottom 200 l containing the frag-
ments (see Note 3b).
8. Examine the settled cells under microscope to see if any clumps
are present. If so, resuspend the settled cells in fresh DMEM,
filter and repeat step 6.
9. Pool the suspension(s) obtained and centrifuge at 1,200 rpm
(300 g) for 10 min.
10. Decant supernatant; resuspend the pellet in 5 ml of ice cold
ammonium chloride (0.17 M pH 7.2) for 5 min on ice.
11. Add 15 ml of serum free DMEM. Count cells to estimate the
number of total cells in the suspension. Spin at 1,200 rpm for
5 min.
12. Resuspend cells in 1 ml of FACS buffer (3 % fetal calf serum in
PBS). Cells are now ready for flow cytometric analysis or fur-
ther isolation procedures.

3.3. Flow Cytometric 1. Reuspend the kidney cells in Fc block (Table 1) for 15 min,
Analysis and Cell adjusting the total suspension volume to approximately 100 l
Sorting per stain. Use a maximum of 1 ml (ten stains) for one whole
kidney.
3.3.1. Flow Cytometry
2. Distribute the cells accordingly into a 96-well V bottom plate
(100 l per well).
3. Add biotinylated antibodies (1/200 dilution), mix gently and
incubate for 30 min on ice. Keep the plates in the dark through-
out the incubation.
4. Meanwhile, mix the next cocktail of antibodies of your choice
together with the streptavidin fluorochrome in a single tube
(1/200 dilution of each antibody). Include a set of stains using
isotype controls.
5. To remove the unbound or excess stain add 100 l of ice cold
PBS to the wells after 30 min of incubation. Centrifuge the
plate for 5 min at 1,200 rpm (300 g) at 4 C.
6. Discard the liquid by inverting the plate once without disturb-
ing the cell pellet.
7. Add the cocktail of antibodies from step 4 to the pellet (100 l/
well) and gently resuspend using a multichannel pipette.
Incubate the plate for another 30 min on ice.
8. Repeat steps 5 and 6.
9. Resuspend the cells in 200 l of 2 % paraformaldehyde, trans-
fer and store in FACS tubes in the dark until the time of acqui-
sition on the flow cytometer (within 24 h).
220 R. Bethunaickan et al.

Table 1
Antibodies used for flow cytometric characterization of kidney macrophages
and DCs

No. Raised in Antibody Fluorochrome Company Catalog no.

1 Rat B220 PE BD Biosciences 553090

2 Rat CD11b PE BD Biosciences 553311
3 Rat CD11b APC eBiosciences 17011282
4 Rat CD11b FITC BD Biosciences 553310
5 Rat CD11b Magnetic beads Miltenyi Biotech 130-049-601
6 Hamster CD11c APC eBiosciences 17011480
7 Hamster CD11c FITC BD Biosciences 553801
8 Hamster CD11c Biotin BD Biosciences 553800
9 Hamster CD11c Magnetic beads Miltenyi Biotech 130-052-001
10 Rat CD16/CD32 BD Biosciences 553142
11 Rat CD19 APC BD Biosciences 550992
12 Rat CD19 AF700 eBiosciences 56019382
13 Rat CD209 Biotin BD Biosciences 55873
14 Rat CD4 FITC BD Biosciences 553047
15 Rat CD43 Biotin BD Biosciences 553269
16 Rat CD44 APC BD Biosciences 559250
17 Rat CD5 PECY5 BD Biosciences 553024
18 Rat CD62L PE BD Biosciences 553151
19 Hamster CD69 PE BD Biosciences 553237
20 Rat CD8 Biotin BD Biosciences 553029
21 Hamster CD80 FITC BD Biosciences 553768
22 Hamster CD80 PE BD Biosciences 553769
23 Rat CD86 Biotin BD Biosciences 553690
24 Rat CD86 FITC BD Biosciences 553691
25 Rat DEC 205 PE Miltenyi Biotech 130092286
26 Rat DECTIN 2 FITC Serotech MCA2415F
27 Rat F4/80 Biotin Caltag MF48015
28 Rat F4/80 FITC Serotech MCA497FB
29 Rat F4/80 PE Caltag MF48004
30 Rat GR1 (Ly6C/G) Biotin eBiosciences 13593181
(continued)
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 221

Table 1
(continued)

No. Raised in Antibody Fluorochrome Company Catalog no.

31 Rat Ly-6C FITC BD Biosciences 553104

32 Goat IGG2A Biotin Southern Biotech 108002
33 Rat MHC II AF 700 eBiosciences 56532182
34 Rat PDCA APC Miltenyi Biotech 130091963
35 Rat VLA4 (CD49d) FITC Southern Biotech 152002
36 Hamster CD49b PE BD Biosciences 558759

3.3.2. Cell Sorting
10. From the CD11b cell gate, sort the CD11chi, F4/80hi and
1. Suspend the cells from both kidneys from one mouse in 2 ml
of FACS buffer with Fc block and incubate on ice for 20 min.
2. Add 7 l of CD11c-biotin antibody to the tube and incubate
for 30 min on ice.
3. Centrifuge the cells at 1,200 rpm (300 g) for 5 min and
decant the supernatant.
4. Resuspend the cell pellet in 2 ml of FACS buffer containing
anti-CD11b APC, F4/80 FITC, and Streptavidin PerCP.
The cocktail should also include a combination of antibodies
to facilitate the exclusion of unwanted cells, such as PE anti-
CD3, CD5, B220, CD138 (optional) and CD49b (57 l
each); incubate for another 30 min.
5. Wash the cells with 2 ml of ice cold PBS and centrifuge at
1,200 rpm (300 g) for 5 min.
6. Resuspend the cell pellet in 500 l of FACS buffer and filter
the suspension using a strainer cap FACS tube. Later, adjust
the volume for the cells based on the number of events acquired
per second on the cell sorter (FACSAria IIu).
7. Just before sorting the cells, add 2 l of DAPI (1 g/ml) to
exclude the dead cells.
8. Pass the cells through the sorter with a flow rate optimized to
5,0007,000 events per second and maintain the efficiency of
the sort at between 85 and 95 %.
9. Use FSC and SSC to set a monocyte/lymphocyte gate and
exclude the unwanted cells in the dump gate and the dead cells
in the DAPI+ gate. Gate on the CD11b+ and/or CD11c+
cells from the live cell gate (see Note 3c).
F4/80lo cells (Fig. 2) into FACS buffer and process them based
on further study requirements (RNA isolation, morphological
and functional characterization, western blot analysis).
222 R. Bethunaickan et al.

3.4. Enrichment 1. Resuspend the pellet obtained from Subheading 3.2, step 11
of Renal Macrophages in PEB containing Fc Block (1:200) for 30 min on ice. Use
and Dendritic Cells 1 ml volume for two kidneys.
Using Magnetic Beads 2. Add required quantity of CD11c coated MACS beads from
Milltenyi Biotec (as per manufacturers recommendation) to
the suspension and incubate for 15 min followed by CD11b
coated MACS beads for 15 min. (For a cell count of ten mil-
lion, add 10 l of each type of bead.)
3. After the incubation period, set aside a few microliters of the
suspension for use as unstained control during sorting.
4. Add 1015 ml PEB to the sample, and centrifuge for 10 min
at 1,500 rpm (570 g).
5. Discard the supernatant by aspiration and resuspend pellet in
5001,000 l of antibody cocktail for 15 min on ice. Filter the
suspension through a 30 m filter and load in Automacs. Set
aside a few microliters for quantifying the percentage of desired
cells in the total suspension.
6. Separate the labeled cells using either POSSEL_S or POSSEL_D
program in Automacs. (POSSEL-S is a sensitive mode in posi-
tive selection, used when we need to have more recovery of the
sample. To achieve high purity, a double positive selection
mode, POSSEL-D is used.)
7. Add DAPI 12 l (1 g/l), to the cell suspension before
sorting to exclude the dead cells.
8. Purify populations from the positively selected fraction using
the FACS-ARIA cell sorter as described in Subheading 3.3.2,
step 10.
9. Cells gated from live cells can be sorted at a flow rate of 2.5
3.0 with a sheath pressure of 34.0. Evaluate purity of the sorted
populations by a post sort analysis.
10. Collect the sorted cells in 1 ml DMEM containing 10 % FBS
for better viability.
11. For calibration of different fluorochromes, use spleen cells as a
reference. This alleviates the need to stain kidney cells for pre-
paring single color controls. Appropriate color controls are
CD4-FITC, CD4-PE, and B220-APC.
12. Spin the post sort cells at 1,500 rpm (570 g) for 10 min and
discard the supernatant.
13. Add 1 ml of fresh medium and re-suspend the cells. Perform a
cell count and plate for cell culture in desired media at required
cell density.
14. Wash the pellet several times in PBS and then use the cells as
starting material for biochemical analyses or for purification of
RNA.
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE
15. Resuspend cells in a small volume of PBS. These can be used
intrinsic macrophages respectively from nephritic mice (two kid-
neys) and 0.10.5 106 cells from a healthy young mouse.
3.5. Morphological
Studies
3.5.1. Cytospin
3.5.2. Scanning Electron
Microscopy SAMPLE PREP
Osmium Quick Fix
3.5.3. Transmission
Electron Microscopy
Sample Preparation
3.6. Cellular Turn Over
Studies
223
for morphologic studies, RNA purification, tissue culture, fur-
ther functional studies or protein analysis.
Yield should be 0.51.5 106 CD11chi dendritic cells or F4/80hi
1. Wash sorted kidney cells (100,000 cells) in cold PBS once and
dilute in 1 ml of cold PBS. Keep all samples on ice.
2. Use 100 l of the cells for each cytospin funnel. Centrifuge for
35 min at a maximum speed of 600 rpm (see Note 4).
3. Fix and process the slides using the desired stain such as
WrightGiemsa stain or Diff-Quick stain.
1. Quick fix the pellet of sorted cells in 1 % osmium tetroxide,
0.1 M sodium cacodylate, 0.2 M sucrose, 5 mM MgCl2 pH 4
(SEM buffer) for 10 s, followed by two changes of 2.5 % glu-
taraldehyde in 1 SEM buffer. Secondary fix cells with 1 %
osmium tetroxide, in 1 SEM buffer for 30 min.
2. Dehydrate through a graded series of ethanol.
3. Critical-point-dry using liquid carbon dioxide in a Tousimis
Samdri 795 Critical Point Drier (Rockville MD).
4. Sputter-coat with gold-palladium in a Denton Vacuum Desk-2
Sputter Coater (Cherry Hill, NJ).
5. Examine in a JEOL JSM6400 Scanning Electron Microscope
(Peabody, MA), using an accelerating voltage of 10 kV.
1. Fix cells with 2.5 % glutaraldehyde and 2 % paraformaldehyde
in 0.1 M sodium cacodylate buffer.
2. Postfix with 1 % osmium tetroxide followed by 2 % uranyl
acetate.
3. Dehydrate through a graded series of ethanol and embed in
LX112 resin (LADD Research Industries, Burlington, VT).
4. Cut ultrathin sections and stain with uranyl acetate followed by
lead citrate and view on a JEOL 1200EX transmission electron
microscope at 80 kV.
1. Freshly prepare a solution of 1 mg BrdU/100 l of PBS. Filter-
sterilize using a 0.45-m filter. Inject 1 mg per mouse i.p. as a
loading dose.
2. Prepare a filter-sterilized solution of 0.8 mg BrdU/ml in drink-
ing water, using 0.45-m filters, and provide it ad libitum to
mice in dark glass water bottles or wrapped with aluminum foil
until time of analysis (typically 760 days).
224 R. Bethunaickan et al.
3.7. Functional Studies In vivo assessment of Cathepsins (B, L, and S) and matrix metal-
of Renal loproteinases (MMPs; MMP2, MMP3, MMP9, and MMP13).
Macrophages/
Dendritic Cells
3.7.1. Cathepsin and
Metalloproteinase Activity
3.7.2. Culturing
of BM-Derived
Macrophages (Mf)
3. Replace with fresh BrdU-containing drinking water every
alternate day.
4. Euthanize mouse and collect the organs of interest after perfu-
sion as above.
5. Single cell suspensions from the organs are made as directed in
Subheading 3.2.
6. Continue with the staining for flow cytometry as in
Subheading 3.3.1. Leave the FITC channel open since this chan-
nel is used for BrdU staining. Do not fix the cells at this stage.
7. Carry out the BrdU FITC staining as described in the manu-
facturers protocol.
8. Fix the cells with 2 % paraformaldehyde. For kidneys, acquire
at least 100,000200,000 cells.
1. Dilute 5 nmol of the probe in 100 l of sterile PBS. Filter-
sterilize using a 0.45-m filter. Perform i.p. injection into the
mouse 24 h before sacrifice.
2. Anesthetize the mice, perfuse, and collect the kidneys. Prepare
single cell suspensions of kidney cells as described in
Subheading 3.2.
3. Stain the cells for flow cytometry with the cocktail of antibod-
ies against CD11b, CD11c and F4/80. Acquire the fixed cells
on the LSRII with the following instructions:
(a) Use the 695/40 filter and 685/LP dichroic filter con-
figuration to detect Prosense-680 and MMPsense-680.
(b) Analyze the cells positive for Prosense-680 and
MMPsense-680 in the Alexafluor 700 channel. Acquire at
least 100,000200,000 cells per sample (see Note 5).
1. Flush mice bone marrow in DMEM followed by RBC lysis
using 5 ml of ice-cold 0.17 M NH4Cl for 5 min over ice.
2. Culture cells in M-CSF (10 ng/ml) or GM-CSF (10 ng/ml)
(negative control).
3. Replace media after 6 days. Harvest and reculture cells in fresh
media in triplicates with M-CSF at a cell density of 2.55.0 105
per well.
4. Treat cells with different cytokines as per requirement.
5. Add IFN- at 10 ng/ml for 48 h.
6. Next day wash one set of IFN- treated cells and add LPS at
10 ng/ml for 30 min. This gives classically activated (CA-) M.
7. Add IL-4 at the required concentration and incubate for 24 h.
This gives alternatively activated (AA-) M.
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 225

8. Leave untreated cells as unstimulated M . A blank well

containing culture media alone is also used as a reference.
9. Culture cells up to 48 h with cytokines.
10. Perform assays for i-NOS or arginase (see Note 6).

3.7.3. Assay for i-NOS
Activity
1. Culture sorted cells with M-CSF (10 ng/ml) with IFN-
gamma (10 ng/ml) LPS (100 ng/ml) for 48 h as above.
2. Carefully remove the media from the wells and spin at
5,000 rpm (2300 g) for 5 min to remove any cell/debris.
3. In a 96-well plate add 130 l of deionized water followed by
130 l of cell supernatant.
4. In a set of wells set up standard nitrite solutions with a water
blank (alternatively, use media for diluting nitrite solutions) to
obtain dilutions between 5 and 100 M concentration.
5. Add 20 l of Griess reagent to the wells and incubate at room
temperature in the dark for 30 min.
6. Read the absorbance at 540 nm in a microplate reader. Use
standards for calculating the amount of nitrite.
7. Wash the cells in the wells several times (at least three times)
with PBS. Aspirate out any remaining solution.
8. Add 100150 l of 0.1 % triton-X with protease inhibitors and
incubate at room temperature for 30 min.
9. Collect the cell lysate and proceed to protein quantification
and arginase assay.

3.7.4. Arginase Assay
1. Take 2550 l of the cell lysate and add equal volume of
10 mM MnCl2 in Tris buffer (50 Mm, pH 7.5).
2. Use a reference set of lysates and add 10 mM EDTA in Tris
buffer as above.
3. Incubate the solutions for 10 min at 55 C to activate the argi-
nase enzyme.
4. Add 2512.5 l of 0.5 M L-Arginine (pH 9.7) solutions to
each tube. Prepare a blank with only water and L-arginine.
5. Incubate for 60 min at 37 C.
6. Remove 50 l of the mix and add 200 l of acid mix
(H2SO4:H3PO4: H2O, 1:3:7).
7. Add 25 l of 9 % -isonitrosopropiophenone (ISPF) prepared
in absolute alcohol.
8. Prepare urea solution in the range of 525 g/ml.
9. Take 50 l of urea solution and add the acid mix and ISPF
solution.
226 R. Bethunaickan et al.

10. Incubate the tubes at 95 C for 45 min. Then cool for 10 min.
to RT.
11. Take 50 l of the solution and assay at 540 nm in a microplate
reader.
12. There should be no significant absorbance in samples with
EDTA incubation.
13. Subtract the EDTA values which account for any non-specific
reaction.
14. Use the urea standards to calculate the amount of urea
released.
15. Express activity in terms of moles of urea released per hour or
per minute.

3.8. Gene Expression 1. Snap freeze one perfused kidney from the mouse immediately
Studies on dry ice, add 3 ml of Trizol and homogenize at once (take
care that the tissue is not thawed during this procedure).
3.8.1. RNA Isolation from
the Kidney by TRIZOL 2. Transfer the homogenate to a 5 ml polypropylene round-bot-
Method tom tube. In a fume hood, add 0.6 ml of chloroform (0.2/ml
per ml of Trizol), mix well by shaking for 15 s and incubate at
room temperature for 5 min.
3. Centrifuge the tube containing the homogenate at a high
speed of 12,000 rpm (16,000 g) for 15 min at 4 C.
4. Transfer the aqueous phase to a new RNase free 5 ml polypro-
pylene round-bottom tube. Add 1,500 l of isopropyl alcohol
(500 l for 1 ml of Trizol reagent).
5. Incubate the sample at RT for 10 min.
6. Centrifuge at 12,000 rpm (16,000 g) for 10 min.
7. Remove supernatant and wash the pellet once with 75 % etha-
nol in RNase free water.
8. Vortex and mix the sample and centrifuge at 7,500 rpm
(6,300 g) for 5 min at 4 C.
9. Remove supernatant from the tube without disturbing the
pellet and air-dry the pellet completely.
10. Resuspend the pellet with 1 ml of 70 % ethanol and transfer it
to an 1.5 ml Eppendorf.
11. Centrifuge at 13,000 rpm (19,000 g) for 10 min at 4 C.
12. Air-dry the pellet in the tube and dissolve it with 100 l of
RNase/DNase free water. Incubate at 55 C for 10 min.
13. Analyze the quality of the isolated RNA by Bioanalyser and
store it at 80 C.
14. To isolate RNA from sorted kidney cells, use RNA isolation
kits such as the RNeasy micro kit from Qiagen or PicoPure
RNA isolation kit which yield high quality and quantity of
RNA.
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 227

3.8.2. cDNA Synthesis 1. Use 5 g of RNA from the whole kidney for cDNA synthesis.
If using sorted cells from the kidney, use 25100 ng as starting
material (see Note 7).
2. Use Superscript III, Invitrogen (Cat #18080-051) for cDNA
synthesis and follow the manufacturers instructions.

3.8.3. Real-Time PCR 1. Identify the optimal concentration of cDNA and perform the
reaction on a 384 well plate run on the LightCycler 480
(Roche).
2. Prepare the reaction mix for a total of 8 l volume per well.
The details of the mix are as follows (see Note 8):
2 Mastermix SYBR Green 4 l per well
Primer (both forward 2 l per well
and reverse) (1 l forward + 1 l reverse)
cDNA 2 l per well

3. Add the above reagents to the wells, seal the plate and centri-
fuge at 1,200 rpm (300 g) for 5 min.
4. Load the plate onto the machine and run using a standard
protocol for SYBR Green. Briefly, the PCR cycle has the fol-
lowing steps:
Denaturation 1 cycle95 C for 5 min
Amplification 45 cycles95 C for 10 s
55 C for 10 s
72 C for 10 s
Melt curve 1 cycle95 C for 20 s
60 C for 30 s
95 C for continuous mode
Cool down 1 cycle40 C for 10 s

3.9. Immunohistologic 1. Cut slides from OCT samples at 80 C and freeze until use.
Studies 2. Thaw slides in a closed chamber at RT until the slides are
thawed (approximately 20 min) (see Note 9).
3. Immerse the slides in cold acetone (pre chilled at 20 C) for
5 min in a Coplin jar. Keep the slides in the dark by covering
the Coplin jar with aluminum foil.
4. Transfer the slides to another Coplin jar and rinse with cold
PBS for 5 min. Leave covered.
5. Repeat step 3 to ensure complete removal of acetone.
6. Prepare Blocking solution (3 % BSA in PBS with Fc
block50 l/10 ml).
7. Apply blocking solution (about 200 l/sample) over the slide
and incubate for 30 min inside the humidified chamber.
228 R. Bethunaickan et al.

8. Discard blocking buffer.

9. Prepare 500 l antibody of choice per sample with desired
Fluorochrome at 1/50 dilution in blocking solution.
10. Apply 300400 l of the diluted antibody over the sample and
incubate for 1 h at RT inside the humidified chamber.
11. Wash slides three times in PBS (5 min each) in dark.
12. For nuclear staining add 200 l of DAPI (1 g/ml of PBS)
and incubate in the dark at RT for 5 min.
13. Wash once with PBS in the dark. Remove PBS from the slides
using filter paper to blot the edges gently.
14. Apply mounting solution (preheated at 65 C water bath) over
the slide and cover with a coverslip taking care not to trap air
bubbles.
15. Store the slides in dark at 4 C until viewed under the
microscope.

3.10. Protein 1. Wash sorted cells 34 times in PBS to remove the BSA (or
Quantification media if cells collected in media) and pellet by centrifuging at
and Analysis 2,500 rpm (600 g) for 5 min.
2. Resuspend the cell pellet in lysis buffer on ice for an hour.
3. After lysis, centrifuge at 14,000 rpm (16,000 g) for 20 min.
4. Discard the pellet and carefully remove the supernatant for fur-
ther analysis.
5. Quantify protein in lysates using Bradford dye binding (1975)
assay using immunoglobulin or bovine serum albumin as the
standard reference.
6. Perform SDS-PAGE using a 12 % resolving and 5 % stacking
gel using the protocol of Laemilli (1976).
7. Carry out immunoblot of proteins after transfer to nitrocellu-
lose membranes using desired antibodies after blocking the
membrane with 5 % skimmed milk.

4. Notes

1. Measuring proteinuria: bladder massage followed by Multitstix

is the most convenient way to follow large cohorts of mice on
a regular basis. Spurious 3+ proteinuria may occur if the mice
become dehydrated but not fixed. ELISA can be used to
confirm the amount of proteinuria before mouse sacrifice
(Subheading 3.1).
10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 229

2. BUN: this tends to go up late in the disease process so cannot

be used to predict disease onset (Subheading 3.1.3).
3. Cell purification:
(a) Kidneys need to be perfused with PBS to remove blood
before processing since large numbers of CD11b+ cells
appear in the blood in SLE models. Perfusion should be
begun with the heart still beating to improve circulation of
the PBS. If the liver is uniformly pale after perfusion then
blood removal has been adequate. Blood removal also
improves the quality of immunohistochemistry
(Subheading 3.2).
(b) Removal of fragments is crucial to avoid blocking the col-
umn and ensuring cell purity. Gravity sedimentation is
very effective (Subheading 3.2).
(c) When performing cell sorting tight gating on the lympho-
cyte/monocyte gate and use of DAPI will exclude cell
fragments and improve purity (Subheading 3.3.2).
4. Cytospin: when performing the cytospin, higher centrifuge
speed (more than 600 rpm) will destroy the morphology of the
cells (Subheading 3.5.1).
5. Flow cytometry: best results for routine staining are obtained
when the data are acquired within 24 h. Phagocytosis assays
should be acquired immediately. MMP and Cathepsin assays
are best when acquired immediately and reagents are fresh
(Subheading 3.7.1).
6. Arginase and iNOS assays: bone marrow derived macrophages
can be used as controls for these assays. Cells and superna-
tants can be frozen in aliquots. Wash cells in PBS before
freezing. Microplates should be centrifuged before reading
to remove air bubbles. The urea reaction is stable and plates
can be read up to 24 h later but the nitrite assay changes
color after an hour. If plated cell density is low (50,000
100,000) add 60 l lysis buffer to the wells after washing
with PBS and take the cell lysate directly for assays
(Subheadings 3.7.3 and 3.7.4).
7. cDNA synthesis: cDNA obtained from 5 g of RNA from
whole kidney (20 l) was diluted 25 times up to 500 l and
from this 2 l was used per well for the real-time PCR
(Subheading 3.8.2).
8. Real-time PCR: the primer concentration used was optimized
at 10 nm for Invitrogen primers and 1.5 M for primers
obtained from SuperArray (Subheading 3.8.3).
9. Immunohistochemistry: make sure not to allow the edges of
the sections to dry, as this will increase the background stain-
ing (Subheading 3.9).
230 R. Bethunaickan et al.
References
1. Davidson A, Aranow C (2006) Pathogenesis
and treatment of systemic lupus erythematosus
nephritis. Curr Opin Rheumatol 18:468475
2. Chan TM (2005) Preventing renal failure in
patients with severe lupus nephritis. Kidney Int
Suppl 94:S116S119
3. Contreras G, Pardo V, Leclercq B, Lenz O,
Tozman E, ONan P, Roth D (2004) Sequential
therapies for proliferative lupus nephritis. N
Engl J Med 350:971980
4. Contreras G, Tozman E, Nahar N, Metz D
(2005) Maintenance therapies for proliferative
lupus nephritis: mycophenolate mofetil, aza-
thioprine and intravenous cyclophosphamide.
Lupus 14(Suppl 1):s33s38
5. Costenbader KH, Solomon DH, Winkelmayer
W, Brookhart MA (2008) Incidence of end-stage
renal disease due to lupus nephritis in the U.S.,
19952004. Arthrit Rheum Abstract 1927
6. Schwartz MM (2007) The pathology of lupus
nephritis. Semin Nephrol 27:2234
7. Hill GS, Delahousse M, Nochy D, Mandet C,
Bariety J (2001) Proteinuria and tubulointer-
stitial lesions in lupus nephritis. Kidney Int 60:
18931903
8. Hill GS, Delahousse M, Nochy D, Remy P,
Mignon F, Mery JP, Bariety J (2001) Predictive
power of the second renal biopsy in lupus
nephritis: significance of macrophages. Kidney
Int 59:304316
9. Li QZ, Xie C, Wu T, Mackay M, Aranow C,
Putterman C, Mohan C (2005) Identification
of autoantibody clusters that best predict lupus
disease activity using glomerular proteome
arrays. J Clin Invest 115:34283439
10. Bagavant H, Fu SM (2005) New insights from
murine lupus: disassociation of autoimmunity
and end organ damage and the role of T cells.
Curr Opin Rheumatol 17:523528
11. Christensen SR, Kashgarian M, Alexopoulou
L, Flavell RA, Akira S, Shlomchik MJ (2005)
Toll-like receptor 9 controls anti-DNA autoan-
tibody production in murine lupus. J Exp Med
202:321331
12. Turnberg D, Cook HT (2005) Complement
and glomerulonephritis: new insights. Curr
Opin Nephrol Hypertens 14:223228
13. Clynes R, Dumitru C, Ravetch JV (1998)
Uncoupling of immune complex formation
and kidney damage in autoimmune glomerulo-
nephritis. Science 279:10521054
14. Anders HJ, Schlondorff D (2007) Toll-like
receptors: emerging concepts in kidney disease.
Curr Opin Nephrol Hypertens 16:177183
15. Sesin CA, Yin X, Esmon CT, Buyon JP, Clancy
RM (2005) Shedding of endothelial protein C
receptor contributes to vasculopathy and renal
injury in lupus: in vivo and in vitro evidence.
Kidney Int 68:110120
16. Chan OT, Hannum LG, Haberman AM,
Madaio MP, Shlomchik MJ (1999) A novel
mouse with B cells but lacking serum antibody
reveals an antibody-independent role for B cells
in murine lupus. J Exp Med 189:16391648
17. Lewis EJ, Schwartz MM (2005) Pathology of
lupus nephritis. Lupus 14:3138
18. Anders HJ, Ninichuk V, Schlondorff D (2006)
Progression of kidney disease: blocking leuko-
cyte recruitment with chemokine receptor
CCR1 antagonists. Kidney Int 69:2932
19. Holdsworth SR, Tipping PG (2007) Leukocytes
in glomerular injury. Semin Immunopathol
29:355374
20. Foster MH (2007) T cells and B cells in lupus
nephritis. Semin Nephrol 27:4758
21. Schiffer L, Sinha J, Wang X, Huang W, von
Gersdorff G, Schiffer M, Madaio MP, Davidson
A (2003) Short term administration of costim-
ulatory blockade and cyclophosphamide
induces remission of systemic lupus erythema-
tosus nephritis in NZB/W F1 mice by a mech-
anism downstream of renal immune complex
deposition. J Immunol 171:489497
22. Daikh DI, Wofsy D (2001) Cutting edge:
reversal of murine lupus nephritis with
CTLA4Ig and cyclophosphamide. J Immunol
166:29132916
23. Timoshanko JR, Sedgwick JD, Holdsworth
SR, Tipping PG (2003) Intrinsic renal cells are
the major source of tumor necrosis factor con-
tributing to renal injury in murine crescentic
glomerulonephritis. J Am Soc Nephrol 14:
17851793
24. Wang Y, Wang Y, Cai Q, Zheng G, Lee VW,
Zheng D, Li X, Tan TK, Harris DC (2008) By
homing to the kidney, activated macrophages
potently exacerbate renal injury. Am J Pathol
172:14911499
25. Ferenbach D, Hughes J (2008) Macrophages
and dendritic cells: what is the difference?
Kidney Int 74:57
26. Kurts C, Heymann F, Lukacs-Kornek V, Boor
P, Floege J (2007) Role of T cells and dendritic
cells in glomerular immunopathology. Semin
Immunopathol 29:317335
27. Mosser DM (2003) The many faces of mac-
rophage activation. J Leukoc Biol 73:209212
28. Mantovani A, Sica A, Sozzani S, Allavena P,
Vecchi A, Locati M (2004) The chemokine sys-
tem in diverse forms of macrophage activation
and polarization. Trends Immunol 25:
677686
 10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE
29. Mosser DM, Edwards JP (2008) Exploring the
full spectrum of macrophage activation. Nat
Rev Immunol 8:958969
30. Hume DA (2008) Differentiation and hetero-
geneity in the mononuclear phagocyte system.
Mucosal Immunol 1:432441
31. Tacke F, Randolph GJ (2006) Migratory fate
and differentiation of blood monocyte subsets.
Immunobiology 211:609618
32. Li L, Huang L, Sung SS, Vergis AL, Rosin DL,
Rose CE Jr, Lobo PI, Okusa MD (2008) The
chemokine receptors CCR2 and CX3CR1
mediate monocyte/macrophage trafficking in
kidney ischemia-reperfusion injury. Kidney Int
74:15261537
33. Swaminathan S, Griffin MD (2008) First
responders: understanding monocyte-lineage
traffic in the acutely injured kidney. Kidney Int
74:15091511
34. Auffray C, Fogg D, Garfa M, Elain G, Join-
Lambert O, Kayal S, Sarnacki S, Cumano A,
Lauvau G, Geissmann F (2007) Monitoring of
blood vessels and tissues by a population of
monocytes with patrolling behavior. Science
317:666670
35. Geissmann F, Auffray C, Palframan R, Wirrig
C, Ciocca A, Campisi L, Narni-Mancinelli E,
Lauvau G (2008) Blood monocytes: distinct
subsets, how they relate to dendritic cells, and
their possible roles in the regulation of T-cell
responses. Immunol Cell Biol 86:398408
36. Skold M, Behar SM (2008) Tuberculosis trig-
gers a tissue-dependent program of differentia-
tion and acquisition of effector functions by
circulating monocytes. J Immunol 181:
63496360
37. Martinez FO, Sica A, Mantovani A, Locati M
(2008) Macrophage activation and polariza-
tion. Front Biosci 13:453461
38. Wu L, Liu YJ (2007) Development of den-
dritic-cell lineages. Immunity 26:741750
39. Liu K, Waskow C, Liu X, Yao K, Hoh J,
Nussenzweig M (2007) Origin of dendritic
cells in peripheral lymphoid organs of mice.
Nat Immunol 8:578583
40. Auffray C, Emre Y, Geissmann F (2008)
Homeostasis of dendritic cell pool in lymphoid
organs. Nat Immunol 9:584586
41. Kamath AT, Henri S, Battye F, Tough DF,
Shortman K (2002) Developmental kinetics
and lifespan of dendritic cells in mouse lym-
phoid organs. Blood 100:17341741
42. Kruger T, Benke D, Eitner F, Lang A, Wirtz
M, Hamilton-Williams EE, Engel D, Giese B,
Muller-Newen G, Floege J, Kurts C (2004)
Identification and functional characterization
of dendritic cells in the healthy murine kidney
231
and in experimental glomerulonephritis. J Am
Soc Nephrol 15:613621
43. Soos TJ, Sims TN, Barisoni L, Lin K, Littman
DR, Dustin ML, Nelson PJ (2006) CX3CR1+
interstitial dendritic cells form a contiguous
network throughout the entire kidney. Kidney
Int 70:591596
44. Segerer S, Heller F, Lindenmeyer MT, Schmid H,
Cohen CD, Draganovici D, Mandelbaum J,
Nelson PJ, Grone HJ, Grone EF, Figel AM,
Nossner E, Schlondorff D (2008) Compartment
specific expression of dendritic cell markers in
human glomerulonephritis. Kidney Int 74:
3746
45. Kurts C (2006) Dendritic cells: not just another
cell type in the kidney, but a complex immune
sentinel network. Kidney Int 70:412414
46. Bethunaickan R, Berthier CC, Ramanujam M,
Sahu R, Zhang W, Sun Y, Bottinger EP,
Ivashkiv L, Kretzler M, Davidson A (2011)
A unique hybrid renal mononuclear phago-
cyte activation phenotype in murine systemic
lupus erythematosus nephritis. J Immunol:
186:49945003
47. Ramanujam M, Davidson A (2008) Targeting
of the immune system in systemic lupus erythe-
matosus. Expert Rev Mol Med 10:e2
48. Singh RR, Saxena V, Zang S, Li L, Finkelman
FD, Witte DP, Jacob CO (2003) Differential
contribution of IL-4 and STAT6 vs STAT4 to
the development of lupus nephritis. J Immunol
170:48184825
49. Santiago ML, Fossati L, Jacquet C, Muller W,
Izui S, Reininger L (1997) Interleukin-4 pro-
tects against a genetically linked lupus-like auto-
immune syndrome. J Exp Med 185:6570
50. Matsumoto K, Watanabe N, Akikusa B,
Kurasawa K, Matsumura R, Saito Y, Iwamoto
I, Saito T (2003) Fc receptor-independent
development of autoimmune glomerulone-
phritis in lupus-prone MRL/lpr mice. Arthritis
Rheum 48:486494
51. Ehlers M, Fukuyama H, McGaha TL, Aderem
A, Ravetch JV (2006) TLR9/MyD88 signal-
ing is required for class switching to pathogenic
IgG2a and 2b autoantibodies in SLE. J Exp
Med 203:553561
52. Wu X, Peng SL (2006) Toll-like receptor 9 sig-
naling protects against murine lupus. Arthritis
Rheum 54:336342
53. Fu Y, Du Y, Mohan C (2007) Experimental
anti-GBM disease as a tool for studying sponta-
neous lupus nephritis. Clin Immunol 124:
109118
54. Perry D, Sang A, Yin Y, Zheng YY, Morel L
(2011) Murine models of systemic lupus erythe-
matosus. J Biomed Biotechnol 2011:271694
232 R. Bethunaickan et al.
55. Ramanujam M, Wang X, Huang W, Liu Z,
Schiffer L, Tao H, Frank D, Rice J, Diamond
B, Yu KO, Porcelli S, Davidson A (2006)
Similarities and differences between selective
and nonselective BAFF blockade in murine
SLE. J Clin Invest 116:724734
56. Rudofsky UH, Lawrence DA (1999) New
Zealand mixed mice: a genetic systemic lupus
erythematosus model for assessing environ-
mental effects. Environ Health Perspect 107:
713721
57. Ramanujam M, Davidson A (2008) BAFF
blockade for systemic lupus erythematosus
will the promise be fulfilled? Immunol Rev
223:156174
58. Pisitkun P, Deane JA, Difilippantonio MJ,
Tarasenko T, Satterthwaite AB, Bolland S
(2006) Autoreactive B cell responses to RNA-
related antigens due to TLR7 gene duplication.
Science 312:16691672
59. Subramanian S, Tus K, Li QZ, Wang A, Tian
XH, Zhou J, Liang C, Bartov G, McDaniel
LD, Zhou XJ, Schultz RA, Wakeland EK
(2006) A Tlr7 translocation accelerates sys-
temic autoimmunity in murine lupus. Proc Natl
Acad Sci USA 103:99709975
60. Haywood ME, Rogers NJ, Rose SJ, Boyle J,
McDermott A, Rankin JM, Thiruudaian V,
Lewis MR, Fossati-Jimack L, Izui S, Walport
MJ, Morley BJ (2004) Dissection of BXSB
lupus phenotype using mice congenic for chro-
mosome 1 demonstrates that separate intervals
direct different aspects of disease. J Immunol
173:42774285
61. Akkerman A, Huang W, Wang X, Ramanujam
M, Schiffer L, Madaio M, Factor SM, Davidson
A (2004) CTLA4Ig prevents initiation but not
evolution of anti-phospholipid syndrome in
NZW/BXSB mice. Autoimmunity 37:445451
62. Kahn P, Ramanujam M, Bethunaickan R,
Huang W, Tao H, Madaio MP, Factor SM,
Davidson A (2008) Prevention of murine
antiphospholipid syndrome by BAFF blockade.
Arthritis Rheum 58:28242834
63. Hang LM, Izui S, Dixon FJ (1981) (NZW
BXSB)F1 hybrid. A model of acute lupus and
coronary vascular disease with myocardial
infarction. J Exp Med 154:216221
64. Watanabe-Fukunaga R, Brannan CI, Copeland
NG, Jenkins NA, Nagata S (1992)
Lymphoproliferation disorder in mice explained
by defects in Fas antigen that mediates apopto-
sis. Nature 356:314317
65. Andrews BS, Eisenberg RA, Theofilopoulos
AN, Izui S, Wilson CB, McConahey PJ, Murphy
ED, Roths JB, Dixon FJ (1978) Spontaneous
murine lupus-like syndromes. Clinical and
immunopathological manifestations in several
strains. J Exp Med 148:11981215
66. Menke J, Rabacal WA, Byrne KT, Iwata Y,
Schwartz MM, Stanley ER, Schwarting A,
Kelley VR (2009) Circulating CSF-1 promotes
monocyte and macrophage phenotypes that
enhance lupus nephritis. J Am Soc Nephrol
20:25812592
67. Hoi AY, Hickey MJ, Hall P, Yamana J,
OSullivan KM, Santos LL, James WG, Kitching
AR, Morand EF (2006) Macrophage migra-
tion inhibitory factor deficiency attenuates
macrophage recruitment, glomerulonephritis,
and lethality in MRL/lpr mice. J Immunol
177:56875696
68. Schiffer L, Bethunaickan R, Ramanujam M,
Huang W, Schiffer M, Tao H, Madaio MP,
Bottinger EP, Davidson A (2008) Activated
renal macrophages are markers of disease
onset and disease remission in lupus nephritis.
J Immunol 180:19381947
 Chapter 11

A Murine Autoimmune Model of Rheumatoid Arthritis

and Systemic Lupus Erythematosus Associated
with Deregulated Production of IL-17 and IL-21
Partha S. Biswas, Kyuho Kang, Sanjay Gupta,
Govind Bhagat, and Alessandra B. Pernis

Abstract
T-helper cell 17 (Th17) cells play an important role in the pathogenesis of many autoimmune disorders
including Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). In this chapter we
describe a murine model where deregulated production of IL-17 and IL-21 can lead to either lupus-like
disease or RA-like symptoms depending on the genetic background. We delineate the key techniques that
can be used to dissect the mechanisms responsible for the pathogenesis of these diseases at both a cellular
and molecular level including in vitro Th17 cell differentiation, chromatin immunoprecipitation assays,
and retroviral transduction experiments. We also describe the methodologies that can be utilized to moni-
tor the classic clinical findings of RA and SLE in murine models. Given the broad involvement of deregu-
lated production of IL-17 and IL-21 in autoimmunity, many of these techniques could also be valuable for
the investigation of these pathways in murine models of other autoimmune diseases.

Key words: Arthritis, Systemic lupus erythematosus, Autoimmunity, Animal models, Interleukin-17,
Interleukin-21

1. Introduction

T-helper cell 17 (Th17) cells play an important role in the patho-

genesis of many autoimmune disorders including Rheumatoid
Arthritis (RA) (14). The ability of Th17 cells to produce IL-17 is
critical to RA pathogenesis since IL-17 induces the production of
proinflammatory cytokines and stimulates MMP activity, matrix
catabolism, and bone resorption (5, 6). Th17 cells also produce
IL-21 (79), which can amplify the differentiation of Th17 cells as
well as control T-dependent humoral responses (10, 11).
Deregulation of IL-17 and IL-21 production plays a role not only

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_11, Springer Science+Business Media New York 2012

233
234 P.S. Biswas et al.

in RA but also in systemic lupus erythematosus (SLE) pathogenesis

(12). Elevated levels of IL-17 and IL-21 have been observed in
murine models of RA and SLE and in patients affected by these
disorders (1, 1315). Furthermore, blockade of the IL-21/IL-21R
pathway is efficacious in ameliorating disease in murine models of
both RA and SLE (14, 16).
The factors that regulate the differentiation of Th17 cells and
the production of IL-17 and IL-21 have been extensively investi-
gated (1720). In particular, TGF- and IL-6 have been shown to
play an important role in the development of Th17 cells. Induction
of IL-17 production depends on the presence of Stat3 and RORt
(2123), while IL-21 expression requires the presence of Stat3 and
either RORt or ROR (8, 24). Interestingly, studies have demon-
strated that another transcription factor, IRF4, is also absolutely
required for Th17 differentiation (25, 26). Indeed the absence of
IRF4 results in a complete lack of IL-17 and IL-21 production
(2527). IRF4 controls the production of IL-17 and IL-21 by
regulating the expression of RORt and ROR and by directly
binding to the IL-17 and IL-21 promoters (2527).
Unlike other master regulators of T helper cell differentiation,
the expression of IRF4 is upregulated by TCR stimulation and is
not restricted to a specific T helper subset (28, 29) suggesting that
regulatory mechanisms exist to control the function of IRF4 in a T
helper subset-specific manner. During a search for proteins interact-
ing with IRF4, our laboratory isolated a protein, termed Def6 (also
known as IBP = IRF4 Binding Protein or SLAT) (3032). We have
found that Def6 serves a crucial but complex immunoregulatory
role in vivo. Indeed, by 5 months of age 60 % of Def6-deficient
female mice on a mixed 129/B6 background, but none of the con-
trol mice, start developing multiple enlarged lymph nodes as well as
splenomegaly (33). Serologic analysis has demonstrated that aging
(>5 m old) Def6-deficient female mice display markedly elevated
serum IgG levels and antibodies against dsDNA and Sm proteins
(33). Aging Def6-deficient female mice also develop proteinuria
and glomerulonephritis, which is associated with the deposition of
IgG and C3 (33). Thus on a mixed 129/B6 background the lack
of Def6 leads to the development of a lupus-like syndrome.
To facilitate our studies on the role of Def6 in T cell function,
Def6-deficient mice were extensively backcrossed onto the Balb/c
background and then crossed to DO11.10 mice, which carry an
I-Ad-restricted transgenic T cell receptor for ovalbumin (34).
Surprisingly, by 3 months of age, 90 % of Def6-deficient DO11.10
mice, but none of the control DO11.10 mice, spontaneously
developed symmetrical joint swelling with hyperemia (26). The
symptoms were chronic and progressive eventually leading to
impaired mobility. Histopathologic analysis revealed pannus for-
mation with destruction of the adjacent cartilage and the subchon-
dral bone (26). Serologic analysis demonstrated that Def6-deficient
 11 A Murine Autoimmune Model of Rheumatoid Arthritis 235

DO11.10 mice displayed elevated titers of Rheumatoid Factor

(RF), anti-CCP, and anti-Collagen II antibodies while anti-dsDNA
titers were not significantly increased (26). Thus on a BALB/c
background the lack of Def6 combined with the presence of a
specific TCR transgene leads to the development of RA-like
arthritis.
The finding that the absence of Def6 in mice leads to either
SLE or RA in distinct genetic backgrounds suggests that, similarly
to what is observed in humans, Def6 controls a common patho-
genic pathway whose final manifestations are shaped by the genetic
(and environmental) milieu of an individual. Indeed an extensive
analysis has demonstrated that autoimmunity in both sets of Def6-
deficient mice is characterized by the expansion of peripheral CD4+
T cells, which aberrantly produce IL-17 and IL-21 under neutral
(or Th0) conditions ((26, 35) and unpublished observations).
A detailed molecular characterization has furthermore demon-
strated that the aberrant production of IL-17 and IL-21 observed
in the absence of Def6 results from an enhanced ability of IRF4 to
target the RORt as well as the IL-17 and IL-21 promoters and
drives the production of these cytokines (26, 35). In addition, we
have found that Def6 controls the function of IRF4 by a dual
mechanism: it physically sequesters IRF4 and it prevents phospho-
rylation of IRF4 by the serinethreonine kinase ROCK2 (26, 35).
In this chapter we outline in detail the key techniques that we
have utilized to dissect disease pathogenesis in this model at both
a cellular and molecular level. In particular we describe the meth-
ods for in vitro Th17 cell differentiation, chromatin immunopre-
cipitation (ChIP) assays, and retroviral transduction experiments.
We also discuss the methodologies that can be utilized to monitor
the classic clinical findings of RA and SLE in murine models.
Although our emphasis will be on these two diseases, given the
broad involvement of deregulated production of IL-17 and IL-21
in many autoimmune disorders (4), much of this information
should also be valuable for the investigation of these pathways in
murine models of other autoimmune diseases.

2. Materials

2.1. T-Helper Cell 17 1. Complete Clicks media (IrvineScientific).

Differentiation of 2. PBS (Mediatech Inc.).
Mouse Nave CD4+
3. RBC lysis buffer (R&D System).
T Cells
4. CD4+ T cells Isolation kit (Miltenyi Biotech).
5. Fluorochrome conjugated antibodies (anti-mouse CD4 PeCy7
(Clone: RM4-5), anti-mouse CD25 PE (Clone: PC61.5),
236 P.S. Biswas et al.
10. PMA and Ionomycin (CalBiochem).
2.2. Chromatin
Immunoprecipitation
Assays
2.3. Retroviral
Infection of CD4+
T Cells
10. Recombinant murine IL-2 (BD Pharmingen).
11. Polybrene (Sigma-Aldrich).
2.4. Flow Cytometric
Analyses of T and B
Cell Compartments
in Spleen and LNs
2.5. Clinical
Assessment of
Arthritis: Serum Levels
of Rheumatoid Factor
anti-mouse CD44 FITC (Clone: IM7), and anti-mouse
CD62L APC (Clone: MEL-14) (eBioscience)).
6. Anti-mouse CD3 (Clone: 17A2) and anti-mouse CD28
(Clone: 37.51) antibody (BioXcell).
7. Anti-mouse IFN- (Clone: XMG1.2) and anti-mouse IL-4
(Clone: 11B-11) antibody (BioXcell), recombinant mouse
IL-12 (Peprotech), recombinant mouse IL-4 (Peprotech),
recombinant mouse IL-6 (Peprotech), and recombinant
human TGF- (Peprotech).
8. Mouse IL-17A ELISA Kit (eBioscience) and Mouse IL-21
ELISA Kit (R&D Systems).
9. BD Cytofix/Cytoperm Kit (BD Pharmingen).
1. Formaldehyde (Sigma-Aldrich).
2. Glycine (Pierce).
3. DNA purification kit (QIAGEN).
4. Rabbit IgG (Sigma-Aldrich).
5. SYBR green real-time PCR premix (Applied Biosystems).
1. 293T cells.
2. Complete DMEM media (Mediatech Inc.).
3. DNA vector.
4. Mammalian cell transfection kit (Millipore).
5. Complete Clicks media (Irvine Scientific).
6. PBS (Mediatech Inc.).
7. RBC lysis buffer (R&D System).
8. CD4+ T cells Isolation kit (Miltenyi Biotech).
9. Anti-mouse CD3 (Clone: 17A2) and anti-mouse CD28
(Clone: 37.51) antibody (BioXcell).
1. Complete Clicks media (IrvineScientific).
2. PBS (Mediatech Inc.).
3. RBC lysis buffer (R&D System).
4. FACS Buffer (1 % BSA and 0.017 % sodium azide).
1. Mouse Rheumatoid Factor ELISA Kit (Alpha Diagnostic
International).
 11 A Murine Autoimmune Model of Rheumatoid Arthritis 237

2.6. Clinical 1. Mouse Anti-dsDNA IgG ELISA Kit (Alpha Diagnostic

Assessment of Lupus: International).
Serum Levels of
Anti-dsDNA Antibody

2.7. Clinical 1. Positive control serum: Serum from diseased MRL/lpr mice.
Assessment of Lupus: 2. Negative control serum: Serum from healthy wild-type mice.
ANA Staining
3. MBL-BION ANA (HEp-2) Antigen substrate slides (MBL
Bion).
4. FITC conjugated anti-mouse IgG (Jackson Immunoresearch).
5. Phosphate Buffer Saline (Mediatech Inc.).
6. Vectashield Mounting medium for Fluorescence (Vector
Lab).

2.8. Clinical 1. Tissue-Tek OCT Compound (Sakura).

Assessment of Lupus: 2. FITC conjugated mouse anti-mouse IgG (Jackson
Immunofluorescence Immunoresearch Lab) and mouse anti-mouse C3 (MP
Staining of Immune- Cappel).
Complex Deposition
3. Vectashield Mounting medium for Fluorescence (Vector
in Kidney Lab).
4. DAPI (Invitrogen).
5. Fluorescence microscope (Nikon).

3. Methods

3.1. T-Helper Cell 17 1. Sacrifice 810-week-old mice and collect spleen and LNs in
Differentiation of ice-cold media.
Mouse Nave CD4+ T 2. Prepare single cell suspension of spleen and LNs by mashing
Cells (See Notes 14) between two frosted microscopic slides followed by passing the
3.1.1. Isolation and cell suspension through 70-m nylon mesh to remove any cell
Purification of Total CD4+ clumps and debris.
T Cells from Mouse Spleen 3. Centrifuge cell suspension for 10 min at 300 g at 4 C.
and Peripheral Lymph 4. Discard the supernatant and lyse the RBCs using RBC lysis
Nodes buffer. Add ice-cold PBS (ten times the volume of RBC lysis
buffer) and centrifuge for 10 min at 300 g at 4 C.
5. Wash the cell pellets twice with ice-cold PBS.
6. Determine viable cell count by Trypan blue exclusion.
7. Proceed to negative selection of total CD4+ T cells using CD4+
T cell Isolation kit following the manufacturers protocol.
238 P.S. Biswas et al.
3.1.2. FACS Sorting
of Nave CD4+ T Cells
from Total CD4+ T Cells
3.1.3. TH17 Differentiation
of Nave CD4+ T Cells
3.1.4. Detection of IL-17
by ELISA and Intracellular
FACS Analyses
1. Wash MACS-purified total CD4+ T cells with ice-cold MACS
Buffer.
2. Determine viable cell count by Trypan blue exclusion.
3. Resuspend the cell pellet in complete Clicks medium at a cell
concentration of ten million cells/ml.
4. Stain the cells with fluorochrome-conjugated antibodies (anti-
mouse CD4 PeCy7, anti-mouse CD25 PE, anti-mouse CD44
FITC, and anti-mouse CD62L APC) for 30 min on ice.
5. Wash the cells twice with ice-cold MACS buffer and finally
resuspend in complete Clicks media at a cell concentration of
1020 million cells/ml.
6. Proceed to cell sorting.
1. Coat 24-well tissue culture plates with anti-CD3 antibody at a
concentration of 1 g/ml in PBS at 37 C for 2 h.
2. In the meantime wash the FACS-sorted nave (CD4+CD25-
CD44intCD62Lhi) CD4+ T cells with ice-cold PBS.
3. Resuspend the cell pellet in complete Clicks media.
4. For Th0, Th1, Th2, and Th17 differentiating conditions, use
the following antibody and cytokine cocktails in complete
Clicks medium:
Th0: Anti-CD28 (2 g/ml).
Th1: Anti-CD28 (2 g/ml), Anti-IL-4 (10 g/ml), recombi-
nant mouse IL-12 (10 ng/ml).
Th2: Anti-CD28 (2 g/ml), Anti-IFN- (10 g/ml), recom-
binant mouse IL-4 (10 ng/ml).
Th17: Anti-CD28 (2 g/ml), Anti-IFN- (10 g/ml),
Anti-IL-4 (10 g/ml), recombinant mouse IL-6 (20 ng/
ml), and recombinant human TGF- (5 ng/ml).
5. Wash the anti-CD3-coated plates with PBS to remove any
unbound antibody.
6. Plate the cells with respective differentiating medium at a con-
centration of 11.5 million cells/ml and culture for 35 days.
Following stimulation of nave CD4+ T cells in Th17 skewing con-
ditions, production of IL-17 can be quantified by two methods:
1. Supernatant ELISA: After 35 days of stimulation in Th17 dif-
ferentiating conditions, harvest the supernatants and subject
them to an IL-17 and IL-21 ELISA using a commercially avail-
able kit from eBioscience and R&D Systems, respectively, as
per the manufacturers protocol.
 11 A Murine Autoimmune Model of Rheumatoid Arthritis 239

2. Intracellular FACS staining following restimulation with PMA

and ionomycin:
(a) Harvest the cells and wash 3 times with ice-cold PBS.
(b) Count the cells with Trypan Blue staining.
(c) Restimulate 2 106 cells/well in a 24-well plate with PMA
(50 ng/ml) and ionomycin (1 M) for 46 h in the pres-
ence of GolgiStop (1 g/ml).
(d) After restimulation collect the cells and wash several times
with PBS.
(e) Proceed for surface staining with fluorochrome-conjugated
anti-mouse CD4+ antibody for 30 min.
(f) After several washing with FACS buffer fix and permeabi-
lize the cells with Fix/Perm Buffer as per the manufac-
turers protocol.
(g) Resuspend the cells in 1 Perm Buffer and proceed to
intracellular staining with anti-mouse IL-17 PE, anti-
mouse IFN- APC, and anti-mouse IL-4 FITC for 60 min
on ice.
(h) Wash the cells twice with 1 Perm Buffer and finally resus-
pend in FACS Buffer and proceed to FACS analyses.

3.2. Chromatin 1. Stimulate mouse CD4+ T cells (10 ~ 20 106 cells) in the con-
Immunoprecipitation dition of interest.
Assays (See Note 5) 2. Cross-link chromatin by incubating the cells with 1 % formal-
dehyde in PBS for 10 min at 37 C. Stop the cross-linking by
adding glycine to a final concentration of 0.1 M. Rinse the cells
twice with ice-cold PBS.
3. Resuspend the cell pellets in 0.4 ml of cell lysis buffer (5 mM
Pipes [pH 8.0], 85 mM KCl, and 0.5 % NP40) containing pro-
tease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 g/ml
aprotinin, 10 g/ml leupeptin, and 1 g/ml pepstatin A) and
incubate on ice for 10 min. Centrifuge the samples for 5 min
at 2,700g at 4 C to pellet the nuclei.
4. Resuspend the nuclear pellets in 0.4 ml of nuclear lysis buffer
(50 mM TrisHCl [pH 8.0], 1 % SDS, and 10 mM EDTA)
supplemented with the above-stated protease inhibitors. Keep
the samples on ice for 10 min.
5. Sonicate the nuclear lysates for four to five 30 s pulses at the
maximum setting of 5, with 30 s of cooling on ice between
the pulses, to shear DNA into small fragments. Centrifuge the
nuclear lysate for 10 min at 18,000g at 4 C.
6. Collect the supernatant fraction (containing cross-linked chro-
matin) and dilute in Chromatin dilution buffer (1 % Triton
X-100, 0.01 % SDS, 2 mM EDTA, 150 mM NaCl, 20 mM
240 P.S. Biswas et al.

TrisHCl [pH 8.0]) containing protease inhibitors. Split the

chromatin solution into two equal aliquots for immunopre-
cipitation with a control antibody or with an antibody against
the DNA-binding protein of interest. Save 100 l of chromatin
solution for input control and store at 20 C.
7. Preclear the chromatin solutions with 50 l of protein G-agarose
beads (pre-blocked with Salmon Sperm DNA) for 1 h at 4 C
with agitation.
8. Incubate the precleared chromatin solutions with 12 g of
control antibody or with an antibody against the DNA-binding
protein of interest overnight at 4 C with rotation.
9. Collect the immune complexes with 50 l of protein G-agarose
beads (pre-blocked with Salmon sperm DNA) for 1 h at 4 C
with constant rotation.
10. Wash the beads sequentially twice for 10 min each with rota-
tion with 1 ml each of low-salt wash buffer (0.1 % SDS, 1 %
Triton X-100, 2 mM EDTA, 20 mM TrisHCl [pH 8.0],
150 mM NaCl), high-salt wash buffer (0.1 % SDS, 1 % Triton
X-100, 2 mM EDTA, 20 mM TrisHCl [pH 8.0], 500 mM
NaCl), and LiCl wash buffer (0.25 M LiCl, 1 % NP-40, 1 %
deoxycholate, 1 mM EDTA, 10 mM TrisHCl [pH 8.0]).
11. Wash the beads again three times with TE buffer (10 mM Tris
HCl [pH 8.0] and 1 mM EDTA).
12. Elute the immune complexes twice by incubating the beads
with 250 l of elution buffer (1 % SDS, 0.1 M NaHCO3) for
15 min each time at room temperature with rotation.
13. Reverse the cross-linking by heating the eluates and input sam-
ples at 65 C for 4 h, followed by removal of proteins by
Proteinase K digestion at 45 C for 2 h.
14. Purify the DNA from the input and the immunoprecipitated
samples by Phenol/Chloroform extraction and ethanol pre-
cipitation, and resuspend the DNA in 50 l of TE buffer.
15. Detect the presence of specific DNA sequence(s) of interest in
immunoprecipitated samples by quantitative PCR. Include
input DNA samples as loading controls and control antibody
immunoprecipitates as negative control. Perform quantitative
PCR reactions in duplicates with 510 l of DNA samples.

3.3. Retroviral 1. Plate 293T cells 24 h before transfection at a concentration of

Infection of CD4+ T 22.5 million cells/100 mm dish/10 ml complete DMEM
Cells (See Notes 610) media.
3.3.1. Preparation of 293T 2. Feed cells with fresh pre-warmed complete media 2 h before
Cells for Transfection transfection.

3.3.2. Preparation of DNA
for Transfection
3.3.3. Formation
of Calcium Phosphate
and DNA Precipitate
3.3.4. Transfection of 293T
Cells
3.3.5. Isolation and
Activation of CD4+ T Cells
3.3.6. Retroviral Infection
of CD4+ T Cells
11 A Murine Autoimmune Model of Rheumatoid Arthritis 241
1. Thaw the DNA and spin at 20,000g for 2 min.
2. Set up two sterile polypropylene tubes for each DNA to be
precipitated and label the tubes Tube # 1 and Tube # 2.
3. To Tube # 1 add 0.5 ml 1 HBSS and 10 l of phosphate
solution.
4. To Tube # 2, add 0.43 ml of ultrapure water minus the volume
of DNA. Total DNA should equal 20 g.
5. Gently mix the DNAs in the water.
6. Add 60 l of calcium solution and mix gently.
1. Bubble air through solution in Tube # 1.
2. Add solution from Tube # 2 slowly and drop-wise into the
bubbling solution of Tube # 1 for slow mixing. As the two
solutions mix slowly they appear milky and form a white
precipitate.
3. Allow the suspension to sit at room temperature for 20 min
before adding to the 293T cells.
1. After 20 min, mix the precipitate well by repeated pipetting
and vortexing.
2. Add 1 ml of DNA suspension to 100 mm plate containing
293T cells. The suspension must be added slowly and drop-
wise while gently swirling the media in the plate.
3. Put the plates back in the incubator for 24 h.
4. Twenty-four hours post transfection remove the media con-
taining precipitate and replace it with 6 ml pre-warmed com-
plete media.
1. Forty-eight hours post transfection purify total CD4+ T cells
from 8- to 10-week-old mouse spleen and LNs using CD4+ T
cells isolation kit as per manufacturers protocol.
2. Activate the CD4+ T cells in the presence of plate-bound anti-
CD3 (1 g/ml) and soluble anti-CD28 (2 g/ml) at a cell
concentration of 1.5 million cells/ml in a 24-well plate.
1. Seventy-two hours post transfection, check for reporter gene
expression under fluorescent microscope (YFP+, GFP+, or
RFP+). Collect the virus-containing media from 293T cells and
add Polybrene (10 g/ml) to it before passing it though a
0.22 m syringe filter.
2. Add 6 ml of pre-warmed complete media and put the plates
containing 293T cells back in the incubator for 2 h.
3. Two hours later very carefully and slowly replace complete
media on CD4+ T cells with virus-containing media from 293T
cells.
242 P.S. Biswas et al.
3.3.7. Resting
of Retrovirally Infected
CD4+ T Cells
3.3.8. Restimulation
of Retrovirally Infected
CD4+ T Cells
3.4. Flow Cytometric
Analyses of T and B
Cell Compartments
in Spleen and Lymph
Nodes (See Notes
1113)
4. Spin the plate at 600700g at 30C for 90 min. After spinning,
place the plate at room temperature for 60 min.
5. Very carefully replace the media containing viruses with 1 ml
of pre-warmed complete Clicks media and put back the cells
in the incubator for 24 h.
6. Ninety-six hours post transfection repeat steps 15.
1. Twenty four hours following the last infection (i.e., 120 h post
transfection of 293T cells), collect the activated CD4+ T cells
and wash three times with ice-cold PBS.
2. Rest the CD4+ T cells in complete Clicks media containing
recombinant murine IL-2 (20 ng/ml) in 12-well plates for
3 days.
1. Following resting in IL-2, collect the cells and wash several
times with ice-cold PBS.
2. Proceed to sorting the retrovirally infected CD4+ T cells on the
basis of reporter gene expression (YFP+, GFP+, or RFP+ cells).
3. Restimulate sorted CD4+ T cells in complete Clicks media in
the presence of plate-bound anti-CD3 (1 g/ml) and soluble
anti-CD28 (2 g/ml) for 48 h.
4. Collect the supernatants for cytokine ELISAs.
5. Alternatively, 96 h post transfection cells can be restimulated
with PMA (50 ng/ml) and ionomycin (1 M) for 46 h in the
presence of Golgi Stop (1 M) and subject to intracellular
staining for the production of cytokines.
1. Sacrifice mice and collect spleen and peripheral LNs in ice-cold
media.
2. Prepare single cell suspension of spleen and LNs by mashing
between two frosted microscopic slides followed by passing the
cell suspension through 70-m nylon mesh to remove any cell
clumps and debris.
3. Centrifuge cell suspension for 10 min at 300 g at 4 C.
4. Discard the supernatant and lyse the RBCs using RBC lysis
buffer. Add ice-cold PBS (ten times the volume of RBC
lysis buffer) and centrifuge for 10 min at 300 g at 4 C.
5. Wash the cell pellets twice with ice-cold PBS.
6. Determine viable cell count by Trypan blue exclusion.
7. For surface staining of T and B cells take one million cells/well
in a 96-well plate.
8. Spin the plate at 300 g for 3 min and discard the super-
natant.
 11 A Murine Autoimmune Model of Rheumatoid Arthritis 243

9. Incubate the cells in the presence of anti-mouse CD16/CD32

antibody (1 g/million cells) dilution in FACS buffer for
20 min.
10. Proceed to stain with the following fluorescent-labeled anti-
body as mentioned below:
Cell type Purpose Staining

B cells GC B cells B220 (BD, Clone: RA3-6B2), PNA

Plasma cells
Marginal zone and follicular B cells
Mature and immature B cells
Transitional B cells (T1, T2, and
T3 B cells)
CD4 T cells Effector and memory
Follicular T helper cells
Extra-follicular T helper cells
Regulatory T cells
(Jackson Immunoresearch Laboratories),
IgG1 (BD, Clone: A65-1), and IgM
(eBioscience, Clone: 11/41)/B220, Fas
(CD95) (BD; Clone: Jo2) and T and B
cell Activation antigen (BD; Clone:
GL7), GL7
B220, CD138 (BD, Clone: 281-2)
B220, CD21 (eBioscience, Clone:
eBio8D9), CD23 (BD, Clone: B3B4)
B220, IgM, IgD (eBioscience, Clone:
11-26)
B220, IgM, IgD, CD23, CD93
(eBioscience, Clone: AA4.1)
CD4 (eBioscience, Clone: RM4-5),
CD62L (eBioscience, Clone: MEL-14),
CD44 (eBioscience, Clone: IM7), CD69
(eBioscience, Clone: H1.2F3)
CD4, CD44, CXCR5 (BD, Clone: 2G8),
PD1 (BD, Clone: J43)
CD4, CD44, CXCR5, PD1, PSGL1 (BD,
Clone: 2PH1), CXCR4 (eBioscience,
Clone: 2B11)
CD4, CD25 (eBioscience, Clone: PC61.5),
Foxp3 (Intracellular stain) (eBioscience,
Clone: FJK-16s)

3.5. Clinical 1. Collect blood by retro-orbital bleeding and keep at 4 C

Assessment of overnight.
Arthritis: Serum Levels 2. Next morning carefully collect the serum and proceed to per-
of Rheumatoid Factor form an ELISA to detect the serum levels of RF using a com-
(See Notes 1416) mercially available kit (Alpha Diagnostics for RF).
3. Dilute (1:100) the serum in Reagent diluent and add 100 l/
well in duplicates for each sample. Incubate at room tempera-
ture for 60 min.
4. Wash five times with wash buffer.
5. Add 100 l/well Anti-Mouse IgG-HRP conjugate (1:100
diluted in Sample reagent) and incubate at room temperature
for 30 min.
6. Wash five times with wash buffer.
7. Add 100 l of TMB substrate to each well and incubate for
15 min in the dark.
244 P.S. Biswas et al.

Fig. 1. Representative macroscopic images of the arthritis that develops in Def6-deficient DO11.10 mice (representative
clinical scores of 4 are shown).

8. Stop the reaction by adding 100 l of stop solution to each

well. Tap gently to mix.
9. Read absorbance of the plate at 450 nm using a single wave-
length within 30 min after Stop Solution is added. If available,
program to subtract OD at 630 nm to normalize well
background.

3.6. Clinical Evaluate joint swelling and extent of inflammation by routine

Assessment of inspection and score as per standard methods (36). Briefly score
Arthritis: Arthritis every week in the following way:
Score (Fig. 1) 0, No evidence of erythema and swelling.
1, Erythema and mild swelling confined to the wrist or ankle.
2, Erythema and mild swelling extending from the wrist or ankle
to the mid-paw.
3, Erythema and moderate swelling extending from the wrist or
ankle to the mid-paw.
4, Erythema and severe swelling encompassing the wrist or ankle,
paws, and digits.
Score for all four paws is totaled for each mouse.
 11 A Murine Autoimmune Model of Rheumatoid Arthritis 245

3.7. Clinical 1. Collect blood by retro-orbital bleeding and keep at 4 C

Assessment of Lupus: overnight.
Serum Levels of 2. Next morning carefully collect the serum and proceed to per-
Anti-dsDNA Antibody form an ELISA to detect the serum levels of anti-dsDNA anti-
(See Notes 1416) body levels using commercially available kit (Alpha Diagnostics
for RF).
3. Dilute (1:100) the serum in Reagent diluent and add 100 l/
well in duplicates for each sample. Incubate at room tempera-
ture for 60 min.
4. Wash five times with wash buffer.
5. Add 100 l/well Anti-Mouse IgG-HRP conjugate (1:100
diluted in Sample reagent) and incubate at room temperature
for 30 min.
6. Wash five times with wash buffer.
7. Add 100 l of TMB substrate to each well and incubate for
15 min in the dark.
8. Stop the reaction by adding 100 l of stop solution to each
well. Tap gently to mix.
9. Read absorbance of the plate at 450 nm using a single wave-
length within 30 min after Stop Solution is added. If available,
program to subtract OD at 630 nm to normalize well
background.

3.8. Serologic 1. Using separate pipette tips, apply one drop (2030 l) of each
Assessment of Lupus: mouse serum dilution and positive and negative controls to
ANA Staining (See individual wells of the slide. Make sure not to touch the anti-
Notes 1721) gen surface with the pipette tip and prevent mixing of samples
between wells.
2. Incubate in a moist chamber at room temperature (2025 C)
for 30 min.
3. Remove slides from moist chamber and rinse gently with PBS
using a squeeze wash bottle.
4. Place the slides in coplin jars and wash the slides three times
with PBS.
5. Remove slides from the wash one at a time, shake off excess
PBS, dry around outside edges if necessary, and return each
slide to the moist chamber.
6. Apply 1020 l of FITC conjugated anti-mouse IgG (Jackson
Immunoresearch Lab) (1:1,000 dilution in PBS) with DAPI
counterstain (1:5,000 dilution) (Invitrogen) to each well of
each slide.
7. Incubate in a moist chamber at room temperature (2025 C)
for 30 min in the dark.
8. Remove slides from moist chamber and rinse gently with PBS
using a squeeze wash bottle.
246 P.S. Biswas et al.

9. Place the slides in coplin jars and wash the slides five times with
PBS.
10. Shake off the excess PBS and immediately mount the slides with
Vectashield Mounting medium for fluorescence (Vector Labs).
11. Examine stained slides as soon as possible using a properly
equipped fluorescence microscope.
12. The slides are graded on the basis of fluorescence intensity
based on the following protocol:
Fluorescent intensity may be semi-quantitated by following
the guidelines established by the Centers for Disease Control,
Atlanta, Georgia:
4+ = Maximal fluorescence; brilliant yellow-green.
3+ = Less brilliant yellow-green fluorescence.
2+ = Definite but dull yellow-green fluorescence.
1+ = Very dim subdued fluorescence.
The degree of fluorescent intensity is not clinically relevant and
has only limited value as an indicator of titer.

3.9. Immuno- 1. Sacrifice mice and collect kidney.

fluorescence Staining 2. Place the kidney in OCT compound and freeze it.
to Evaluate Immune-
3. Make 68 m thick frozen sections of kidney.
Complex Deposition
in Kidney (Fig. 2) (See 4. Air-dry the sections followed by fixation in ice-cold acetone for
Notes 22 and 23) 10 min.
5. Air-dry the slides.
6. Wash the slides with PBS-Tween20 three times.
7. Block the sections with 1 % BSAPBS for 60 min at room
temperature.
8. Stain the sections with anti-mouse IgGFITC (1:200 dilution
in 1 % BSAPBS) and anti-mouse C3-FITC (1:200 dilution in
1 % BSAPBS) for 60 min at room temperature.
9. Wash the slides with PBS-Tween20 six times.
10. Counterstain the slides with DAPI (1:5,000 dilution in 1 %
BSAPBS) for 20 min at room temperature.
11. Wash the slides with PBS-Tween20 three times and mount
them with Vectashield Mounting medium for fluorescence.

4. Assessing
Histologic Severity
of Immune-
Complex-Mediated The various histopathologic alterations in different compartments
Glomerulonephritis of the kidney, including the severity of inflammation, can be graded
in a semiquantitative manner on microscopic examination of
 11 A Murine Autoimmune Model of Rheumatoid Arthritis 247

Fig. 2. Representative images of the renal histopathologic findings observed in Def6-deficient mice (on a 129/B6 back-
ground). Left panel : Representative H&E-stained sections from the kidneys of WT and Def6-deficient mice; photomicro-
graphs were taken at a magnification of 400. Deposition of immunoglobulin complexes in the glomeruli was detected by
immunofluorescence with anti-IgG (middle panel ) and anti-C3 (right panel ) staining. Magnification of 400 is shown.

Hematoxylin and Eosin (H&E)-stained sections (3 m thickness).

The severity of glomerulonephritis (GN) is graded in a three-tiered
fashion, mild, moderate, or severe (score 13) by assessing the size
of glomeruli, glomerular cellularity (mesangial, capillary, and pres-
ence of crescents), immune-complex deposition-associated changes
(capillary loop/glomerular basement membrane thickening,
mesangial widening/nodularity), and extent of glomerulosclerosis.
The presence of cellular crescents, with or without other glomeru-
lar changes, automatically leads to a score of 3 or severe GN desig-
nation. The severity of interstitial and perivascular chronic
inflammation (lymphocytes and plasma cells) is also graded from
mild to severe (score 13). Presence of vasculitis leads to the assign-
ment of a score of 1. The scores of each component can be added
to generate a global kidney injury score or restricted to assess-
ment of the severity of glomerulonephritis alone.
248 P.S. Biswas et al.

5. Notes

1. Sorted nave CD4+ T cells should be >95 % pure.

2. Do not reuse cytokine aliquots.
3. Addition of fresh differentiating media after day 3 increases the
frequency of IL-17+ cells.
4. Do not add IL-2 in Th17 differentiating medium.
5. ChIP assays:
(a) ChIP amplicon size should be around 100 bp so that it is
possible to get high-resolution mapping.
(b) Use normal IgG as a negative control.
(c) The amount of antibody for IP varies and needs to be
empirically determined. Antibodies used for ChIP should
be fully characterized. In general, polyclonal antibodies
are preferred because they may recognize a number of dif-
ferent epitopes, rather than monoclonal antibodies, which
only recognize a single epitope. Usually, if an antibody
works in a normal immunoprecipitation, it is a good can-
didate for ChIP.
(d) Before performing ChIP, optimize the sonication proto-
col to shear the DNA at a size of 2001,000 bp. This is
important to get good resolution for ChIP.
(e) Cross-linking is a time-critical procedure and should be
carried out for 10 min. Excessive cross-linking can lead to
a decrease in the amount of protein bound to the DNA
and reduction in the availability of epitopes/changes in
epitopes for antibody binding.
6. Every time use freshly thawed 293T cells for transfection.
7. Maxiprep preparations of DNA should be endotoxin free.
8. Be careful about loss of cells while taking out media from CD4+
T cell culture wells.
9. Always supplement IL-2 when media is changed.
10. Check to see whether the protein of interest is expressed prop-
erly in 293T cells using Western Blot technique.
11. Optimum dilution of each fluorochrome-conjugated antibody
should be determined depending on the application.
12. Surface staining should always be performed on ice.
13. Following staining cells can be fixed in 1 % PFA for 20 min on
ice and stored for 35 days at 4 C in the dark.
14. Bring all reagents to room-temperature (1830 C) equilibra-
tion (at least 30 min).
 11 A Murine Autoimmune Model of Rheumatoid Arthritis 249

15. After each reagent addition gently tap the plate to mix the well
contents prior to beginning incubation.
16. Dilute serum samples in working sample diluent according to
expected autoantibody levels.
17. Nonspecific binding may occur if the reagent is allowed to dry
on the slide.
18. Do not touch the pipette tip in the wells.
19. Take care not to mix the samples between wells.
20. Do not focus the PBS stream directly onto the wells. To prevent
cross contamination tilt slide first toward wells 16 and, run-
ning PBS stream along the midline of the slide, allow the PBS
to run off the top edge of the slide. Then tilt the slide toward
wells 712, and repeat this procedure, allowing the PBS to run
off the bottom edge of the slide.
21. Examine stained slides as soon as possible using a properly
equipped fluorescence microscope. It is recommended that
slides be examined on the same day they are stained.
22. To prevent nonspecific staining never let the slides to dry after
the blocking step.
23. Optimal dilution of fluorochrome-conjugated antibody should
be worked out to prevent background staining.

Acknowledgments

This work was supported by the Alliance for Lupus Research, the
Mary Kirkland Center for Lupus Research, and the NIH (HL62215
and AI076474 to A.P.).

References
1. McInnes IB, Schett G (2007) Cytokines in the
pathogenesis of rheumatoid arthritis. Nat Rev
Immunol 7:429442
2. Toh ML, Miossec P (2007) The role of T cells
in rheumatoid arthritis: new subsets and new
targets. Curr Opin Rheumatol 19:284288
3. Sarkar S, Cooney LA, Fox DA (2010) The role
of T helper type 17 cells in inflammatory arthri-
tis. Clin Exp Immunol 159:225237
4. Tesmer LA, Lundy SK, Sarkar S, Fox DA
(2008) Th17 cells in human disease. Immunol
Rev 223:87113
5. Koenders MI, Joosten LA, van den Berg WB
(2006) Potential new targets in arthritis therapy:
interleukin (IL)-17 and its relation to tumour
necrosis factor and IL-1 in experimental arthri-
tis. Ann Rheum Dis 65(Suppl 3):iii29iii33
6. Stamp LK, James MJ, Cleland LG (2004)
Interleukin-17: the missing link between T-cell
accumulation and effector cell actions in rheu-
matoid arthritis? Immunol Cell Biol 82:19
7. Korn T, Bettelli E, Gao W, Awasthi A, Jager A,
Strom TB, Oukka M, Kuchroo VK (2007)
IL-21 initiates an alternative pathway to induce
proinflammatory T(H)17 cells. Nature 448:
484487
8. Nurieva R, Yang XO, Martinez G, Zhang Y,
Panopoulos AD, Ma L, Schluns K, Tian Q,
Watowich SS, Jetten AM, Dong C (2007)
Essential autocrine regulation by IL-21 in the
generation of inflammatory T cells. Nature
448:480483
9. Zhou L, Ivanov II, Spolski R, Min R, Shenderov
K, Egawa T, Levy DE, Leonard WJ, Littman
250 P.S. Biswas et al.
DR (2007) IL-6 programs T(H)-17 cell
differentiation by promoting sequential
engagement of the IL-21 and IL-23 pathways.
Nat Immunol 8:967974
10. Leonard WJ, Spolski R (2005) Interleukin-21:
a modulator of lymphoid proliferation, apopto-
sis and differentiation. Nat Rev Immunol 5:
688698
11. Mehta DS, Wurster AL, Grusby MJ (2004)
Biology of IL-21 and the IL-21 receptor.
Immunol Rev 202:8495
12. Crispin JC, Kyttaris VC, Terhorst C, Tsokos
GC (2010) T cells as therapeutic targets in
SLE. Nat Rev Rheumatol 6:317325
13. Afzali B, Lombardi G, Lechler RI, Lord GM
(2007) The role of T helper 17 (Th17) and
regulatory T cells (Treg) in human organ trans-
plantation and autoimmune disease. Clin Exp
Immunol 148:3246
14. Herber D, Brown TP, Liang S, Young DA,
Collins M, Dunussi-Joannopoulos K (2007)
IL-21 has a pathogenic role in a lupus-prone
mouse model and its blockade with IL-21R.Fc
reduces disease progression. J Immunol 178:
38223830
15. Sawalha AH, Kaufman KM, Kelly JA, Adler
AJ, Aberle T, Kilpatrick J, Wakeland EK, Li
QZ, Wandstrat AE, Karp DR, James JA, Merrill
JT, Lipsky P, Harley JB (2008) Genetic asso-
ciation of IL-21 polymorphisms with systemic
lupus erythematosus. Ann Rheum Dis 67:
458461
16. Young DA, Hegen M, Ma HL, Whitters MJ,
Albert LM, Lowe L, Senices M, Wu PW, Sibley
B, Leathurby Y, Brown TP, Nickerson-Nutter
C, Keith JC Jr, Collins M (2007) Blockade of
the interleukin-21/interleukin-21 receptor
pathway ameliorates disease in animal models
of rheumatoid arthritis. Arthritis Rheum 56:
11521163
17. Bettelli E, Oukka M, Kuchroo VK (2007)
T(H)-17 cells in the circle of immunity and
autoimmunity. Nat Immunol 8:345350
18. Ivanov II, Zhou L, Littman DR (2007)
Transcriptional regulation of Th17 cell differ-
entiation. Semin Immunol 19:409417
19. Dong C (2008) TH17 cells in development: an
updated view of their molecular identity and
genetic programming. Nat Rev Immunol 8:
337348
20. Takatori H, Kanno Y, Chen Z, OShea JJ
(2008) New complexities in helper T cell fate
determination and the implications for autoim-
mune diseases. Mod Rheumatol 18: 533541
21. Ivanov II, McKenzie BS, Zhou L, Tadokoro
CE, Lepelley A, Lafaille JJ, Cua DJ, Littman
DR (2006) The orphan nuclear receptor
RORgammat directs the differentiation program
of proinflammatory IL-17+ T helper cells. Cell
126:11211133
22. Laurence A, Tato CM, Davidson TS, Kanno Y,
Chen Z, Yao Z, Blank RB, Meylan F, Siegel R,
Hennighausen L, Shevach EM, OShea J
(2007) Interleukin-2 signaling via STAT5 con-
strains T helper 17 cell generation. Immunity
26:371381
23. Yang XO, Panopoulos AD, Nurieva R, Chang
SH, Wang D, Watowich SS, Dong C (2007)
STAT3 regulates cytokine-mediated generation
of inflammatory helper T cells. J Biol Chem
282:93589363
24. Yang XO, Pappu BP, Nurieva R, Akimzhanov
A, Kang HS, Chung Y, Ma L, Shah B,
Panopoulos AD, Schluns KS, Watowich SS,
Tian Q, Jetten AM, Dong C (2008) T helper
17 lineage differentiation is programmed by
orphan nuclear receptors ROR alpha and ROR
gamma. Immunity 28:2939
25. Brustle A, Heink S, Huber M, Rosenplanter C,
Stadelmann C, Yu P, Arpaia E, Mak TW,
Kamradt T, Lohoff M (2007) The develop-
ment of inflammatory T(H)-17 cells requires
interferon-regulatory factor 4. Nat Immunol
8:958966
26. Chen Q, Yang W, Gupta S, Biswas P, Smith P,
Bhagat G, Pernis AB (2008) IRF-4 Binding
Protein inhibits interleukin-17 and interleu-
kin-21 production by controlling the activity of
IRF-4 transcription factor. Immunity 299:
899911
27. Huber M, Brustle A, Reinhard K, Guralnik A,
Walter G, Mahiny A, von Low E, Lohoff M
(2008) IRF4 is essential for IL-21-mediated
induction, amplification, and stabilization of
the Th17 phenotype. Proc Natl Acad Sci U S A
105:2084620851
28. Matsuyama T, Grossman A, Mittrucker H,
Siderovski D, Kiefer F, Kawakami T, Richardson
C, Taniguchi T, Yoshinaga S, Mak T (1995)
Molecular cloning of LSIRF, a lymphoid-
specific member of the interferon regulatory
factor family that binds the interferon-stimu-
lated response element (ISRE). Nucl Acid Res
23: 21272136
29. Rengarajan J, Mowen KA, Mcbride KD, Smith
ED, Singh H, Glimcher LH (2002) Interferon
regulatory factor 4 (IRF4) interacts with
NFATc2 to modulate interleukin 4 gene expres-
sion. J Exp Med 195:10031012
30. Gupta S, Lee A, Hu C, Fanzo J, Goldberg I,
Cattoretti G, Pernis AB (2003) Molecular
cloning of IBP, a SWAP-70 homologous GEF,
which is highly expressed in the immune sys-
tem. Hum Immunol 64:389401
31. Hotfilder M, Baxendale S, Cross MA, Sablitzky
F (1999) Def-2, -3, -6, -8, novel mouse genes
 11 A Murine Autoimmune Model of Rheumatoid Arthritis
differentially expressed in the hematopoietic
system. Br J Haematol 106:335344
32. Tanaka Y, Bi K, Kitamura R, Hong S, Altman
Y, Matsumoto A, Tabata H, Lebedeva S,
Bushway PJ, Altman A (2003) SWAP-70-like
adapter of T cells, an adapter protein that regu-
lates early TCR-initiated signaling in Th2 lin-
eage cells. Immunity 18:403414
33. Fanzo JC, Yang W, Jang SY, Gupta S, Chen Q,
Siddiq A, Greenberg S, Pernis AB (2006) Loss
of IRF-4-binding protein leads to the sponta-
neous development of systemic autoimmunity.
J Clin Invest 116:703714
251
34. Murphy KM, Heimberger AB, Loh DY (1990)
Induction by antigen of intrathymic apoptosis
of CD4 + CD8 + TCRlo thymocytes in vivo.
Science 250:17201723
35. Biswas PS, Gupta S, Chang E, Song L, Stirzaker
RA, Liao JK, Bhagat G, Pernis AB (2010)
Phosphorylation of IRF4 by ROCK2 regulates
IL-17 and IL-21 production and the develop-
ment of autoimmunity in mice. J Clin Invest
120:32803295
36. Brand DD, Kang AH, Rosloniec EF (2004)
The mouse model of collagen-induced arthri-
tis. Methods Mol Med 102:295312
 Chapter 12

The Parent-into-F1 Murine Model in the Study of Lupus-Like

Autoimmunity and CD8 Cytotoxic T Lymphocyte Function
Kateryna Soloviova, Maksym Puliaiev, Anthony Foster,
and Charles S. Via

Abstract
The transfer of homozygous C57Bl/6 (B6) or DBA/2 (DBA) parental strain T cells into normal B6D2F1
mice in the parent-into-F1 (p F1) model results in a graft-vs.-host disease (GVHD) that takes one of the
following two forms: (a) acute GVHD seen with B6 F1 mice and mediated by donor CD8 cytotoxic T
cells that eliminate host lymphocytes and (b) a chronic lupus-like GVHD seen with DBA F1 mice and
mediated by donor CD4 T cell cognate help to autoreactive B cells resulting in autoantibody production
and renal disease similar to human lupus. Importantly, these two phenotypes can be distinguished by flow
cytometry as early as 2 weeks after donor cell transfer. The p F1 model can be used to screen for agents
that alter lupus development. Additionally, the model is useful for preclinical screening of biologic agents
with immunomodulatory potential. Agents that selectively inhibit CD8 T cell function will convert acute
GVHD to chronic GVHD in B6 F1 mice. Conversely, agents that promote CD8 CTL function will
convert chronic GVHD to acute GVHD in DBA F1 mice. Agents that completely suppress T cell function
will block both phenotypes. The model is also useful for examining the effects of T cell mutations by transferring
mutant T cells into wild-type hosts and assessing the effects on disease phenotype. Differences observed
from wild-type T cells F1 can be directly ascribed to alterations in mutant T cell function. Because of the
early 2-week phenotype development, the p F1 model is well suited to screening of potential immuno-
modulatory therapeutic compounds and the assessment of T cell mutations on in vivo function.

Key words: Graft-vs.-host disease, T cells, Cytotoxic T lymphocytes, Lupus

1. Introduction

In the parent-into-F1 (p F1) model, homozygous parental strain

murine T lymphocytes are transferred into normal unirradiated
semi-allogeneic F1 recipients by either intravenous (i.v.) or intrap-
eritoneal routes. Following transfer, donor T cells recognize and
respond to the F1 alloantigens of the host and give rise to one of
the two graft-vs.-host disease (GVHD) phenotypes, originally
named based on mortality: (a) acute GVHD with death occurring

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_12, Springer Science+Business Media New York 2012

253
254 K. Soloviova et al.

at ~24 weeks or (b) chronic GVHD with death occurring after

several months. Although many p F1 strain combinations have
been studied, this chapter focuses on our experience using the
(C57Bl/6 DBA/2)F1 (BDF1) recipient and either C57Bl/6
(B6) or DBA/2 (DBA) parental donor cells. In this strain combi-
nation, B6 BDF1 mice exhibit a robust acute GVHD and
DBA BDF1 mice exhibit a chronic GVHD with features of
human lupus to include lupus-specific autoantibodies and lupus-
like immune complex glomerulonephritis (ICGN).
The kinetics of donor T cell activation, expansion, and
contraction during the first 2 weeks after transfer are detailed
elsewhere (1, 2). Briefly, chronic GVHD is initiated by donor CD4
T cell activation in response to host allogeneic MHC II resulting
in cognate donor CD4 T cell help to host B cells that in turn pro-
motes host lymphocyte expansion, autoantibody production, and
eventually lupus-like ICGN. In DBA F1 mice, long-term renal
disease is more severe using female donor and host mice (f F)
than in male (m M) mice; however, both exhibit ICGN (3).
Acute GVHD occurring in B6 F1 mice exhibits the same
initiating mechanism as in DBA F1 mice, i.e., donor CD4 T cell
recognition of host alloantigens and cognate help to host B cells;
however, the subsequent activation of B6 donor CD8 T cells in
response to host allogeneic MHC I in conjunction with help from
donor B6 CD4 T cells separates acute from chronic GVHD by
promoting the maturation of donor CD8 CTL effectors that then
eliminate host splenocytes, thereby preventing donor CD4 T cell-
driven B cell expansion and the path to humoral autoimmunity.
Confirming this pivotal role of donor CD8 T cells is the observation
that depletion of donor CD8 T cells prior to transfer in B6 F1
mice results in chronic GVHD rather than the expected acute
GVHD phenotype (reviewed in (2)). Conversely, chronic GVHD
in DBA F1 mice occurs despite the transfer of DBA donor CD8
T cells due to defective in vivo CD8 CTL effector development
(4), thereby making DBA F1 mice useful for screening of
compounds that promote CD8 CTL activity (see below).
Acute and chronic GVHD phenotypes can be reliably distin-
guished at 2 weeks after donor cell transfer by flow cytometric
analysis of F1 splenic lymphocyte populations. Typically acute
GVHD mice exhibit a profound reduction in host splenic B cells
(<10 % of control) and chronic GVHD mice exhibit a ~1.5- to
2-fold increase in host B cells (reviewed in (5)). Intermediate
phenotypes can be seen following the transfer of donor T cells with
altered function, particularly perforin or FasL defective T cells as
these two molecules account for >90 % of host killing by donor
T cells (5). Intermediate 2-week phenotypes require long-term
observation to determine which of the two phenotypes will
ultimately emerge.
 12
1.1. Applications
1.1.1. Analysis of
Biological Agents with
Immunomodulatory
Potential
1.1.2. Testing of T Cell
Mutations (Knockouts or
Transgenics)
The Parent-into-F1 Murine Model in the Study of Lupus-Like 255
The p F1 model is a useful preclinical in vivo model for
examination of agents or manipulations with immunomodula-
tory potential, particularly T cell-targeted agents. Successfully
tested agents by our group include recombinant cytokines,
cytokine blockers, cytokine inducers, and agents targeted to
molecules important in immune responses (2). For example,
agents that globally suppress T cell function such as the costimu-
latory blocker CTLA4Ig prevent both acute and chronic GVHD
phenotype (6).
A novel aspect of the p F1 model is its value in identifying
agents that specifically target CD8 T cell function. Agents that pro-
mote CD8 CTL maturation (e.g., rIL-12) (7) are best examined in
DBA F1 mice because unlike chronic GVHD occurring in CD8-
depleted B6 BDF1 mice, donor CD8 T cells are transferred in
the DBA donor inoculum; however, their maturation into effector
CTL is defective. Thus, agents that promote CTL in vivo will
convert chronic GVHD phenotype to acute GVHD in DBA F1
mice (7). Conversely, agents that inhibit CD8 CTL function are
best examined in B6 F1 mice because selective inhibition of B6
donor CD8 CTL maturation as seen with anti-TNF mAb treatment
will convert the 2-week phenotype from acute to chronic GVHD
(8). The mechanism of action by which CD8 CTL function is
altered can be further dissected by timing the administration of the
agent under investigation to (a) the initial activation phase (days
05); (b) effector maturation phase (days 610); or (c) the homeo-
static contraction phase (days 1014) (1).
As outlined above, immunopathogenesis in the p F1 model is T
cell driven. The functional consequences of T cell genetic mutations
or alterations can be readily determined by transferring mutant
T cells into normal F1 mice and comparing the resulting pheno-
type to that observed in F1 mice receiving wild-type (WT) T cells.
A large number of mutations exist on the B6 background making
the B6 BDF1 model a useful strain combination for this approach.
A dose of 50 106 B6 WT splenocytes is typically just above the
threshold for acute GVHD induction and the expected 2-week
phenotype is reliably seen. Nevertheless, we find it necessary to
determine the percentage of B6 CD4 and CD8 T cells prior to
transfer to make sure that at least 4 106 B6 CD8 T cells and
>6 106 B6 CD4 T cells are transferred. Initial assessments of
mutations on the B6 background are best determined transferring
unfractionated splenocytes normalized for donor CD4 and CD8
T cell number to that seen for ~50 106 B6 WT (age and sex matched)
splenocytes followed by a determination of whether the expected
2-week acute GVHD phenotype is altered (potentiated or inhibited).
Alterations in phenotype can be subsequently confirmed by trans-
ferring donor splenocytes depleted of B cells and APC leaving only
donor CD4 and CD8 T cells, thereby demonstrating that the effect
256 K. Soloviova et al.
1.1.3. Investigating Mutant
T Cell Help to B Cells
is intrinsic to the donor T cells and not to a defect in the non-T cell
donor population. Lastly, donor T cell function can be further dis-
sected by purifying donor CD4 and CD8 T cell subsets separately
from both wild-type and mutant strains and recombining them in
mix-and-match fashion so that all four possible combinations are
transferred. The two control groups are the following: (a) WT
CD4 + WT CD8 F1 and (b) mutant CD4 + mutant CD8 F1,
both of which are used to confirm that neither the WT nor the
mutant GVHD phenotype seen using unfractionated donor sple-
nocytes is altered as a result of the purification and recombination
procedure. The two experimental groups are the following: (c) WT
CD4 + mutant CD8 F1 which tests the ability of mutant CD8
T cells to mature into effector CTL when provided with a normal
source of CD4 T cell help and (d) mutant CD4 + WT CD8 F1
which tests the ability of mutant CD4 T cells to provide help for
normal CD8 CTL maturation. This mix-and-match approach
requires testing of several doses of donor cells to ensure that the
dose is off plateau. Typically, we use a donor CD4:CD8 ratio in the
range of 1.5:1 to 2:1. If we have observed that unfractionated
mutant cells inhibit CD8 CTL function and mitigate acute GVHD
phenotype, then we use a dose of donor cells just above the thresh-
old for acute GVHD induction, i.e., 68 106 CD4 T cells and
45 106 CD8 T cells (9), to demonstrate that at a dose in which
control WT F1 mice exhibit a robust acute GVHD phenotype,
host splenocyte elimination is defective for mutant F1 mice.
Alternatively, if we have observed potentiation of acute GVHD by
mutant T cells, then the donor cell dosage is reduced to levels just
below the threshold of acute GVHD induction, i.e., 4 106 CD4
and 2 CD8 T cells (10), and potentiation is demonstrated if a
typical 2-week acute GVHD phenotype ensues for mutant F1
mice but not for WT F1 mice. For both the inhibition or poten-
tiation analyses, further titration of donor cell numbers up or
down, respectively, should be carried out to determine the donor
cell dose range for the effect.
The previous section outlines the investigation of possible altera-
tions in CD4 T cell help for CD8 CTL maturation. CD4 T cell
function can be further dissected by testing the ability of mutant
CD4 T cells to provide help to B cells and induce chronic GVHD.
In this approach, donor CD8 T cells are depleted prior to transfer
and chronic GVHD phenotype assessed at both 2 weeks and long
term (i.e., lupus-like ICGN). Significant elevation (~20-fold over
control) of autoantibodies such as anti-ssDNA can be seen at
4 weeks following the transfer of as few as 8 106 B6 donor CD4
T cells. More substantial elevations (~75-fold) are seen at 12 106
CD4 T cells (9); however, renal disease is histologically mild at
this dose and typically 1520 106 B6 CD4 T cells are required
for significantly elevated glomerular scores (11). By contrast,
unfractionated DBA splenocytes containing as few as 1214 106
 12
1.1.4. Mutations
in Host Cells
1.2. Major Procedures
1.2.1. GVHD Induction
1.2.2. GVHD Assessment
The Parent-into-F1 Murine Model in the Study of Lupus-Like 257
DBA CD4 T cells will induce a severe membranous GN in females
and a milder but still significant ICGN in males (3).
The BDF1 is less well suited for the study of mutations in B cells or
APC as this requires breeding an F1 with the required mutation on
both the B6 and DBA parental strains. Although many mutations
are available on the B6 background, very few are available on the
DBA strain. A useful substitute is the (Balb/c B6)F1 (CB6F1)
because of the larger number of mutations present on the Balb/c
background vs. the DBA background. B6 CB6F1 and
Balb/c CB6F1 mice exhibit acute and chronic GVHD pheno-
types, respectively; however, the lupus-like renal disease in
Balb/c CB6F1 mice can require 6 months to develop following
a single transfer of donor cells (12) and is not as well characterized
as that seen in DBA BDF1 chronic GVHD mice.
General. All procedures should be approved by the Institutional
Animal Care and Use Committee beforehand. Both donor and
host mice should be young adults preferably close to 810 weeks
of age and of the same sex to avoid donor or host recognition of
H-y antigens, although in acute GVHD transfers this is not an
absolute requirement because donor anti-host MHC alloantigen
recognition will supercede any host anti-donor H-y recognition.
Donor cell transfer: Any source of T cells is acceptable (e.g.,
lymph node, thymus); however, we routinely use splenocytes exclu-
sively for donor transfers because of the ease of preparation.
Additionally, unfractionated splenocytes contain stem cells that can
mitigate mortality by permitting long-term donor repopulation
and stable chimerism (13). Our group exclusively uses a single
injection of unfractionated splenocytes or T cells purified from
splenocytes and transferred intravenously. The use of multiple
transfers and/or intraperitoneal injection is also effective; however,
the single transfer permits a kinetic evaluation of a single popula-
tion of donor T cells and standardization of the phases of donor
T cell activation and contraction kinetics (1). Additionally, using the
single dose allows correlation of both the short-term and long-
term phenotypes to the single population of donor cells.
Because the spleen is the major filter for the blood and because our
transfers are i.v., we primarily analyze F1 spleens after transfer.
Liver and lymph node also exhibit donor T cell infiltration within
the first 2 weeks (14); however, the spleen is well suited for early
analysis of the effects of biologics or mutant donor T cells and liver
and lymph nodes offer no experimental advantages. Acute murine
GVHD can also exhibit attacks on a variety of other organs similar
to human acute GVHD, although this generally requires >2 weeks
for organ damage (14).
Day 14: Flow cytometric analysis of splenic lymphocyte populations.
Acute and chronic GVHD phenotypes can be determined by
258 K. Soloviova et al.

multiparameter flow cytometric assessment of donor CD4 and

CD8 T cell engraftment and by the numbers of host B cells, den-
dritic cells (DC), macrophages, and T cells. Phenotypes are as
described above and in detail previously (5).
Day 10: Peak of donor effector cell maturation. Day 10 is the major
time point for evaluation of whether donor cells have matured into
CD8 CTL effector cells as demonstrated by donor CD8 T cell
expression of typical effector markers to include KLRG-1, CD107a,
and intracellular IFN-g, perforin, and granzyme B.
Days 24: Initial donor T cell activation. Donor T cells home to the
spleen within the first 24 h after transfer and cell division can be
tracked using donor T cells labeled with CFSE. These studies are
indicated when differences in days 10 and 14 suggest that dif-
ferential donor T cell activation may be operative.
Ancillary studies: Short term. Additional parameters of interest are
quantification of donor and host Tregs, T cell proliferation, T cell
apoptosis, and intracellular cytokine expression.
Ancillary studies: Long term. For chronic GVHD mice, we assess
the severity of lupus-like features using the following: (1) ELISA
for ssDNA/dsDNA antibodies; (2) renal histological assessment
of ICGN; (3) proteinuria.
Cytokine gene expression by real-time polymerase chain reaction
(RT-PCR). This can be performed on whole spleen preparations at
any time after donor cell transfer.

2. Materials

2.1. GVHD Induction 1. Polystyrene round-bottom tube, 17 100 mm, BD Bioscience

2.1.1. Donor Cell
Preparation and Transfer
(San Diego, CA).
2. RPMI-1640 Medium without L-Glutamine, Quality Biological
(Gaithersburg, MD).
3. Sterile instruments (medical scissors, forceps).
4. Petri dishes, BD Bioscience (San Diego, CA).
5. Glass mesh, cell strainer, BD Bioscience (San Diego, CA).
6. Transferring pipets, Fisher (Pittsburgh, PA).
7. 50 ml polypropylene conical tubes, BD Bioscience (San
Diego, CA).

2.1.2. Negative 1. Buffer 1: Phosphate-buffered saline (PBS) (without Ca+ and

T Cell Isolation Mg+) with 10 % bovine serum albumin (BSA) and 2 mM
EDTA, pH 7.4.
2. Buffer 2: RPMI-1640 with 10 % heat-inactivated fetal calf
serum (FCS).
 12
2.1.3. Positive
T Cell Isolation
2.1.4. Intravenous Injection
2.2. GVHD Assessment
2.2.1. Flow Cytometry
Miscellaneous Materials
Surface Staining Antibodies
Apoptosis
Intracellular Staining
The Parent-into-F1 Murine Model in the Study of Lupus-Like 259
3. Dynal mouse negative isolation kits for T cells, CD8 T cells,
CD4 T cells (Carlsbad, CA).
4. Dynal Magnets MPC-6, MPC-50, Invitrogen (Carlsbad, CA).
5. Dynal bi-directional rotator.
1. Isolation Buffer: PBS (without Ca+ and Mg+) with 10 % BSA
and 2 mM EDTA, pH 7.4.
2. Culture Media: RPMI-1640 with 10 % FCS.
3. Dynabeads: Mouse CD8 (Lyt2), Mouse CD4 (Lyt2),
Invitrogen (Carlsbad, CA).
4. Dynal MPC-6, Dynal MPC-50, Invitrogen (Carlsbad, CA).
1. Extra cage.
2. Heat lamp.
3. 1 ml insulin Syringe VWR (Bridgeport, NJ). (3 ml syringe with
#27 gauge needles are also acceptable.)
4. Mouse restrainer.
1. 96-well polystyrene plate, U-bottom, non-sterile, USA
Scientific (Ocala, FL).
2. 12 75 mm, 5 ml polystyrene round bottom test tube, BD
Bioscience (San Diego, CA).
3. Facs-Buffer (10 g BSA, Sigma (Atlanta, GA) + 0.1 g Sodium
Azide, minimum 99.5 %, Sigma (Atlanta, GA) in 500 ml
PBS).
4. FC-blocker, Invitrogen (Carlsbad, CA).
5. 1 % Paraformaldehyde, Sigma (Atlanta, GA).
1. The following mAbs are used: CD4, CD8, B220, H-2Kb/H-
2Kd, I-Ab/I-Ad, Cd11b, Cd11c, CD44, CD62L, CXCR5,
ICOS, FAS, PD1, CCR7, KLRG, CD80, CD86, CD107,
FASL, CD107a. Typically, we use 45-color flow cytometry
using the following fluorochromes: Alexa Fluor;
Allophycocyanin-Cy-7-; Biotin, Phycoerythrin, Texas Red,
Peridinin Chlorophyll Protein Complex, Pacific Blue-
conjugated and Pacific orange. All are available from BD
Biosciences (San Jose, CA), BioLegend (San Diego, CA),
eBioscience (San Diego, CA), or Invitrogen (Carlsbad, CA).
1. Annexin V-Pe Apoptosis Detection kit I, BD Bioscience (San
Diego, CA).
Cytokines
1. 10 Permeabilization buffer (eBioscience, San Diego, CA).
2. IC Fixation solution (eBioscience, San Diego, CA).
260 K. Soloviova et al.

3. mAb against TNF, GranzymeB, Perforin, IFN-gamma from

BD Biosciences (San Jose, CA), eBioscience (San Diego, CA),
or Invitrogen (Carlsbad, CA).
Regulatory T cells (Treg) and Proliferation (KI-67)
1. Foxp3 staining buffer set, eBioscience (San Diego, CA).
2. APC anti-mouse/rat Foxp3, eBioscience (San Diego, CA).
3. PE mouse anti-human Ki-67 set, BD Bioscience (San Diego, CA).

CFSE Staining 1. Cell Trace CFSE Cell Proliferation Kit, Molecular Probes, Inc.
(Eugene, OR).

Equipment and Software 1. BD Biosciences LSRII Cell Analyzer, BD Bioscience (San

Diego, CA).
2. WinList 6.0 or FlowJo 7.6.
3. ModFit (Verity Software) for CFSE analysis.

2.2.2. Renal Studies 1. Polystyrene round-bottom tube, 17 100 mm, BD Bioscience

(San Diego, CA).
2. Sterile instruments (medical scissors, forceps).
3. Petri dishes, BD Bioscience (San Diego, CA).
4. 10 % Buffered Formalin.
5. Histoprep SH75-125D, Fisher (Pittsburgh, PA).
6. PBS, Quality Biological (Gaithersburg, MD).
7. Seal-rite 2 ml microcentrifuge tube, natural, USA Scientific
(Ocala, FL).
8. Foil.

2.2.3. ELISA 1. High binding capacity polystyrene flat bottom 96-well micro-
titer plates (Thermo scientific NUNC Maxi-Sorp, Thermo
Fisher Scientific, Rochester, NY).
2. DNA, single stranded from calf thymus, lyophilized powder
(Sigma-Aldrich, Atlanta, GA).
3. Double stranded DNA plates (QuantaLITE, INOVA
Diagnostics, Inc. San Diego, CA).
4. Goat anti-mouse IgGAlkaline Phosphatase antibody (Sigma-
Aldrich, Atlanta, GA).
5. Alkaline Phosphatase substrate kit (Bio-Rad, Hercules, CA).
6. Tween-20, BSA (Sigma-Aldrich, Atlanta, GA).
7. 1 PBS (Quality Biological Inc., Gaithersurg, MD).
8. DNA-stock solution: 1 mg single-stranded DNA, 1 ml DEPC-
treated water with 1 mM EDTA.
9. DNA working solution: 20 l of DNA stock solution in 1 ml
of 1 PBS.
 12 The Parent-into-F1 Murine Model in the Study of Lupus-Like 261

10. Washing buffer: 0.05 % Tween-20, 1 PBS.

11. Blocking buffer: 1 % BSA, 1 PBS.
12. 50300 l multichannel pipette.

Equipment 1. Microplate photometer (Labsystems Multiskan Ascent 354,

Thermo Scientific).

Software 2. Titri version 4.66 http://gestur.tripod.com/programs/titri.htm.

2.2.4. Real-Time 1. RNA STAT-60 (TEL-TEST INC., Friendswood, TX).

Polymerase Chain Reaction
mRNA Isolation
2. Chloroform, ACS grade (Sigma-Aldrich, Atlanta, GA).
3. Isopropanol, ACS grade (Fisher Bio reagents).
4. Ethanol, ACS grade (Sigma-Aldrich, Atlanta, GA).
5. DEPC-treated RNAse-free water (Quality Biological Inc.,
Gaithersburg, MD).
6. 1.5 ml polypropylene Eppendorf tubes.

Reverse Transcription 1. TaqMan Reverse Transcription Reagents kit (N808-0234,

Applied Biosystems, Foster City, CA).
2. TaqMan Universal PCR Master Mix (Applied Biosystems,
Foster City, CA).
3. TaqMan Gene Expression Assays (Applied Biosystems, Foster
City, CA).
4. Molecular biology-grade water (Quality Biological Inc.,
Gaithersburg, MD).
5. Optical 96-well reaction plate (Applied Biosystems, Foster
City, CA).
6. MicroAmp Optical Adhesive Film (Applied Biosystems,
Foster City, CA).

Equipment 1. Thermo cycler (PTC-225 DNA Engine Tetrad, MJ Research,

Waltham, MA).
2. 7500 Real-Time PCR System (Applied Biosystems, Foster
City, CA).

3. Methods

3.1. Tissue Harvesting 1. Euthanize mice according to AALAC recommendations and

under an approved IACUC protocol.
2. Under sterile conditions open the thorax and collect blood by car-
diac puncture first. Afterwards, open the peritoneum and remove
spleen, then identify kidneys retroperitoneally and remove.
262 K. Soloviova et al.
3.2. Splenocyte
Isolation (for Donor Cell
Transfer or Analysis)
3.3. GVHD Induction
3.3.1. Splenocyte
Preparation
3.3.2. Donor T Cell
Purification: Negative
Isolation
11. Wash beads 2 with Buffer 1.
12. Wash cells 3 with RPMI-1640 and then resuspend in desired
13. Determine the purity of depletion by flow cytometry prior to
1. Place spleen in Petri dish with RPMI without FCS.
2. Homogenize spleen by gently pressing capsule with either
proximal end of sterile 3 cc syringe or glass mesh homogenizer.
3. Filter splenocyte mixture through cell strainer into a 50 ml
polypropylene conical tube.
4. Centrifuge for 10 min, resuspend cells in 2 ml RPMI, and
count using hemocytometer. Adjust to desired concentration.
1. For transfers using unfractionated splenocytes use cells as pre-
pared in Subheading 3.2 and determine the relative percentage
of CD4 and CD8 T cells by flow cytometry (see below).
2. Adjust donor inoculum to desired numbers of CD4 and CD8
T cells.
The protocol is based on 108 WBC/ml and follows the manufac-
turers protocol.
1. Wash Dynabeads before use and resuspend in vial.
2. Transfer Dynabeads to a 15 ml conical tube, and add 5 ml
Buffer 1.
3. Place tube in a magnet for 1 min and then discard supernatant
using transferring pipette. Do not remove tube from magnet
during this step.
4. Remove tube from magnet and resuspend Dynabeads in 10 ml
of Buffer 1.
5. Prepare cells from mouse spleen as described above and after
centrifuging resuspend cells in Buffer 1.
6. To the splenocytes, add 200 l of heat-inactivated FCS and
200 l of desired mAb mix.
7. Mix well and incubate for 20 min at between 2 and 8 C with
Dynal bi-directional rotator.
8. Add 30 ml Buffer 1 to each tube and centrifuge at 3,000 rpm
(1876.9 g) between 4 and 8 C for 8 min.
9. Resuspend cells in 15 ml of Buffer 1 and add prewashed beads
at 2 ml beads per 100 106 leucocytes. Incubate for 20 min at
room temperature (RT) with bidirectional rotation.
10. Resuspend cells in 5 ml Buffer 1, place tube into magnet for
2 min, and then transfer supernatant into the clean 50 ml tube.
volume of RPMI-1640.
tail vein injection.
 12
3.3.3. Donor T Cell
Purification: Positive
Isolation
3.3.4. CFSE Staining
of Donor Cells
3.3.5. IV Injection
The Parent-into-F1 Murine Model in the Study of Lupus-Like 263
Dynabeads and splenocyte preparation is as above.
1. Add the appropriate amount of Dynabeads (2 ml beads per 108
WBC).
2. Incubate for 20 min at +2 to +8 C with bidirectional rotation.
3. Discard the supernatant and wash the bead-bound cells 3
times in Isolation Buffer to the original sample volume.
4. Separate using the magnet as described above.
5. Wash cells 3 with RPMI-1640 and then resuspend in desired
volume of RPMI-1640.
6. Determine the purity of selection by flow cytometry prior to
tail vein injection.
1. Prepare a 5 mM CellTrace CFSE stock solution immediately
prior to use.
2. Prepare splenocytes as outlined above.
3. Resuspend cells in prewarmed PBS + 0.1 % BSA to fi nal
concentration 106 cells/ml.
4. Add 2 l of CellTrace CFSE stock solution per milliliter of cells
and incubate at +37 C for 10 min.
5. Quench the staining by the addition of 5 volumes of ice-cold
RPMI-1640 + 10 % heat-inactivated FCS; incubate for 5 min
on ice.
6. Wash cell suspension with RPMI-1640 3 and resuspend cells
in RPMI-1640.
7. Determine the percentage of labeled CD4 and CD8 T cells by
flow cytometry prior to tail vein injection.
Assessment of donor CD4 and CD8 T cells by flow cytometry
should be performed prior to transfer. Live dead gating can be
used but typically the live cells are >90 % and often >95 %. The
donor inocula should be adjusted so that experimental and
controls receive comparable numbers of CD4 and CD8 T cells.
1. Prior to injection, place the cage of mice to be injected under
a heat lamp to increase circulation to the surface blood vessels
and make the tail veins more visible.
2. Prepare syringe for injection and be sure all bubbles are
removed.
3. Place mouse in the restrainer, and hold the tail gently tugging
back to prevent movement during the injection.
4. Line up the needle (bevel side up) exactly in the line with vein.
5. Insert the needle into the vein keeping the needle parallel to
the tail so as not to penetrate out the other side of the vein.
264 K. Soloviova et al.
3.4. GVHD Assessment
3.4.1. Splenocyte Isolation
3.4.2. Splenocyte Flow
Cytometry: General Protocol
for Surface Staining
Surface Staining Protocol
3.4.3. Splenocyte Flow
Cytometry: Intracellular
Staining Protocol (e.g.,
IFN-g, TNF, granzymeB)
6. Slowly inject the cell solution.
7. If the needle is in the vein, the syringe plunger will move easily.
If not, plunger movement is difficult and swelling is observed
around the injection site as the material is injected subcutaneously.
In this event, stop the injection and try the contralateral vein.
Splenocytes are isolated as described in Subheading 3.1.
All mAb should be titrated prior to use for optimal staining.
All of these staining protocols can be performed in either tubes
or plates. The following protocol is for tube staining. For plate
staining, make these substitutions:
1. Use 5 105 cells/well.
2. Decrease concentration of Ab in two times.
3. Use plate-shaker instead of vortex.
4. Do not overload wells during the washing step.
1. Using 1 106 splenocytes/tube, wash 1 with 1 ml Facs-Buffer.
2. Add 10 l FC-blocker; vortex; and incubate for 10 min,
+4 C.
3. Dilute the optimal concentration of mAb in Facs-Buffer; add
10 l to the appropriative tube; vortex; and incubate for
20 min, +4 C.
4. Wash with 1 ml Facs-Buffer.
5. Add 10 l of secondary Ab (if needed); vortex; and incubate
for 20 min, +4 C.
6. Wash with 1 ml Facs-Buffer.
7. Resuspend the cell pellet in 500 l 1 % paraformaldehyde.
8. We usually analyze within 2436 h, although they are stable
for longer.
1. Stain cell-surface markers as above.
2. After last wash, fix cells by adding 100 l Fixation Solution,
vortex, and incubate for 20 min at room temperature.
3. Add 1 ml of 1 Permeabilization Buffer to each well, centri-
fuge for 5 min, and aspirate supernatant.
4. Repeat previous step, then resuspend cells in 100 l 1
Permeabilization Buffer, and incubate in the dark at room
temperature for 5 min.
5. Dilute the optimal concentration of mAb in 20 l 1
Permeabilization Buffer and add to the appropriative tube;
vortex; incubate in the dark at RT for 20 min.
 12
3.4.4. Splenocyte Flow
Cytometry: Intracellular
Staining Protocol for Foxp3
and Ki67
3.4.5. Splenocyte Flow
Cytometry: Annexin V
Staining Protocol
The Parent-into-F1 Murine Model in the Study of Lupus-Like 265
6. Add 1 ml of 1 Permeabilization Buffer to each well, centrifuge
for 5 min, and aspirate supernatant.
7. Resuspend the cell pellet in 500 l Facs Buffer.
8. Run on flow cytometer and analyze within 1 h.
The protocol below can also be used to analyze cellular prolifera-
tion by substituting anti-KI67 ab of anti-Foxp3 ab. Both anti-KI67
and anti-Foxp3 ab can be stained simultaneously using this proto-
col assuming that appropriate fluorochromes are chosen.
1. Stain cell-surface antigen following the Surface Staining
Protocol but do not fix in 1 % PFA, rather proceed after final
washing step to add Fixation/Permeabilization as described
below.
2. Dilute Fixation/Permeabilization Concentrate (one part) into
the Fixation/Permeabilization Diluent (three parts) to make a
Fixation/Permeabilization Working Solution. Do not store
buffer more than 1 day.
3. After last wash of surface staining, resuspend cell pellet, add
1 ml of Fixation/Permeabilization Working Solution, and
vortex.
4. Incubate for 118 h (overnight) at +4 C in the dark. For
convenience, we usually incubate overnight; however shorter
periods will suffice.
5. Wash twice with 2 ml of 1 Permeabilization Buffer. Optional:
If cell yields are a problem at this stage, it may be that fixation
and permeabilization has rendered them more buoyant.
Centrifugation at speeds of 2,000 rpm (834.2 g) on standard
tabletop centrifuge can improve the cell yield. We generally do
not find this necessary.
6. Add anti-Foxp3 ab or isotype control in 100 l total of 1
Permeabilization Buffer; incubate for 20 min, +4 C.
7. Wash 2 with 2 ml of 1 Permeabilization Buffer.
8. Resuspend in 500 l of Facs-buffer and analyze in flow
cytometer.
9. Gates and voltages may need to be modi fi ed due to the
fi xation and permeabilization procedure which can alter the
FSC/SSC distribution of the cell population compared to
that of live cells.
1. Stain cell-surface antigen following the Surface Staining
Protocol.
2. Wash cells 2 with cold PBS.
3. Resuspend in 1 Binding Buffer at concentration 106 cells/ml.
4. Transfer 100 l of the solution to 5 ml culture tube.
266 K. Soloviova et al.
3.4.6. Renal Histology
(Formalin and Liquid
Nitrogen Prep)
3.4.7. ELISA The following is for anti-ssDNA. For anti-dsDNA, the DNA prep-
aration can be treated with S1 endonuclease or commercial dsDNA
plates can be purchased and the assay performed according to the
manufacturers instructions.
5. Add 5 l Annexin V and 5 l 7-AAD; vortex; and incubate for
15 min at RT in the dark.
6. Add 400 l of 1 Binding Buffer to each tube.
7. Run on flow cytometer and analyze within 1 h.
1. Initially, place harvested kidney into polystyrene round-bottom
tube with PBS (one per tube).
2. Once all kidneys are harvested, one at a time place them in
Petri dish and using forceps, remove the capsule and then cut
in axial plane.
3. Place half of kidney into a 2 ml tube with 2 ml of 10 %
formalin.
4. Place the other half of kidney into the center of foil piece
(pre-labeled), and apply Histoprep on the top of kidney. Fold
the foil to create a secure envelope.
5. Place envelope into liquid nitrogen (in paper box)/or dry ice
for 10 min, and then store in 80 C.
1. Coat 96-well plates with 100 l of DNA working solution and
incubate at 4 C overnight.
2. Remove coating solution by tapping the plate face down against
a paper towel. Wash the plate three times with washing buffer,
400 l/well. After each washing, tap the plates against a paper
towel to ensure no residue on the bottom of the wells.
3. Add 100 l of blocking buffer into each well and incubate for
1 h at 37 C, 5 % CO2.
4. Remove the blocking buffer by tapping and wash three times
with washing buffer, 400 l/well.
5. Dilute each serum sample 1:40 in washing buffer in upper
wells of the plate, and make three serial dilutions (1:80, 1:160,
1:320). Do duplicate dilutions for each sample.
6. Dilute standard control serum 1:100 in washing buffer in upper
wells of the plate, and make serial dilutions (1:200, 1:400, 1:800,
1:1,600, 1:3,200, 1:6,400). Do duplicate dilutions of standard
control. Leave the last wells in the standard control columns
blank. Make a separate standard control for each plate.
7. Incubate for 1 h at 37 C, 5 % CO2.
8. Wash three times with washing buffer, 400 l/well.
9. Dilute detection antibody 1:4,000 in blocking buffer and add
100 l/well.
 12 The Parent-into-F1 Murine Model in the Study of Lupus-Like 267

10. Incubate for 1 h at 37 C, 5 % CO2.

11. Wash the plate three times with washing buffer and three times
with 1 PBS, 400 l/well.
12. Prepare the substrate solution according to the manufacturers
instructions (one tablet pNPP, 1 ml 5 concentrate dietha-
nolamine buffer, 4 ml distilled water) and add 100 l/well.
Watch for yellow color development.
13. Read the plate in a spectrophotometer at several time points
during color change using 405 nm wavelength to be sure that
values are obtained that are off plateau.
14. Transfer OD values into the Titri software and analyze accord-
ing to the Titri template.

3.4.8. Real-Time mRNA isolation. The procedure is performed in room temperature

Polymerase Chain Reaction
in the fume hood or well-ventilated area.
1. Transfer 107 mouse splenocytes into a 2 ml sterile polypropyl-
ene microcentrifuge tube.
2. Centrifuge the tubes for 2 min at 1,000 g at room temperature.
3. Decant supernatant and resuspend the pellet by gentle pipetting
or gentle vortexing.
Homogenization: Homogenize the cells by adding 1 ml of RNA
STAT-60 solution to each tube. Vortex each tube thoroughly.
Store the homogenate at room temperature for 5 min. If needed,
the homogenized samples can be stored in 80 C and remain
stable for weeks.
RNA extraction:
1. Add 0.2 ml of chloroform per 1 ml of RNA STAT-60 to each
tube, vortex vigorously for 15 s, and let it stand at room tem-
perature for 23 min.
2. Centrifuge the tubes at 12,000 g for 15 min at 4 C. Upon
centrifugation, the homogenate separates into two phasesa
lower red phenolchloroform phase and upper clear aqueous
phase. RNA remains exclusively in the aqueous phase.
RNA precipitation:
1. Transfer the clear aqueous phase to a fresh 1.5 ml polypropyl-
ene microcentrifuge tube.
2. Add 0.5 ml of isopropanol per 1 ml of RNA STAT-60 used for
homogenization. Vortex. Let stand at room temperature for
510 min.
3. Centrifuge at 12,000 g for 10 min at 4 C. RNA precipitate
forms a white pellet on the bottom of the tube.
4. RNA wash: Remove supernatant and add 1 ml of 75 % ethanol
per 1 ml of RNA STAT-60 used for homogenization. Vortex.
268 K. Soloviova et al.

5. Centrifuge the tubes at 7,500 g for 5 min at 4 C.

6. Remove most of the supernatant and dry the rest using vac-
uum or by leaving the tube open for 1020 min. Do not let the
RNA pellet dry completely.
7. Add 10 l of sterile DEPC-treated water to each tube.
8. The mRNA can be stored in 80 C for weeks.
Reverse transcription. The procedure is performed in the fume hood
to reduce contamination. The reagents are kept on a cold rack or ice.
1. Measure the concentration of RNA in the sample using a
spectrometer. To assess the RNA contamination with protein,
the ratio of absorption at 260 nm vs. 280 nm can be used.
Values in the range of 1.802.00 suggest that the RNA is
sufficiently pure and suitable for cDNA synthesis.
2. Knowing the RNA concentration, calculate the amount of
sample containing 1 g of RNA, add to sterile polypropylene
microcentrifuge 0.2 ml tubes, and then add sterile molecular
biology grade water to the total volume of 9 l.
3. Mix the components of TaqMan Reverse Transcription
Reagents kit according to kit protocol and add 16 l to each
tube up to total volume of 25 l (16 l reagents mix + 9 l of
water and sample mix). Vortex.
4. Place tubes into the thermal cycler. Program it for three cycles:
first at 25 C for 10 min; second at 37 C for 60 min; third at
95 C for 5 min.
5. After the run is finished, the samples can either be used right
away or stored in 80 C for weeks.
Real-time PCR. The procedure is performed in a fume hood, bio-
safety cabinet, or on a designated part of the lab bench to reduce
contamination. The reagents are kept on cold rack or ice.
1. On ice, prepare the total amount of working mix needed for
reaction, separately for every gene of interest and endogenous
control (18s rRNA). For each sample, use 12.5 l of TaqMan
Universal PCR Master Mix, 1.25 l of pre-designed primer
and probe mix from TaqMan Gene Expression Assays or 18s
rRNA for endogenous control, and 9.25 l of sterile molecular
biology grade water.
2. On the optical 96-well PCR plate assign the wells that will be
containing reagents for endogenous control and target gene.
3. Add previously synthesized cDNA into the wells of optical
96-well PCR plate, 2 l for each well.
4. Into the corresponding well of the plate, add 23 l of working
mix containing either the primer and probe mixture for the
gene of interest or the endogenous control.
 12 The Parent-into-F1 Murine Model in the Study of Lupus-Like 269

5. Cover the plate with clear MicroAmp Optical Adhesive Film.

When optical film is secure, vortex gently to mix.
6. Centrifuge for 1 min at 4,000 g at 4 C to bring the mixture
to the bottom of the wells and load the plate into the ABI
Prism 7500 Real-time PCR system. Care should be taken to
prevent air bubbles from forming or remaining in reaction
wells as they may impair proper optical measurement of
fluorescence.
7. Set up and run the PCR reactions according to the manufac-
turers instructions.
8. The calculation of relative gene expression differences is done
using comparative 2CT method. For each gene of interest,
the acquired Ct values from each specimen are normalized to
an endogenous reference gene (18s rRNA) and then compared
to the calibrator (untreated sample). For the gene of interest,
gene expression is described for each treated (experimental)
group as fold change vs. the untreated (control) group.

4. Notes

4.1. Tail Vein Injections
1. Make sure that you have a comfortable position during the
injection. Our injections are performed in a hood but this is
not absolutely required.
This procedure is difficult to master and requires frequent
practice.
2. Do not expose animals too long to the heat lamp. When their
activity level increases, they are ready to inject.
3. Visualization of the tail veins can be increased by curling the
end of the tail. Swabbing the tail with 70 % isopropyl alcohol
immediately prior to injection may also improve visualization
of the tail vein.
4. Keep in mind that the veins are located laterally. A dark line
can often be seen running down the back of the tail. This is
not a vein.
5. Be sure to mix splenocyte preparation before drawing up in
syringe as settling of cells occurs.

4.2. Intracellular We do not use a secondary in vitro restimulation phase or Golgi

Staining for Cytokine blockers for the measurement of intracellular cytokines. As a result,
Production the brightness for positive staining cells may be lower than that
typically seen with restimulation. Nevertheless, clearly defined
positives can be detected. To determine positives, markers are set
such that normal uninjected control F1 or donor splenocytes are
<5 % positive.
270 K. Soloviova et al.
References
1. Puliaev R, Puliaeva I, Welniak LA et al (2008)
CTL-promoting effects of CD40 stimulation
outweigh B cell-stimulatory effects resulting in
B cell elimination and disease improvement in a
murine model of lupus. J Immunol
181:4761
2. Via CS (2010) Advances in lupus stemming
from the parent-into-F1 model. Trends
Immunol 31:236245
3. Foster AD, Haas M, Puliaeva I et al (2010)
Donor CD8 T cell activation is critical for
greater renal disease severity in female chronic
graft-vs.-host mice and is associated with
increased splenic ICOS(hi) host CD4 T cells
and IL-21 expression. Clin Immunol
136:6173
4. Via CS, Sharrow SO, Shearer GM (1987) Role
of cytotoxic T lymphocytes in the prevention of
lupus-like disease occurring in a murine model
of graft-vs-host disease. J Immunol
139:18401849
5. Puliaeva I, Puliaev R, Via CS (2008) Therapeutic
potential of CD8+ cytotoxic T lymphocytes in
SLE. Autoimmun Rev 8:219223
6. Via CS, Rus V, Nguyen P et al (1996)
Differential effect of CTLA4Ig on murine
graft-versus-host disease (GVHD) develop-
ment: CTLA4Ig prevents both acute and
chronic GVHD development but reverses only
chronic GVHD. J Immunol 157:42584267
7. Via CS, Rus V, Gately MK et al (1994) IL-12
stimulates the development of acute graft-ver-
sus-host disease in mice that normally would
develop chronic, autoimmune graft-versus-host
disease. J Immunol 153:40404047
8. Via CS, Shustov A, Rus V et al (2001) In vivo
neutralization of TNF-alpha promotes humoral
autoimmunity by preventing the induction of
CTL. J Immunol 167:68216826
9. Puliaeva I, Puliaev R, Shustov A et al (2008)
Fas expression on antigen-specific T cells has
costimulatory, helper, and down-regulatory
functions in vivo for cytotoxic T cell responses
but not for T cell-dependent B cell responses.
J Immunol 181:59125929
10. Puliaeva I, Soloviova K, Puliaiev M et al (2011)
Enhancement of suboptimal CD8 cytotoxic T
cell effector function in vivo using antigen-
specific CD80 defective T cells. J Immunol
186:291304
11. Rus V, Nguyen V, Puliaev R et al (2007) T cell
TRAIL promotes murine lupus by sustaining
effector CD4 Th cell numbers and by inhibit-
ing CD8 CTL activity. J Immunol
178:39623972
12. Tschetter JR, Mozes E, Shearer GM (2000)
Progression from acute to chronic disease in a
murine parent-into-F1 model of graft-versus-
host disease. J Immunol 165:59875994
13. Hakim FT, Sharrow SO, Payne S et al (1991)
Repopulation of host lymphohematopoietic
systems by donor cells during graft-versus-host
reaction in unirradiated adult F1 mice injected
with parental lymphocytes. J Immunol
146:21082115
14. Niculescu F, Niculescu T, Nguyen P et al
(2005) Both apoptosis and complement mem-
brane attack complex deposition are major fea-
tures of murine acute graft-vs.-host disease.
Exp Mol Pathol 79:136145
 Chapter 13

Genetic Approach to Study Lupus Glomerulonephritis

Yan Ge, Michael G. Brown, Hongyang Wang, and Shu Man Fu

Abstract
Genetic and environmental factors contribute in the pathogenesis of systemic lupus erythematosus (SLE).
Lupus nephritis, the most common and severe manifestation of SLE, involves inflammation in the kidney
leading to loss of renal function. However, it is not clear what controls the progression of lupus nephritis;
this is an important research question, considering its implications in clinical treatment of lupus nephritis.
Finding genes that underlie the development and progression of lupus nephritis will shed light on this
question. NZM2328 is a spontaneous mouse model for SLE. Most NZM2328 female mice develop
autoantibodies (e.g., antinuclear antibody and anti-dsDNA antibody), glomerulonephritis (GN), and
severe proteinuria between 5 and 12 months of age. In contrast, C57L/J mice fail to exhibit similar signs
of autoimmune disease. We used classical genetics to map and identify SLE genes in offspring generated
by backcrossing C57L/J to NZM2328. Quantitative trait loci (QTL) controlling acute (Agnz1 and
Agnz2) and chronic (Cgnz1) GN features were uncovered by the analysis. To verify the Cgnz1 and Agnz1
on distal mouse chromosome 1, we produced the NZM23238.C57Lc1 (Lc1) congenic strain, which
replaced NZM2328 Cgnz1 and Agnz1 alleles with those derived from C57L/J. The development of acute
GN and chronic GN was markedly reduced in Lc1 mice, confirming the linkage findings. Further mapping
by the generation of intrachromosomal recombinants of NZM2328.Lc1 support the thesis that acute GN
and chronic GN are under separate genetic control.

Key words: Congenic strain, End-stage renal disease, Genetic mapping, Genotyping,
Glomerulonephritis, Linkage analysis, Lupus nephritis, Marker-assisted selection protocol,
Microsatellite marker, NZM2328, Quantitative trait loci, QTL mapping, Speed congenic, Systemic
lupus erythematosus

1. Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease; it

is characterized by autoantibody production and complement-
fixing immune complex deposition that result in tissue inflammation
and damage. Lupus nephritis is the most common and severe man-
ifestation of SLE, affecting all renal compartments (i.e., glomeruli,
tubules, interstitium, and renal blood vessels). Lupus nephritis

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_13, Springer Science+Business Media New York 2012

271
272 Y. Ge et al.

contributes significantly to the morbidity and mortality of SLE

patients because of the risk of progressing to end-stage renal dis-
ease (ESRD). Sixty percent of SLE patients develop lupus nephritis
at some stage of the disease (1); moreover, 520 % of the patients
with lupus nephritis progress to ESRD within 10 years of disease
onset (2) despite advances in our approaches in the treatment of
lupus nephritis. Thus, the understanding of the pathogenesis of
lupus nephritis remains a major challenge in the care of patients
with SLE.

1.1. Genetic During the last six decades, extensive data have been accumulated,
Contributions suggesting that both genetic and environmental factors play
in Human SLE significant role in the pathogenesis of SLE. Twin studies have pro-
vided definitive evidence that genetics as well as environment play
important roles in human SLE: the concordance rate of SLE in
monozygotic twins is 2458 %, whereas in dizygotic twins or sib-
lings, the rate was 25 % (3). SLE also displays familial aggrega-
tion, as the risk of disease among siblings of SLE patients is about
29 times higher than that among the general population (4).
Recent genome-wide association studies on lupus susceptibility
genes have identified more than 30 candidate genes that have rela-
tive minor impacts on SLE (58). Evidence has also been obtained
showing ethnic variation in lupus clinical manifestations and the
effects of social-economic factors on this disease (9). These data
suggest the complexity of lupus genetics. The heterogeneity of the
human population is still a major obstacle in genetic studies in
man. It is also evident that to delineate the pathogenetic factors in
a complex disease with protean clinical presentations present a for-
midable challenge. Studies on mouse models on lupus in general
and lupus nephritis in particular have helped significantly in our
understanding of this disorder. They will remain useful comple-
menting human studies (10, 11). In this chapter, the genetic
approaches in mapping genes contributing to lupus nephritis in
mouse are reviewed briefly. Our studies on NZM2328 are dis-
cussed in detail to illustrate the methods for mapping genes that
contribute to the development of lupus nephritis in mouse.

1.2. Mouse Models New Zealand mice were the first mouse model described for human
of Spontaneous lupus (reviewed in (12, 13)). (NZB NZW)F1 females develop
Lupus Nephritis glomerulonephritis (GN) that resembles human proliferative lupus
nephritis with immune complex deposition. New Zealand mice
have been studied extensively. Subsequently, two other models,
MLR/lpr and BXSB have been described. Although these three
mouse models have important similarities, they differ significantly
in their natural history, autoantibody profiles, organ involvement,
and sex predilection (11, 14). It was recognized early that the
background genes influence phenotypic expression. Thus, MRL/lpr
has severe autoimmune phenotypes with autoantibody production,
 13 Genetic Approach to Study Lupus Glomerulonephritis 273

vacuities, skin disease, and severe GN while B6.lpr has very few
autoimmune traits. During the past two decades, recombinant
inbred strains have been established from a (NZB NZW)F2 or
(NZB NZW)F1 NZW mating (15). These strains have approxi-
mately 75 % of NZW with 25 % NZB genes. They have ANA and
varying degree of GN (16). These strains are very useful for map-
ping genes contributing to lupus GN.
The aforementioned spontaneous lupus-prone models have
been used in mapping genes contributing to various autoimmune
phenotypes (reviewed in (10, 11, 1719)). Typically, genetic analy-
sis was carried out to identify genetic segments with quantitative
trait loci (QTL) that contribute specifically to particular autoim-
mune phenotypes as distinguished by a quantitative trait by the
study of either a cohort of (lupus-prone strain non-lupus prone
strain)F1 backcrossed to the non-lupus strain or a cohort of inter-
cross (F2) progenies. The identified QTL needs to be substantiated
by the generation of congenic strains in which the genomic region
corresponding to the QTL of interest is introgressed to the non-
autoimmune strain. This approach was taken by Wakeland, Morel
and colleagues in their studies of Sle1, Sle2, and Sle3. In this case,
Sle 1, Sle2, and Sle3 on chromosome 1, 4, and 7, respectively, were
identified to be linked to GN by the analysis of a cohort of
(NZM2410 C57BL/6)F1 C57BL/6, in which NZM2410 is a
lupus-prone strain while C57BL/6 (B6) is the non-lupus prone
strain. Although autoantibody production was one of the traits of
interest, no such QTL was identified. None of the congenics,
B6.Sle1, B6.Sle2, and B6.Sle3 have GN; however, the tricongenic
B6. Sle1Sle2 Sle3 has severe GN. Although the functions of these
three loci in B6-based lupus cogenics have been studied exten-
sively, the nature of the genes conferring the GN phenotypes
remains elusive (reviewed in (11, 19)).
As an alternative approach, we have chosen NZM2328 as the
lupus-prone strain and C57L as the non-lupus strain. As detailed
below, we have identified a single locus Cgnz1 controlling chronic
GN on chromosome 1, a single locus Adnz1 on chromosome 4
linked to ANA and anti-dsDNA production, and three loci, Agnz1
on chromosome 1, the H-2 complex and a locus, Agnz2, distal to
the H-2 complex on chromosome 17 to be linked to acute GN.
The phenotypes of NZM2328-base congenic strains, NZM2328.
Lc1 and MZN2328.Lc4 confirm the genetic data (20, 21).
These two contrasting approaches underline the importance of
the choice of strains and the defining of traits in the mapping of
genetic traits for lupus nephritis.

1.3. NZM2328 as an NZM2328 female mice spontaneously develop severe proteinuria

SLE Model and lupus nephritis, which leads to early mortality. In contrast, male
mice have ANA and anti-dsDNA antibodies and immune complex
deposit in the kidney without severe proteinuria and early mortality.
274 Y. Ge et al.

Thus, the NZM2328 strain displays a gender bias towards females,

which is a feature of human SLE. Regarding autoantibodies,
NZM2328 mice have ANA and anti-dsDNA antibodies; however,
there are little anti-IgG antibody (rheumatoid factor) activities in
the sera of NZM2328 mice. NZM2328 mice also have hypergam-
maglobulinemia (20). It was noted that severe proteinuria is
correlated with the development of chronic GN and early mortality.
Thus, severe proteinuria has been used as a biomarker for chronic
GN and early mortality.
During the characterization of NZM2328, it was noted that
two distinct stages of GN can be discerned in NZM2328: acute
GN and chronic GN. Acute GN is marked by active proliferative
GN, including signs of mesangial proliferation and glomerular
hypercellularity without global sclerosis and tubular abnormality.
In contrast, chronic GN is characterized by fibrotic changes, such
as glomerulosclerosis, tubular atrophy and dilation, and interstitial
fibrosis. In NZM2328 mice, lupus nephritis starts with acute
inflammation primarily within the glomeruli, and then gradually
progresses to the chronic form involving permanent damage in
both the glomeruli and the interstitium (20). Between the stages
of acute and chronic GN, we have documented a transitional dis-
ease phase in NZM2328 mice, which is histologically distinctive
(22). Interestingly, NZM2328 males develop acute GN without
progression to end-stage renal failure by 12 months of age (20).
However, the mechanisms controlling the progression of lupus
nephritis remain to be determined.

1.4. Systemic Lupus To identify genes that contribute to the development of lupus
Nephritis Loci in nephritis, we conducted a genome-wide linkage analysis using a
NZM2328 backcross cohort derived by crossing SLE-prone NZM2328 and
non-lupus C57L mice. The backcross offspring were PCR geno-
typed with a genome-wide panel of simple sequence length poly-
morphism (SSLP) markers; they were also characterized in terms
of lupus traits. SLE trait analysis involved following individual off-
spring until 12 months of age or when severe proteinuria devel-
oped, whichever occurred first. As stated above, QTL for acute and
chronic GN (SLE traits) were genetically mapped; acute GN QTLs
Agnz1 and Agnz2 were positioned on chromosomes 1 and 17,
respectively, and a chronic GN QTL Cgnz1 was mapped on distal
chromosome 1. In addition, a single locus, Adnz1 was located on
chromosome 4 (20). Because Cgnz1and Adnz1 were monogenic
for association with ANA/anti-dsDNA and anti-nucleosome anti-
body production and chronic GN, respectively, congenic lines,
NZM2328.Lc1 and NZM2328.Lc4 were generated by introgress-
ing the genetic regions containing these two loci into NZM2328.
These congenic lines have been informative and are discussed sepa-
rately (21).
 13 Genetic Approach to Study Lupus Glomerulonephritis 275

1.5. Independent A cohort of NZM2328.Lc1 female mice were monitored over a

Genetic Control period of 12 months for the production of ANA/ANA/anti-
of ANA/Anti-dsDNA dsDNA and anti-nucleosome antibody production and the devel-
and Anti-nucleosome opment of chronic GN. The results showed that these females did
Antibody Production not produce ANA, anti-dsDNA, and anti-nucleosome antibodies
and GN and confirmed the genetic analysis. Despite the lack of production
of these antibodies, these mice had severe proteinuria and chronic
GN. The kinetics of development of severe proteinuria and chronic GN
was similar to that of the parental strain NZM2328. These results
demonstrate that ANA and anti-dsDNA and anti-nucleosome anti-
bodies are not needed for the development of GN. They suggest
that the hypothesis that breaking tolerance to these autoantigens is
the first and crucial step in lupus pathogenesis (19) should be
modified. Thus, a broader view regarding autoantibody role in
lupus nephritis is advocated (23).

1.6. Cgnz1 and Agnz1 The findings of Cgnz1 and Agnz1 agree with previous genetic
Verification and studies on murine SLE, as the Cgnz1 and Agnz1 interval overlap
SLE-Gene Precision with Sle1 and Sle1 was found to be linked to GN in a genome-wide
Mapping on linkage analysis of a (NZM2410 C57BL/6)F1 NZM2410
Chromosome 1 backcross cohort (20).
To verify the biological function of the Cgnz1 and Agnz1 loci,
we generated NZM2328.C57Lc1 (Lc1) congenic mice by intro-
gressing a 24-cM chromosome 1 fragment of C57L/J, which spans
Cgnz1 and Agnz1 loci, onto the NZM2328 background. The Lc1
mice had significantly reduced incidences of both acute and chronic
GN, autoantibody production, severe proteinuria, and early mor-
tality (21); these observations were consistent with our linkage
findings (20). The successful generation of this important congenic
model to study lupus GN in animals with disparate chromosome 1
intervals was largely due to the congenic approach we employed
(i.e., using NZM2328 as the recipient background instead of a
non-lupus strain). On the other hand, GN cannot be directly
examined in congenic strains produced by the opposite approach
as discussed and detailed in Subheading 3.2.
We have generated multiple intrachromosomal recombinant
strains of NZM2328.Lc1 by monitoring cohorts of (NZM2328
NZM2328.Lc1)F2 for recombinations. Preliminary data indicate
that replacing the regions of Agnz1 had no effect on the develop-
ment of both acute and chronic GN and the replacement of the
regions controlling chronic GN inhibited the development of
chronic GN despite the manifestation of acute GN with immune
complex deposition (24). Furthermore, the region controlling
chronic GN has been mapped to a 1.34 Mb segment of chromo-
some 1 (22). This paves the way for the identification of the gene
controlling chronic GN by transgenesis or by knock-in approach.
This aspect of mapping the genes for lupus nephritis is not dis-
cussed further in this chapter.
276 Y. Ge et al.

2. Materials

1. Gentra Puregene Mouse Tail Kit (QIAGEN, Valencia, CA).

2. Isopropanol.
3. 70 % ethanol.
4. 10 PCR buffer (without MgCl2) (Applied Biosystems, Foster
City, CA).
5. Primers for microsatellite markers with or without fluorescence
labeling (Integrated DNA Technologies, Coralville, IA).
6. 25 mM MgCl2.
7. 2.5 mM dNTPs: 2.5 mM dATP, 2.5 mM dTTP, 2.5 mM
dCTP, 2.5 mM dGTP (Invitrogen, Carlsbad, CA).
8. 5 U/ml Taq DNA polymerase (Applied Biosystems).
9. MetaPhor agarose (Cambrex Bio Science Rockland, Inc.,
Rockland, ME).
10. 1 TBE buffer (pH 8.0): 89 mM Tris, 89 mM Boric Acid, and
2 mM EDTA (pH 8.0).
11. Ethidium bromide.
12. 6 gel loading dye containing Bromophenol Blue (New
England BioLabs, Ipswich, MA).
13. 50-bp DNA ladder (New England BioLabs, Ipswich, MA).
14. Applied Biosystems 3130xl Genetic Analyzer (Applied
Biosystems).
15. MicroAmpTM Optical 96-Well Reaction Plate (Applied
Biosystems).
16. Genescan 400HD Rox Standard (Applied Biosystems).
17. GeneMapper Software (Applied Biosystems).
18. Hi-Di Formamide (Applied Biosystems).
19. QTL mapping software.

3. Methods

The methods described here outline (a) classical genetic analysis

and quantitative trait mapping in backcross mice; (b) congenic
strain production; (c) precision genetic mapping for lupus nephri-
tis loci; and (d) histological evaluation of GN. In addition to pro-
viding these protocols, we will utilize our work on NZM2328 to
emphasize important considerations in carrying out mapping stud-
ies for SLE susceptibility genes.
 13 Genetic Approach to Study Lupus Glomerulonephritis 277

3.1. Classical Genetic 1. Breed lupus-prone NZM2328 with non-lupus C57L/J mice
Analysis and SLE Trait to generate (NZM2328 C57L/J)F1 progeny (see Note 1 for
Mapping in NZM2328 discussion of mating direction). F1 males and females should
Backcross Mice be kept for further backcrossing and phenotype analysis. All F1
pedigree records (e.g., ID of breeding cage and ID of both
3.1.1. Generation of
parents) must be maintained for future reference.
NZM2328 Backcross Mice
and SLE Trait Development 2. Breed NZM2328 with (NZM2328 C57L/J)F1 mice to
generate [(NZM2328 C57L/J)F1 NZM2328] backcross
offspring (and see Note 1). Alternatively, use a brother sister
(NZM2328 C57L/J)F1 intercross mating scheme to gener-
ate F2 offspring for genetic analysis (see Note 2).
3. Monitor SLE traits (i.e., severe proteinuria and kidney histol-
ogy) in NZM2328 backcross females over the period of
12 months after birth (see Note 3).

3.1.2. Extraction of Mouse 1. Clip 23 mm tail tissue from control NZM2328 and C57L
Tail Genomic DNA mice and backcross offspring and store in a microcentrifuge
tube (1.5 ml) labeled with the mouse ID at 20 C.
2. Add 300 ml Cell Lysis Solution (Gentra) and 1.5 ml Puregene
Proteinase K (final conc. 0.1 mg/ml) to mouse tail tissue (fresh
or frozen) and mix gently by inverting 25 times.
3. Incubate at 55 C overnight or until the tissue has completely
dissolved. Invert tube periodically during the incubation.
When tail tissue is not completely dissolved after overnight
digestion, additional Proteinase K may be added, followed by
12 h further incubation at 55 C.
4. To quickly cool the tail lysate, incubate on ice for 1 min. Add
100 ml Protein Precipitation Solution (Gentra), and mix well
by vortexing vigorously for 20 s at high speed.
5. Centrifuge sample at ~16,000 g for 3 min at room tempera-
ture. The protein precipitate should form a tight pellet.
However, if the pellet is loose, incubate on ice for 5 min and
repeat the centrifugation.
6. In a clean microcentrifuge tube, add 300 ml isopropanol.
Carefully decant the supernatant from step 5 into the tube
with isopropanol. Note that the protein pellet should not be
dislodged during pouring.
7. Mix sample gently by inverting 50 times, centrifuge sample at
~16,000 g for 1 min at room temperature. The DNA precipi-
tate may form a visible small white pellet. Carefully discard the
supernatant, and drain the tube by inverting on a clean piece of
absorbent paper. Be careful not to lose the DNA pellet during
the process, as it might be loose and easily dislodged.
8. Add 300 ml of 70 % ethanol and mix gently by inverting several
times to wash the DNA pellet.
278 Y. Ge et al.

9. Centrifuge sample at ~16,000 g for 1 min. Carefully discard

the supernatant, and drain the tube by inverting on a clean
piece of absorbent paper. Be careful not to lose the DNA
pellet.
10. Air dry the DNA pellet for 10 min. Add 50 ml DNA Hydration
Solution (Gentra) and vortex 5 s at medium speed to mix.
Incubate tail DNA samples at room temperature overnight to
allow DNA rehydration. The DNA can be safely stored at
48 C (short-term) and at 20 or 80 C (long-term).

3.1.3. Simple Sequence Detection of large and small effect QTLs in a genome-wide link-
Length Polymorphism and age analysis requires genetic markers spaced at ~20-cM distances,
Single Nucleotide and no more than 10-cM from chromosome ends, for all chromo-
Polymorphism Marker somes in the genome (25). Microsatellite repeat sequences in the
Selection for Genome- mouse genome work well for the purpose of generating SSLP
Wide Linkage Analysis markers to readily distinguish allele variants in common laboratory
strains of mice, including NZB and NZW (20, 26). SNP markers
to distinguish NZB and NZW mouse alleles have been published
(20, 26).
1. Published SSLP (also referred to as PCR marker) and SNP
markers for 89 strains of mice may be obtained through the
Mouse Genome Informatics (MGI), which can be accessed at
http://www.informatics.jax.org/. Detailed marker informa-
tion includes mouse chromosome position, product (allele)
size and amplification primers (PCR markers), and nucleotide
differences for SNP markers.
2. When strain-specific allele information is not available in public
databases for potentially informative and useful SSLP and SNP
markers based on their chromosome location, these must be
tested against the control progenitor strains of interest using
the methods described in Subheadings 3.1.4 and 3.1.5.
3. In practice, one should assemble and test the entire panel of
select genetic markers, which can readily distinguish SSLP and
SNP allele differences in select progenitor strains and their
hybrid offspring. We have routinely used SSLP markers to reli-
ably and accurately genotype alleles distinguished by 2+ bp dif-
ferences in backcross and intercross progenies.
4. All hybrid offspring must be genotyped using the approved
panel of genetic markers.

3.1.4. PCR Genotyping Mouse tail DNA genotyping by PCR is carried out with select
Mouse Tail DNA with SSLP SSLP markers as discussed in Subheading 3.1.3.
Markers
1. Prepare PCR master mix sans tail DNA based on number of
mouse tail DNA samples to be genotyped per SSLP marker.
 13 Genetic Approach to Study Lupus Glomerulonephritis 279

2. Aliquot PCR master mix into individual PCR tubes, add tail
DNA, and carry out PCR on a thermal cycler according to the
conditions given in step 3.
Volume per Final
Component reaction (ml) concentration

10 PCR buffer (No MgCl2) 1.0 1

25 mM MgCl2 1.0 2.5 mM
2.5 mM dNTPs 0.8 0.2 mM
2.5 mM forward primer 1.0 0.25 mM
2.5 mM reverse primer 1.0 0.25 mM
ddH2O 4.15
5 U/ml Taq DNA polymerase 0.05 0.25 U/10 ml
Mouse Tail DNA 1.0 2050 ng/10 ml
Total volume 10 ml

3. Amplify SSLP Markers using the following PCR conditions.

Initial denature 94 C for 3 min

PCR cycles 30 cycles of:
94 C for 20 s
55 C for 30 s
72 C for 30 s
72 C for 3 min
Final extension 72 C for 3 min

3.1.5. Size Analysis Two methods to separate and visualize SSLP PCR-amplified mark-
of PCR-Amplified SSLP ers are detailed here.
Markers

Capillary Polymer-Based Electrophoretic mobility in polymer-filled capillaries offers a conve-

SSLP Size Analysis nient method to resolve PCR-amplified SSLP marker size differ-
ences. In this protocol, PCR is run with a fluorescent-labeled (5)
primer so that SSLP markers, which have incorporated a fluorescent
label, can be excited by an argon laser and visualized in the Genetic
Analyzer 3130xl (Applied Biosystems). Compatible fluorescent dyes
for PCR primers include 6-carboxyfluorescein (6-FAM, blue),
hexachlorofluorescein phosphoramidite (HEX, green), NED (yel-
low), and ROX (red). The Genetic Analyzer can separate a complex
mixture of DNA fragments (labeled with different fluorescence dyes)
in one sample and determine the length of each fragment. Thus,
these dyes can be used in multiplex PCR. SSLP marker mobility in
polymer-filled capillaries is compared against ROX-labeled sized
standards to determine allele sizes. This method automates marker
calls and yields outstanding run-to-run consistency and reliability.
280 Y. Ge et al.

1. Run SSLP marker PCR according to Subheading 3.1.4 with

one of the primers (forward or reverse) 5-labeled with a com-
patible dye. Note that SSLP markers can be multiplexed in
PCR with judicious selection and optimization of particular
SSLP markers based on size and/or 5-primer label differences
and when PCR primers for the different markers do not form
primer dimers.
2. Quickly spin down PCR products. Transfer 1 ml of each PCR
product to a MicroAmpTM Optical 96-Well Reaction Plate
(Applied Biosystems).
3. Prepare size standard by diluting Genescan 400HD ROX Size
Standard (Applied Biosystems) 1:40 in Hi-Di Formamide
(Applied Biosystems). Mix well by vortexing.
4. Add 9 ml of the ROX/Formamide mixture to each PCR prod-
uct in the reaction plate from step 2, seal the plate with a septa
(Applied Biosystems), and mix well by vortexing.
5. Quickly spin down the reaction plate, denature samples in the
reaction plate on a thermal cycler at 95 C for 5 min, and then
immediately place the reaction plate with PCR samples on ice
to incubate for 5 min.
6. Load the reaction plate with denatured PCR samples onto the
Genetic Analyzer 3130xl and run samples per manufacturers
instruction.
7. Analyze the electrophoretic mobilities of SSLP markers with
Data Collection (v3.0) and GeneMapper Software (Applied
Biosystems) according to the manufacturers instructions.

MetaPhor Agarose Gel MetaPhor agarose (Cambrex Bio Science Rockland, Inc., Rockland,
Electrophoresis-Based Maine) has an intermediate melting temperature of 75 C and
SSLP Size Analysis offers outstanding resolving capacity for separation of PCR-
amplified SSLP markers which differ by 5+ bp. To insure accurate
genotypes, we routinely run SSLP PCR with progenitor strain (i.e.,
NZM2328 and C57L/J) controls on the same MetaPhor gel with
all offspring PCR products under analysis (27). To achieve opti-
mum resolution, users must adhere strictly to manufacturers
instructions when preparing MetaPhor agarose gels.
1. Add sufficient quantity of MetaPhor agarose slowly to chilled
1 TBE buffer in a beaker with continuous swirling to make a
2 % solution.
2. After soaking agarose in cold 1 TBE buffer for ~15 min,
weigh the beaker and solution, cover the beaker with plastic
wrap with a small hole, and heat the beaker in a microwave
oven until all the particles are dissolved.
3. Add sufficient heated distilled water (75 C) to obtain the
beaker and mix thoroughly. After cooling the solution to

3.1.6. Genetic Mapping of
SLE Quantitative Trait Loci
3.2. Generation of
Non-SLE NZM2328
Congenic Strains
13 Genetic Approach to Study Lupus Glomerulonephritis 281
~60 C, add ethidium bromide (0.3 mg/ml), mix well, and
cast the gel.
4. Allow gel to solidify at room temperature, and then condition
it at 4 C for 20 min before use. This maneuver is to insure
optimal resolution and gel handling characteristics.
5. After adding 2 ml 6 gel loading dye to each 10 ml PCR prod-
uct, load 10 ml of it on the MetaPhor gel submerged in chilled
1 TBE buffer and electrophorese for 34 h at 6 V/cm using
horizontal electrophoresis system (see Note 4).
6. Visualize the PCR products under UV transilluminator and
take photographs for records.
7. Make allelic calls by comparing sizes of PCR products of mice
being genotyped to those of both parental strains.
To map chromosome locations for SLE QTL, NZM2328 back-
cross genotypes and lupus trait data were analyzed using
MapMangerQT (20). Other very useful QTL mapping programs
include QTL Cartographer, R/qtl, and MapQTL (28, 29).
Single-QTL genome scans using regression analysis typically
detect chromosome map locations ranging from 10 to 30 cM based
on likelihood ratio test statistic (LRS) or logarithm of the odds
(LOD) scores. For a QTL, the mapping resolution depends on the
number of recombination events in the mapping cohort. When
assessing whether a QTL is statistically significant, Lander and
Kruglyacks guidelines are standard practice: Highly-significant
refers to p < 0.001, significant p < 0.05, and suggestive p < 0.63,
after correcting for multiple testing in genome-wide scans (30).
For a backcross genome-wide linkage analysis, the LOD score
threshold for suggestive linkage is 1.9, and significant linkage 3.3
(30). When reporting QTL mapping results, the LOD scores, the
peak positions, and the estimated confidence intervals should be
provided. Once identified, one may further refine significant QTL
locations by typing the initial backcross or intercross cohort with
additional SSLP markers targeting these regions of interest, and
then rerunning the QTL mapping program. Additionally, preci-
sion mapping for candidate loci may be carried out as described in
Subheading 3.3.
Following their identification in broad genome-wide linkage stud-
ies, SLE QTL must be verified at the biological level. A useful
method to establish QTL validity is to produce a congenic strain
by introgressing a donor chromosome fragment with its potential
QTL onto a different genetic background which does not display
the QTL effect. This may be accomplished via repeated backcross-
ing and genetic selection for donor alleles flanking a select QTL on
the recipient strain background, but without donor alleles else-
where in the genome.
282 Y. Ge et al.

3.2.1. Selection of Genetic
Background for Congenic
Strains
A critical first step toward precision SLE gene mapping and later
functional studies is to select a desirable genetic background for the
production of congenic strains. This is because background genetic
factors may interact with or contribute to donor QTL allele effects,
which may further influence whether a given QTL effect is manifest.
Recently, we produced NZM2328 chromosome 1-congenic
mice with non-lupus alleles derived from C57L/J mice (21).
An important feature in the NZM2328.C57Lc1 (also referred to
as Lc1) mice is that only the select chromosome 1 interval is
responsible for changes in disease progression normally observed
in NZM2328 females. Thus, we can directly monitor the impact of
Agnz1 and Cgnz1 on SLE traits in these mice. Inhibition of both
acute and chronic GN was observed in Lc1 females, thus confirming
that NZM2328 mice harbor one or more SLE QTL on chromo-
some 1 which contribute to disease (20).
In contrast to our approach, several related studies have used
B6 mice, which do not develop lupus features, as recipients to gen-
erate congenic strains with donor alleles derived from lupus-prone
mouse strains (e.g., NZM2410, NZW, and BXSB) (31, 32). An
inherent weakness of the latter approach is that lupus traits have
not manifested in the B6 background. For example, although Sle1
and Nba2 were initially mapped for GN, neither B6.Sle1 nor
B6.Nba2 congenic strains developed GN. Instead, B6.Sle1 mice
developed increased levels of IgG ANA, and B6.Nba2 congenic
mice developed IgG anti-DNA and anti-chromatin autoantibodies
(33, 34). Due to the lack of GN in B6.Sle1, the subsequent fine
mapping of Sle1 had to use autoantibody production as the trait to
approximate GN (35). This approach has made Morel and col-
leagues to postulate the presence of Sle1d that confers the original
phenotype of GN with unknown location within the Sle1 region
(35). Another weakness of the approach is that it potentially intro-
duced a whole new set of gene interactions between non-lupus
background and donor lupus alleles, complicating an already com-
plex research question. In this regard, the non-lupus prone B6 has
been shown to harbor a gene contributing to autoantibody pro-
duction (36). Thus, we conclude that the introgression of non-
lupus alleles into SLE-prone NZM2328 is highly useful and
informative for the purpose of QTL verification and further study
of acute and chronic GN and ESRD.

3.2.2. Speed Congenic The traditional protocol for generating congenic strains is to back-
Protocol cross a donor locus to a recipient genetic background for 10+ gen-
erations, accompanied by screening and selection for progeny
carrying the donor locus at each generation. The founders are gen-
erated by intercrossing the mice of the last backcross and then
selecting for homozygosity at the target region. Though straight-
forward, completion of this protocol takes 34 years (37). The
marker-assisted selection protocol (MASP)-based breeding strategy,
 13 Genetic Approach to Study Lupus Glomerulonephritis 283

also known as the speed congenic approach, only requires

1.52 years (37). The basis of the speed congenic method is to
select progeny at each backcross that carry the target donor region
(or gene), but with minimal donor genetic contamination (i.e.,
heterozygosity) elsewhere in a recipient animal genome, which can
dramatically accelerate completion of the desired congenic strain.
Another important consideration, when one generates con-
genic strains without phenotypic selection for the trait of interest,
one must make certain that select donor alleles actually flank the
potential QTL. In our experience, it is safer to start with a large
chromosome segment to ensure that this will be the case, since it
will be far simpler and less time-consuming to narrow the target
region by generating subcongenic lines, than to generate a new
congenic strain. The speed congenic protocol below describes how
the chromosome 1 interval of NZM2328 carrying Agnz1 and
Cgnz1 was replaced with a C57L-derived chromosome 1 fragment
to produce the Lc1 congenic strain.
1. Produce 3040 NZM2328 backcross (N2) mice (1520 mice
of both sexes) as described in Subheading 3.1.1 (see Note 5).
2. Genotype NZM2328 backcross (N2) mice with SSLP and SNP
marker panel established in Subheading 3.1.2 to assess zygos-
ity across the genome. SSLP markers for X and Y chromosomes
were not included since a simple breeding step can fix the host
sex chromosomes (see step 5).
3. Genotype NZM2328 backcross (N2) mice with SSLP markers
for the donor target region (i.e., the interval between markers
D1Mit15 and D1Mit155). Offspring were further screened
with additional SSLP markers with ~5-cM spacing per marker
to minimize the potential for intra-region recombination
events. Select several NZM2328 backcross (N2) mice that carry
the donor allele(s) of interest and the highest percentage of
recipient NZM2328 alleles elsewhere in the genome for fur-
ther backcrossing.
4. Breed select NZM2328 backcross (N2) mice with NZM2328
to produce 3040 NZM2328 backcross (N3) offspring and
repeat step 2 with this new generation of mice.
5. Repeat steps 3 and 4 with NZM2328 backcross (N3N5) in
place of N2 backcross offspring until select NZM2328 back-
cross (N5) are obtained, which should contain the donor target
region (heterozygous) in the homozygous NZM2328 back-
ground. Theoretically, donor genome contamination should
be less than 0.5 % for N5 backcross offspring by this method
(37). To fix the sex chromosomes, make sure that at any one of
the first three backcross generations (N2N4), female backcross
mice were crossed with NZM2328 males, and that at the
immediate following generation, male backcross mice were
crossed with NZM2328 females.
284 Y. Ge et al.

6. Select a single pair of heterozygous NZM2328 backcross (N5)

mice that were from the same parents. Perform a complete
genome-wide genotype scan on both animals to ensure that
each retains the donor target region, but without donor genetic
contamination elsewhere. Cross the pair to produce Lc1
homozygous offspring.
7. Genotype the offspring with the same set of informative SSLP
markers. Select a breeding pair of animals that are homozygous
for Lc1, and use them for all subsequent inbreeding (i.e., a
brother sister mating scheme) of Lc1 congenic mice.

3.3. Precision Mapping A major goal in precision mapping is to identify a minimal critical
for SLE Genes interval spanning the gene(s) responsible for a QTL effect, which
on Chromosome 1 is flanked or bounded by recombination crossovers. We have used
this approach before to establish critical genomic regions needed
in viral resistance, which subsequently allowed us to identify the
relevant genes (3843). More recently, we used this approach to
pinpoint distinct locations for Agnz1 and Cgnz1 SLE loci within
the larger (24-cM) chromosome 1 interval based on crossovers in
a panel of Lc1 recombinant congenic lines (20, 22). Importantly,
these results demonstrate that precision mapping is an important
step in positional cloning and gene identification, even in narrow
gene regions, based on findings obtained for three different loca-
tions in the mouse genome. As an example, the following method
describes the generation of Lc1 recombinant congenic sublines.
1. Breed NZM2328.C57Lc1 mice, which are heterozygous for
the Lc1 interval, in a brother x sister mating scheme. Most
offspring produced in this breeding scheme will exhibit
heterozygosity or homozygosity over the entire 24-cM Lc1
congenic interval. However, a fraction of the offspring will
have a crossover on one or both chromosomes.
2. Genotype all offspring with select SSLP markers according to
protocol in Subheading 3.1.4 to screen for recombination
crossovers within the Lc1 congenic interval. Subheading 3.1.3
provides detailed information about how to obtain informative
SSLP markers to distinguish alleles in the relevant strains.
(a) When useful markers are unavailable, novel SSLP markers
can be generated as we described previously (26, 42, 44).
(b) In brief, download relevant genomic sequence covering
the region of interest from the National Center for
Biotechnology Information and manually inspect it for
microsatellite repeat sequences.
(c) Design sequence-specific primers to PCR amplify select
microsatellite sequences.
(d) Test SSLP marker primers to verify specific amplification
of the select microsatellite, the capacity to distinguish the
 13 Genetic Approach to Study Lupus Glomerulonephritis 285

relevant alleles and genetic linkage (i.e., novel SSLP markers

can be tested for linkage with other Lc1 markers in back-
cross or intercross offspring DNA samples previously
generated).
3. Examine Lc1 SSLP marker profiles for all offspring. Offspring,
which display a potential recombinant crossover profile, must
be verified with SSLP genotype analysis of newly prepared tis-
sue DNA from the relevant animal.
4. Backcross the select and validated recombinant congenic
mouse to NZM2328 to fix a new line. Repeat this step with
each recombinant congenic animal to generate a mapping
panel to precisely locate the QTL/gene(s) of interest.
5. Pinpoint recombination crossover boundaries with further
SSLP marker PCR analysis to refine the size of the recombi-
nant interval.
6. Monitor and record lupus features in the panel of recombinant
congenic females over a period of 12 months.
7. Define Agnz1 and Cgnz1 critical regions based on recombina-
tion crossover locations and trait analyses.

3.4. Histological [(NZM2328 C57L/J)F1 X NZM2328] backcross mice were dis-

Scoring of Murine sected at 12 months of age or at the time of being severely protei-
Glomerulonephritis nuric for two consecutive weeks as determined by Multistix 10 SG
(Bayer Diagnostics Division, Elkhart, Indiana). Signs of both acute
and chronic renal pathology were scored separately by light
microscopy.

3.4.1. Acute GN Acute GN was scored blindly using a scale of 04, based on the
severity of glomerular hypercellularity, mesangial matrix expansion,
focal necrosis, and epithelial cell crescents (Fig. 1 B1, C1, D1, and
E1). Grade 0 indicates the absence of signs of acute GN. Grade 1
indicates mild focal lesions with proliferative changes that are pri-
marily in the mesangium. Grades 2 and 3 represent diffuse involve-
ment of moderate severity in the glomeruli, whereas grade 4
indicates widespread changes of more severity (20).

3.4.2. Chronic GN Chronic GN was scored blindly using a scale of 04, based on the
severity of glomerulosclerosis (focal to global), tubular atrophy,
dilated tubules with hyaline casts, and interstitial fibrosis (Fig. 1
B2, C2, D2, and E2). Grade 0 indicates no signs of chronic GN.
Grade 1 represents mild focal glomerulosclerosis and interstitial
inflammation. Grades 2 and 3 represent moderate glomerulo-
sclerosis, tubule atrophy, and interstitial fibrosis. Grade 4 indi-
cates severe glomerulosclerosis, tubule atrophy, and interstitial
fibrosis (20).
Fig. 1. Stages of acute and chronic GN: Normal Kidney (A) from a 12-week-old NZM2328 female mouse. Kidneys from
24- to 28-week-old NZM2328 female mice with acute GN: (B1) stage 1 acute GN kidney showing mesangial expansion
and some hypercellularity with normal tubular cells. Note that the glomerulus is bigger than that in normal kidneys,
 13 Genetic Approach to Study Lupus Glomerulonephritis 287

4. Notes

1. Whether to breed NZM2328 males or females with C57L/J

for the F1 progeny is a practical choice that depends on repro-
ductive ability and availability of males and females of both
NZM2328 and C57L/J. In our experience, NZM2328 females
are poor breeders, whereas NZM2328 males breed well even
above 6 months of age.
For the breeding of backcross mice, use F1 females for
mating if the QTL could be X-linked. However, if the goal is
simply to map an autosomal mutation, the direction of the
backcross can be chosen arbitrarily. Since F1 female mice tend
to be very prolific, it is preferable to use them for the
backcrossing.
In cases where the possibility of X-chromosome-linkage
has not been excluded previously, it is wise to carefully pedigree
all F1 and subsequent matings and progeny, in case genetic het-
erogeneity is encountered. In addition, both male and female
F1 mice should be phenotyped to assess whether X-chromosome-
linkage exists for the traits under investigation.
2. In general, if there is evidence of directional dominance (e.g.,
a particular phenotype in one strain caused by a combination
of two or more recessive factors), it is preferable to backcross
F1 mice to the recessive parental strain. On the other hand, the
intercross design provides better precision in mapping, and is
more powerful for detecting QTLs that lack dominance (i.e.,
when heterozygotes have intermediate phenotype between the
two homozygous parental strains). Therefore, when little can
be assumed about the genetic control of a trait of interest, the
intercross design is preferred.
3. We selected only female backcross mice for the linkage analysis
due to the gender bias toward NZM2328 females in the develop-
ment of severe proteinuria and fatal GN (20). The required num-
ber of backcross mice depends on the strength of the QTL effect.
In addition, time and cost are other factors to be considered. See
reference (25) for guidance in determining useful cohort sizes.

Fig. 1. (continued) (C1) stage 2 acute GN showing more cellular infiltration in the glomeruli, (D1) stage 3 acute GN showing
loci of interstitial infiltration with hypercellular glomerulus, and (E1) stage 4 acute GN showing segmental scleroses in the
glomerulus without collapsing of the Bowmans capsule. The tubules are essentially normal. Kidneys from NZM2328
female mice older than 40 weeks with severe proteinuria and chronic GN: (B2) stage 1 chronic GN showing affected glom-
erulus with tubule dilatation and noncellular cast in the tubules, (C2) stage 2 chronic GN showing area of collapsed
Bowmans capsule and a red cell cast in one of the tubules, (D2) stage 3 chronic GN showing generalized tubular dilatation
and more extensive collapse of the Bowmans capsules, and (E2) stage 4 chronic GN showing sclerotic and necrotic glom-
erulus, with tubular dilatation and interstitial fibrosis. All photos were taken at 400 magnification.
288 Y. Ge et al.

4. A 5-bp difference in PCR-amplified products (e.g., 95- and

100-bp SSLP markers) requires ~4 h to resolve by MetaPhor
gel electrophoresis (27).
5. Genome-wide screening of 16 progeny per backcross genera-
tion with a low marker density (2025 cM distance) is a cost-
effective strategy (37).

Acknowledgments

This work was supported in part by NIH grants AI050072

(M.G.B.), P50-AR04522, R01-AR047988, R01-AR049449 and
R01-AI079621 (S.M.F.) and a grant from Alliance for Lupus
Research (S.M.F.).

References

1. Cameron JS (1999) Lupus nephritis. J Am Soc
Nephrol 10(2):413424
2. Houssiau FA, Vasconcelos C, DCruz D,
Sebastiani GD, de Ramon GE et al (2004)
Early response to immunosuppressive therapy
predicts good renal outcome in lupus nephritis:
lessons from long-term followup of patients in
the euro-lupus nephritis trial. Arthritis Rheum
50(12):39343940
3. Deapen D, Escalante A, Weinrib L, Horwitz D,
Bachman B et al (1992) A revised estimate of
twin concordance in systemic lupus erythema-
tosus. Arthritis Rheum 35(3):311318
4. Alarcon-Segovia D, Alarcon-Riquelme ME,
Cardiel MH, Caeiro F, Massardo L et al (2005)
Familial aggregation of systemic lupus erythe-
matosus, rheumatoid arthritis, and other auto-
immune diseases in 1,177 lupus patients from
the GLADEL cohort. Arthritis Rheum 52(4):
11381147
5. Deng Y, Tsao BP (2010) Genetic susceptibility
to systemic lupus erythematosus in the genomic
era. Nat Rev Rheumatol 6(12):683692
6. Gualtierotti R, Biggioggero M, Penatti AE,
Meroni PL (2010) Updating on the pathogen-
esis of systemic lupus erythematosus.
Autoimmun Rev 10(1):37
7. Delgado-Vega A, Sanchez E, Lofgren S,
Castillejo-Lopez C, Alarcon-Riquelme ME
(2010) Recent findings on genetics of systemic
autoimmune diseases. Curr Opin Immunol
22(6):698705
8. Kaiser R, Criswell LA (2010) Genetics research
in systemic lupus erythematosus for clinicians:
methodology, progress, and controversies.
Curr Opin Rheumatol 22(2):119125
9. Borchers AT, Naguwa SM, Shoenfeld Y,
Gershwin ME (2010) The geoepidemiology of
systemic lupus erythematosus. Autoimmun Rev
9(5):A277A287
10. Jorgensen TN, Gubbels MR, Kotzin BL (2004)
New insights into disease pathogenesis from
mouse lupus genetics. Curr Opin Immunol
16(6):787793
11. Morel L (2010) Genetics of SLE: evidence
from mouse models. Nat Rev Rheumatol 6(6):
348357
12. Theofilopoulos AN, Dixon FJ (1985) Murine
models of systemic lupus erythematosus. Adv
Immunol 37:269390
13. Borchers A, Ansari AA, Hsu T, Kono DH,
Gershwin ME (2000) The pathogenesis of
autoimmunity in New Zealand mice. Semin
Arthritis Rheum 29(6):385399
14. Andrews BS, Eisenberg RA, Theofilopoulos
AN, Izui S, Wilson CB et al (1978) Spontaneous
murine lupus-like syndromes. Clinical and
immunopathological manifestations in several
strains. J Exp Med 148(5):11981215
15. Rudofsky UH, Evans BD, Balaban SL, Mottironi
VD, Gabrielsen AE (1993) Differences in
expression of lupus nephritis in New Zealand
mixed H-2z homozygous inbred strains of mice
derived from New Zealand black and New
 13 Genetic Approach to Study Lupus Glomerulonephritis
Zealand white mice. Origins and initial charac-
terization. Lab Invest 68(4):419426
16. Rudofsky UH, Lawrence DA (1999) New zea-
land mixed mice: a genetic systemic lupus ery-
thematosus model for assessing environmental
effects. Environ Health Perspect 107(Suppl 5):
713721
17. Kono DH, Theofilopoulos AN (2006) Genetics
of SLE in mice. Springer Semin Immunopathol
28(2):8396
18. Theofilopoulos AN, Kono DH (2001) Genetics
of systemic autoimmunity and glomerulone-
phritis in mouse models of lupus. Nephrol Dial
Transplant 16(Suppl 6):6567
19. Fairhurst AM, Wandstrat AE, Wakeland EK
(2006) Systemic lupus erythematosus: multiple
immunological phenotypes in a complex
genetic disease. Adv Immunol 92:169
20. Waters ST, Fu SM, Gaskin F, Deshmukh US,
Sung SS et al (2001) NZM2328: a new mouse
model of systemic lupus erythematosus with
unique genetic susceptibility loci. Clin Immunol
100(3):372383
21. Waters ST, McDuffie M, Bagavant H,
Deshmukh US, Gaskin F et al (2004) Breaking
tolerance to double stranded DNA, nucleosome,
and other nuclear antigens is not required for
the pathogenesis of lupus glomerulonephritis. J
Exp Med 199(2):255264
22. Ge Y, Fu SM. MS in preparation. In press.
23. Bagavant H, Fu SM (2005) New insights from
murine lupus: disassociation of autoimmunity
and end organ damage and the role of T cells.
Curr Opin Rheumatol 17(5):523528
24. Ge Y, Jiang C, Gaskin F, Sung SJ, Bagavant H
et al (2009) Pathogenesis of proliferative lupus
nephritis: different genetic control for acute
and chronic glomerulonephritis and new insight
into the mechanism of immune complex medi-
ated nephritis. Arthritis Rheum 60:2019
25. Darvasi A (1998) Experimental strategies for
the genetic dissection of complex traits in ani-
mal models. Nat Genet 18(1):1924
26. Rodriguez MR, Lundgren A, Sabastian P, Li
Q, Churchill G et al (2009) A Cmv2 QTL on
chromosome X affects MCMV resistance in
New Zealand male mice. Mamm Genome
20(7):414423
27. Asif M, Rahman M, Mirza JI, Zafar Y (2008)
High resolution metaphor agarose gel elecctro-
phoresis for genotyping with microsatellite
markers. Pak J Agric Sci 45(1):7579
28. Manly KF, Olson JM (1999) Overview of QTL
mapping software and introduction to map
manager QT. Mamm Genome 10(4):327334
29. Broman KW, Wu H, Sen S, Churchill GA
(2003) R/qtl: QTL mapping in experimental
crosses. Bioinformatics 19(7):889890
289
30. Lander E, Kruglyak L (1995) Genetic dissec-
tion of complex traits: guidelines for interpret-
ing and reporting linkage results. Nat Genet
11(3):241247
31. Morel L, Rudofsky UH, Longmate JA,
Schiffenbauer J, Wakeland EK (1994) Polygenic
control of susceptibility to murine systemic
lupus erythematosus. Immunity 1(3):219229
32. Drake CG, Rozzo SJ, Hirschfeld HF,
Smarnworawong NP, Palmer E et al (1995)
Analysis of the new zealand black contribution
to lupus-like renal disease. Multiple genes that
operate in a threshold manner. J Immunol
154(5):24412447
33. Mohan C, Alas E, Morel L, Yang P, Wakeland
EK (1998) Genetic dissection of SLE patho-
genesis. Sle1 on murine chromosome 1 leads to
a selective loss of tolerance to H2A/H2B/
DNA subnucleosomes. J Clin Invest 101(6):1
3621372
34. Rozzo SJ, Allard JD, Choubey D, Vyse TJ, Izui
S et al (2001) Evidence for an interferon-
inducible gene, Ifi202, in the susceptibility to
systemic lupus. Immunity 15(3):435443
35. Morel L, Blenman KR, Croker BP, Wakeland
EK (2001) The major murine systemic lupus
erythematosus susceptibility locus, Sle1, is a
cluster of functionally related genes. Proc Natl
Acad Sci U S A 98(4):17871792
36. Heidari Y, Fossati-Jimack L, Carlucci F, Walport
MJ, Cook HT et al (2009) A lupus-susceptibil-
ity C57BL/6 locus on chromosome 3 (Sle18)
contributes to autoantibody production in 129
mice. Genes Immun 10(1):4755
37. Wakeland E, Morel L, Achey K, Yui M,
Longmate J (1997) Speed congenics: a classic
technique in the fast lane (relatively speaking).
Immunol Today 18(10):472477
38. Scalzo AA, Brown MG, Chu DT, Heusel JW,
Yokoyama WM et al (1999) Development of
intra-natural killer complex (NKC) recombi-
nant and congenic mouse strains for mapping
and functional analysis of NK cell regulatory
loci. Immunogenetics 49(3):238241
39. Scalzo AA, Wheat R, Dubbelde C, Stone L,
Clark P et al (2003) Molecular genetic charac-
terization of the distal NKC recombination
hotspot and putative murine CMV resistance
control locus. Immunogenetics 55(6):370378
40. Brown MG, Zhang J, Du Y, Stoll J, Yokoyama
WM et al (1999) Localization on a physical
map of the NKC-linked Cmv1 locus between
Ly49b and the prp gene cluster on mouse chro-
mosome 6. J Immunol 163(4):19911999
41. Brown MG, Dokun AO, Heusel JW, Smith
HR, Beckman DL et al (2001) Vital involve-
ment of a natural killer cell activation receptor
in resistance to viral infection. Science 292(5518):
934937
290 Y. Ge et al.
42. Xie X, Stadnisky MD, Brown MG (2009)
MHC class I dk locus and Ly49G2+ NK cells
confer H-2k resistance to murine cytomegalo-
virus. J Immunol 182(11):71637171
43. Xie X, Stadnisky MD, Coats ER, Ahmed
Rahim MM, Lundgren A et al (2010) MHC
class I D(k) expression in hematopoietic and
nonhematopoietic cells confers natural killer
cell resistance to murine cytomegalovirus.
Proc Natl Acad Sci U S A 107(19):
87548759
44. Rodriguez M, Sabastian P, Clark P, Brown MG
(2004) Cmv1-independent antiviral role of NK
cells revealed in murine cytomegalovirus-
infected New Zealand white mice. J Immunol
173(10):63126318
 Chapter 14

Animal Models of Primary Biliary Cirrhosis:

Materials and Methods *
Patrick S.C. Leung, Guo Xiang Yang, Amy Dhirapong,
Koichi Tsuneyama, William M. Ridgway, and M. Eric Gershwin

Abstract
Primary biliary cirrhosis (PBC) is a female-predominant autoimmune disease of the liver characterized by
immune-mediated destruction of the intrahepatic bile ducts and the presence of antimitochondrial anti-
bodies (AMAs). There have been limited advances in understanding the molecular pathogenesis of the
disease because of the difficulty in accessing human tissues and the absence of appropriate animal models.
Recently, several unique murine models that manifest the serological, biochemical, and histological fea-
tures similar to human PBC have been described. In this article, we discuss the current data on three
spontaneous and two induced murine models of PBC. The spontaneous models are: (a) NOD.c3c4,
(b) dominant negative TGF- receptor II (dnTGFRII), and (c) IL-2R/ mouse line models. The two
induced models are: (a) xenobiotic and (b) Novosphingobium aromaticivorans immunized mice. These
animal models provide various important platforms to further investigate the etiology and mechanisms of
pathogenesis in PBC. Laboratory methodologies and the protocols that are used in evaluating these animal
models are described. Finally, we stress the importance of realizing the strengths and limitations of the
animal models are essential in data analysis and their application in therapeutic studies.

Key words: Antimitochondrial antibodies, Biliary epithelial cells, Cytokine analysis, E2 subunit of
pyruvate dehydrogenase, ELISA, Flow cytometry, Immunohistochemical staining, Liver lesions,
Primary biliary cirrhosis, Western Blot

Abbreviations
PBC Primary biliary cirrhosis
AMA Antimitochondrial antibody
PDC-E2 E2 subunits of pyruvate dehydrogenase
BCOADC-E2 2-Oxo acid dehydrogenase
OGDC-E2 2-Oxo-glutarate dehydrogenase
NOD Non-obese diabetic
dnTGFRII Dominant negative TGF- receptor II

* Supported in part by National Institutes of Health grants DK074768 DK090019, DK067003, and
DK39588.

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_14, Springer Science+Business Media New York 2012

291
292 P.S.C. Leung et al.

IDDM Insulin-dependent diabetes mellitus

BSA Bovine serum albumin
2-OA 2-Octynoic acid
NKT Natural killer T cells
MHC Major histocompatibility complex

1. Introduction

Primary biliary cirrhosis (PBC) is a liver-specific autoimmune

disease characterized by antimitochondrial autoantibodies (AMAs)
and progressive destruction of intrahepatic bile ducts (1). The
major mitochondrial autoantigens are identified as the E2 subunits
of pyruvate dehydrogenase (PDC-E2), branched chain 2-oxo acid
dehydrogenase (BCOADC-E2), and 2-oxo-glutarate dehydroge-
nase (OGDC-E2) (24). Over the past two decades, extensive
efforts in defining the target mitochondrial autoantigens, T and B
cell epitopes, the innate and adaptive immune responses, the
immunobiology of the biliary epithelium, and the pathology of
destruction of biliary cells have greatly advanced the knowledge of
the molecular mechanisms in the pathogenesis of PBC (512).
However, cognizance on early events in the induction of tissue
inflammation and autoimmunity in PBC has been greatly hampered
by the cryptic onset of the disease, limitations in accessing human
liver samples and the lack of a suitable animal model. Fortunately,
this picture has changed dramatically within the last 5 years with
the reports of several murine models that manifest characteristic
clinical features of human PBC. Here, we discuss the current data
on three spontaneous and two induced murine models of PBC.
The spontaneous models are: (a) NOD.ABD, (b) dominant nega-
tive TGF- receptor II (dnTGFRII), and (c) IL-2R/ mouse
line models. The two induced models are: (a) xenobiotic and
(b) Novosphingobium aromaticivorans immunized mice. Each of
these animal models demonstrates the AMA profile that is specific
to human PBC and contains lymphocyte infiltration with biliary
epithelial cell pathology (Table 1). The availability of these PBC
animal models provides unique systems to further dissect the
immunological, genetic, and environmental components that
are intrinsic for the development of clinical PBC.

2. Mouse Models

2.1. NOD.ABD Mouse Non-obese diabetic (NOD) mice exhibit susceptibility to sponta-
Lines neous development of autoimmune type I diabetes (T1D) (13).
Genetic loci associated with susceptibility to T1D have been
defined through the development of congenic mouse strains, which
Table 1
Comparison between human PBC and murine models of PBC

Human Spontaneous models Induced models

2-OA-
BSA + alpha
14

PBC IL-2Ra/ 2-OA-BSA glyceramide Novosphingobium aromati-

patients NOD.ABD mice dnTGF-bRII mice mice immunization immunization civorans immunization

Background/ N/A NOD.c3c4 C57BL/6 C57BL/6 C57BL/6 C57BL/6 NOD 1101

strain Over expression of
a dn form of
TGFRII under
CD4 promoter
B cell immunity
AMA 9095 % 5060 % 100 % 100 % 100 % 100 % 100 %
Dominant AMA PDC-E2 PDC-E2 PDC-E2 PDC-E2 PDC-E2 PDC-E2 PDC-E2
target protein
Dominant Lipoyl Lipoyl domain Lipoyl domain Lipoyl domain Lipoyl domain Lipoyl domain Lipoyl domain
PDC-E2 domain
epitope
Liver histology
Portal lymphoid +++ +++ +++ +++ + ++ +
infiltrates
CD4 cell + ++ + + + + +
CD8 cell ++ + ++ ++ ++ +++ +
B cell + - + + + + +
Bile duct +-+++ + ++ +++ + ++ +
destruction
Animal Models of Primary Biliary Cirrhosis: Materials and Methods

Granuloma +-++ + + +
Eosinophilia + +
(continued)
293
Table 1
294

(continued)

Human Spontaneous models Induced models

2-OA-
BSA + alpha
PBC IL-2Ra/ 2-OA-BSA glyceramide Novosphingobium aromati-
patients NOD.ABD mice dnTGF-bRII mice mice immunization immunization civorans immunization
P.S.C. Leung et al.

Remarks Also develop Spontaneously Also develop Proof of hypothesis The only The remarkable sequence
common bile develop inflammatory that molecular murine homology between
duct dilation inflammatory bowel mimics of model of lipoylated proteins
and biliary bowel disease disease, lipoylated PBC that of xenobiotic metaboliz-
epithelial cell unless main- 2050 % die PDC-E2 in the exhibits ing N. aromaticivorans
proliferation tained by a from environment can fibrosis in combinationwith
Helicobacter free hemolytic lead to loss of the natural liver tropism
diet and anemia at tolerance to of NKT cells and the
antibiotics in 820 weeks PDC-E2 and accumulation of
drinking water of age autoimmune N. aromaticivorans
cholangitis in the liver likely explains
the liver specificity
of destructive responses
and AMA.
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 295

have identified several insulin-dependent diabetes (Idd) loci and

candidate genes (1416). In addition to T1D, NOD mice are also
prone to develop other autoimmune syndromes (17). NOD.c3c4
mice, the first spontaneous mouse model of autoimmune biliary
disease (ABD) resembling PBC, were serendipitously discovered
during the process of identifying gene segments responsible for
T1D. NOD.c3c4 mice have large chromosome 3 and 4 B6/B10-
derived regions of their genome introgressed onto the NOD
background (18). In 2006, Irie et al. (19) reported that NOD.
c3c4 mice developed liver lesions and AMAs similar to that of
patients with PBC. These mice exhibit liver histopathological
abnormalities including liver lymphocytic infiltrates, portal tract
lesions, and epithelial granuloma-like formation. 5060 % sponta-
neously develop autoantibodies to PDC-E2 at a relatively young
age of 910 weeks (19), and 8090 % develop antinuclear antibodies.
Histochemical analysis demonstrated that affected areas of the
biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T
cells. Furthermore, the treatment of NOD.c3c4 mice with mono-
clonal antibody (mAb) to CD3 partially protects them from ABD.
NOD.c3c4-scid mice are also protected from disease (they do not
develop clinical symptoms) but develop disease after adoptive
transfer of splenocytes, demonstrating a central role for T cells in
the pathogenesis in this model. Recently we published a second
NOD.ABD line with smaller regions from B10 on chromosomes 3
and 4 (20). Memory CD8+ T cells were increased in the intrahe-
patic lymphocyte fraction of these mice. Careful analysis using
transfer models demonstrated some surprising genetic restrictions
on ABD. First, whole spleen from NOD.ABD mice could transfer
both T1D and ABD into NOD.c3c4-scid recipients, but could not
transfer biliary disease into NOD-scid recipients. This showed that
the recipient mice must express the genes important for ABD in
the target tissue, not only the hematopoietic cells (20). One aspect
of NOD.ABD disease that differs from human PBC is the presence
of biliary epithelial proliferation. The spleen transfer model,
however, showed that ductule damage and lymphocytic infiltration
preceded biliary epithelial proliferation in this model. Finally,
CD8+ cells alone from NOD.ABD donors transferred severe bil-
iary diseasebut NOD CD8 cells did not (20). There were two
possible explanations for this finding: (1) that the genetic regions
responsible for ABD must be found in the donor CD8 cells, or
(2) that CD8 memory cells are stimulated in ABD mice by tissue
abnormalities in the target organ (which are lacking in NOD mice
and hence memory autoreactive T cells cannot develop). This
important distinction is being explored in ongoing studies but
overall these studies indicate a major role for CD8 T cells in the
pathogenesis of ABD.
296 P.S.C. Leung et al.

2.2. Dominant Negative Shortly after the discovery of the NOD.c3c4 mouse model, Oertelt
TGF-b Receptor II Mice et al. described another mouse model with PBC-like liver pathology
and AMAs, viz. the dnTGFRII mice (21). The dnTGFII mice
have an over-expression of a dominant-negative form of TGF-
receptor type II under the control of the CD4 promoter (22).
TGF- receptor II is critical for signal transduction of TGF-,
which regulates the activation of lymphocytes (23, 24). Deficiency
in TGF- results in various pleiotrophic immunological abnormali-
ties including colitis and early death (2527). dnTGFRII mice are
maintained by mating male dnTGFRII mice with normal B6
females (since female dnTGFRII mice experience reproductive
failure), to produce heterozygote dnTGFRII mice and normal
B6 offspring. To maintain the colony, genotyping is performed at
4 weeks of age by purifying DNA collected from ear punches.
dnTGFRII mice are fed a sterile rodent helicobacter diet and sterile
drinking water including sulfratrim, and are maintained individu-
ally in ventilated cages under specific pathogen-free conditions.
dnTGFRII mice exhibit major serologic and histological
characteristics of human PBC suggesting that the dnTGFRII
pathway is important in the pathogenesis of PBC (21, 28). They are
100 % AMA positive with autoantibodies directed against PDC-E2,
BCOADC-E2, and OGDC-E2, the major mitochondrial autoanti-
gens in human PBC. The liver and serum cytokine levels reflect a
Th1 profile. The liver histology of dnTGFRII mice manifests
lymphoid cell infiltration in the portal tracts of 100 % of the mice
including CD4, CD8, and CD19 positive cells as seen in human
PBC. This is accompanied by bile duct injury in 2550 % of mice up
to 22 weeks of age, which is also seen in human PBC (28).
Similar to patients with PBC, there is an elevated level of CD8/
CD4 T cells in the livers of dnTGFRII mice (21). To understand
the role of CD4+ and CD8+ T cells in liver pathology in these
mice, we performed adoptive transfer studies by transferring dnT-
GFRII mice-derived splenic CD4+ and/or CD8+ T cells derived
into Rag1/ recipients. Rag1/ recipients of dnTGFRII mice
unfractionated splenocytes developed features of liver disease
similar to human PBC, suggesting that splenic T and B cell loss of
tolerance causes autoimmune cholangitis. Furthermore, the transfer
of dnTGFRII-derived CD8+ T cells into Rag1/ recipients
resulted in liver-specific autoimmunity, whereas CD4+ T cell transfer
led to colitis, indicating that CD8+ T cells are the primary contribu-
tors for bile duct destruction in this model (28).
Although the presence of high titers of AMAs is present in
95 % of patients with PBC, there is no direct correlation between
AMAs and pathogenesis (29, 30). We have investigated the role of
AMAs in disease pathology in the dnTGFRII mice model of PBC.
Briefly, dnTGFRII mice were crossed with B cell-deficient mice
(Ig/), and were evaluated for the development of liver
inflammation, as well as the severity of accompanying colitis.
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 297

Surprisingly, Ig/ dnTGFRII mice developed a more severe

cholangitis and colitis compared to dnTGFRII mice, indicating a
suppressive effect of B cells on the inflammatory response in the
dnTGFRII mice (31). To further determine the role of B cells in
tissue pathology in dnTGFRII mice, we examined the effects of
therapeutic B cell depletion. Young (46 weeks) and old (20
22 weeks) dnTGFRII mice were injected intraperitoneal with
anti-mouse CD20 mAb every 2 weeks, and then the disease phe-
notype was compared with that of the control Ab-treated mice
(32). Treatment of young mice demonstrated a fully depleted
serum AMAs, a lower incidence of liver inflammation, and a fewer
number of activated hepatic CD8+ T cells, whereas colon
inflammation was significantly exacerbated. In contrast, anti-CD20
treatment of animals with established disease was ineffective, sug-
gesting that B cells play both a positive and negative regulatory
role in the pathogenesis of PBC.
Previous studies have shown that CD1d expression and the
frequency of CD1d-restricted NKT cells were increased in the
livers of patients with PBC (33). Thus, to examine the role of
CD1d-restricted NKT cells in the pathogenesis of PBC, we generated
CD1d/ dnTGFRII mice and showed that these mice had
decreased mononuclear cell infiltration in the liver and lower INF-
serum levels, which ultimately ameliorated liver injury compared
to that of dnTGFRII mice (34). Data from this work suggests
that CD1d-restricted NKT cells have a primarily proinflammatory
phenotype with a Th1 cytokine bias, and promote deprivation of
TGF signaling.
In addition to INF-, IL-12 has also been implicated in
autoimmune inflammatory diseases. Interestingly, deletion of
IL-12p40 in dnTGFRII mice resulted in lower levels of
inflammatory cytokines, immune infiltrates, and bile duct damage
but does not alter AMA levels, whereas deletion of IFN- has no
effect on autoimmune cholangitis in the dnTGFRII mice (35).
Although, the dnTGFRII mice exhibit features resembling
that seen in PBC, it is also important to note that there are some
differences from human PBC, such as the lack of a female bias,
eosinophilic infiltration, and granuloma formation (36).
Nevertheless, the association with a decrease in peripheral Tregs in
PBC patients (37) and the role of TGF- in immunomodulation
makes the dnTGFRII mice an interesting model for PBC.

2.3. IL-2Ra / Mice IL-2 is critical for the development and peripheral expansion of
CD4+ CD25+ Tregs, which promote self-tolerance by suppressing
T cell responses in vivo (38, 39). Previously, it was reported that a
child with a genetic deficiency of IL-2R developed clinical
manifestations similar to PBC (40). Interestingly, C57BL/6 J
IL-2R/ mice had serological and pathological characteristics
resembling those of chronic nonsuppurative destructive cholangitis,
298 P.S.C. Leung et al.

which is also seen in human PBC. Serologically, IL-2R/ mice

are 100 % PDC-E2 positive and 80 % ANA positive. Their portal
tracts exhibited an abundance of CD4+ and CD8+ infiltrates when
compared with control mice, and yielded a higher percentage of
CD8+ than CD4+ T cells. There is also evidence of decreased
frequency of CD4+ Fox3+ Treg cells in the blood samples of
IL-2R/ mice suggesting that the inflammatory lesions in the
liver are due to a reduction in Tregs (41).
The role of T cells in autoimmune cholangitis is demonstrated
through a series of experiments. This IL-2R/ model showed
exacerbated intrahepatic biliary ductular destruction, but had
diminished colitis in IL-2R/-CD4/ mice, and lacked biliary
pathology in IL-2R/-CD8/ mice (42). These observations are
in agreement with adoptive transfer studies in dnTGFRII mice,
where CD8+, but not CD4+ T cells, were found to be the major
T cell player responsible for bile duct destruction. It should be
noted that IL-2R/ mice exhibit autoimmune cholangitis, con-
comitant inflammatory bowel disease, and that 2550 % of
IL-2R/ mice die from severe hemolytic anemia between 8 and
20 weeks of age.

3. 2-Octynoic
Acid-BSA-
Immunized Mice
Our laboratory has hypothesized that xenobiotic modification of
the native lipoyl moiety of the major mitochondrial autoantigen
PDC-E2, may lead to the loss of self-tolerance in PBC (43). This
thesis is based on the findings of readily detectable levels of immu-
noreactivity of PBC sera against extensive panels of protein microarrays,
which mimics the inner lipoyl domain of PDC-E2 and subsequent
quantitative structureactivity relationships (QSARs) (44). Previous
studies showed that rabbits immunized with one such xenobiotic,
6-bromohexanoate conjugated to bovine serum albumin (BSA),
produced AMAs to PDC-E2, BCOADC-E2, and OGDC-E2, but
without any PBC-like liver pathology (45). In 2008, Wakabayashi
et al. reported that murine immunization with 2-octynoic acid
(2-OA) coupled to BSA induced AMAs and cholangitis. When
using 2-OA-BSA as an immunogen in B6 mice and NOD.1101
(NOD.B6 Idd10 Idd18r2) mice, it led to high titer AMAs, portal
inflammation, and autoimmune cholangitis similar to human PBC
(46). Using this mouse model, we investigated the role of B cells
in PBC by depleting B cells using two different monoclonal anti-
bodies, CD20 and CD79. The results of the experiment revealed
that B cell depletion led to exacerbated cholangitis, with higher
T cell infiltrates and inflammatory cytokines, indicating a protec-
tive role of B cells in PBC (32).
Taking advantage of our experience in this xenobiotic induced
model of PBC, we have investigated the role of innate immunity
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 299

and natural killer T (NKT) cells on modulating disease activity in

this xenobiotic-induced mouse model. Briefly, we immunized mice
with and without the addition of -galactosylceramide (-GalCer),
an invariant NKT cell activator. 2-OA-BSA-immunized mice
exposed to -GalCer developed a profound exacerbation of their
autoimmune cholangitis, including significant increases in CD8+
T cell infiltrates, portal inflammation, granuloma formation, and
bile duct damage. More excitingly, these mice produced increased
levels of AMAs and have evidence of fibrosis, a feature not previ-
ously reported in any other murine models of PBC (47). These
results are critical and emphasize the role of innate immunity in the
natural history of PBC. Furthermore, the data also provides clues
to the mechanisms by which biliary disease becomes perpetuated in
humans as well as explaining the recurrence of PBC following liver
transplantation in the absence of major histocompatibility complex
(MHC) compatibility. Thus, in the absence of MHC restriction,
disease reoccurrence would depend on a nonadaptive cellular
mechanism, i.e., innate immunity, suggesting that biliary epithelial
cells are more than simply an innocent victim of an immune attack.
Rather, they attract immune attack by virtue of the unique biochem-
ical mechanisms by which they process PDC-E2 during apoptosis
(11, 48). Results from these recent works also explain the success
of ursodiol, a drug that appears to have anti-apoptotic properties
and also may modulate innate responses. Our data would also
explain the relative failure of immunosuppressive drugs to alter
PBC, because such agents are ineffective against innate mechanisms.
Finally, the induction of fibrosis in 2-OA-BSA-immunized mice
exposed to -GalCer permits not only dissection of its induction,
but also has the potential to be useful in studies of intervention.

3.1. Novosphingobium N. aromaticivorans is a proteobacterium of the Sphingomonodaceae

aromaticivorans family of gram-negative alphaproteobacteria, and is found commonly
Immunized Mice in soil, water, and coastal plain sediments (49). The distinctive
xenobiotic metabolizing properties and remarkable amino acid
sequence homology between its lipoyl containing proteins and the
human PDC-E2 (50) suggests that exposure to N. aromaticivorans,
perhaps the xenobiotic-modified lipoyl proteins, could lead to a
break in tolerance to PDC-E2 and can trigger PBC (5155).
Interestingly, instead of possessing the common lipopolysac-
charide on their cell walls, N. aromaticivorans express glycosphin-
golipids such as -glycuronosylceramides, which are recognized by
CD1d restricted NKT cells (56, 57). This is important for the
activation of NKT cells and the subsequent release of Th1 and Th2
cytokines and chemokines (58, 59). It has been observed that PBC
patients have an increase of NKT cells and the expression of CD1d,
leading to the hypothesis and subsequent experimental discovery
that PBC-like liver disease can be caused by the infection of mice
with N. aromaticivorans and requires NKT cells (60). Briefly, infection
300 P.S.C. Leung et al.

of mice strains (NOD, C57BL/6, and SJL) with N. aromaticivorans

at 5 107 cfu intravenously at week 0 and week 2 not only induced
signature antibodies against microbial PDC-E2 and its mitochondrial
counterpart, but also triggered chronic T cell-mediated autoim-
munity against small bile ducts. Further work was directed to the
role of NKT cells in autoimmune cholangitis and has focused on
the NOD.1101 mouse strain because it exhibited particularly
severe liver disease similar to PBC. NOD.1101 belongs to a set of
NOD subcongenic strains originating from NOD.c3c4 mice and
was produced by introgressing Idd loci 10 and 18r2 from B6
(chromosome 3) onto an NOD background (19, 6164). In this
model, disease induction requires NKT cells, which specifically
respond to the N. aromaticivorans cell wall, -glycuronosylceramides,
presented by CD1d molecules. Combined with the natural liver
tropism of NKT cells, the accumulation of N. aromaticivorans in
the liver likely explains the liver specificity of destructive responses.
Furthermore, once established, liver disease could be adoptively
transferred by T cells independently of NKT cells and microbes,
illustrating the importance of early microbial activation of NKT cells
in the initiation of autonomous organ-specific autoimmunity (60).

3.2. Summary From these models, it is evident that autoimmune cholangitis

results from multiorchestrated immune responses, and points to
the thesis that loss of tolerance to PDC-E2 (antimitochondrial
response) is secondary to a genetic predisposition and environ-
mental influences of either xenobiotic chemicals or bacteria. This
multilineage loss of tolerance to PDC-E2 would be clinically
insignificant were it not for the unique apotopes found on biliary
cells. Further, the perpetuation of disease, and perhaps the initia-
tion from the asymptomatic serologically positive patients, may be
dependent on the activation of NKT cells. CD8+ memory T cells
play a critical role in many of the models. Finally, the use of -GalCer
promotes the development of hepatic fibrosis, more akin to the
human disease. The availability of these models of murine autoim-
mune cholangitis will allow us opportunities that were not
available before, to dissect the immunological mechanisms under-
lying the initiation stages of PBC and explore new therapeutic
options for patients with PBC.

4. Materials

4.1. Preparation of Luria Broth (USB Corporation, Clevaland, OH).

Recombinant Proteins Ampicillin (Sigma Aldrich, St. Louis, MO).
Isopropyl -D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, St.
Louis, MO).
Phosphate buffered saline (PBS, Sigma Aldrich, St. Louis, MO).
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods
4.2. Resolving
Recombinant PDC-E2
by Polyacrylamine Gel
Electrophoresis
4.3. Western Blot
4.4. Enzyme-Linked
Immunosorbent Assay
4.5. Paraffin Tissue
Preparation
301
Triton X-100 (Sigma Aldrich, St. Louis, MO).
Tween 20 (Sigma Aldrich, St. Louis, MO).
Dithiothreitol (Sigma Aldrich, St. Louis, MO).
Protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO).
Glutathione agarose (Sigma Aldrich, St. Louis, MO).
L-Glutathione, reduced (Sigma Aldrich, St. Louis, MO).
NUPAGE sample buffer (Invitrogen, Carlsbad, CA).
Dithiothreitol (Sigma Aldrich, St. Louis, MO).
BisTris NUPAGE system 10 % gel (Invitrogen, Carlsbad, CA).
MOPs SDS running buffer (Invitrogen, Carlsbad, CA).
Ponceau S (Sigma Aldrich, St. Louis, MO).
Simply Blue Safe Stain (Invitrogen, Carlsbad, CA).
NuPage transfer buffer (Invitrogen, Carlsbad, CA).
Methanol (Fischer Scientific, Pittsburg, PA).
Non-fat dry milk (Fischer Scientific, Pittsburg, PA).
Tris-buffered saline (TBS, Sigma Aldrich, St. Louis, MO).
SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific, Pittsburg, PA).
PBS (Mediatech, Manassas, VA).
Tween 20 (Sigma Aldrich, St. Louis, MO).
Na2CO3 (Sigma Aldrich, St. Louis, MO).
NaHCO3 (Sigma Aldrich, St. Louis, MO).
NaN3 (Sigma Aldrich, St. Louis, MO).
NaCl (Sigma Aldrich, St. Louis, MO).
KCL (Sigma Aldrich, St. Louis, MO).
Na2HPO4 (Sigma Aldrich, St. Louis, MO).
Horseradish peroxidase-conjugated anti-mouse IgG.
BD OptEIA TMB Substrate Reagent Set (BD Biosciences, San
Jose, CA).
Dissecting instruments (forceps, scissors, tweezers, etc.).
10 % buffered formalin (Sigma Aldrich, St. Louis, MO).
4 % paraformaldehyde (PFA) fixative, freshly prepared at 4 C.
Paraffin wax Tissue Prep (Fischer Scientific, Pittsburg, PA).
50, 70, 95, and 100 % ethanol (Sigma Aldrich, St. Louis, MO).
Xylenes (Wako, Osaka, Japan).
Embedding molds (Sakura Finetek, Tokyo, Japan).
302 P.S.C. Leung et al.

4.6. Frozen Tissue OCT embedding compound (Sakura Finetek, Tokyo, Japan).
Preparation 2-Methylbutane (Sigma Aldrich, St. Louis, MO).

4.7. H&E Staining Xylene (Wako, Osaka, Japan).

Meyers hematoxylin solution.
Hematoxylin (Merck, Darmstadt, Germany).
Potassium alum (Sigma Aldrich, St. Louis, MO).
Sodium iodate (Sigma Aldrich, St. Louis, MO).
Chloral hydrate (Sigma Aldrich, St. Louis, MO).
Citric acid (Sigma Aldrich, St. Louis, MO).
Eosin solution.
Eosin Y (Waldeck, Munster, Germany).
95 % Ethanol (Sigma Aldrich, St. Louis, MO).
Acetic acid (Sigma Aldrich, St. Louis, MO).
Permount slide mounting fluid (Daigger, Vernon Hills, IL).

4.8. Immunohisto Antigen retrieval solution (DAKO, Carpinteria, CA).

chemical Staining for Microprocessor-controlled pressure chamber (Pascal: DAKO).
CD4 and CD8 Cells
Rat mAb against mouse CD4 (BioLegend, San Diego, CA).
Rat mAb against mouse CD8 (BioLegend, San Diego, CA).
Polymered secondary antibodies for rat monoclonal antibodies
with peroxidase (Histofine, Nichirei, Tokyo).
DAB/H2O2 solution: Peroxidase substrate kit (Vector,
Burlingame, CA).
Permount slide mounting fluid (Daigger, Vernon Hills, IL).

4.9. Cell Preparation Sterile 60 15 mm polystyrene petri dish (Fisher scientific,

for Flow Cytometry Pittsburgh, PA).
Analysis 100-m Nylon cell strainer (BD Bioscience, Bedford, MA).
70 m Nylon Spectra/Mesh N (Fisher scientific, Pittsburgh, PA).
Disposable polyethylene transfer pipet (Fisher scientific, Pittsburgh,
PA).
Gibco HBSS (Hanks balanced salt solution 1, Invitrogen,
Carlsbad, CA).
PBS (Mediatech, Manassas, VA).
Histopaque-1.077 (Sigma Aldrich, St. Louis, MO).
1.5 ml Natural polypropylene microcentrifuge tube (E&K
Scientific, Santa Llara, CA).
15 ml Nonpyrogenic polypropylene centrifuge tube (Corning
Incorporated, Corning, NY).
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods
4.10. Flow Cytometry
of Liver and Spleen
Cell Populations
4.11. Serum Cytokine
Analysis
4.12. Liver Cytokine
and Chemokine
Analysis
5. Methods
5.1. Preparation of
Recombinant Proteins
303
7 100 mm, 14 ml polystyrene round bottom test tube (BD
Biosciences, Bedford, MA).
14 ml round bottom tube (BD Biosciences, Bedford, MA).
PBS (Sigma Aldrich, St. Louis, MO).
BSA (Sigma Aldrich, St. Louis, MO).
Sodium azide (Sigma Aldrich, St. Louis, MO).
FACS tube (12 75 mm culture tube, VWR North American).
Anti-CD16/32, CD4, CD8, TCR, B220, NK1.1, CD11c,
CD11b, Gr-1 mAbs (Biolegend, San Diego, CA).
Anti-CD44, CD69, CD62L, CD43, IgM, IgD mAbs (BD
Biosciences, Bedford, MA).
Anti-CD19 mAb, CD25, CD1d, CD27 (eBioscience, San Diego,
CA).
Mouse Inflammatory (Containing IL-6, IL-10, MCP-1, IFN-,
TNF, and IL-12p70) and Th1/Th2/Th17 (Containing IL-2,
IL-4, IL-6, IFN-, TNF, IL-17A, IL-10) Cytometric Bead
Array kit (BD Biosciences, San Jose, CA).
Mouse IL-12p40, IL-23p19 ELISA kit (R&D system, Minneapolis,
MN).
Mouse IL-27 (p28//EBI3, IL27) ELISA Kit (eBioscience, San
Diego, CA).
Mouse IL-18 ELISA Kit (MBL International, Woburn, MA).
TNE buffer (1 % NP 40, 10 mM TrisHCl pH 7.5, 150 mM NaCl,
1 mM EDTA) containing protease inhibitor and phosphatase
inhibitor cocktail (Roche, Indianapolis, IN).
BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
Mouse Inflammatory (Containing IL-6, IL-10, MCP-1, IFN-,
TNF, and IL-12p70) and Th1/Th2/Th17 (Containing IL-2,
IL-4, IL-6, IFN-, TNF, IL-17A, IL-10) Cytometric Bead
Array kit (BD Biosciences, San Jose, CA).
1. Inoculate a single colony of an E. coli clone that expresses a
GST fusion protein, human PDC-E2 in pGEX 4T-1, into 5 ml
of LB supplemented with 200 g/ml of ampicillin. Incubate
the cells in a 37 C incubator with shaking for 8 h until the
A 450 is between 0.4 and 0.8.
2. Transfer the 5 ml culture into 500 liter of LB supplemented
with 200 g/ml ampicillin into a 1 liter Erlenmeyer flask and
304 P.S.C. Leung et al.

incubate the cells at 37 C incubator with shaking until the

A450 is between 0.4 and 0.5.
3. Add IPTG (final concentration of 1 mM) to the media and
continue to incubate the cells for 34 h in a 37 C incubator
with shaking.
4. Spin down the cells at 10,000 g for 5 min at 4 C. When the
spinning is complete, decant the supernatant and save the cell
pellet.
5. Wash the pellet with 50 ml of PBS.
6. Spin the cells at 10,000 g for 5 min at 4 C. When the spinning
is complete, decant the supernatant and save the cell pellet.
7. Resuspend the cells in 2 ml of PBS and subject the cells to 34
freeze thaw cycles by freezing them on dry ice and then thawing
them in a 65 C water bath.
8. Lyse the cells with a solution containing 20 ml of PBS, 1 %
Triton X-100, 1 % Tween 20, 1 mM of dithiothreitol, and
protease inhibitor cocktail, followed by sonication.
9. Spin the cell lysate at 10,000 g for 15 min at 4 C.
10. Transfer the supernatant to 2 ml of preswollen glutathione
agarose in a 50 ml conical tube and incubate for 1 h at room
temperature.
11. Centrifuge the agarose-cell lysate mixture at 5,000 g for
5 min at 4 C and remove the supernatant.
12. Wash the agarose with PBS 34 times until the A260 of the
supernatant is close to zero.
13. Elute the fusion protein with a solution containing 5 mM
reduced glutathione in 50 mM TrisHCl, pH 8.0.
14. Measure the concentration of the fusion protein.

5.2. Determination 1. Resolving recombinant PDC-E2 by polyacrylamide gel

of Immunoreactivity electrophoresis
to Mitochondrial (a) Mix 10 g of purified recombinant protein PDC-E2 with
Autoantigens by 50 l of 4 gel loading buffer (Novagen), 20 l of DTT
Western Blotting and (0.5 M stock) and distilled water to a final volume of
ELISA 200 l.
(b) Heat for 5 min in a boiling water bath, and then centrifuge
for 10 s in a microfuge at room temperature.
(c) Load the sample prepared above onto a 10 % gel
(Invitrogen, BisTris NUPAGE system).
(d) Run the gel in MOPs SDS buffer at 100 V constant
voltage for about 1.5 h or until the tracking dye is about
1 mm from the bottom of the gel.
(e) Remove the gel cassette from the gel apparatus and
carefully separate the gel from the plates.
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 305

(f) Transfer the protein onto a nitrocellulose filter.

(g) Stain the nitrocellulose filter with Ponceau S to determine if
the transfer is successful. You should see a single red band.
(h) Using a sharp razor blade, cut the nitrocellulose filter into
3 mm strips such that each strip contains a protein band of
the resolved PDC-E2.
2. Western blot
(a) Cut Whatman filter sheets and nitrocellulose membrane
the same dimensions.
(b) Soak filter paper in NuPage buffer.
(c) Wet the nitrocellulose membrane in methanol.
(d) Hydrate in H2O, and then equilibrate in transfer buffer.
(e) Separate glass plates containing the gel that was electro-
phoresed.
(f) Create a transfer sandwich (order from bottom to top).
Bottom of cassette.
Sponge.
Filter paper.
Gel.
Membrane.
Filter paper.
Sponge.
Top of cassette.
(g) Insert the sandwich cassette into the transfer apparatus.
Ensure to place the cassette in the correct orientation with
the gel side of the cassette facing the () and the mem-
brane towards the (+) electrode.
(h) Transfer for 1 h at 100 mA at 4 C.
(i) When transfer is complete, remove sandwich cassette from
electro-blotter apparatus.
(j) Separate the membrane and for orientation, nick the cor-
ner above MW markers.
(k) Block membrane with 3 % non-fat dry milk in TBS for 1 h
or overnight at 4 C
(l) Incubate the membrane with antibody as follows (after
each step rinse well with three 10 min changes of buffer):
Primary antibody diluted in 3 % milk in TBS for 1 h.
Secondary antibody diluted in 3 % milk in TBS for 1 h.
(m) Develop using Chemiluminescence and immediately
expose to film away from light for 5 min.
306 P.S.C. Leung et al.

1.84 1.29 1.14

Tg+
1.44 Tg-
OD of PDC-E2 by ELISA

OD of OGDC by ELISA

OD of BCKD by ELISA
0.99 0.94

1.04
0.69 0.74
0.64

0.24 0.39 0.54

0.24 0.39 0.54

0.00 0.00 0.00

4-10w 12-16w 22-39w 4-10w 12-16w 22-39w 4-10w 12-16w 22-39w

Fig. 1. Serological Ig Reactivity to PDC-E2, OGDC-E2, or BCOADC-E2. Serum samples from dnTGFRII mice and control B6
littermates were analyzed against recombinant proteins of PDC-E2, OGDC-E2, or BCOADC-E2 by ELISA. Serum samples
were diluted 1/500. Sera from 4- to 10-week-old mice (dnTGFRII n = 11, B6 n = 10), 12- to 16-week-old mice (dnTGFRII
n = 10, B6 n = 9), and 22- to 39-week-old mice (dnTGFRII n = 19, B6 n = 10) were tested. Threshold values are set by
mean + 2 SD of values for B6 controls. p < 0.001. Tg+ dnTGFRII transgenic mice, Tg littermate controls.

3. ELISA
Note: A typical comparison of antibody titer to PDC-E2 in AMA
positive mice versus control mice is shown in Fig. 1.
(a) Prepare coating antigens from stock antigen (recombinant
PDC-E2) such that the final concentration of coating anti-
gens is 10 g/ml in carbonate coating buffer (0.5 M
carbonate bicarbonate buffer, pH 9.6).
(b) To a 96-well plate, add 100 l of coating antigen to each
well and store at 4 C.
(c) Remove coating solution. Wash plate three times with
300 l of 0.05 % PBST (0.15 M PBS, pH 7.2 with 0.05 %
Tween 20) per well. Then wash two times with 300 l
of PBS.
(d) Remove liquid by flicking inverted plate over sink and pat
dry on paper towels.
(e) Block entire plate with 150 l of 3 % skim milk in PBS and
incubate for 1 h at room temperature.
(f) Decant blocking solution from the wells and pat dry on
paper towels.
(g) Add 100 l of serum samples (diluted 1/250 with 3 % milk
in PBS) to plate and incubate for 1 h at room temperature.
Make sure to include sera from nave mice as negative con-
trols and blank wells without sera for background.
(h) Decant serum samples from plate. Wash plate three times
with 300 l of 0.05 % PBST per well. Then wash two times
with 300 l of PBS, and pat dry on paper towels
(i) Add 100 l of horseradish peroxidase-conjugated anti-
mouse IgG (at a predetermined dilution in 3 % milk with
PBS) per well and incubate for 1 h at room temperature.
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 307

(j) Remove solution from plate. Wash plate three times with
300 l of 0.05 % PBST per well. Then wash two times
with 300 l of PBS, and pat dry on paper towels.
(k) Add 100 ml of ELISA developing substrate. (For BD
OptEIA, mix substrate A and B in 1:1 ratio.) Incubate at
room temperature for 10 min.
(l) Stop the reaction by adding 50 l of 2N H2SO4 to plate.
(m) Read the optical density of the plate at 450 nm using a
plate reader.

5.3. Paraffin Tissue
Preparation
1. Excise the liver from the peritoneal cavity immediately after
animal is sacrificed.
2. Using sterile scissors, dissect the liver into approximately
46 mm cubes or other desired portion and immediately fix
the tissues by transferring the liver cubes to 10 % neutral
buffered formalin or 4 % PFA solution.
3. Fix the liver with fixative for 2 days at room temperature.
4. Wash the tissue with 0.1 M PBS several times.
5. Place the sliced tissue in a histology cassette.
6. Dehydrate the tissue through a series of ascending ethanol
percentage, via 70100 % ethanol, xylene, and paraffin
infiltration by automatic tissue processor (Sakura).
1 5 min 70 % ethanol.
1 5 min 100 % ethanol.
5 60 min 100 % ethanol.
3 60 min xylene.
1 420 min paraffin infiltration.
7. Embedded in paraffin using embedding molds and make
paraffin-embedded tissue blocks with histology cassettes.
8. Cut the tissue block on a microtome in 34 m thickness.

5.4. Frozen Tissue 1. Label the plastic tissue mold with proper tissue identification
Preparation and fill partly with OCT embedding compound.
2. Note: If freezing unfixed tissue, remove tissue from animal,
dab on towel to remove any fluid, and immediately place tissue
mold in with OCT and continue with protocol.
3. Note: Choose the best orientation for the tissue to be frozen
in, and let it settle to the bottom. The bottom of the mold is
where the cutting will begin.
4. Add more OCT on top of tissue to cover it completely and fill
the mold.
5. Fill a plastic or metal container half way with 2-methylbutane.
6. Add several small pieces of dry ice and wait a few moments for
the temperature to drop (40 C).
308 P.S.C. Leung et al.

7. Grasp the edge of the mold with forceps and dip into
2-methylbutane.
8. Note: Dip only the most bottom part of the plastic mold. Do
not submerge the mold yet. The OCT will begin to turn white
as it freezes.
9. When all of the OCT is frozen, drop the mold into the cold
2-methylbutane to freeze thoroughly (~5 min).
10. Remove the mold from the freezing liquid, wrap the mold in
foil, and immediately store at 80 C.

5.5. H&E Staining 1. Place paraffin specimens in a slide holder and place the slide
holder into a staining jar.
2. Deparaffinize the specimen via xylene, 10080 % ethanol and
rehydrate with distilled water.
3 3 min xylene (blot excess xylene before going into ethanol).
3 3 min 100 % ethanol.
1 3 min 95 % ethanol.
1 3 min 80 % ethanol.
1 5 min deionized H2O.
3. Blot excess water from slide holder.
4. Apply Meyers hematoxylin solution for 5 min.
5. Rinse with running water, warm water, followed by distilled
water.
6. Blot excess water from slide holder.
7. Apply Eosin solution for 5 min.
8. Dehydrate via ethanol and xylene.
3 5 min 95 % ethanol.
3 5 min 100 % ethanol (blot excess ethanol before going into
xylene).
3 15 min xylene.
9. Place coverslip over the slides by placing a drop of Permount
(xylene based) on the slide using a glass rod, taking care to
leave no bubbles.
10. Angle the coverslip and let it fall gently onto the slide.
11. Allow the Permount to spread beneath the coverslip, covering
all the tissue.
12. Dry overnight in the hood.

5.6. Immunohisto- Note: A histological staining of liver portal tracts with lymphocytic
chemical Staining infiltrations, specifically CD4+ and CD8+, is illustrated in Fig. 2.
for CD4 and CD8 Cells
1. Place paraffin specimens in a slide holder.
2. Place slide holder with paraffin specimens in a 60 C oven
beforehand.
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 309

Fig. 2. Histological features of the 2428 week liver of dnTGFRII mice. (ac) Different degrees of lymphocytic infiltration
(red arrows) surrounding the small bile ducts were detected within the portal tracts (H&E staining); (df) individual cells
(red arrows) of cell types composing the portal tract infiltrates were stained with Abs against cell markers: (d) biliary cells
identified by cytokeratin 7; (e) CD4+ lymphocytes; (f) CD8+ lymphocytes.

3. Deparaffinize the specimen via xylene, 10080 % ethanol and

rehydrate with distilled water.
1 3 min xylene (blot excess xylene before going into ethanol).
1 3 min 100 % ethanol.
1 3 min 95 % ethanol.
1 3 min 80 % ethanol.
1 5 min deionized H2O.
4. Place slide holder with specimens into a plastic jar with antigen
retrieval solution (DAKO).
5. Place plastic jar on microprocessor-controlled pressure
chamber (Pascal: DAKO) for target/epitope retrieval in tissue
sections and heat at 120 C for 10 min.
6. Rinse with running water, followed by distilled water.
7. Apply 5 % BSA in TBS for 5 min for blocking of nonspecific
reaction.
8. Place specimens on a moist plastic chamber.
9. Apply appropriately diluted primary antibodies (CD4 and
CD8) with 1 % BSA in TBS for 1 h or longer.
10. Place specimens in a slide holder. Rinse with TBS-T with shaking
(5 1 min).
11. Apply 100 % methanol with 3 % H2O2 for 5 min to block endo-
geneous peroxidase reaction.
310 P.S.C. Leung et al.

12. Rinse with running water, distilled water, and TBS-Tween.

13. Place specimens on a moist plastic chamber.
14. Apply ready-to-use polymered secondary antibodies for rat
monoclonal antibodies with peroxidase (Histofine, Nichirei,
Tokyo) for 1 h or longer.
15. Place specimens in a slide holder. Rinse with TBS with shaking
(5 1 min).
16. Place specimens on a moist plastic chamber.
17. Apply DAB/H2O2 solution (Vector) for 510 min.
18. Place specimens in a slide holder. Rinse with TBS and running
water.
19. Apply hematoxylin for counter staining for 30 s.
20. Rinse with running water and distilled water.
21. Dehydrate via ethanol and xylene.
3 5 min 95 % ethanol.
3 5 min 100 % ethanol (blot excess ethanol before going into
xylene).
3 15 min xylene.
22. Place coverslip over the slides by placing a drop of Permount
(xylene based) on the slide using a glass rod, taking care to
leave no bubbles.
23. Angle the coverslip and let it fall gently onto the slide.
24. Allow the Permount to spread beneath the coverslip, covering
all of the tissue.
25. Dry overnight in the hood.

5.7. Cell Preparation
for Flow Cytometry
Analysis
1. Asphyxiate a mouse by CO2 overdose, and then dip the entire
mouse in a beaker containing 70 % ethanol.
2. In a sterile hood, carefully cut the skin of the mouse with sterile
instruments. Make an incision through the peritoneal layer and
remove the whole spleen (and other lymphoid organs if need).
3. Place the entire spleen in a 60 mm petri dish containing 5 ml
of 0.2 % BSA-PBS.
4. Transfer the spleen into a 100-m nylon cell strainer. Use the
plunger end of the syringe to gently mash and press the spleen
through the cell strainer into the petri dish.
5. Pipet up and down several times to separate the cells to the
single cell suspension using a 1 ml transfer pipette.
6. Filter the suspended cells through a nylon mesh mounted on a
15 ml conical tube in order to remove the debris and other
connective tissues.
7. Pellet the cells at 1,500 rpm (or 514 g) for 5 min in a table
top centrifuge. Discard the supernatant and then resuspend
the cells in 8 ml HBSS (0.2 % BSA).
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 311

a Liver b Spleen
Tg+ Tg- Tg+ Tg-

59.5% 34.8% 39.2% 30.7%

CD3
CD3

c FSC d FSC

23.0 44.7 30.2 47.4

CD4
CD4

34.1 22.5 59.4 35.3

CD8 CD8

Fig. 3. Flow cytometry analysis of lymphoid cells from liver and spleen of dnTGFRII transgenic mice (Tg+) and littermate
controls (Tg). Lymphoid cells isolated from liver and spleen of dnTGFRII mice were stained with fluorochrome-conjugated
mAbs and analyzed by flow cytometry. (a, b) Frequency of CD3+ lymphocytes in total lymphocyte population. (c, d)
The percentage of CD4+ and CD8+ cells in total CD3+ population.

8. Gently overlay the single cell suspension on top of 3 ml

Histopaque-1.077 density gradient and centrifuge at 2,000 rpm
(or 750 g) for 20 min.
9. Carefully collect the mononuclear cells from the interface with
a pipette, wash with PBS (2,500 rpm, 10 min) (or 1,430 g for
10 min.), and then resuspend in 10 ml PBS.
10. Determine the cell number.

5.8. Flow Cytometry
of Liver and Spleen
Cell Populations
Note: A representative analysis of the liver and spleen CD3+, CD4+,
and CD8+ cells are shown in Fig. 3.
1. Aliquot 106 cells from liver or spleen samples into a 1.5 ml
Eppendorf tube.
2. Centrifuge at 6,000 rpm (or 3,300 g) for 2 min.
3. Aspirate supernatant.
4. Add 25 l of staining buffer (PBS containing 0.2 % BSA and
0.1 % sodium azide) that includes 0.5 l of the blocking Ab
(anti-CD16/32 Ab, Biolegend) to each tube.
5. Vortex the tube to resuspend cells.
6. Incubate at 4 for 10 min.
7. Add 25 l of staining buffer that includes Ab to each tube and
vortex the tube.
312 P.S.C. Leung et al.

8. Incubate at 4 C for 30 min.

9. Add 1 ml of PBS and centrifuge at 6,000 rpm (or 3,300 g) for
2 min.
10. Aspirate supernatant and resuspend cells in 400 l of PBS.
11. Transfer cells into a 5 ml FACS tube (12 75 mm culture tube).
12. Run FACS.

5.9. Serum Cytokine 1. Warm the mouse with heating lamp for about 10 min.
Analysis 2. Prepare cotton soaked with alcohol, a razor, and tubes in the
mean time.
3. Place a mouse in a restraining tube. Apply alcohol cotton to
the ventral side of the tail to clean.
4. After visualizing the ventral artery, use a razor to make a shallow
incision on the ventral side of the tail from the tip of the tail
(about 23 cm from the tip of the tail).
5. Collect the blood in an Eppendorf tube as drops appear. About ten
drops will be sufficient for serum AMA and cytokine detection.
6. Apply pressure to stop the bleeding.
7. Keep the collected blood for 34 h at room temperature.
8. Centrifuge at 10,000 rpm (or 9,200 g) for 5 min.
9. Collect serum in a new Eppendorf tube.
10. Measure cytokine by ELISA kit following the manufacturers
directions.

5.10. Liver Cytokine and 1. Sacrifice mice by CO2 overdose in a CO2 chamber.
Chemokine Analysis 2. Cut open the abdominal cavity and remove the liver and place
in a 60 mm culture dish.
3. Make TNE buffer and keep the buffer on ice prior use.
4. Cut 100200 mg liver tissue off and put the liver tissue into a
14 ml round bottom tube, keep the sample on ice.
5. Add 500 l of TNE buffer into the tube.
6. Homogenize tissue on ice using a homogenizer.
7. Clean, rinse, and dry the probe after homogenizing each sample.
8. Centrifuged sample at 12,000 rpm (or 13,300 g) for 20 min
at 4 C.
9. Collect the supernatant in a new tube for protein concentra-
tion assay (store protein at 80 C if not use).
10. Measure protein concentration using BCA protein assay kit
following the manufacturers directions.
11. Use 50100 mg of total protein to determine the cytokine
expression levels by an inflammatory cytokine and Th1/Th2/
Th17 CBA kit, or an ELISA kit according to the instructions
provided by the manufacturers.
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods
References
1. Gershwin ME, Ansari AA, Mackay IR,
Nakanuma Y, Nishio A, Rowley MJ, Coppel
RL (2000) Primary biliary cirrhosis: an orches-
trated immune response against epithelial cells.
Immunol Rev 174:210225
2. Leung PS, Chuang DT, Wynn RM, Cha S,
Danner DJ, Ansari A, Coppel RL, Gershwin
ME (1995) Autoantibodies to BCOADC-E2
in patients with primary biliary cirrhosis recog-
nize a conformational epitope. Hepatology
22:505513
3. Moteki S, Leung PS, Dickson ER, Van Thiel
DH, Galperin C, Buch T, Alarcon-Segovia D,
Kershenobich D, Kawano K, Coppel RL et al
(1996) Epitope mapping and reactivity of autoan-
tibodies to the E2 component of 2-oxoglutarate
dehydrogenase complex in primary biliary cir-
rhosis using recombinant 2-oxoglutarate dehy-
drogenase complex. Hepatology 23:436444
4. Van de Water J, Ansari A, Prindiville T, Coppel
RL, Ricalton N, Kotzin BL, Liu S, Roche TE,
Krams SM, Munoz S, Gershwin ME (1995)
Heterogeneity of autoreactive T cell clones
specific for the E2 component of the pyruvate
dehydrogenase complex in primary biliary cir-
rhosis. J Exp Med 181:723733
5. Ishibashi H, Shimoda S, Gershwin ME (2005)
The immune response to mitochondrial autoan-
tigens. Semin Liver Dis 25:337346
6. Chuang YH, Ridgway WM, Ueno Y, Gershwin
ME (2008) Animal models of primary biliary
cirrhosis. Clin Liver Dis 12:333347, ix
7. Folci M, Meda F, Gershwin ME, Selmi C
(2011) Cutting-edge issues in primary biliary
cirrhosis. Clin Rev Allergy Immunol DOI
10.1007/s12016-011-82533
8. Gershwin ME, Mackay IR (2008) The causes
of primary biliary cirrhosis: convenient and
inconvenient truths. Hepatology 47:737745
9. Leung PS, Coppel RL, Gershwin ME (2005)
Etiology of primary biliary cirrhosis: the search
for the culprit. Semin Liver Dis 25:327336
10. Liu X, Invernizzi P, Lu Y, Kosoy R, Bianchi I,
Podda M, Xu C, Xie G, Macciardi F, Selmi C,
Lupoli S, Shigeta R, Ransom M, Lleo A, Lee
AT, Mason AL, Myers RP, Peltekian KM,
Ghent CN, Bernuzzi F, Zuin M, Rosina F,
Borghesio E, Floreani A, Lazzari R, Niro G,
Andriulli A, Muratori L, Muratori P, Almasio
PL, Andreone P, Margotti M, Brunetto M,
Coco B, Alvaro D, Bragazzi MC, Marra F,
Pisano A, Rigamonti C, Colombo M, Marzioni
M, Benedetti A, Fabris L, Strazzabosco M,
Portincasa P, Palmieri VO, Tiribelli C, Croce L,
Bruno S, Rossi S, Vinci M, Prisco C, Mattalia
A, Toniutto P, Picciotto A, Galli A, Ferrari C,
313
Colombo S, Casella G, Morini L, Caporaso N,
Colli A, Spinzi G, Montanari R, Gregersen PK,
Heathcote EJ, Hirschfield GM, Siminovitch
KA, Amos CI, Gershwin ME, Seldin MF
(2010) Genome-wide meta-analyses identify
three loci associated with primary biliary cir-
rhosis. Nat Genet 42:658660
11. Lleo A, Bowlus CL, Yang GX, Invernizzi P,
Podda M, Van de Water J, Ansari AA, Coppel
RL, Worman HJ, Gores GJ, Gershwin ME
(2010) Biliary apotopes and anti-mitochondrial
antibodies activate innate immune responses in
primary biliary cirrhosis. Hepatology
52:987998
12. Oertelt S, Rieger R, Selmi C, Invernizzi P,
Ansari AA, Coppel RL, Podda M, Leung PS,
Gershwin ME (2007) A sensitive bead assay for
antimitochondrial antibodies: chipping away at
AMA-negative primary biliary cirrhosis.
Hepatology 45:659665
13. Kikutani H, Makino S (1992) The murine
autoimmune diabetes model: NOD and related
strains. Adv Immunol 51:285322
14. Chen YG, Scheuplein F, Osborne MA, Tsaih
SW, Chapman HD, Serreze DV (2008)
Idd9/11 genetic locus regulates diabetogenic
activity of CD4 T-cells in nonobese diabetic
(NOD) mice. Diabetes 57:32733280
15. Fox CJ, Paterson AD, Mortin-Toth SM,
Danska JS (2000) Two genetic loci regulate T
cell-dependent islet inflammation and drive
autoimmune diabetes pathogenesis. Am J Hum
Genet 67:6781
16. Fraser HI, Dendrou CA, Healy B, Rainbow
DB, Howlett S, Smink LJ, Gregory S, Steward
CA, Todd JA, Peterson LB, Wicker LS (2010)
Nonobese diabetic congenic strain analysis of
autoimmune diabetes reveals genetic complex-
ity of the Idd18 locus and identifies Vav3 as a
candidate gene. J Immunol 184:50755084
17. Aoki CA, Borchers AT, Ridgway WM, Keen
CL, Ansari AA, Gershwin ME (2005) NOD
mice and autoimmunity. Autoimmun Rev
4:373379
18. Koarada S, Wu Y, Fertig N, Sass DA, Nalesnik
M, Todd JA, Lyons PA, Fenyk-Melody J,
Rainbow DB, Wicker LS, Peterson LB, Ridgway
WM (2004) Genetic control of autoimmunity:
protection from diabetes, but spontaneous
autoimmune biliary disease in a nonobese dia-
betic congenic strain. J Immunol 173:
23152323
19. Irie J, Wu Y, Wicker LS, Rainbow D, Nalesnik
MA, Hirsch R, Peterson LB, Leung PS, Cheng
C, Mackay IR, Gershwin ME, Ridgway WM
(2006) NOD.c3c4 congenic mice develop
314 P.S.C. Leung et al.
autoimmune biliary disease that serologically
and pathogenetically models human primary
biliary cirrhosis. J Exp Med 203:12091219
20. Yang GX, Wu Y, Tsukamoto H, Leung PS,
Lian ZX, Rainbow DB, Hunter KM, Morris
GA, Lyons PA, Peterson LB, Wicker LS,
Gershwin ME, Ridgway WM (2011) CD8 T
cells mediate direct biliary ductule damage in
nonobese diabetic autoimmune biliary disease.
J Immunol 186:12591267
21. Oertelt S, Lian ZX, Cheng CM, Chuang YH,
Padgett KA, He XS, Ridgway WM, Ansari AA,
Coppel RL, Li MO, Flavell RA, Kronenberg
M, Mackay IR, Gershwin ME (2006) Anti-
mitochondrial antibodies and primary biliary
cirrhosis in TGF-beta receptor II dominant-
negative mice. J Immunol 177:16551660
22. Gorelik L, Flavell RA (2000) Abrogation of
TGFbeta signaling in T cells leads to spontane-
ous T cell differentiation and autoimmune dis-
ease. Immunity 12:171181
23. Taylor AW (2009) Review of the activation of
TGF-beta in immunity. J Leukoc Biol
85:2933
24. Yoshimura A, Wakabayashi Y, Mori T (2010)
Cellular and molecular basis for the regulation
of inflammation by TGF-beta. J Biochem
147:781792
25. Ebert EC, Panja A, Das KM, Praveen R, Geng
X, Rezac C, Bajpai M (2009) Patients with
inflammatory bowel disease may have a trans-
forming growth factor-beta-, interleukin (IL)-
2- or IL-10-deficient state induced by intrinsic
neutralizing antibodies. Clin Exp Immunol
155:6571
26. Kel JM, Girard-Madoux MJ, Reizis B, Clausen
BE (2010) TGF-beta is required to maintain
the pool of immature Langerhans cells in the
epidermis. J Immunol 185:32483255
27. Perruche S, Zhang P, Maruyama T, Bluestone
JA, Saas P, Chen W (2009) Lethal effect of
CD3-specific antibody in mice deficient in
TGF-beta1 by uncontrolled flu-like syndrome.
J Immunol 183:953961
28. Yang GX, Lian ZX, Chuang YH, Moritoki Y,
Lan RY, Wakabayashi K, Ansari AA, Flavell RA,
Ridgway WM, Coppel RL, Tsuneyama K,
Mackay IR, Gershwin ME (2008) Adoptive
transfer of CD8(+) T cells from transforming
growth factor beta receptor type II (dominant
negative form) induces autoimmune cholangi-
tis in mice. Hepatology 47:19741982
29. Invernizzi P, Crosignani A, Battezzati PM,
Covini G, De Valle G, Larghi A, Zuin M, Podda
M (1997) Comparison of the clinical features
and clinical course of antimitochondrial anti-
body-positive and -negative primary biliary cir-
rhosis. Hepatology 25:10901095
30. Kim WR, Poterucha JJ, Jorgensen RA, Batts
KP, Homburger HA, Dickson ER, Krom RA,
Wiesner RH, Lindor KD (1997) Does antimi-
tochondrial antibody status affect response to
treatment in patients with primary biliary cir-
rhosis? Outcomes of ursodeoxycholic acid ther-
apy and liver transplantation. Hepatology
26:2226
31. Moritoki Y, Zhang W, Tsuneyama K, Yoshida
K, Wakabayashi K, Yang GX, Bowlus C,
Ridgway WM, Ueno Y, Ansari AA, Coppel RL,
Mackay IR, Flavell RA, Gershwin ME, Lian ZX
(2009) B cells suppress the inflammatory
response in a mouse model of primary biliary
cirrhosis. Gastroenterology 136:10371047
32. Dhirapong A, Lleo A, Yang GX, Tsuneyama K,
Dunn R, Kehry M, Packard TA, Cambier JC,
Liu FT, Lindor K, Coppel RL, Ansari AA,
Gershwin ME (2011) B cell depletion therapy
exacerbates murine primary biliary cirrhosis.
Hepatology 53:527535
33. Chuang YH, Lian ZX, Tsuneyama K, Chiang
BL, Ansari AA, Coppel RL, Gershwin ME
(2006) Increased killing activity and decreased
cytokine production in NK cells in patients
with primary biliary cirrhosis. J Autoimmun
26:232240
34. Chuang YH, Lian ZX, Yang GX, Shu SA,
Moritoki Y, Ridgway WM, Ansari AA,
Kronenberg M, Flavell RA, Gao B, Gershwin
ME (2008) Natural killer T cells exacerbate
liver injury in a transforming growth factor
beta receptor II dominant-negative mouse
model of primary biliary cirrhosis. Hepatology
47:571580
35. Yoshida K, Yang GX, Zhang W, Tsuda M,
Tsuneyama K, Moritoki Y, Ansari AA, Okazaki
K, Lian ZX, Coppel RL, Mackay IR, Gershwin
ME (2009) Deletion of interleukin-12p40 sup-
presses autoimmune cholangitis in dominant
negative transforming growth factor beta
receptor type II mice. Hepatology 50:
14941500
36. Nakamura A, Yamazaki K, Suzuki K, Sato S
(1997) Increased portal tract infiltration of
mast cells and eosinophils in primary biliary cir-
rhosis. Am J Gastroenterol 92:22452249
37. Lan RY, Cheng C, Lian ZX, Tsuneyama K,
Yang GX, Moritoki Y, Chuang YH, Nakamura
T, Saito S, Shimoda S, Tanaka A, Bowlus CL,
Takano Y, Ansari AA, Coppel RL, Gershwin
ME (2006) Liver-targeted and peripheral blood
alterations of regulatory T cells in primary bil-
iary cirrhosis. Hepatology 43:729737
38. Buckner JH (2010) Mechanisms of impaired
regulation by CD4(+)CD25(+)FOXP3(+) reg-
ulatory T cells in human autoimmune diseases.
Nat Rev Immunol 10:849859
 14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods
39. Malek TR, Castro I (2010) Interleukin-2
receptor signaling: at the interface between tol-
erance and immunity. Immunity 33:153165
40. Aoki CA, Roifman CM, Lian ZX, Bowlus CL,
Norman GL, Shoenfeld Y, Mackay IR,
Gershwin ME (2006) IL-2 receptor alpha
deficiency and features of primary biliary cir-
rhosis. J Autoimmun 27:5053
41. Wakabayashi K, Lian ZX, Moritoki Y, Lan RY,
Tsuneyama K, Chuang YH, Yang GX, Ridgway
W, Ueno Y, Ansari AA, Coppel RL, Mackay IR,
Gershwin ME (2006) IL-2 receptor alpha(/)
mice and the development of primary biliary
cirrhosis. Hepatology 44:12401249
42. Hsu W, Zhang W, Tsuneyama K, Moritoki Y,
Ridgway WM, Ansari AA, Coppel RL, Lian
ZX, Mackay I, Gershwin ME (2009) Differential
mechanisms in the pathogenesis of autoim-
mune cholangitis versus inflammatory bowel
disease in interleukin-2Ralpha(/) mice.
Hepatology 49:133140
43. Long SA, Quan C, Van de Water J, Nantz MH,
Kurth MJ, Barsky D, Colvin ME, Lam KS,
Coppel RL, Ansari A, Gershwin ME (2001)
Immunoreactivity of organic mimeotopes of
the E2 component of pyruvate dehydrogenase:
connecting xenobiotics with primary biliary
cirrhosis. J Immunol 167:29562963
44. Rieger R, Leung PS, Jeddeloh MR, Kurth MJ,
Nantz MH, Lam KS, Barsky D, Ansari AA,
Coppel RL, Mackay IR, Gershwin ME (2006)
Identification of 2-nonynoic acid, a cosmetic
component, as a potential trigger of primary
biliary cirrhosis. J Autoimmun 27:716
45. Leung PS, Quan C, Park O, Van de Water J,
Kurth MJ, Nantz MH, Ansari AA, Coppel
RL, Lam KS, Gershwin ME (2003)
Immunization with a xenobiotic 6-bromo-
hexanoate bovine serum albumin conjugate
induces antimitochondrial antibodies. J
Immunol 170:53265332
46. Wakabayashi K, Lian ZX, Leung PS, Moritoki
Y, Tsuneyama K, Kurth MJ, Lam KS, Yoshida
K, Yang GX, Hibi T, Ansari AA, Ridgway WM,
Coppel RL, Mackay IR, Gershwin ME (2008)
Loss of tolerance in C57BL/6 mice to the
autoantigen E2 subunit of pyruvate dehydro-
genase by a xenobiotic with ensuing biliary
ductular disease. Hepatology 48:531540
47. Wu SJ, Yang YH, Tsuneyama K, Leung PS,
Illarionov P, Gershwin ME, Chuang YH (2011)
Innate immunity and primary biliary cirrhosis:
Activated invariant natural killer T cells exacer-
bate murine autoimmune cholangitis and
fibrosis. Hepatology 53:915925
48. Lleo A, Invernizzi P, Gao B, Podda M,
Gershwin ME (2009) Definition of human
315
autoimmunityautoantibodies versus autoim-
mune disease. Autoimmun Rev 9:A259A266
49. Takeuchi M, Hamana K, Hiraishi A (2001)
Proposal of the genus Sphingomonas sensu
stricto and three new genera, Sphingobium,
Novosphingobium and Sphingopyxis, on the
basis of phylogenetic and chemotaxonomic
analyses. Int J Syst Evol Microbiol
51:14051417
50. Padgett KA, Selmi C, Kenny TP, Leung PS,
Balkwill DL, Ansari AA, Coppel RL, Gershwin
ME (2005) Phylogenetic and immunological
definition of four lipoylated proteins from
Novosphingobium aromaticivorans, implica-
tions for primary biliary cirrhosis. J Autoimmun
24:209219
51. Brodie EL, DeSantis TZ, Parker JP, Zubietta
IX, Piceno YM, Andersen GL (2007) Urban
aerosols harbor diverse and dynamic bacterial
populations. Proc Natl Acad Sci U S A
104:299304
52. Cavicchioli R, Fegatella F, Ostrowski M, Eguchi
M, Gottschal J (1999) Sphingomonads from
marine environments. J Ind Microbiol
Biotechnol 23:268272
53. Kaplan MM (2004) Novosphingobium aromati-
civorans: a potential initiator of primary biliary
cirrhosis. Am J Gastroenterol 99:21472149
54. Olafsson S, Gudjonsson H, Selmi C, Amano K,
Invernizzi P, Podda M, Gershwin ME (2004)
Antimitochondrial antibodies and reactivity to
N. aromaticivorans proteins in Icelandic
patients with primary biliary cirrhosis and their
relatives. Am J Gastroenterol 99:21432146
55. Selmi C, Balkwill DL, Invernizzi P, Ansari AA,
Coppel RL, Podda M, Leung PS, Kenny TP,
Van De Water J, Nantz MH, Kurth MJ,
Gershwin ME (2003) Patients with primary
biliary cirrhosis react against a ubiquitous xen-
obiotic-metabolizing bacterium. Hepatology
38:12501257
56. Kawahara K, Moll H, Knirel YA, Seydel U,
Zahringer U (2000) Structural analysis of two
glycosphingolipids from the lipopolysaccha-
ride-lacking bacterium Sphingomonas capsu-
lata. Eur J Biochem 267:18371846
57. Kosako Y, Yabuuchi E, Naka T, Fujiwara N,
Kobayashi K (2000) Proposal of
Sphingomonadaceae fam. nov., consisting of
Sphingomonas Yabuuchi et al. 1990,
Erythrobacter Shiba and Shimidu 1982,
Erythromicrobium Yurkov et al. 1994,
Porphyrobacter Fuerst et al. 1993, Zymomonas
Kluyver and van Niel 1936, and Sandaracinobacter
Yurkov et al. 1997, with the type genus
Sphingomonas Yabuuchi et al. 1990. Microbiol
Immunol 44:563575
316 P.S.C. Leung et al.
58. Kinjo Y, Wu D, Kim G, Xing GW, Poles MA,
Ho DD, Tsuji M, Kawahara K, Wong CH,
Kronenberg M (2005) Recognition of bacterial
glycosphingolipids by natural killer T cells.
Nature 434:520525
59. Mattner J, Debord KL, Ismail N, Goff RD,
Cantu C 3rd, Zhou D, Saint-Mezard P, Wang
V, Gao Y, Yin N, Hoebe K, Schneewind O,
Walker D, Beutler B, Teyton L, Savage PB,
Bendelac A (2005) Exogenous and endogenous
glycolipid antigens activate NKT cells during
microbial infections. Nature 434:525529
60. Mattner J, Savage PB, Leung P, Oertelt SS,
Wang V, Trivedi O, Scanlon ST, Pendem K,
Teyton L, Hart J, Ridgway WM, Wicker LS,
Gershwin ME, Bendelac A (2008) Liver auto-
immunity triggered by microbial activation of
natural killer T cells. Cell Host Microbe
3:304315
61. Lyons PA, Hancock WW, Denny P, Lord CJ,
Hill NJ, Armitage N, Siegmund T, Todd JA,
Phillips MS, Hess JF, Chen SL, Fischer PA,
Peterson LB, Wicker LS (2000) The NOD
Idd9 genetic interval influences the pathoge-
nicity of insulitis and contains molecular vari-
ants of Cd30, Tnfr2, and Cd137. Immunity
13:107115
62. Wicker LS, Leiter EH, Todd JA, Renjilian RJ,
Peterson E, Fischer PA, Podolin PL, Zijlstra
M, Jaenisch R, Peterson LB (1994) Beta
2-microglobulin-deficient NOD mice do not
develop insulitis or diabetes. Diabetes
43:500504
63. Koarada S, Wu Y, Yim YS, Wakeland EW,
Ridgway WM (2004) Nonobese diabetic CD4
lymphocytosis maps outside the MHC locus
on chromosome 17. Immunogenetics
56:333337
64. Podolin PL, Denny P, Armitage N, Lord CJ,
Hill NJ, Levy ER, Peterson LB, Todd JA,
Wicker LS, Lyons PA (1998) Localization of
two insulin-dependent diabetes (Idd) genes to
the Idd10 region on mouse chromosome 3.
Mamm Genome 9:283286
 Chapter 15

Modeling Innate Immunity in Murine Skin: Utilization

of Subcutaneous Osmotic Pumps for Inflammatory
and Fibrotic Skin Disease
Michael Dimarzio, Giuseppina Farina, and Robert Lafyatis

Abstract
Current models for systemic sclerosis have many limitations for mimicking human disease. We have recently
described a novel model based on chronic stimulation of skin by poly(I:C), a form of double-stranded
RNA known to stimulate innate immune responses. This model shows inflammation and extracellular
matrix deposition, and upregulated expression of genes associated with fibrosis and vascular injury. We
detail here the methodology for this model, utilizing osmotic pumps to deliver innate immune stimuli to
subcutaneous tissue. Subcutaneous osmotic pumps provide a flexible system for studying innate immunity
in the skin as well as the affects of other ligands on skin inflammation and fibrosis.

Key words: Skin, Innate immunity, Osmotic pump, Toll-like receptor, Systemic sclerosis, Scleroderma

1. Introduction

1.1. Subcutaneous Investigating the effect of biologically active ligands in murine

Osmotic Pumps and
Utility in Modeling
Skin Disease
tissues is a common goal in understanding many aspects of disease
pathogenesis. Transgenic mice have provided major insights into
normal and pathological functions of protein ligands, such as
cytokines and other proteins involved in cellcell signaling in vivo.
A major advantage of transgenic approaches is that the effects of
extended relatively stable protein expression can be assessed.
Limitations of transgenic mice include the time and expense
required to create the mice, the widespread expression of the
transgenic protein, the expression of the protein throughout devel-
opment potentially confusing effects of mediators active only during
adult disease, and the time and expense of backcrossing to an
appropriate strain that might be required for a given disease model.

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_15, Springer Science+Business Media New York 2012

317
318 M. Dimarzio et al.

These limitations can generally be addressed using tissue-specific

and/or inducible promoters, and investigator persistence. However,
and particularly important for this discussion, only gene products,
i.e., RNAs and proteins can be overexpressed.
Osmotic pumps provide a vehicle for continuous delivery of
ligands systemically or, when placed subcutaneously, to the skin.
These pumps, known as Alzet osmotic pumps and manufactured
by Durect Corporation, have no moving part, rather pump fluid
instilled into the pump interior by passive absorption of surround-
ing interstitial fluids (1). The pumps are designed to deliver the
fluid continuously and steadily over a period of time between
3 days and 6 weeks. Thus, these pumps allow delivery of virtually
any water-soluble ligand, biological or nonbiological provided that
the ligand is stable at body temperature (see Note 1). The advantage
of this approach is that no special mouse breeding is required as the
pumps can be placed in any strain, and innate immune stimuli, in
addition to protein ligands, can be studied.

1.2. Modeling Innate Skin in patients with systemic sclerosis (SSc) is characterized by
Immune-Mediated collagen deposition and varying degrees of inflammation. Poly(I:C)
Inflammation and is a synthetic double-stranded RNA that acts as a ligand for toll-like
Fibrosis receptor 3 (TLR3), but is also a cytosolic sensor, melanoma differ-
entiation-associated protein-5 (MDA5) (2). Poly(I:C) is a particularly
potent stimulus of innate immune responses by dermal fibroblasts,
upregulating both pro-inflammatory and pro-fibrotic gene expres-
sion (3, 4). We have recently described utilization of osmotic pump
delivery of poly(I:C) as a model for understanding the effect of
chronic innate immune activation in the skin. We have shown using
this model that poly(I:C) delivered subcutaneously over 7 days
induces expression of both interferon- and transforming growth
factor--regulated genes (3, 4). This pattern of gene expression is
similar to what we have seen in the skin of patients with SSc,
suggesting that this model reproduces some of the relevant patho-
logical features of this disease. In addition, if 28-day osmotic pumps
are used matrix deposition, fat loss, and myofibroblast accumula-
tion is seen, thus reproducing all the typical features of SSc skin.
Although we will be focusing on the methodology used for the
poly(I:C) model of SSc, we have used the same basic approach
with other ligands, in particular TGF, but also in ongoing work
using other protein and nucleic acid ligands (ref. 4 and R. Lafyatis
unpublished).

1.3. Summary The simplicity of this system is its major advantage. However, careful
of Methodology attention to sterility and consideration of outcomes to be assessed
(histology, immunofluorescence, and gene expression) are key
features to success. The goal of this chapter is to describe how
specific placement of poly(I:C) in osmotic pumps can be employed
 15 Modeling Innate Immunity in Murine Skin 319

to study innate immune responses. However, the flexibility of this

system allows its ready application to a broad array of other media-
tors. The first two Subheadings (3.1 and 3.2) thus discuss specifically
how osmotic pumps are loaded and placed. However, as important
as pump placement is analysis of skin. Technical aspects of mouse
skin preparation for RT-PCR, histology, and immunoflorescence
are considered in Subheadings 3.3 and 3.4.

2. Materials

For pump placement:

Syringes: 3 ml, and individually packaged sterile 1 ml.
Needles: 25 gauge, 1/2 length.
HMW Poly(I:C) (Invivogen).
Osmotic pumps (Alzet): Alzet is the only supplier of these pumps
and provides them in a variety of capacities and pumping rates.
For young adult mice: model 2001 (1 l/h for 7 days).
100 mm diameter sterile Petri dishes.
Sterile gloves.
Kitchen scale.
Surgical instruments: scissors, forceps (two sets), hemostat, or nee-
dle holder.
Surgical stapler (BD AUTOCLIP, Wound clip applier, cat. #
427630).
Staples (BD AUTOCLIP, Wound clips 9 mm, cat. # 427631).
Betadine swabs.
~40 ml 70 % ethanol in 50 ml conical.
Phosphate buffered saline.
Spray bottle containing 70 % ethanol.
Small animal shaver (Oster Golden A5 Clipper).
For skin analyses:
Histology cassettes.
Biopsy foam pads (Fisher cat.# 22038221).
Formalin.
O.C.T. (Tissue-Tek).
Tissue homogenizer (Brinkman Polytron).
Trizol (Invitrogen).
320 M. Dimarzio et al.

3. Methods

3.1. Preparation Prior These procedures are carried out in a tissue culture hood.
to Surgery: Selection
1. Pumps and modulators are removed from supplied packaging
and Loading Osmotic
and dropped into sterile petri dish(es). Typically five pumps
Pumps, and can be placed into one 100 mm petri dish. Pumps should be
Preparation of divided so that pumps in each petri dish will be filled with the
Anesthesia/Analgesia same ligand (see Note 2).
2. Poly(I:C) 0.5 mg/ml or other ligand at the appropriate con-
centration (to be determined empirically) to be placed into the
pump is prepared by dissolving in sterile PBS in sterile
Eppendorf or equivalent.
3. The person to load pumps dons sterile gloves.
4. A second person opens a sterile 1 ml syringe, handling only the
package and delivers the syringe sterilely to the pump loader.
5. While the sterile syringe is held steady by the pump loader, the
filling tube included with the pumps is opened and pushed
onto the sterile syringe by holding and then removing the needle
cap by the second person.
6. The pump loader draws up the sterile solution of PBS, poly(I:C)
or other ligand sufficient to fill one or several pumps (~250 l/
pump).
7. The needle-like filling tube is carefully inserted into the top
of the pump and the pump filled (see Note 3).
8. The regulator is carefully inserted into the pump top.
9. Steps 68 are repeated, filling all pumps with the same ligand
solution.
10. If pumps containing another ligand are to be loaded, then
steps 49 are repeated with other ligands and/or control PBS
(see Note 4), using a new filing tube for each ligand.
11. During the procedure above, the second person assists the
gloved pump loader by opening and closing petri dishes and
vials of ligands.
12. Petri dishes with loaded pumps are all covered, gloves are
removed.
13. 0.5 ml Xylazine (20 mg/ml stock), 1.0 ml ketamine (100 mg/
ml stock), and 4 ml buprenorphine (0.3 mg/ml stock) are
drawn by syringe/needle sterilely from vials and mixed into
14.5 ml PBS.
14. The diluted anesthetic/analgesic solution is sterilely drawn
into 3 ml syringes (~2.5 ml/syringe).
 15 Modeling Innate Immunity in Murine Skin 321

3.2. Induction These procedures are carried out in a laminar flow hood in the
of Anesthesia animal facility. The hood surface is cleaned with 70 % ethanol.
and Pump Insertion
1. Mice are weighed (see Note 5).
2. 0.02 ml/g mouse of the diluted anesthetic/analgesic solution
(0.5 ml for typical 25 g, 68 week mouse), is injected intrap-
eritoneally, low on the abdomen directed caudally, after brief
spray sterilization of the mouse abdomen with 70 % ethanol.
3. Mice typically fall to sleep in 23 min.
4. While mice are falling asleep, the sterilized instruments are
placed into the 50 ml conical holding ~40 ml 70 % ethanol.
5. After the mice are sleeping, an area approximately 1 cm 2 cm
across the mid scapula is shaved (see Note 6). Typically pumps
can be inserted in up to five mice in parallel (a batch). After
shaving, the hair needs to be completely removed from the
hood so that it does not contaminate the operating field. Mice
should be placed on a towel to help maintain body heat during
the remainder of the procedure.
6. The shaved area on each mouse is generously sterilized with
betadine solution either on a swab or gauze. In young mice the
ears might also be sterilized.
7. Using forceps and scissors, a 78 mm incision is made over the
upper back tangentially to spine and laterally to the midline
(see Fig. 1). The incision needs to pierce the subcutaneous
tissue, but not enter the underlying muscle (see Note 7).

Fig. 1. Pump insertion. A region over the midscapula is shaved and an incision made with scissors (panel a). Hemostats or
needle holders are used for blunt dissection, forming a pocket for the pump (panel b). The pump is inserted through the
incision into the subcutaneouspocket (panel c) and stapled closed with 23 staples (panel d). Panel (e) shows pump in
place with wound completely closed.
322 M. Dimarzio et al.

8. Using forceps and the needle holder or hemostat, gently tease

apart a pocket the size of the pump into the subcutaneous tis-
sue in the caudal direction, by repeatedly inserting the tip of
the needle holder into the wound and then opening the jaws
(see Note 8). Repeat steps 7 and 8 with other mice in this
batch, using the 70 % ethanol to re-sterilize the hemostat
between each insertion and between animals.
9. Open the petri dish containing the pumps to be inserted, and
don sterile gloves.
10. Grasping the caudal edge of the wound with forceps (in left
hand if right handed), pick up a pump in the right hand and
insert the pump into the wound, gently pushing it caudally
into the pocket. The pump outlet should be most caudal, i.e.,
away from the wound edge. The pump must not touch sur-
rounding tissues or ears as it is being inserted. Repeat this step
with other mice in this batch (see Note 9).
11. Using two pairs of forceps appose the interior wound edges.
12. Holding the wound edges together with one hand/forceps,
staple the wound closed. This typically requires 23 staples.
13. House mice in separate cages for the duration of the experi-
ment, observing for pain behavior (see Note 10) or wound
dehiscence (see Note 11).

3.3. Histological 1. Sacrifice the mouse by CO2 narcosis

Assessment of Skin 2. Grasping the pump and, taking care not to move the pumps
at Pump Outlet position, mark through the fur with a marker the point where
the pump outlet can be anticipated (see Note 12).
3. Cut open the skin at the surgical wound site and remove the
pump.
4. Shave the skin around the region of the pump outlet.
5. Cut the skin in ~1 cm diameter circle, centered at the marked
pump outlet site.
6. For routine histology, mouse skin must be placed between
biopsy foam pads before placing in fixative, formalin or similar
overnight (see Note 13). After overnight fixation biopsy foam
pads should be removed, the skin should be trimmed into
~4 mm 8 mm piece for embedding and proper orientation for
cross-sectioning using standard histological technique. Skin
surrounding pumps containing poly(I:C) show inflammation
when 7-day pumps are used and mild inflammation and fibrosis
when 28-day pumps are used (Fig. 2).
7. For frozen sections, a rectangular piece of skin must be tightly
jelly-rolled and then frozen in OCT. The uneven density of
mouse skin makes it virtually impossible to cut frozen sections
unless first jelly-rolled. Frozen sections can then be cut using
standard techniques.
 15 Modeling Innate Immunity in Murine Skin 323

Fig. 2. Histologic evaluation of skin from poly(I:C) treated mice. Formalin fixed, H&E stained skin 7 days after placement of
7-day osmotic pumps containing PBS (panel a) or poly(I:C) (panel b, bold arrows shows inflammation of fat, narrow arrow
shows early fibrosis), or 28 days after placement of 28-day pump: low power (panel c) or high power (panel d, arrows
indicate area of fibrosis).

3.4. Assessment 1. The critical step for RNA preparation from skin is vigorous and
of Gene Expression immediate homogenization of the tissue. Tissue cut from the
in Skin area of the pump outlet is placed on aluminum foil and minced
thoroughly with scissors.
2. The minced skin is submerged in 2.0 ml Trizol in a 15 ml coni-
cal tube and homogenized vigorously, typically 1520 s until
no visible pieces of skin are visible. At this point the homoge-
nized tissue solution may be stored at 80 C.
3. Thaw the homogenized Trizol/tissue homgenate at 37 C for
>5 min to permit complete dissociation of nucleoprotein com-
plexes and then centrifuge at >2,000 rpm (1,800 g) in clinical
centrifuge to pellet debris.
4. Aliquot and refreeze 1 ml of the clarified tissue/Trizol solution for
back-up if needed. Transfer the remaining 1.0 ml tissue/Trizol
solution to a 1.5 ml Eppendorf, and continue RNA purification
according to the protocol included with Trizol reagent.
5. Resuspend RNA in 50 l RNase-free H2O and store at
80 C.
6. Prepare cDNA and analyze for gene expression by real-time
q-PCR using standard methods.
324 M. Dimarzio et al.

4. Notes

1. Ligand stability can be easily tested by placing the ligand in the

buffer to be used in the pump in a 37 C incubator for the
period of the anticipated experiment, and then testing biological
activity.
2. We do not use sterile gloves for this process. It does take
significant time as the pumps are enclosed in inconvenient
packaging. Although the manufacturer indicates that the
pumps may be sterilized after removal by wiping with ethanol,
we have no experience with this. Removing pumps thus just
takes time and patience to maintain sterility of the pump and
modulator.
3. Although it would seem an ordinary needle could be used to
replace the needle supplied by the manufacturer, these lead to
problems. The filling tube should be gently introduced until
reaching the bottom of the pump. As the liquid fills the pump,
it is possible to see the top part of the pump being filled, making
it relatively easy to fill pumps without overfilling and spilling
solution on the pump exterior.
4. It is essential to have mice treated with pumps containing the
vehicle (PBS). Not surprisingly, there is a clear effect of pump
insertion on gene expression (R. Lafyatis, unpublished obser-
vations), so that untreated skin is not an adequate control. The
effect of pump insertion, one of mild inflammation, largely
subsides after about 3 days (R. Lafyatis, unpublished
observations).
5. Mouse weights are very difficult to estimate accurately and
proper anesthesia assures animal well-being without mortality
related to excess anesthesia. We have found that the least
expensive, nonelectric scale available in kitchen stores is the
best (batteries do not run down) and adequately precise for
assessing mouse weight for anesthesia dosing.
6. The area shaved ultimately determines where the incision for
pump placement will be made. In young mice the working
space is relatively small, in particular because the ears have a
tendency to get in the way during pump insertion. Resist the
temptation to insert the pumps rostrally from a more caudal
incision as the mice will pick at and sometimes dehisce the
wound.
7. The incision must be adequately large to permit pump inser-
tion. Trying to force the pump through an inadequate incision
causes the surrounding tissues to close around and contami-
nate the pump.
 15 Modeling Innate Immunity in Murine Skin 325

8. Much aggravation with pump insertion can be avoided by

adequate preparation of the pocket for the pump, hoping to
force the pump through the subcutaneous tissues without first
creating this pocket will be unsuccessful.
9. To reinforce sterility, we generally have a second person spray
70 % ethanol onto the gloves of the surgeon between each
inserted pump in a batch, changing gloves entirely with each
batch of mice operated on. Sterile handling and insertion of
the pump is of critical importance for this model, as although
the mice may survive an infection of the pump pocket, results
will be uninterpretable.
10. We have not generally witnessed evidence of pain behavior
after insertion of osmotic pumps. The dose of buprenorphine
given with the anesthetic lasts up to 12 h. Further dosing is
problematic because the location of the surgical wound makes
it difficult to grasp the mouse for further administration of
narcotics, and is painful for the mouse as it inevitable tears at
the wound. Based on our observations, we do not think it
makes sense to try to administer further narcotics. Pain behavior
probably indicates a surgical complication, possibly indicating
that the mouse needs to be sacrificed.
11. Wound dehiscence should be very rare if pumps are inserted
through an incision on the upper back and mice are housed
separately, indicating that dehiscence most often results from
mice or their littermates picking at the wound. Although it is
costly to house mice separately, wound dehiscence is a com-
mon problem for mice housed together postoperatively, and
we have had limited success trying to salvage pumps (by resta-
pling, etc.) that are falling out through dehiscent wounds.
12. Using Evans Blue dye we have verified that in general most
pump contents disperse over an area within about cm sur-
rounding the pump outlet, sometimes also tracking up the
edge of the pump (though doubtless systemic absorption of
small molecules also occurs). The Evans Blue dye unfortunately
also leads to some inflammation on its own, so we do not advise
using this routinely in experiments. However, for a first test of
this protocol, adding Evans Blue dye makes it easy to see the
pump fill, providing confidence that the protocol is working
well, and showing that you are taking skin from the correct
location.
13. As mouse skin is very thin, fixation without histology sponges
leads to curled tissues and extremely distorted morphology.
Thus, biopsy foam pads are essential for routine murine skin
fixation for histology.
326 M. Dimarzio et al.
References
1. http://www.alzet.com/products/guide_to_
use/pump_selection.html
2. Kawai T, Akira S (2008) Toll-like receptor and
RIG-I-like receptor signaling. Ann NY Acad
Sci 1143:120
3. Farina G, York M, Collins C, Lafyatis R (2010)
dsRNA activation of endothelin-1 and markers
of vascular activation in endothelial cells and
fibroblasts. Ann Rheum Dis 70:544550
4. Farina GA, York MR, Di Marzio M, Collins
CA, Meller S, Homey B, Rifkin IR, Marshak-
Rothstein A, Radstake TR, Lafyatis R (2010)
Poly(I:C) drives type I IFN- and TGFbeta-
mediated inflammation and dermal fibrosis
simulating altered gene expression in sys-
temic sclerosis. J Invest Dermatol 130:
25832593
 Chapter 16

Flow Cytometric Identification of Fibrocytes

in Scleroderma Lung Disease
Thomas M. Russell, Erica L. Herzog, and Richard Bucala

Abstract
Scleroderma is an autoimmune disease characterized by the progressive and dysregulated accumulation of
collagen in the skin and internal organs. Pulmonary complications including interstitial lung disease have
emerged as the greatest cause of mortality in this disease. Because treatments are limited, new areas of
investigation are sorely needed. An emerging area of interest in this field is a potential role for fibrocytes as
biomarkers or mediators of disease. Fibrocytes are monocyte-derived mesenchymal progenitor cells that
exhibit features of extracellular matrix production and wound contraction in addition to immunologic
functions such as cytokine and chemokine production, antigen presentation, leukocyte trafficking, and
modulation of angiogenesis. Fibrocytes could participate in the pathogenesis of scleroderma lung disease
through any or all of these functions and may be useful biomarkers of disease activity. This chapter presents
protocols that have been developed for the study of fibrocytes obtained from human circulation and tis-
sues. Protocols for the quantification of fibrocytes in murine models also are described, along with discus-
sion of common technical challenges. It is hoped that this information will allow further investigation of
the role that fibrocytes might play in Scleroderma-related lung disease and perhaps lead to new areas of
study in this difficult-to-treat and deadly disease.

Key words: Biomarker, Collagen, Extracellular matrix, Fibrocyte, Flow cytometry, Scleroderma,
Pulmonary fibrosis

1. Introduction

Scleroderma, or systemic sclerosis (SSc), is a multisystem disease

characterized by cutaneous and visceral fibrosis. Of the approxi-
mately 500,000 Americans with scleroderma, nearly 70 % have
some form of lung disease. Due to advances in the treatment of
Sclerodermas renal manifestations, pulmonary involvement has
emerged as the greatest cause of mortality for patients with this
disease (1). The lungs of patients with scleroderma-associated
interstitial lung disease (SSc-ILD) exhibit replacement of the normal

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_16, Springer Science+Business Media New York 2012

327
328 T.M. Russell et al.

lung architecture with inflamed and fibrotic tissue that is ineffective

for gas exchange. Approximately 42 % of patients with SSc-ILD
will die of disease progression within 10 years of diagnosis (1).
There is emerging evidence that some patients progress very slowly
while others follow a more accelerated clinical course (2). There is
currently no way to predict which patients will progress [and thus
are candidates for more intensive therapy with highly cytotoxic (3)
or lymphocyte modulating agents (4, 5) and/or referral for lung
transplantation (6, 7)] and which patients will follow a more indo-
lent course (potentially requiring less intense follow-up). Thus, the
development of a clinically predictive measure of pathologic pro-
gression would be of great benefit to physicians caring for patients
with this disease. The ideal biomarker would be present in easily
accessible specimens such as the peripheral blood, would exhibit
biological plausibility in terms of contribution to disease develop-
ment, and would be easily studied in murine models of disease. For
this reason, fibrocytes have emerged as an exciting new area of study
in the field of scleroderma.

1.1. Fibrocytes: fibrocytes might play important roles in tissue repair and remodel-
Disease Associations ing. These cells are identified by their co-expression of leukocyte
markers such as CD45, extracellular matrix proteins such as
Collagen-1, and stem cell markers such as CD34 (8). An increas-
ing amount of data highlight the association between peripheral
blood fibrocyte abnormalities in diverse forms of autoimmune dis-
ease such as rheumatoid arthritis (9) autoimmune thyroiditis (10),
amyopathic antisynthetase syndrome (11), and scleroderma (12,
13). Fibrocytes also are present in many forms of chronic
inflammatory disorders that are not classically associated with auto-
immunity including idiopathic pulmonary fibrosis (IPF) (1416),
asthma (1719), nephrogenic systemic fibrosis (20), cardiovascular
disease (21), pulmonary hypertension (22), and even normal aging
(12). Furthermore, animal modeling implicates fibrocytes in the
development of organ fibrosis for which fibroblasts, and their acti-
vated counterpart, the myofibroblast, are believed to be centrally
involved including the kidney (23, 24), liver (25), heart (2628),
vasculature (29), and lungs (15, 16, 30, 31). In many of these
studies fibrocytes appeared to function as a biomarker of disease
activity, which has led to speculation that similar parallels may be
drawn in patients with SSc-ILD. For all of these reasons, the study
of fibrocytes has become particularly important and timely.

1.2. Identification The general approach to flow cytometric identification of fibrocytes

of Fibrocytes in the circulation or in diseased organs is based upon the combina-
in the Circulation tion of characteristic cell surface marker expression with intracellular
staining for collagens or extracellular matrix components. Human
fibrocytes express many markers reflecting their hematopoietic
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 329

origin such as CD45 (8) and Leukocyte-specific protein-1 (LSP-1)

(32) as well as markers of monocyte lineage and function including
adhesion and motility (CD11b, CD11c, CD11d) (33), chemokine
receptors such as CXCR4 (15), proteins important in host defense
and scavenger receptors (CD16/32, CD163) (33), antigen pre-
sentation (Major Histocompatibility Complex (MHC) I and II,
CD80, and CD86) (34), and cell surface enzymes such as CD10
and CD13 (33). Fibrocytes typically lack markers of lymphocytes
(33, 35). Circulating and cultured fibrocytes also express CD34
(8), a motility protein that is also expressed on certain stem cell
populations that allows fibrocytes to be distinguished from other
cell types such as mature macrophages and fibroblasts. However,
because CD34 is frequently lost upon entry into target tissue (11,
31) its absence does not rule out a cell as being a fibrocyte (espe-
cially in the setting of tissue analysis). In addition to these cell
surface markers, fibrocytes also produce a wide array of ECM com-
ponents including structural proteins and glycosaminoglycans
(GAGs) (33, 35, 36). A listing of the markers attributed to
fibrocytes is shown in Table 1.

1.3. Fibrocytes: Insight into fibrocyte function may be gleaned from an under-
Differentiation standing of the factors promoting their differentiation and recruit-
and Homing ment. Fibrocytes differentiate from a precursor population present
within the CD14+ monocyte fraction of peripheral blood (37).
Further enrichment for CD11b(+) CD115(+) Gr1(+) expressing
monocytes increases the monocyte-to-fibrocyte transition in cul-
tured murine cells; these effects are promoted by direct contact
with activated CD4+ lymphocytes via an mTOR-PI3 kinase-depen-
dent pathway (38). Other studies have determined that the fibrocyte
precursor expresses the Fc receptor (39) and that inhibition of this
receptor with the short pentraxin protein serum Amyloid P
significantly reduces fibrocyte outgrowth in human (39, 40), rat
(41), and murine samples (30) via an ITIM-dependent mechanism
(42). Fibrocyte differentiation from CD14+ precursors is also
reduced by the TH1 cytokines IFN, TNF, and IL-12 and is aug-
mented by the TH2 cytokines IL-4 and IL-13 (43), TGF-1, and
engagement of the 1 integrin subunit (13, 22). These latter effects
require Erk phosphorylation (22). However, careful lineage
tracing studies will be required to confirm the monocyte origin of
fibrocytes under different fibrogenic stimuli.
Murine fibrocytes express the chemokine receptors CCR2,
CCR7, and CXCR4 which mediate the recruitment of fibrocytes to
injured tissue (31, 44, 45). Human fibrocytes also express the
chemokine receptors CCR3 (eotaxin receptor) and CCR5 (MCP-1
receptor) as well as CD29 and Semaphorin 7a (46). While there are
only limited data regarding the mediators that affect fibrocyte
recruitment in humans, our own work demonstrates an association
between concentrations of soluble factors such as TNF, IL-10,
330 T.M. Russell et al.

Table 1
Fibrocyte marker expression

Marker Expression Reference

Adhesion and motility
CD9, CD11a, CD11b, CD11c, +/++ (8, 9, 32, 33)
CD43, CD164, Mac2, LSP-1
CD34 + (8, 12, 33, 56)
CD29, CD44, CD81, ICAM-1, + (8, 12, 22, 33)
CD49 complex, CD81
Cell surface enzymes
CD10, CD172a + (8)
CD13, Prolyl-4-hydroxylase +
FAP +
Scavenging receptors and host defense
CD14, CD68, CD163, CD206, +/ (8)
CD209, CD35, CD36
Fc receptors
CD16, CD32a, CD32b, CD32c +/++ (8)
Chemokine receptors
CCR2, CCR5, CCR4, CCR7, CCR9, +/++ (15, 33)
CXCR1, CXCR4, CXC3R1
Antigen presentation
CD80, CD86, MHCI, MCHII + (34)
Extracellular matrix
Collagen-I/III/IV, vimentin, + (8, 17, 33)
tenascin
Fibronectin, -SMA +/ (8, 17, 33, 36)
Collagen V ++ (36)
Glycosaminoglycans
Perlecan, Veriscan, Hyaluronan ++/+ (36)
Decorin + (36)
Miscellaneous
Semaphorin 7a + (12)
CD115 (33)
Thy1.1 + (10)
CD105 + (33)
++ high level, + moderate, +/ conflicting reports or equivocal, no expression

MCP-1, and IL-1 receptor antagonist (IL-1Ra) in the blood of

patients with scleroderma, suggesting that fibrocytes may be mobi-
lized into the circulation in response to one or more of these fac-
tors. Similarly, high levels of CXCL12, which is the cognate ligand
for CXCR4, have been found in the lungs and blood of patients
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 331

with IPF and these levels correlate with circulating fibrocyte

concentrations (15). The blood of patients with SSc-ILD also
demonstrates high-level expression of Plexin C1, an inhibitory
receptor for Sema 7a (47). Interestingly, in vitro inhibition of this
receptor leads to marked increases in fibrocyte outgrowth, suggest-
ing that the increase in Plexin C1 detected in these patients is a
counterregulatory response (47). The relationship between these
trafficking molecules and fibrocyte accumulation is an active area
of investigation with the potential to lead to new therapies for
fibrosing diseases.

1.4. Fibrocytes: It has been postulated that the ultimate phenotype of fibrocytes is
Functions the contractile myofibroblast (31, 37, 46, 48), a hypothesis that is
based on the finding that cultured fibrocytes respond to TGF-1
by expressing alpha-Smooth Muscle Actin (-SMA) and contract-
ing collagen gels in vitro (31, 37, 46, 48). However, the ability of
fibrocytes to differentiate into myofibroblasts in vivo is less clear.
Studies using bone marrow transplantation show only a minimal
contribution of fibrocytes to -SMA production in some models
(25, 49, 50), implying that this differentiation pathway may not
necessarily be a dominant feature of fibrocytes in the tissue remod-
eling response.
While a specific contribution to disease pathogenesis has yet to
be established, fibrocytes exhibit many functions that would be
expected to play a role in different autoimmune diseases (Fig. 1).
Human fibrocytes respond to Interleukin-1 beta (IL-1) by
increasing secretion of Interleukin-6 (IL-6), Interleukin-8 (IL-8),
Chemokine (C-C motif) ligand 2 (CCL2, also called monocyte
chemotactic protein-1 or MCP-1), and Chemokine (C-C motif)
ligand 3 (CCL3, also called Macrophage inflammatory protein-
1) and by increasing expression of Intercellular adhesion mole-
cule-1 (ICAM-1) which would be expected to recruit and activate
leukocytes (51). Porcine fibrocytes respond to Toll-like receptor
(TLR) agonists and viral infection to increase expression of MHC
I and II, and the costimulatory proteins CD80 and CD86 which
allow efficient antigen presentation and subsequent activation of
cytotoxic CD8+ cells (52). Human fibrocytes demonstrate these
properties as well (34). In addition to these proinflammatory func-
tions, fibrocytes also respond to IL-1 by increasing Interleukin-10
(IL-10) production (51) which would be expected to reduce
inflammation. Fibrocytes could influence repair and remodeling
through their ability to express -SMA and enact wound contrac-
tion in ex vivo wound chambers (8). Fibrocytes also exhibit a dis-
tinctive pattern of ECM production characterized by high levels of
Collagen V, hyaluronan, versican, and perlecan (36); this profile
would be expected to promote recruitment of inflammatory cells
332 T.M. Russell et al.

Fig. 1. Potential contributions of fibrocytes to autoimmune pathogenesis. In the setting of autoantigen exposures, acute
injury, IL-1, serum factors, and innate immune stimuli with TLRs and viral infection fibrocytes adopt a proinflammatory
phenotype characterized by secretion of IFN, IL-6, IL-8, CCL3, and CCL4. Leukocyte trafficking is enhanced via ICAM-1.
Production of ECM components is reduced by exposure to SAP via Fc-mediated effects. Antigen presentation to T cells is
performed by CD80, CD86, MHCI, and MHCII. Local tissue destruction may be increased by expression of MMPs. As the
local milieu begins to favor repair and remodeling (or perhaps concurrent with ongoing injury in the right biological context)
fibrocytes evolve into a more reparative cell, perhaps in response to exogenous or autocrine secretion of IL-10. Here
TGF-1 stimulates fibrocyte development via noncanonical pathways mediated by Semaphorin 7a and CD29, though other
TGF-1 signaling pathways may also be involved. Sema 7a could activate monocytes and dendritic cells while dampening
T cell responses. ECM production is further stimulated by TH2 cytokines such as IL-4 and IL-13 as well as by exposure to
apoptotic cells. Transformation of tissue-resident fibroblasts into activated myofibroblasts is promoted by TGF-1 pro-
duced by fibrocytes, though fibrocytes may also adopt -SMA expression in response to TGF-1 and ET-1. PDGF, IL-10,
VEGF, HGF, and b-HGF support neoangiogenesis, and recruitment to sites of injury is promoted via expression of chemokine
receptors such as CXCR4.

and tissue repair. Fibrocytes secrete paracrine factors such as plate-

let-derived growth factor (PDGF) and TGF-1 that induce
myofibroblast transformation in culture and contribute to angio-
genesis in part through their secretion of matrix metalloproteinases
(MMPs) vascular endothelial growth factor (VEGF), PDGF-A,
hepatocyte growth factor (HGF), granulocytemacrophage col-
ony-stimulating factor (GM-CSF), basic fibroblast growth factor
(b-FGF), IL-8, and IL-1 (53). Fibrocytes express Semaphorin 7a
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 333

(Sema 7a or CD108W) (13), which can activate macrophages

and dendritic cells (54) and negatively regulate T cell responses
(55). Fibrocytes are identified in human malignancies (56, 57) and
promote tumor metastasis in rodent models via suppression of TH1
cytokines such as interferon-gamma (IFN) and tumor necrosis
factor (TNF) (58), as well as via overexpression of MMP9 (59).
When viewed in combination, these data suggest that fibrocytes
respond to inflammatory stimuli by adopting diverse functional
characteristics characterized by both inflammation and ECM pro-
duction. Any or all of these functions could contribute to the dys-
regulated immune activation and tissue remodeling seen in
autoimmune diseases.

2. Materials

1. Kendall Monoject Blood Collection Tubes (Tyco/Healthcare,

Mansfield, MA) (human only).
2. Histopaque (Sigma-Aldrich, St. Louis, Mo) (human only).
3. PerCP conjugaated Anti-human CD45 (eBioscience), FITC
conjugated Anti-human CD45 (eBioscience) (BD Biosciences,
San Jose, CA) (human only).
4. IsoPerCP Mouse IgG1, Isointracellular Purified Rat IgG1 (BD
Biosciences Pharmingen) (human only).
5. Alexa Fluor 488 conjugated Goat Anti-rat (Invitrogen, Eugene,
Oregon) (human only).
6. FCR Blocking Reagent Human (MACS Miltenyi Biotec)
(human only).
7. Pro-Collagen I Rat anti Human (Millipore, Temecula, CA)
(human only).
8. 10 Phosphate-buffered saline (PBS) pH 7.4: 1.4 M NaCl,
0.1 M phosphate, pH 7.4, 0.03 M KCl. Mixed with dH2O to
get 1 PBS (American Bioanalytical, Natick, MA).
9. Ethylenediaminetetraacetate (EDTA) pH 8.0 (American
Bioanalytical, Natick, MA).
10. Fetal bovine serum (FBS).
11. Normal goat serum (NGS).
12. FACS Buffer: PBS with 2 % FBS, 0.01 % NaN3, and 1 mM
EDTA.
13. Paraformaldehyde (PFA) (JT Baker, Phillipsburg, NJ).
14. FACS Calibur (Becton, Dickinson).
334 T.M. Russell et al.

15. CellQuest (BD Biosciences).

16. FlowJo v.8.7.3.
17. Spray bottle containing 70 % ethanol (mouse only).
18. Styrofoam board covered with foil (mouse only).
19. Rubber bands, tape, or needles (mouse only).
20. Paper towels (mouse only).
21. Surgical instruments (sharp scissors, sharp forceps, blunt for-
ceps) (mouse only).
22. Surgical Gauze (mouse only).
23. 25-gauge needles (mouse and human).
24. 1 and 10 ml syringe (mouse and human).
25. 14 and 50 ml Falcon Tubes.
26. Collagenase (mouse and human).
27. 42 C water bath (mouse and human).
28. 1 % low melt agarose at 42 C.
29. Ice in ice bucket (mouse and human).
30. 23 gauge angiocatheter (mouse only).
31. Suture material (mouse only).
32. VWR marker.
33. Anesthetic (mouse only).
34. PBS.
35. 1.5 ml Eppendorf tubes.
36. 100 mm Petri dishes (mouse and human).
37. PerCP conjugated Anti-mouse CD45 and FITC conjugated
Anti-mouse CD45.
38. PerCP isotype control.
39. Collagen I Rabbit anti-Mouse (Millipore, Temecula, CA).
40. Purified Mouse anti-Rabbit IgG (BD Pharmingen).
41. Fluorescein Goat Anti-rabbit IgG (Invitrogen).
42. Dulbeccos Modified Eagle Medium High Glucose 1
(DMEM) (Invitrogen, Grand Island, NY).
43. TRIzol (Invitrogen).
44. Lung dissociation medium: DMEM supplemented with 10 %
NGS, 100 U/ml DNase I (mouse and human).
45. PBS, 1 % BSA.
46. 4 % Paraformaldehyde: Heat 100 ml of PBS in flask (no more
than 55 C) with stir bar. Add 4 g of PFA. Add 1 N NaOH
with dropper until solution clears and adjust pH to 7.4.
47. 10 Pharm Lyse (BD Biosciences Pharmingen).
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 335

3. Methods

The methods described here outline (a) human peripheral

mononuclear cell collection and staining for identification of
fibrocytes; (b) harvest and isolation of single-cell suspensions of
cells from human lung; (c) mouse sacrifice and collection of blood
and lungs; (d) preparation of murine samples; (e) staining of pri-
mary murine lung cells for the identification of fibrocytes; (f) flow
cytometry of stained samples; and (g) analysis of flow cytometry
data.

3.1. Human Peripheral
Blood Mononuclear
Cell Collection and
Staining
3.1.1. Separation
of Peripheral Blood
Mononuclear Cells
from Plasma
1. Collect peripheral blood in sterile sodium heparin tubes (10 ml
draw).
2. Perform the following procedures in hood with sterile
technique.
3. Dilute blood with PBS 1:2 blood to PBS.
4. Layer diluted blood over histopaque: 1:3 histopaque to blood/
PBS (typically 10 ml of histopaque to 30 ml blood/PBS).
5. Centrifuge at 10,625 g for 22 min at 12 C with acceleration,
deceleration curves at (1,1) (most gradual).
6. Remove peripheral blood mononuclear cells (PBMCs) in the
buffy coat layer (layer between plasma and histopaque) with
pipettor.
7. Wash with PBS twice, and centrifuge at 10,625 g for 5 min at
4 C with acceleration, deceleration curves at (9,9).
8. Count cells with hemocytometer and trypan blue and resus-
pend at one million cells/ml.
9. If needed, separate cells for RNA and DNA analysis prior to
FACS Staining (one million cells/Eppendorf, individual
Eppendorfs for RNA and DNA).
(a) RNA and DNA analysis (non-sterile).
(b) Centrifuge cells at 10,625 g for 1 min.
(c) Pipette off supernatant.
(d) Store DNA pellet at 80 C.
(e) Resuspend RNA pellet in 350 l of TRIzol buffer and
store at 80 C.

3.1.2. Staining of Cells
for CD45 and Pro-
Collagen-1 Expression
1. Place approximately 1 106 cells/tube. Prepare the following
tubes:
(a) No Stain.
(b) CD45-FITC + PerCP isotype antibody.
(c) CD45 PerCP (2 l) + Isointracellular + 2Ab.
(d) CD45 PerCP (2 l), Rat Anti-human Procol-I FITC.
336 T.M. Russell et al.

2. Centrifuge cells at 10,625 g for 5 min at 4 C with acceleration,

deceleration curves at (9,9).
3. Resuspend cells in 100 l of 10 % NGS FACS Buffer and 5 l/
sample of FCR blocker.
4. Add appropriate antibodies (amount noted above) for extra-
cellular staining.
5. Incubate at 4 C for 30 min.
6. Wash with FACS Buffer twice, and centrifuge at 10,625 g for
5 min at 4 C with acceleration, deceleration curves at (9,9).
7. Add 100200 l of cytofix/cytoperm to the remaining samples
and incubate at 4 C for 15 min.
8. Wash with Permeabilizing wash (P/W), and centrifuge at
10,625 g for 5 min at 4 C with acceleration, deceleration
curves at (9,9).
9. Wash extracellular stains with FACS Buffer and intracellular
stains with P/W, and centrifuge at 10,625 g for 5 min at 4 C
with acceleration, deceleration curves at (9,9).
10. Resuspend intracellular samples in 100 l of P/W and add 1 l
of Pro-collagen I to all applicable samples and one 1 l of iso-
type intracellular control antibody to all applicable samples.
11. Incubate covered at 4 C for 30 min.
12. Wash all with P/W, and centrifuge at 10,625 g for 5 min at
4 C with acceleration, deceleration curves at (9,9).
13. Add 100 l of Alexa Fluor 488 dilution (1:500 Alexa Fluor
488 to P/W) to all intracellular samples.
14. Incubate at 4 C for 30 min.
15. Wash with P/W, then wash with FACS Buffer, and centrifuge
at 10,625 g for 5 min at 4 C with acceleration, deceleration
curves at (9,9).
16. Fix all intracellular samples with 100200 l of 4 % PFA and
store at 4 C until FACS analysis. Representative flow cytom-
etry images of human PBMCs are shown in Fig. 2.

3.1.3. Human Lung Cell Lung biopsy is rarely performed for the diagnosis of lung disease in
Isolation and Staining patients with scleroderma. However, the following protocol is suit-
able for use in explanted scleroderma lungs, lung samples obtained
from nonfibrotic controls and from lung biopsies performed for
other types of fibrotic lung disease (autoimmune and idiopathic).
1. Lung sample should be placed in DMEM + 10 % FBS and trans-
ported to the laboratory from the pathology department.
2. Upon arrival in laboratory remove from medium, place in
100 mm Petri Dish, and cut into 1 cubes.
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 337

Fig. 2. Flow cytometric identification of fibrocytes in human peripheral blood. (a) Live cells are selected based on FSC vs.
SSC. (b, c) FITC-detected intracellular isotype control (X axis) vs. anti-CD45-PerCP (Y axis). The negative gate for PerCP is
set (b). PerCP-stained CD45+ve cells are selected and the negative gate for intracellular FITC staining is chosen (c).
(d) This gate is then applied to the sample stained with FITC-detected Pro-Collagen-I (X axis) vs. anti-CD45-PerCP. The
dual-positive cells in the right upper quadrant represent fibrocytes. The proportion of dual-positive cells in the right upper
quadrant in the negative control (c) is subtracted from the proportion of dual-positive cells in the sample stained with Pro-
Collagen-I (d) to determine the overall percentage of double-positive cells. (eh) Importance of choosing correct controls.
(e) Extracellular FITC isotype control (X axis) vs. anti-CD45-PerCP (Y axis) on an unpermeabilized sample from the subject
shown in panel (d). As can be seen, there is only minimal shift of the CD45+ve cells to the right. When this gate is applied
to the sample shown in (d), nearly 100 % of the cells fall into the right upper quadrant (f). This problem demonstrates the
importance of permeabilizing all of the control samples. Another commonly encountered problem is the comparison of
samples from different individuals run at the same time. Panel (g) demonstrates a sample from a normal control that was
stained with FITC-detected intracellular isotype control (X axis) vs. anti-CD45-PerCP (Y axis). There is a much less pro-
nounced rightward shift of the CD45+ve cells. When this gate is applied to the scleroderma subject, again a very large
number of cells fall into the double-positive gate. This commonly encountered problem can be avoided by preparing a
CD45/intracellular isotype control for each subject.

3. Using a 23 gauge needle, inflate the lung cubes with prewarmed

Collagenase and allow to sit at room temperature for 30 min.
Repeat the Collagenase inflation every 57 min.
4. Using a razor blade, mince digested lung into 2 mm sections.
5. Place lung pieces in 100 mm Petri dish containing 7 ml DMEM
supplemented with 10 % NGS and 100 U DNAseI/ml and
gently swirl plate to dissociate cells.
6. Use pipet to gently take up lung and transfer to a 50 ml Falcon
tube over which a 70 m filter has been placed.
7. Rinse Petri dish with DMEM + Hepes + DNAse to Petri dish in
increments and then pour over filter (gently pushing tissue
through between increments).
8. Filter cell suspension through a 40 m filter and then 22 m
Nytex mesh to remove large aggregates.
338 T.M. Russell et al.

9. Centrifuge at 10,625 g for 5 min at 4 C with acceleration,

deceleration curves at (9,9).
10. Resuspend in x ml (based on the estimate of cell count) of
FACS Buffer and use a hemocytometer or Coulter Counter to
perform cell count.
11. Resuspend cells so that concentration is 1 106 cells/ml.
12. Proceed to Subheading 3.1.2 for FACS staining. Representative
flow cytometry images for human lung are shown in Fig. 3.

3.2. Mouse Sacrifice Murine modeling allows mechanistic studies of the pathways regu-
and Isolation of Blood lating fibrocyte biology. For this reason, assessment of circulating
and Lung Cells and/or tissue fibrocytes has emerged as an important tool in mouse
models of scleroderma lung disease caused by either TGF-1 over-
expression (30) or Bleomycin administration (31). Presented here
is a detailed approach to harvest and preparation of the murine
blood and lung for flow cytometric analysis of circulating and intra-
pulmonary fibrocytes. Users should modify the detailed outline
below for their own particular tissue.

3.2.1. Sacrifice of Mouse
1. Anesthetize mouse so that it is unresponsive to paw pinch and
is breathing deeply. Do not perform cervical dislocation.
2. Place mouse in supine position with limbs extended and immo-
bilized on a foil covered Styrofoam board that has been cov-
ered with paper towels. Spray mouse with 70% ethanol.
3. With the mouse head at the top of the board, use scissors and
sharp forceps to create a 1 cm incision above trachea.
4. Use blunt forceps to separate tissue covering trachea. Be care-
ful not to transect the blood vessels that run vertically along
the trachea.
5. Use sharp forceps to cleanly and carefully lift exposed trachea.
With opposite hand use the second set of forceps to pass suture
material underneath.
6. Loosely tie suture in knot around trachea.
7. Insert 23 gauge angiocatheter directly into exposed trachea,
advance 1 cm, remove metal stylet, and tie suture tightly to
stabilize catheter.
(a) Note: Advancing the catheter too far will puncture the tra-
chea. For this reason, it may be useful to make a small
mark on the angiocatheter about 1 cm from the bottom.
When this mark is reached, the catheter should be advanced
no further.

3.2.2. Bronchoalveolar 1. To perform bronchoalveolar lavage (BAL), instill 750 l ice-

Lavage and Perfusion cold PBS gently into trachea, remove gently, place on ice, and
repeat the procedure for a total of 1.5 ml of BAL return.
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 339

Fig. 3. Flow cytometric analysis of fibrocytes in fibrotic human lungs. (a) Live cells are gated on based on FSC (X axis) vs.
SSC (Y axis). (b) Unstained cells demonstrate mild to moderate autofluorescence in the FL1 (X axis) and FL2 (Y axis) chan-
nels. (c) FITC-positive control (X axis) vs. PE-negative control (Y axis). This gate is used to set the negative gate for PE.
(d, e) FITC-detected intracellular isotype control (X axis) vs. anti-CD45-PE (Y axis). CD45+ve cells are gated on (d) and the
negative gate for FITC is set (e). (f) FITC-detected intracellular Pro-Collagen-I staining (X axis) vs. anti-CD45-PE (Y axis).
The dual-positive cells in the right upper quadrant meet flow cytometric criteria for fibrocytes. These panels demonstrate
acceptable staining for fibrocytes. Panels (gi) demonstrate unacceptable staining. (g) Unstained cells from a poorly pre-
pared specimen demonstrate high autofluorescence in the FL-1 (X axis) and FL-2 (Y axis) channels. (h) FITC-detected
intracellular isotype control (X axis) vs. anti-CD45-PE (Y axis) on this sample. The diagonal line evident in the left upper
quadrant indicates that the baseline autofluorescence of the sample has made compensation difficult. (i) FITC-detected
intracellular Pro-Collagen-I staining (X axis) vs. anti-CD45-PE (Y axis). The dual-positive population, which represents more
than 50 % of the CD45+ve cells, demonstrates a similar diagonal morphology and represents false positive staining.

2. Rotate mouse 180 so that feet are now at the top of the board
and make vertical incision from below xyphoid/rib cage up to
and including the incision in the neck, cut through ribs and
expose heart (still beating) and lungs, and transect inferior
vena cava. Exsanguination will occur.
3. Insert a 25 gauge needle attached to a 10 ml syringe containing
50 l of EDTA into the left ventricle. Remove as much blood
340 T.M. Russell et al.

as possible (usually approximately 300400 l), transfer to a

1.5 ml Eppendorf tube, and leave at room temperature until
ready to process.
4. Insert a 25 gauge needle attached to a 10 ml syringe containing
10 ml of ice-cold PBS into right ventricle and perfuse pulmo-
nary circulationlungs will blanche when done properly.
5. Rotate mouse 180 again so that head is facing you and insert
750 l collagenase through cannula into trachea.
6. Inflate lungs with 2 ml warm agarose.
(a) Note: If histologic analysis of lungs is desired, it is possible
to tie off one lung and fix in formalin. In this case, use of
a 60 ml syringe that is filled with 20 ml prewarmed aga-
rose is used. The syringe is mounted 20 cm above the
mouse. The syringe is mounted above the mouse and
pressure tubing is used to connect the syringe to the
angiocatheter. When the tubing is connected to the angio-
catheter, agarose will slowly flow in and inflate the lungs
to 20 cm water pressure. Then tie off the lung to be used
for histologic analysis and place in 10 % formalin for 4 h.
The remaining lung can be removed for use in the
digestion protocol below.

3.2.3. Removal 1. Tie off lungs, use scissors to remove, and place on ice for
and Enzymatic Digestion 2 min.
of Lungs 2. Following incubation on ice, transfer lungs to a 14 ml Falcon
tube eppendorf tube containing 3 ml prewarmed collagenase
and incubate at 37 C 25 min.
3. Remove lungs from collagenase and mince into small pieces
with scissors.
4. Place lung pieces in 100 mm Petri dish containing 7 ml DMEM
supplemented with 10 % NGS and 100 U DNAseI/ml (lung
dissociation buffer) and gently use surgical instruments to
homogenize tissue.
5. Add an additional 510 ml of lung dissociation buffer to Petri
dish in increments and then pour over filter (gently pushing
tissue through between increments).
(a) Note: In the fibrotic lung there will be some areas that
do not digest as well as others; these are remodeled areas
containing increased extracellular matrix proteins.
6. Centrifuge at 10,625 g for 5 min at 4 C with acceleration,
deceleration curves at (9,9).
7. Resuspend in x ml (based on the estimate of cell count) of
FACS Buffer and use a hemocytometer or Coulter Counter to
perform cell count.
8. Resuspend cells so that concentration is 1 106 cells/ml.
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 341

3.2.4. Preparation of Blood 1. Dilute 10 pharmlyse to 1 with water.

for Flow Cytometry
2. In a 14 ml Falcon tube, incubate blood in 1 ml of 1
pharmlyse 10 min.
3. Centrifuge at 10,652g for 5 min at 4 C with acceleration,
deceleration curves at (9,9).
4. Resuspend in x ml (based on the estimate of cell count) of
FACS Buffer and use a hemocytometer or Coulter Counter to
perform cell count.
5. Resuspend cells so that concentration is 1 106 cells/ml and
proceed to step 3 below.

3.2.5. Staining of Murine 1. Divide cells up to 1 106 cells/test tube using 22 m Nytex
Blood and Dissociated mesh to help de-aggregate the cells. Samples are as follows:
Lung Cells for Fibrocyte (a) Unstained.
Identification
(b) CD45 FITC (0.5 l) and PerCP isotype control.
(c) CD45 PerCP + isointracellular + 2Ab.
(d) Stain 1 [CD45 PercP (0.5 l)], Collagen-1 FITC.
2. Centrifuge cells at 10,625 g for 5 min at 4 C with accelera-
tion, deceleration curves at (9,9).
3. Resuspend cells in 100 l of 10 % NGS FACS Buffer and 5 ml/
sample of FCR blocker.
4. Add appropriate antibodies (amount noted above) for extra-
cellular staining.
5. Incubate at 4 C for 30 min.
6. Wash with FACS Buffer twice, and centrifuge at 10,625 g for
5 min at 4 C with acceleration, deceleration curves at (9,9).
7. Add 100200 l of cytofix/cytoperm to the remaining samples
and incubate at 4 C for 15 min.
8. Wash with P/W, and centrifuge at 10,625 g for 5 min at 4 C
with acceleration, deceleration curves at (9,9).
9. Fix all samples that were only stained with extracellular anti-
bodies in 100200 l of 4 % PFA and store at 4 C.
10. Resuspend intracellular samples in 100 l of P/W and add 1 l
of Anti-collagen antibody.
11. Incubate in fridge for 30 min.
12. Wash all with P/W and centrifuge at 10,625 g for 5 min at
4 C with acceleration, deceleration curves at (9,9).
13. Add 100 l of Fluorescein dilution (1:1,000 Fluorescein to
P/W) to all intracellular samples [Secondary antibody step
(2Ab)].
14. Incubate at 4 C for 30 min.
342 T.M. Russell et al.
15. Wash with P/W, then wash with FACS Buffer, and centrifuge
16. Fix all intracellular samples with 100200 l of 4 % PFA and
3.3. Flow Cytometry
3.3.1. Flow Cytometry of
Stained Samples
3.4. Analysis of Flow
Cytometry Data
3.4.1. FACS Analysis for
Fibrocytes
at 10,625 g for 5 min at 4 C with acceleration, deceleration
curves at (9,9).
store at 4 C.
1. Open Cell Quest, connect to the cytometer, and make sure
that the setup box is checked.
2. Voltages for the FACS Calibur are set using the non-stain
control.
(a) Adjust the Forward Scatter (FSC) (controls lateral move-
ment of events on graph if FSC is X-axis) and Side Scatter
(SSC) (controls vertical tilt if SSC is Y-axis) to record as
many of the cells of interest as possible.
(b) Using histograms of each of the channels (FL1, FL3) or a
dot plot, adjust voltages so that the majority of events
occur between 0 and 101.
3. Compensations for the FACS Calibur are set using the single-
color controls.
(a) Using the FITC control, adjust the compensation so that
the majority of events occur in the FITC channel (it will
bleed into PE).
(b) Using the PerCP control, adjust the compensations so
that the majority of events occur in the PerCP channel
(it will bleed into PE and APC).
4. Uncheck the setup box, set the location to record all samples,
and acquire all samples (including controls).
1. Open the flow cytometry analysis software (in this case, Flowjo)
and import FACS files.
2. First a gate is set for all of the live cells based on the FSC and
SSC and applied to all samples (Fig. 2a). It is important to
include only live cells as dead/dying cells can increase
autofluorescence. In addition, it is important to be consistent
in the populations being analyzed throughout samples.
3. Quadrant gating is established through comparing the single-
color positive controls to the isotype controls (ideally single-color
positive controls should be about 90 % positive, and isotype con-
trols should be greater than 97 % negative) (Fig. 2b, c).
4. The negative gate for fibrocyte analysis is determined by stain-
ing a CD45-stained sample with intracellular isotype control
and secondary antibody (Fig. 2c).
5. Once the negative gate for Col-I staining of CD45+ cells is
established, this gating strategy should be applied to the
sample(s) of interest (Fig. 2d).
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 343

4. Notes

1. When assessing fibrocyte quantities in PBMCs from patients

with scleroderma-related lung disease, parallel processing of
controls who have no autoimmune disease and who have scle-
roderma but no lung disease is important. We have typically
established baseline values for controls that are matched in terms
of age and demographics (which we have found to significantly
affect fibrocyte values in the circulation) and process cells from
two to three controls and test subjects at each processing. We
have found that freezing cells at 80 C decreases viability and
increases autofluorescence and for this reason we never perform
the assay on defrosted cells. (E. Herzog, unpublished data)
2. Fibrocytes have also been identified as CXCR4+ Pro-Col-I+ve
cells. In general, the combination of hematopoietic cell surface
marker expression with intracellular staining detecting produc-
tion of ECM components such as collagen, vimentin, or pro-
lyl-4 hydroxylase is considered sufficient for the detection of
circulating fibrocytes (60).
3. A number of important issues should be mentioned. While in
normal individuals the inherent autofluorescence of the unstained
cells is usually low, patients suffering from autoimmunity may
exhibit increased baseline autofluorescence, presumably due to
differences in phenotypes of circulating inflammatory cells. In
this setting, it may be necessary to prepare an isotype control
from the diseased individual, as this shift in autofluorescence
may be incorrectly interpreted as intracellular staining (Fig. 2).
Similar principles apply for the lung samples (Fig. 3) (12).
4. Fibrocyte levels can be reported as either percentage of total
PBMCs or as total quantities per milliliter of blood. The for-
mer is derived simply by subtracting the CD45/Pro-Col stain-
ing from CD45/intracellular isotype staining. The latter is
derived by multiplying the live cell gate based on FSC vs. SSC
by the CD45/Pro-Col-subtracted CD45/intracellular isotype
staining and multiplying this by the Post-Ficoll cell count
divided by the milliliters of blood put into Ficoll (12).
5. Pro-Collagen and the other ECM proteins used to identify
fibrocytes are intracellular proteins that require fixation and
permeabilization for detection. Thus, in vitro functional stud-
ies cannot be performed on fibrocytes detected in this manner.
For a detailed protocol for the isolation of live fibrocytes for
in vitro analysis, the reader is referred to ref. 61.
6. With the exception of the antibodies all reagents are prepared as
stock solution and stored at 4 C or at 80 C. We do not store
reagents in a 20 C frost-free freezer due to the freezethaw
temperature fluctuation that can occur in these units (62).
344 T.M. Russell et al.

7. In mouse studies, the importance of proper lung processing

cannot be overstated. Use of perfusion and lavage to remove
contaminating inflammatory cells will greatly enhance stain-
ing. Failure to perform perfusion and BAL will almost certainly
result in uninterpretable results (63).

Acknowledgements

This work was supported in part by grants K08 HL079066 from

the National Institutes of Health, the National Scleroderma
Foundation, the American Thoracic Society, and funds from the
Yale Department of Internal Medicine.

References
1. Steen VD, Medsger TA (2007) Changes in
causes of death in systemic sclerosis, 1972
2002. Ann Rheum Dis 66:940944
2. Goh NS, Desai SR, Veeraraghavan S, Hansell
DM, Copley SJ, Maher TM, Corte TJ, Sander
CR, Ratoff J, Devaraj A et al (2008) Interstitial
lung disease in systemic sclerosis: a simple stag-
ing system. Am J Respir Crit Care Med 177:
12481254
3. Tashkin DP, Elashoff R, Clements PJ, Goldin J,
Roth MD, Furst DE, Arriola E, Silver R, Strange
C, Bolster M et al (2006) Cyclophosphamide
versus placebo in scleroderma lung disease. N
Engl J Med 354:26552666
4. Swigris JJ, Olson AL, Fischer A, Lynch DA,
Cosgrove GP, Frankel SK, Meehan RT, Brown
KK (2006) Mycophenolate mofetil is safe, well
tolerated, and preserves lung function in
patients with connective tissue disease-related
interstitial lung disease. Chest 130:3036
5. Daoussis D, Liossis SN, Tsamandas AC,
Kalogeropoulou C, Kazantzi A, Sirinian C,
Karampetsou M, Yiannopoulos G, Andonopoulos
AP (2010) Experience with rituximab in sclero-
derma: results from a 1-year, proof-of-principle
study. Rheumatology (Oxford) 49:271280
6. DOvidio F, Singer LG, Hadjiliadis D, Pierre
A, Waddell TK, de Perrot M, Hutcheon M,
Miller L, Darling G, Keshavjee S (2005)
Prevalence of gastroesophageal reflux in
end-stage lung disease candidates for lung
transplant. Ann Thorac Surg 80:12541260
7. DOvidio F, Mura M, Tsang M, Waddell TK,
Hutcheon MA, Singer LG, Hadjiliadis D,
Chaparro C, Gutierrez C, Pierre A et al (2005)
Bile acid aspiration and the development of
bronchiolitis obliterans after lung transplanta-
tion. J Thorac Cardiovasc Surg 129:11441152
8. Bucala R, Spiegel LA, Chesney J, Hogan M,
Cerami A (1994) Circulating fibrocytes define
a new leukocyte subpopulation that mediates
tissue repair. Mol Med 1:7181
9. Galligan CL, Siminovitch KA, Keystone EC,
Bykerk V, Perez OD, Fish EN (2010) Fibrocyte
activation in rheumatoid arthritis. Rheumatology
(Oxford) 49:640651
10. Douglas RS, Afifiyan NF, Hwang CJ, Chong
K, Haider U, Richards P, Gianoukakis AG,
Smith TJ (2010) Increased generation of
fibrocytes in thyroid-associated ophthalmopa-
thy. J Clin Endocrinol Metab 95:430438
11. Peng XP et al (2011) Local apoptosis promotes
collagen production by monocyte derived cells.
Fibrogen Tissue Repair 4:12
12. Mathai SK, Gulati M, Peng X, Russell TR,
Shaw AC, Rubinowitz AN, Murray LA, Siner
JM, Antin-Ozerkis DE, Montgomery RR et al
Circulating monocytes from systemic sclerosis
patients with interstitial lung disease show an
enhanced profibrotic phenotype. Lab Invest
6:812823
13. Gan Y, Reilkoff RA, Peng X, Russell TR, Chen
QC, Mathai SK, Gulati M, Homer RJ, Elias JA,
Bucala RJ et al (2011) Role of Semaphorin 7a
in TGF 1 induced lung fibrosis, fibrocyte dif-
ferentiation, and scleroderma-related intersti-
tial lung disease 63:24842494
14. Moeller A, Gilpin SE, Ask K, Cox G, Cook D,
Gauldie J, Margetts PJ, Farkas L, Dobranowski
J, Boylan C et al (2009) Circulating fibrocytes
are an indicator for poor prognosis in idiopathic
pulmonary fibrosis. Am J Respir Crit Care Med
7:588594
15. Mehrad B, Burdick MD, Zisman DA, Keane
MP, Belperio JA, Strieter RM (2007)
Circulating peripheral blood fibrocytes in
 16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease
human fibrotic interstitial lung disease. Biochem
Biophys Res Commun 353:104108
16. Mehrad B, Burdick MD, Strieter RM (2009)
Fibrocyte CXCR4 regulation as a therapeutic
target in pulmonary fibrosis. Int J Biochem
Cell Biol 41:17081718
17. Schmidt M, Sun G, Stacey MA, Mori L, Mattoli
S (2003) Identification of circulating fibrocytes
as precursors of bronchial myofibroblasts in
asthma. J Immunol 171:380389
18. Wang CH, Huang CD, Lin HC, Lee KY, Lin
SM, Liu CY, Huang KH, Ko YS, Chung KF,
Kuo HP (2008) Increased circulating fibrocytes
in asthma with chronic airflow obstruction. Am
J Respir Crit Care Med 178: 583591
19. Nihlberg K, Larsen K, Hultgardh-Nilsson A,
Malmstrom A, Bjermer L, Westergren-
Thorsson G (2006) Tissue fibrocytes in patients
with mild asthma: a possible link to thickness of
reticular basement membrane? Respir Res 7:50
20. Vakil V, Sung JJ, Piecychna M, Crawford JR,
Kuo P, Abu-Alfa AK, Cowper SE, Bucala R,
Gomer RH (2009) Gadolinium-containing
magnetic resonance image contrast agent pro-
motes fibrocyte differentiation. J Magn Reson
Imaging 30:12841288
21. Falk E (2006) Pathogenesis of atherosclerosis.
J Am Coll Cardiol 47:C7C12
22. Nikam VS, Wecker G, Schermuly R, Rapp U,
Szelepusa K, Seeger W, Voswinckel R (2011)
Treprostinil inhibits adhesion and differentia-
tion of fibrocytes via cAMP and Rap dependent
ERK inactivation. Am J Respir Cell Mol Biol
45:692703
23. Niedermeier M, Reich B, Rodriguez Gomez
M, Denzel A, Schmidbauer K, Gobel N, Talke
Y, Schweda F, Mack M (2009) CD4+ T cells
control the differentiation of Gr1+ monocytes
into fibrocytes. Proc Natl Acad Sci USA 106:
1789217897
24. Katebi M, Fernandez P, Chan ES, Cronstein
BN (2008) Adenosine A2A receptor blockade
or deletion diminishes fibrocyte accumulation
in the skin in a murine model of scleroderma,
bleomycin-induced fibrosis. Inflammation 31:
299303
25. Kisseleva T, Uchinami H, Feirt N, Quintana-
Bustamante O, Segovia JC, Schwabe RF,
Brenner DA (2006) Bone marrow-derived
fibrocytes participate in pathogenesis of liver
fibrosis. J Hepatol 45:429438
26. Haudek SB, Cheng J, Du J, Wang Y, Hermosillo-
Rodrigues J, Trial J, Taffet GE, ML E (2010)
Monocytic fibroblast precursors mediate fibrosis
in angiotensin-II-induced cardiac hypertrophy.
J Mol Cell Cardiol 49:499507
27. Haudek SB, Xia Y, Huebener P, Lee JM,
Carlson S, Crawford JR, Pilling D, Gomer RH,
Trial J, Frangogiannis NG et al (2006) Bone
345
marrow-derived fibroblast precursors mediate
ischemic cardiomyopathy in mice. Proc Natl
Acad Sci USA 103:1828418289
28. Haudek SB, Trial J, Xia Y, Gupta D, Pilling D,
Entman ML (2008) Fc receptor engagement
mediates differentiation of cardiac fibroblast
precursor cells. Proc Natl Acad Sci USA 105:
1017910184
29. Buday A, Orsy P, Godo M, Mozes M, Kokeny
G, Lacza Z, Koller A, Ungvari Z, Gross ML,
Benyo Z et al (2010) Elevated systemic TGF-
beta impairs aortic vasomotor function through
activation of NADPH oxidase-driven superox-
ide production and leads to hypertension, myo-
cardial remodeling, and increased plaque
formation in apoE(/) mice. Am J Physiol
Heart Circ Physiol 299:H386H395
30. Murray LA, Chen Q, Kramer MS, Hesson DP,
Argentieri RL, Peng X, Gulati M, Homer RJ,
Russell T, van Rooijen N et al TGF-beta driven
lung fibrosis is macrophage dependent and
blocked by serum amyloid P. Int J Biochem
Cell Biol 43:154162
31. Phillips RJ, Burdick MD, Hong K, Lutz MA,
Murray LA, Xue YY, Belperio JA, Keane MP,
Strieter RM (2004) Circulating fibrocytes
traffic to the lungs in response to CXCL12 and
mediate fibrosis. J Clin Invest 114:438446
32. Yang L, Scott PG, Giuffre J, Shankowsky HA,
Ghahary A, Tredget EE (2002) Peripheral
blood fibrocytes from burn patients:
identification and quantification of fibrocytes in
adherent cells cultured from peripheral blood
mononuclear cells. Lab Invest 82:11831192
33. Pilling D, Fan T, Huang D, Kaul B, Gomer RH
(2009) Identification of markers that distin-
guish monocyte-derived fibrocytes from mono-
cytes, macrophages, and fibroblasts. PLoS One
4:e7475
34. Chesney J, Bacher M, Bender A, Bucala R
(1997) The peripheral blood fibrocyte is a
potent antigen-presenting cell capable of prim-
ing naive T cells in situ. Proc Natl Acad Sci
USA 94:63076312
35. Bellini A, Mattoli S (2007) The role of the
fibrocyte, a bone marrow-derived mesenchymal
progenitor, in reactive and reparative fibroses.
Lab Invest 87:858870
36. Bianchetti L, Barczyk M, Cardoso J, Schmidt
M, Bellini A, Mattoli S (2011) Extracellular
matrix remodeling properties of human
fibrocytes. J Cell Mol Med 3:483495
37. Abe R, Donnelly SC, Peng T, Bucala R, Metz
CN (2001) Peripheral blood fibrocytes: differ-
entiation pathway and migration to wound
sites. J Immunol 166:75567562
38. Niedermeier M, Reich B, Gomez MR, Denzel
A, Schmidbauer K, Gobel N, Talke Y, Schweda
F, Mack M (2009) CD4+ T cells control the
346 T.M. Russell et al.
differentiation of Gr1+ monocytes into
fibrocytes. Proc Natl Acad Sci USA 106:
1789217897
39. Pilling D, Buckley CD, Salmon M, Gomer RH
(2003) Inhibition of fibrocyte differentiation by
serum amyloid P. J Immunol 171:55375546
40. Pilling D, Tucker NM, Gomer RH (2006)
Aggregated IgG inhibits the differentiation of
human fibrocytes. J Leukoc Biol 79:
12421251
41. Pilling D, Roife D, Wang M, Ronkainen SD,
Crawford JR, Travis EL, Gomer RH (2007)
Reduction of bleomycin-induced pulmonary
fibrosis by serum amyloid P. J Immunol 179:
40354044
42. Castano AP, Lin SL, Surowy T, Nowlin BT,
Turlapati SA, Patel T, Singh A, Li S, Lupher
ML Jr, Duffield JS (2009) Serum amyloid P
inhibits fibrosis through Fc gamma R-dependent
monocyte-macrophage regulation in vivo. Sci
Transl Med 1:5ra13
43. Shao DD, Suresh R, Vakil V, Gomer RH, Pilling
D (2008) Pivotal advance: Th-1 cytokines
inhibit, and Th-2 cytokines promote fibrocyte
differentiation. J Leukoc Biol 83:13231333
44. Moore BB, Murray L, Das A, Wilke CA,
Herrygers AB, Toews GB (2006) The role of
CCL12 in the recruitment of fibrocytes and lung
fibrosis. Am J Respir Cell Mol Biol 35:175181
45. Sakai N, Wada T, Yokoyama H, Lipp M, Ueha S,
Matsushima K, Kaneko S (2006) Secondary lym-
phoid tissue chemokine (SLC/CCL21)/CCR7
signaling regulates fibrocytes in renal fibrosis.
Proc Natl Acad Sci USA 103:1409814103
46. Quan TE, Cowper S, Wu SP, Bockenstedt LK,
Bucala R (2004) Circulating fibrocytes: colla-
gen-secreting cells of the peripheral blood. Int
J Biochem Cell Biol 36:598606
47. Lazova R, Gould Rothberg BE, Rimm D, Scott
G (2009) The semaphorin 7A receptor Plexin
C1 is lost during melanoma metastasis. Am J
Dermatopathol 31:177181
48. Gomperts BN, Strieter RM (2007) Fibrocytes
in lung disease. J Leukoc Biol 82:449456
49. Lin SL, Kisseleva T, Brenner DA, Duffield JS
(2008) Pericytes and perivascular fibroblasts
are the primary source of collagen-producing
cells in obstructive fibrosis of the kidney. Am J
Pathol 173:16171627
50. Hashimoto N, J H, Liu T, Chensue SW, Phan
SH (2004) Bone marrow derived progenitor
cells in pulmonary fibrosis. J Clin Invest 113:
243252
51. Chesney J, Metz C, Stavitsky AB, Bacher M,
Bucala R (1998) Regulated production of type
I collagen and inflammatory cytokines by
peripheral blood fibrocytes. J Immunol 160:
419425
52. Balmelli C, Alves MP, Steiner E, Zingg D,
Peduto N, Ruggli N, Gerber H, McCullough
K, Summerfield A (2007) Responsiveness of
fibrocytes to toll-like receptor danger signals.
Immunobiology 212:693699
53. Hartlapp I, Abe R, Saeed RW, Peng T, Voelter
W, Bucala R, Metz CN (2001) Fibrocytes
induce an angiogenic phenotype in cultured
endothelial cells and promote angiogenesis
in vivo. FASEB J 15:22152224
54. Suzuki K, Okuno T, Yamamoto M, Pasterkamp
RJ, Takegahara N, Takamatsu H, Kitao T,
Takagi J, Rennert PD, Kolodkin AL et al
(2007) Semaphorin 7A initiates T-cell-mediated
inflammatory responses through alpha1beta1
integrin. Nature 446:680684
55. Czopik AK, Bynoe MS, Palm N, Raine CS,
Medzhitov R (2006) Semaphorin 7A is a nega-
tive regulator of T cell responses. Immunity
24:591600
56. Barth PJ, Ebrahimsade S, Hellinger A, Moll R,
Ramaswamy A (2002) CD34+ fibrocytes in
neoplastic and inflammatory pancreatic lesions.
Virchows Arch 440:128133
57. Barth PJ, Koster H, Moosdorf R (2005)
CD34+ fibrocytes in normal mitral valves and
myxomatous mitral valve degeneration. Pathol
Res Pract 201:301304
58. Kraman M, Bambrough PJ, Arnold JN, Roberts
EW, Magiera L, Jones JO, Gopinathan A,
Tuveson DA, Fearon DT (2010) Suppression
of antitumor immunity by stromal cells express-
ing fibroblast activation protein-alpha. Science
330:827830
59. van Deventer HW, Wu QP, Bergstralh DT,
Davis BK, OConnor BP, Ting JP, Serody JS
(2008) C-C chemokine receptor 5 on pulmonary
fibrocytes facilitates migration and promotes
metastasis via matrix metalloproteinase 9. Am J
Pathol 173:253264
60. Herzog EL, Bucala R (2010) Fibrocytes in
health and disease. Exp Hematol 38:548556
61. Quan TE, Bucala R (2007) Culture and analy-
sis of circulating fibrocytes. Methods Mol Med
135:423434
62. Lawrence JM, Umekubo MA, Chiu V, Petitti
DB (2000) Split sample analysis of serum folate
levels after 18 days in frozen storage. Clin Lab
46:483486
63. Garn H (2006) Specific aspects of flow cyto-
metric analysis of cells from the lung. Exp
Toxicol Path 57:S21S24
 Chapter 17

Oxidative Stress and Beta Cell Dysfunction

Yama L. Lightfoot, Jing Chen, and Clayton E. Mathews

Abstract
Autoimmune Type 1 A Diabetes (T1D) is characterized by dependence on exogenous insulin consequential
to the autoimmune attack and destruction of insulin-producing islet beta cells. Pancreatic islet cell
inflammation, or insulitis, precedes beta cell death and T1D onset. In the insulitic lesion, innate immune
cells produce chemokines and cytokines that recruit and activate adaptive immune cells (Eizirik D et al.,
Nat Rev Endocrinol 5:219226, 2009). Locally produced cytokines not only increase immune surveillance
of beta cells (Hanafusa T and Imagawa A, Ann NY Acad Sci 1150:297299, 2008), but also cause beta cell
dysfunction and decreased insulin secretion due to the generation of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) by the beta cells. This, coupled to the high levels of ROS and RNS
secreted by activated macrophages and the low antioxidant capacities of beta cells (Huurman VA, PLoS
One 3:e2435, 2008; Schatz D, Pediatr Diabetes 5:7279, 2004; Verge CF, Diabetes 44:11761179,
1995), implicates free radicals as important effectors in T1D pathogenesis (Eizirik D et al., Nat Rev
Endocrinol 5:219226, 2009; Hanafusa T and Imagawa A, Ann NY Acad Sci 1150:297299, 2008;
Eisenbarth GS and Jeffrey J, Arq Bras Endocrinol Metabol 52:146155, 2008; Pietropaolo M et al.,
Pediatr Diabetes 6:184192, 2005).

Key words:, Reactive oxygen species, Reactive nitrogen species, Pancreatic beta cells, Diabetes,
Mitochondria, Apoptosis, Necrosis

1. Introduction

Autoimmune Type 1 A Diabetes (T1D) results from responses of

an unbalanced immune system against proteins expressed in the
insulin-secreting pancreatic beta cells, leading ultimately to the
destruction of these cells, a dearth of insulin secretion, and hyper-
glycemia. Autoimmune responses against autoantigens in T1D are
associated with aberrant cellular as well as humoral responses.
Autoantibodies have been used to predict those at risk for T1D
development (36), and T cell responses are seen as predictive of
eminent risk for T1D (7). Antibodies mark both initial loss of
tolerance as well as progression or potential to progress to T1D.

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_17, Springer Science+Business Media New York 2012

347
348 Y.L. Lightfoot et al.

A decline in first-phase insulin release (FPIR) can be measured in

those at risk for developing T1D, subsequent to the measurement
of autoantibodies against beta cell antigens. Metabolic measures,
such as changes in circulating c-peptide (8) or FPIR (9), allow for
additional risk to be assessed. Reductions in markers of beta cell
function likely signal metabolic dysregulation in beta cells resulting
from the islet inflammation.
Prior to the advent of animal models that develop spontaneous
autoimmune diabetes, investigators seeking to destroy beta cells
and induce insulin-requiring diabetes would do so using alloxan or
streptozotocin (STZ). Both alloxan and STZ are free radical gen-
erators that selectively toxic to beta cells due to their structural
resemblance to glucose. These compounds gain entry into beta
cells via Glucose transporter 2 (GLUT2) [Fig. 1: #1] (10). Once
inside the beta cell, oxidative reactions occur with thiol-containing
enzymes such as glucokinase [Fig. 1: #1 and #2] and aconitase
resulting in impaired glucose sensing, mitochondrial dysfunction,
and necrotic cell death [Fig. 1: #3] (1114). The findings that
alloxan-induced beta cytotoxicity could be prevented by antioxi-
dants (1517) and that beta cell death resulting from exposure to
STZ could be partially prevented with superoxide dismutase (18)
were instrumental in our understanding of beta cell resistance to
both oxidative and immune-mediated stress. This led to the obser-
vation that in comparison to other tissues, beta cells had reduced
or absent activity of antioxidants (1924). The explanation for the
low levels of antioxidants in beta cell remains unclear, as upregula-
tion of antioxidants in beta cells does not have a significant impact
on glucose-stimulated insulin secretion (22, 2527). However, the
reduction of defenses to oxidative stress results in beta cells being
exquisitely sensitive to reactive oxygen species (ROS) damage
caused by inflammation.
There is a preponderance of evidence that beta cell dysfunction
can result from inflammation-induced oxidative stress. Free radi-
cal-mediated beta cell dysfunction and death can be due to either
murder (exogenous) or suicide (endogenous). The source of exog-
enous ROS or reactive nitrogen species (RNS) production is likely
activated macrophages, which are present in high numbers in the
preclinical insulitic infiltrates (28, 29). These cells act locally, releas-
ing high concentrations of highly ROS [e.g., superoxide ( SO 2 ),
hydroxyl radical (OH), nitric oxide (NO)]. Oxidative bursts
induced from macrophages activated in vitro via the xanthine oxi-
dase system (mainly generating NO) destroy cocultured islet cells
(30, 31). Cytotoxic effects of NO are thought to be mediated via
destruction of intracellular iron-containing enzymes, including
members of the Krebs cycles and the electron transport chain,
resulting in the reduction of energy metabolism (3235). The
impact of NO in reducing ATP production blunts insulin secretion
and induces necrosis [Fig. 1: #4] (36, 37). Potent tissue-damaging
 17 Oxidative Stress and Beta Cell Dysfunction 349

Fig. 1. Oxidative stress-induced beta cell dysfunction and death. (1) Alloxan (A) is a cytotoxic ROS generating glucose
analogue that preferentially accumulates in beta cells via the GLUT2 glucose transporter. Alloxan prevents glucose-
stimulated insulin secretion by inhibiting glucokinase activity, the enzyme responsible for the rate-limiting step of glucose
catabolism, as well as enzymes associated with mitochondrial ATP production. ROS are generated in a cyclic reaction
between alloxan and its reduced product, dialuric acid (AH2). Autoxidation of dialuric acid generates superoxide radicals

( O 2 ), hydrogen peroxide (H2O2), and, in the presence of a metal catalyst through the Fenton reaction, hydroxyl radicals
(OH). (2) Glutathione (GSH) is consumed within the cell for redox cycling, thereby producing oxidized glutathione (GSSG).
However, because beta cells display low glutathione reductase activity, beta cells are unable to maintain redox balance and
undergo necrotic cell death in response to ROS challenge. (3) Beta cells also exhibit low levels of the H2O2-inactivating
enzymes catalase and glutathione peroxidase. This contributes to the high susceptibility of beta cells to ROS. (4) Exogenous
Nitric Oxide (NO) production promotes beta cell dysfunction by preventing increases in ATP/ADP ratios through the inhibi-
tion of aconitase, a tricarboxylic acid (TCA) cycle enzyme, and electron transport chain complex 4. NO causes necrotic beta
cell death. (5) The combination of IL1 and IFN leads to the induction of NFB-responsive stress genes such as inducible
Nitric Oxide Synthase (iNOS) by the beta cell, further driving dysfunction and necrosis by endogenous NO production.
(6) When added together, IL1, IFN, and TNF cause changes in the mitochondrial membrane potential (m) and
mitochondrial outer membrane permeabilization (MOMP), which allows the release of pro-apoptotic proteins such as
cytochrome c (Cyt c) and subsequent activation of the caspase cascade via the apoptosome (Cyt c, Apaf-1, and Caspase
9). Ultimately, effector caspases (Caspase 3) activate Caspase-Activated Deoxyribonuclease (CAD), a DNase enzyme. DNA
cleavage promotes apoptotic cell death. (7) Mitochondrial ROS production induced by the interaction of TNF with TNFR1
or FasL with Fas contributes to Caspase 8 activation, potentiating the caspase cascade.

oxygen radicals can be derived from free cytosolic Fe2+ by the

Fenton reaction as well as through arachidonic acid metabolism,
destroying organelle membranes and membrane-associated
enzymes via lipid peroxidation (38).
350 Y.L. Lightfoot et al.

Additional cytotoxic free radicals in the prediabetic state are

generated within the beta cells themselves in response to cytotoxic
mixtures of monokines and lymphokines (3941). Islet-infiltrating
CD4+ and CD8+ T cells are the source of cytokines, particularly the
potent macrophage activator, interferon gamma (IFN). IFN
potentiates the effects of the monokines IL-1 and TNF, which
impair function of rodent and human beta cells individually, and
are highly cytotoxic when combined (39, 42, 43). This toxicity
mediated by the combined action of monokines and IFN has been
attributed to both the induction of NO synthase (iNOS) and the
subsequent production of NO (44, 45). Cytokines synergize to
activate NF-kB, the major transcription factor for iNOS expression
[Fig. 1: #5] (24). Use of transgenic mouse models has shown that
beta cell overexpression of iNOS and the ensuing elevation of islet
NO kill beta cells independent of insulitis (46) [Fig. 1: #4]. Recent
work suggests that the superoxide-producing phagocyte NADPH
Oxidase (NOX) is expressed in beta cells (4749). NOX can be
activated in response to proinflammatory cytokines resulting in
ROS production and beta cell damage and death (47). Cytokines
can also generate potent cytotoxic aldehyde moieties (malondial-
dehyde, butanal, pentanal, 4-hydroxynonenal, and hexanal) capa-
ble of lipid peroxidation (50), perhaps through the activation of
NOX or excessive mitochondrial ROS production.
In addition to the effect cytokines have in inducing beta cells
to produce ROS, these same molecules increase the potential for
beta cells to be surveyed by CD8+ cytotoxic T lymphocytes (CTLs).
IFN, in particular, increases the expression of the Major
Histocompatibility Complex (MHC) Complex I proteins on the
cell surface as well as Fas and adhesion molecules, such as ICAM1.
This predisposes the beta cells to CTL-mediated destruction by
FAS or perforin/granzyme B. A significant body of literature has
implicated a role for Fas in beta cell destruction that results in
T1D. When autoreactive CTLs recognize their cognate antigen in
the context of Class I MHC, these cells engage Fas Ligand with
Fas on the beta cell surface. This interaction activates Fas-mediated
apoptosis, which includes the activation of caspase 8 and then
downstream effector caspases, such as caspase 3 [Fig. 1: #6]. While
this pathway has not been completely detailed, it is known that
mitochondrial ROS production is an essential step in the activation
of caspase 8 [Fig. 1: #7] (51).
Clearly ROS are important mediators of beta cell dysfunction
in response to inflammation. However, recent evidence points to a
role for ROS in glucose-stimulated insulin secretion. Both phar-
macological inhibition of NOX as well as knockdown of the essen-
tial p47phox subunit resulted in a significant reduction of
glucose-stimulated insulin secretion (48, 49). Therefore, it is likely
that optimal beta cell function is only achieved at a redox balance
that is neither too reduced nor too oxidized.
 17 Oxidative Stress and Beta Cell Dysfunction 351

Our knowledge of the impact of ROS in beta cell function and

dysfunction is incomplete. The purpose of this chapter is to serve
as a guide to aid in designing experiments for exploring hypotheses
concerning ROS and the beta cell.

2. Materials

1. Surgical supplies: 16-, 18-, 20-, 22-, and 27-gauge needles,

10 mL syringes, 20-gauge blunt-end needle, Tygon R-3603
Laboratory Tubing (Saint-Gobain Performance Plastics),
Straight-tip Iris Scissors, Straight Bulldog Clamp, Angled
London Forceps (Fine Science Tools).
2. 50 mL Conical Tubes (BD Biosciences).
3. Hanks Balanced Salt Solution modified with calcium, with
magnesium, with phenol red (HBSS, Sigma), and supple-
mented with 1 % HEPES 1 M Buffer Solution (Sigma), 100 U/
mL Penicillin and 0.1 mg/mL Streptomycin Solution (Pen/
Strep; Gemini Bio-Products).
4. Collagenase P (Roche): 0.5 U/mL in HBSS.
5. Histopaque-1119 (Sigma), Histopaque-1100 [mix 5 Histopaque-
1119:1 HBSS (v/v)], Histopaque-1080 [mix 2 Histopaque-1119:
1 HBSS (v/v)], Histopaque-1060 [mix 1 Histopaque-1119:1
HBSS (v/v)].
6. Dissecting microscope with external light source.
7. 60 15-mm Falcon tissue culture dishes (BD Biosciences).
8. 96-Well flat and round bottom cell culture plates (Corning).
9. Very Complete High Glucose Dulbeccos Modified Eagles
Medium (VC-HG-DMEM): 16.7 mM D-glucose DMEM
(Sigma), 1 % Minimum Essential Medium Non-Essential
Amino Acids Solution 10 mM (MEM, Sigma), 1 % HEPES
1 M Buffer Solution (Sigma), 100 U/mL Penicillin and
0.1 mg/mL Streptomycin Solution (Pen/Strep, Gemini Bio-
Products), 5 105 M 2-Mercaptoethanol (2-ME, Sigma),
10 % Fetal Bovine Serum (FBS, Fisher Scientific).
10. Very Complete Low Glucose Dulbeccos Modified Eagles
Medium (VC-LG-DMEM): 5.5 mM D-glucose DMEM
(Sigma), constitute as above.
11. Phenol red-free Low Glucose DMEM: 5.5 mM D-glucose
DMEM without phenol red (Sigma), constitute as above.
12. 20 mM L-Arginine DMEM: VC-Low Glucose DMEM supple-
mented with 20 mM L-Arginine (Sigma).
13. 30 mM KCl DMEM: VC-Low Glucose DMEM supplemented
with 30 mM KCl.
352 Y.L. Lightfoot et al.

14. Very Complete Very Low Glucose Dulbeccos Modified Eagles

Medium (VC-VLG DMEM): 500 mL 0 mM Glucose DMEM
(Sigma), 7 mg Phenol Red, 1 g NaHCO3, 50 mg L-Glutamine,
500 mL VC-LG-DMEM.
15. Cytomix: 1,000 U/mL recombinant mouse IFN (Sigma),
1,000 U/mL recombinant mouse TNF (eBioscience),
100 U/mL recombinant mouse IL-1 (BioLegend).
16. NG-Methyl-L-arginine acetate salt (L-NMMA; Sigma).
17. tert-Butyl hydroperoxide solution (tBHP; Sigma).
18. Alloxan Monohydrate (AL; Sigma).
19. N-Acetyl-L-cysteine (NAC; Sigma).
20. Metalloporphyrin superoxide dismutase mimic (FBC-007;
kindly provided by Dr. Jon D. Piganelli, University of Pittsburgh
College of Medicine, Pittsburgh, PA).
21. AcidEthanol: 1.5 % 12 mM HCl, 75 % Ethanol, and 23.5 %
H2O.
22. 3 N NaOH: 12 g NaOH in 100 mL H2O.
23. ALPCO Insulin (Mouse) Ultrasensitive EIA (ALPCO
Diagnostics).
24. Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen).
25. 96-Well flat, clear bottom, black-wall cell culture plates
(Corning).
26. Thiazolyl Blue Tetrazolium Bromide Solution (MTT, Sigma):
5 mg/mL in Phosphate Buffered Saline prepared right before
using (PBS; Sigma).
27. AcidIsopropanol: 0.04 N HCl in isopropanol.
28. Spectrophotometer and fluorescent spectrophotometer:
SpectraMax M5 plate reader.
29. Caspase-3 and -8 Fluorometric Assay Kits (R&D Systems).
30. BCA Protein Assay kit (Pierce Chemical Co.)
31. ApoGlow Assay Kit (Lonza).
32. Zeiss Axioskop Fluorescent Microscope (Carl Zeiss, Inc.).
33. Allophycocyanin conjugated (APC) annexin V (Invitrogen).
34. Propidium iodide (PI; Invitrogen).
35. GCH-Glo Glutathione Assay (Promega).
36. Luminometer: SpectraMax M5 plate reader.
37. Orbital microplate shaker with 150900 rpm capabilities.
38. Griess Reagents and Standards: Greiss Reagent 1 (0.1 g N-1-
napthylethylenediamine dihydrocloride QS to 50 mL H2O),
Greiss Reagent 2 (1 g sulfanilamide, 5.9 mL of 85 % phosphoric
acid, QS to 50 mL H2O), make 1, 2, 4, 8, 16, 32, 64, 128 M
NaNO2 standards diluted in same media as samples to be tested.
 17 Oxidative Stress and Beta Cell Dysfunction 353

39. Oxidation sensitive fluorescent probes MitoSox Red

(Invitrogen) and 5-[(and-6)-chloromethyl-5-[and -6]-chlo-
romethyl-2, 7-dichloridihydrofluorescein diacetate, acetyl
ester (CM-H2DCFDA; Invitrogen).
40. Hydrogen peroxide solution (H2O2, Sigma).

3. Methods

The methods detailed here describe (a) isolation and culture of

mouse pancreatic islets with oxidative stress induction; (b) mea-
surement of stimulated insulin secretion and content normalized
to total DNA; (c) viability and caspase activity assays; (d) measure-
ment of intracellular ATP and ADP:ATP ratio; (e) luminescence-
based quantification of reduced glutathione (GSH); and (f)
detection of mediators of oxidative stress.

3.1. Islet Cell Isolation, 1. Perform common bile duct cannulation with a 27-gauge nee-
Culture, and Oxidative dle connected with tygon R-3603 laboratory tubing to a
Damage Induction 10 mL syringe containing the collagenase solution.
3.1.1. Isolation and Culture 2. Inflate pancreas with collagenase solution (2 mL per mouse); pre-
of Mouse Pancreatic Islets vent leakage into the intestine by using a straight bulldog clamp
to seal the entrance of the common bile duct into duodenum.
3. Remove pancreata and place no more than two in a 50 mL
conical tube.
4. Incubate in a 37 C water bath for 19 min.
5. Add 30 mL of HBSS and shake vigorously; mix by pipeting
with a 10 mL pipette several times.
6. Wash five times with HBSS by centrifugation at 200 g for
5 min.
7. Resuspend in 15 mL Histopaque 1119 and place in a clean
50 mL conical tube; use a 10 mL syringe with a 20-gauge
blunt-end needle for the gradient layering steps.
8. Gently layer on 10 mL of Histopaque 1100.
9. Gently layer on 10 mL of Histopaque 1080.
10. Gently layer on 10 mL of Histopaque 1060.
11. Centrifuge at room temperature for 18 min at 1,000 g.
12. Take caution as not to disturb the layers; islets will be between
the two middle layers.
13. Remove and wash these two layers with HBSS in a 50 mL con-
ical tube.
14. Hand pick for purity, place in tissue culture dishes containing
VC-LG-DMEM, and maintain at 37 C in a humidified atmo-
sphere with 5 % CO2.
354 Y.L. Lightfoot et al.
3.2. Induction
of Oxidative Stress
3.2.1. Treatment with
Cytomix and Inhibitors of
Oxidative Stress
3.2.2. Treatment with
Peroxide and Inhibitors
of Oxidative Stress
3.2.3. Treatment with
Alloxan and Inhibitors
of Oxidative Stress
1. Preculture islets for 24 h in VC-LG-DMEM.
2. Prepare fresh cytomix in VC-LG-DMEM at 2 final
concentration.
3. Prepare 10 mM L-NMMA solution in VC-LG-DMEM; final
concentration will be 5 mM.
4. Prepare 10 mM NAC solution in VC-LG-DMEM; final con-
centration will be 5 mM.
5. Prepare 68 M FBC-007 solution in VC-LG-DMEM; final
concentration will be 34 M.
6. Place ten islets per well in 100 L VC-LG-DMEM, L-NMMA,
NAC, or FBC-007 VC-LG-DMEM; seed triplicate wells per
treatment and control group.
7. Add an equal volume of VC-LG-DMEM (control) or 2
cytomix.
8. Incubate islets at 37 C, 5 % CO2 for the desired period of time
(24 h for functional assays, 36 or more hours for viability
assays) and harvest.
1. Preculture islets for 24 h in VC-LG-DMEM.
2. Prepare 0, 5, 10, and 50 M tBHP solutions in VC-LG-
DMEM.
3. Prepare 5 mM L-NMMA solution in VC-LG-DMEM.
4. Prepare 5 mM NAC solution in VC-LG-DMEM.
5. Prepare 34 M FBC-007 solution in VC-LG-DMEM.
6. Place ten islets per well; seed triplicate wells per treatment and
control group.
7. Incubate islets in tBHP solutions at 37 C, 5 % CO2 for 5 h.
8. Collect and wash free of tBHP with VC-LG-DMEM.
9. Culture in VC-LG-DMEM, L-NMMA, NAC, or FBC-007
VC-LG-DMEM for an additional 24 h and harvest.
1. Preculture islets for 6 days in VC-LG-DMEM; change into
fresh medium every 48 h.
2. Prepare a 1 M stock alloxan solution in PBS pH 2.0, and dilute
to target concentrations with culture medium. Prepare 0, 1, 2,
and 3 mM alloxan solutions.
3. Prepare 5 mM L-NMMA solution in VC-LG-DMEM.
4. Prepare 5 mM NAC solution in VC-LG-DMEM.
5. Prepare 34 M FBC-007 solution in VC-LG-DMEM.
6. Place ten islets per well; seed triplicate wells per treatment and
control group.
7. Incubate islets in alloxan solutions at 37 C, 5 % CO2 for 5 min.
 17 Oxidative Stress and Beta Cell Dysfunction 355

8. Collect and wash free of alloxan with culture medium.

9. Culture in VC-LG-DMEM, L-NMMA, NAC, or FBC-007
VC-LG-DMEM for 6 additional days with 48-h changes and
harvest.

3.3. Measurement 1. Place harvested islets in VC-VLG-DMEM for 1 h.

of Stimulated Insulin
Secretion and Content
3.3.1. Glucose-Stimulated
Insulin Secretion
2. Collect half volume of supernatant and freeze for testing at a
later time or test immediately.
3. Add same volume (half of original) of VC-HG-DMEM and
culture for 2 h.
4. Collect and freeze supernatant for testing at a later time or test
immediately.

3.3.2. Arginine-Stimulated 1. Place harvested islets in VC-VLG-DMEM for 1 h.

Insulin Secretion
2. Collect and freeze supernatant for testing at a later time or test
immediately.
3. Incubate in 20 mM L-Arginine DMEM for 2 h.
4. Collect and freeze supernatant for testing at a later time or test
immediately.

3.3.3. KCl-Stimulated 1. Place harvested islets in VC-VLG-DMEM for 1 h.

Insulin Secretion
2. Collect and freeze supernatant for testing at a later time or test
immediately.
3. Incubate in 30 mM KCl DMEM for 30 min.
4. Collect and freeze supernatant for testing at a later time or test
immediately.

3.3.4. Insulin Content
of Islet Cells
1. Incubate stimulated islets from above in acidethanol for 18 h
at 4 C.
2. Extract protein with TCA.
3. Neutralize with 3 N NaOH (10 L 3 N NaOH to 200 L
acidethanol) before testing for insulin and DNA
concentration.

3.3.5. Insulin ELISA Quantification of the stimulated insulin secreted as well as the
insulin content of the harvested islets is performed using the
ALPCO Insulin (Mouse) Ultrasensitive EIA Kit according to
the manufacturers instructions, and read with a SpectraMax M5
plate reader. Insulin standard curves are established in each experi-
ment; low and high controls should also be included. Samples
often need to be diluted in the range of 10 to 50; it is recom-
mended that the required dilution factors be established before
running all the samples.
356 Y.L. Lightfoot et al.
3.3.6. DNA Quantification
Assay
3.4. Measurement
of Intracellular ATP
and Relative ADP:
ATP Ratio
1. Pipette 5 L of each standard, control, or diluted sample into
assigned wells of the microplate strips.
2. Dispense 75 L of Enzyme Conjugate to each well.
3. Maintain at room temperature for 2 h while shaking at 800 rpm
on an orbital microplate shaker.
4. Wash microplate strips six times with Wash Buffer.
5. Add 100 L of Substrate to each well.
6. Incubate, while shaking at 800 rpm on an orbital microplate
shaker, for 30 min.
7. Pipette 100 L of Stop Solution to each well and mix gently.
8. Read absorbance at 450 nm with a reference wavelength of
620 nm. Yellow color intensity is directly proportional to the
protein concentration.
9. Calculate concentrations and normalize to DNA concentra-
tions determined below.
The Quant-iT PicoGreen dsDNA reagent system is used to quan-
tify the total DNA in acidethanol extracted samples, according to
the manufacturers instructions. Fluorescence is then read with a
SpectraMax M5 plate reader.
1. Prepare a low-range standard curve of dsDNA from 25 pg/
mL to 25 ng/mL using a sample of known concentration.
2. Add 100 L of standard curve or neutralized test sample to
their corresponding wells in a 96-well, clear bottom, black
plate.
3. Add 100 L of working solution of Quant-iT PicoGreen
reagent to each well.
4. Incubate for 5 min at room temperature in the dark.
5. Read fluorescence and calculate concentrations based on the
prepared standard curve.
6. Normalize insulin concentration measurements from above
per nanogram of DNA.
The ApoGlow Assay Kit employs the luciferase reaction, which uti-
lizes ATP to catalyze the oxidation of luciferin and generate light.
Thus light intensity is directly proportional to the ATP concentra-
tion. Conversion of ADP to ATP, and subsequent detection with
luciferase, allows the measurement of ADP levels in the test sam-
ples. The reactions are performed according to the manufacturers
instructions and luminescence read with a 1-s integrated reading
using a SpectraMax M5 plate reader.
1. Place 50 islets per well; seed triplicate wells per treatment and
control groups.
 17 Oxidative Stress and Beta Cell Dysfunction 357

2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-inducing

stimuli for the specified time period.
3. Prepare kit reagents as indicated and allow reagents and
samples to equilibrate to room temperature.
4. Pipette 100 L of nucleotide-releasing reagent into each test
well and incubate for 5 min.
5. Add 20 L of nucleotide-monitoring reagent and immediately
read luminescence (Reading A).
6. Read luminescence again after 10 min (Reading B).
7. Add 20 L of ADP-converting reagent and incubate for
5 min.
8. Read luminescence (Reading C).
9. Reading A corresponds to the amount of ATP and the
ADP:ATP ratio is calculated by (C B)/A.

3.5. Quantification GSH availability within the islets is detected and quantified with
of Glutathione the GSH-Glo Glutathione Assay kit following the manufacturers
in the Islet Cells instructions. The luminescence-based assay uses the GSH-
dependent conversion of a luciferin derivate to luciferin and the
latter is detected as described before.
1. Place 30 islets per well; seed triplicate wells per treatment and
control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
ing stimuli for the specified time period.
3. Prepare kit reagents, including standard curve, as indicated
and allow reagents and samples to equilibrate to room
temperature.
4. Harvest islets by centrifugation and collect the supernatant for
other studies.
5. Resuspend in 50 L of PBS and add to their corresponding
wells.
6. Add 50 L of GSH-Glo reagent 2 to each well and mix by
shaking at 150 rpm on an orbital microplate shaker for 30 s.
7. Incubate at room temperature for 30 min.
8. Pipette 100 L of luciferin detection reagent to each well and
mix by shaking at 150 rpm on an orbital microplate shaker for
30 s.
9. Incubate at room temperature for 15 min.
10. Read luminescence. The standard curve should demonstrate a
linear relationship between luminescence and GSH
concentration.
11. Convert relative luminescence units (RLUs) to M of GSH.
358 Y.L. Lightfoot et al.
3.6. Measurement
of Nitric Oxide
and Reactive Oxygen
Species Production
3.6.1. Nitrate Assay
3.6.2. MitoSox Red
and CM-H2DCFDA-Based
Detection of ROS
3.7. Viability
and Caspase Enzyme
Activity Assays
3.7.1. MTT Assay
Nitric oxide production within the islets is indirectly quantified by
measuring nitrite ( N O 2), a stable end product of NO, via the
Greiss Reagent System. Absorbance is read with a SpectraMax M5
plate reader.
1. The supernatants collected in Subheading 3.4, step 4, will be
used for analysis.
2. Add 100 L of sample media and standards (0, 1, 2, 4, 8, 16,
32, 64, 128 M NaNO2) to plate in duplicate.
3. Prepare a 1:1 solution of Greiss Reagent 1:Greiss Reagent 2.
4. Add 100 L of this solution to each well.
5. Read absorbance at 550 nm and calculate concentration.
1. Place 50 islets per well in a 96-well, clear bottom, black plate;
seed triplicate wells per treatment and control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
ing stimuli for the specified time period.
3. As positive control, include islet cells treated with 1.5 % H2O2
10 min before performing assay.
4. Harvest islets and wash with phenol red-free LG-DMEM.
5. Add MitoSox Red at a final concentration of 5 M or
CM-H2DCFDA at a final concentration of 10 M.
6. Allow dye loading for 30 min in the dark at 37 C, 5 % CO2.
7. Read MitoSox Red fluorescence with an excitation wavelength
of 520 nm and emission wavelength of 590 nm.
8. Read CM-H2DCFDA fluorescence with an excitation wave-
length of 488 nm and emission wavelength of 560 nm.
1. Place 100 islets per well; seed triplicate wells per treatment and
control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
ing stimuli for the specified time period in a total volume of
200 L.
3. Remove 20 L and add 20 L of MTT solution for a final
concentration of 0.5 mg/mL.
4. Incubate at 37 C, 5 % CO2 for 15 h to allow MTT to be
metabolized to insoluble formazan crystals.
5. Remove media and blot on paper towels.
6. Dissolve crystals with acidisopropanol by shaking at 150 rpm
on an orbital microplate shaker for 5 min.
7. Read absorbance at 560 nm and subtract background at
670 nm. Intensity of purple color is directly proportional to
live cell number.
 17 Oxidative Stress and Beta Cell Dysfunction 359

3.7.2. Annexin V and PI 1. Place 50 islets per well; seed triplicate wells per treatment and
Staining of Islet Cells control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
ing stimuli for the specified time period.
3. Harvest and wash in PBS.
4. Incubate for 10 min at 37 C in Ca2+- and Mg2-free PBS con-
taining 0.2 mg/mL EDTA to disperse into single cells.
5. Inject islet cells through 16- to 22-gauge needles to further
disperse islets into a single-cell suspension (16-, 18-, 20-,
22-gauge needles).
6. Incubate for 15 min with PI (10 g/mL) and APC-annexin V
(5 L) in the dark.
7. Visualize and estimate cell death using a Zeiss Axioskop
Microscope with fluorescence capabilities.

3.7.3. Caspase-8 and -3 Protein-based fluorometric assay kits are used in parallel to mea-
Fluorometric Assays sure caspase-8 and -3 activity inductions. Active caspases cleave
caspase-specific peptides conjugated to a fluorochrome, thereby
releasing the molecules that, when excited at 400 nm, fluoresce at
505 nm. Fluorescence is read with a SpectraMax M5 plate reader.
1. Place 1,000 islets per well; seed triplicate wells per treatment
and control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-inducing
stimuli for the specified time period.
3. Harvest islet cells and discard supernatant.
4. Add 60 L of cold lysis buffer.
5. Incubate on ice for 10 min.
6. Centrifuge at 10,000 g for 1 min and collect supernatant.
7. Determine protein concentration.

3.7.4. BCA Protein Assay 1. Prepare working reagent by mixing 50 parts of BCA Reagent
A with 1 part BCA Reagent B.
2. Prepare diluted albumin (BSA) standards with a concentration
range of 02,000 g/mL.
3. Remove 10 L of test sample from above and dilute to run in
triplicate wells.
4. Pipette 10 L of each standard or test sample into a well;
include two replicates for each.
5. Add 200 L of working reagent to each well and mix by shak-
ing at 150 rpm on an orbital microplate shaker for 30 s.
6. Incubate at 37 C for 30 min.
7. Cool plate to room temperature and read absorbance at 562 nm.
8. Calculate protein concentration and dilute in the range of
24 mg/mL using lysis buffer.
360 Y.L. Lightfoot et al.

3.7.5. Caspase Activity 1. Add 50 L of cell lysate (100200 g protein) to a 96-well,

Assay clear bottom, black plate.
2. Add 50 L of prepared 2 Reaction Buffers 8 or 3 to their cor-
responding wells.
3. Add 5 L of Caspase-8 or -3 fluorogenic substrates to the cor-
responding reaction wells.
4. Incubate at 37 C for 2 h.
5. Read fluorescence with an excitation wavelength of 400 nm
and emission wavelength of 505 nm. Analyze results as fold
activity increase over untreated samples.

References

1. Eizirik DL, Colli ML, Ortis F (2009) The role
of inflammation in insulitis and beta-cell loss in
type 1 diabetes. Nat Rev Endocrinol 5:
219226
2. Hanafusa T, Imagawa A (2008) Insulitis in
human type 1 diabetes. Ann N Y Acad Sci
1150:297299
3. Eisenbarth GS, Jeffrey J (2008) The natural
history of type 1A diabetes. Arq Bras Endocrinol
Metabol 52:146155
4. Pietropaolo M, Yu S, Libman IM, Pietropaolo
SL, Riley K, LaPorte RE et al (2005)
Cytoplasmic islet cell antibodies remain valu-
able in defining risk of progression to type 1
diabetes in subjects with other islet autoanti-
bodies. Pediatr Diabetes 6:184192
5. Verge CF, Gianani R, Kawasaki E, Yu L,
Pietropaolo M, Jackson RA et al (1996)
Prediction of type I diabetes in first-degree
relatives using a combination of insulin, GAD,
and ICA512bdc/IA-2 autoantibodies. Diabetes
45:926933
6. Wang J, Miao D, Babu S, Yu J, Barker J,
Klingensmith G et al (2007) Prevalence of
autoantibody-negative diabetes is not rare at all
ages and increases with older age and obesity. J
Clin Endocrinol Metab 92:8892
7. Huurman VA, Hilbrands R, Pinkse GG, Gillard
P, Duinkerken G, van de Linde P et al (2008)
Cellular islet autoimmunity associates with
clinical outcome of islet cell transplantation.
PLoS One 3:e2435
8. Schatz D, Cuthbertson D, Atkinson M, Salzler
MC, Winter W, Muir A et al (2004) Preservation
of C-peptide secretion in subjects at high risk
of developing type 1 diabetes mellitusa new
surrogate measure of non-progression? Pediatr
Diabetes 5:7279
9. Verge CF, Gianani R, Yu L, Pietropaolo M,
Smith T, Jackson RA et al (1995) Late progres-
sion to diabetes and evidence for chronic beta-
cell autoimmunity in identical twins of patients
with type I diabetes. Diabetes 44:
11761179
10. Lenzen S, Brand FH, Panten U (1988)
Structural requirements of alloxan and ninhy-
drin for glucokinase inhibition and of glucose
for protection against inhibition. Br J Pharmacol
95:851859
11. Lenzen S, Tiedge M, Panten U (1987)
Glucokinase in pancreatic B-cells and its inhibi-
tion by alloxan. Acta Endocrinol (Copenh)
115:2129
12. Lenzen S, Freytag S, Panten U (1988)
Inhibition of glucokinase by alloxan through
interaction with SH groups in the sugar-bind-
ing site of the enzyme. Mol Pharmacol 34:
395400
13. Lenzen S, Munday R (1991) Thiol-group reac-
tivity, hydrophilicity and stability of alloxan, its
reduction products and its N-methyl deriva-
tives and a comparison with ninhydrin. Biochem
Pharmacol 42:13851391
14. Lenzen S, Mirzaie-Petri M (1992) Inhibition
of aconitase by alloxan and the differential
modes of protection of glucose,
3-O-methylglucose, and mannoheptulose.
Naunyn Schmiedebergs Arch Pharmacol 346:
532536
15. Grankvist K, Marklund S, Sehlin J, Tljedal I-B
(1979) Superoxide dismutase, catalase and
scavengers of hydroxyl radicals protect against
toxic action of alloxan on pancreatic islet cells
in vitro. Biochem J 182:1725
16. Grankvist K, Marklund SL, Taljedal IB (1981)
CuZn-superoxide dismutase, Mn-superoxide
dismutase, catalase and glutathione peroxidase
in pancreatic islets and other tissues in the
mouse. Biochem J 199:393398
17. Grankvist K, Marklund S, Taljedal IB (1981)
Superoxide dismutase is a prophylactic against
alloxan diabetes. Nature 294:158160
 17 Oxidative Stress and Beta Cell Dysfunction
18. Asplund K, Grankvist K, Marklund S, Taljedal
IB (1984) Partial protection against streptozo-
tocin-induced hyperglycaemia by superoxide
dismutase linked to polyethylene glycol. Acta
Endocrinol (Copenh) 107:390394
19. Lenzen S, Drinkgern J, Tiedge M (1996) Low
antioxidant enzyme gene expression in pancre-
atic islets compared with various other mouse
tissues. Free Radical Bio Med 20:463466
20. Tiedge M, Lortz S, Drinkgeer J, Lenzen S
(1997) Relation between antioxidant enzyme
gene-expression and antioxidative defense sta-
tus of insulin-producing cells. Diabetes 46:
17331742
21. Tiedge M, Lortz S, Munday R, Lenzen S
(1998) Complementary action of antioxidant
enzymes in the protection of bioengineered
insulin producing rinm5f cells against the tox-
icity of reactive oxygen species. Diabetes 47:
15781585
22. Mathews CE, Leiter EH (1999) Constitutive
differences in anti-oxidant defense status dis-
tinguish Alloxan Resistant (ALR/Lt) and
Alloxan Susceptible (ALS/Lt) mice. Free
Radical Bio Med 27:449455
23. Mathews CE, Leiter EH (1999) Resistance of
ALR/Lt Islets to free radical mediated diabeto-
genic stress is inherited as a dominant trait.
Diabetes 48:21892196
24. Mathews CE, Suarez-Pinzon WL, Baust JJ,
Strynadka K, Leiter EH, Rabinovitch A (2005)
Mechanisms Underlying Resistance of Pancreatic
Islets from ALR/Lt Mice to Cytokine-Induced
Destruction. J Immunol 175:12481256
25. Chen H, Li X, Epstein PN (2005) MnSOD and
catalase transgenes demonstrate that protection
of islets from oxidative stress does not alter
cytokine toxicity. Diabetes 54:14371446
26. Li X, Chen H, Epstein PN (2006)
Metallothionein and catalase sensitize to diabe-
tes in nonobese diabetic mice: reactive oxygen
species may have a protective role in pancreatic
beta-cells. Diabetes 55:15921604
27. Mathews CE, Graser RT, Savinov AY, Serreze
DV, Leiter EH (2001) Unusual resistance of
ALR/Lt beta cells to autoimmune destruction:
Role for beta cell expressed resistance determi-
nants. Proc Natl Acad Sci 98:235240
28. Foulis AK, McGill M, Farquharson MA (1991)
Insulitis in type 1 (insulin-dependent) diabetes
mellitus in manmacrophages, lymphocytes,
and interferon-gamma containing cells. J
Pathol 165:97103
29. Foulis AK (1996) The pathology of the endo-
crine pancreas in type 1 (insulin-dependent)
diabetes mellitus. APMIS 104:161167
30. Kroncke KD, Kolb-Bachofen V, Berschick B,
Burkart V, Kolb H (1991) Activated mac-
361
rophages kill pancreatic syngeneic islet cells via
arginine-dependent nitric oxide generation.
Biochem Biophys Res Commun 175:752758
31. Schwizer RW, Leiter EH, Evans R (1984)
Macrophage mediated cytotoxicity against cul-
tured pancreatic islet cells. Transplantation
37:539544
32. Sandler S, Eizirik DL, Sternesjo J, Welsh N
(1994) Role of cytokines in regulation of pan-
creatic B-cell function. Biochem Soc Trans
22:2630
33. Corbett JA, McDaniel ML (1994) Reversibility
of interleukin-1 beta-induced islet destruction
and dysfunction by the inhibition of nitric oxide
synthase. Biochem J 299(Pt 3):719724
34. Scarim AL, Heitmeier MR, Corbett JA (1997)
Irreversible inhibition of metabolic function
and islet destruction after a 36-hour exposure
to interleukin-1beta. Endocrinology 138:
53015307
35. Augstein P, Heinke P, Salzsieder E, Grimm R,
Giebel J, Salzsieder C et al (2008) Dominance
of cytokine- over FasL-induced impairment of
the mitochondrial transmembrane potential
(Deltapsim) in the pancreatic beta-cell line
NIT-1. Diab Vasc Dis Res 5:198204
36. Steer SA, Scarim AL, Chambers KT, Corbett
JA (2005) Interleukin-1 Stimulates beta-Cell
Necrosis and Release of the Immunological
Adjuvant HMGB1. PLoS Med 3:e17
37. Hughes KJ, Chambers KT, Meares GP, Corbett
JA (2009) Nitric oxides mediates a shift from
early necrosis to late apoptosis in cytokine-
treated {beta}-cells that is associated with irre-
versible DNA damage. Am J Physiol Endocrinol
Metab 297:E1187E1196
38. Rabinovitch A, Suarez WL, Thomas PD,
Strynadka K, Simpson I (1992) Cytotoxic
effects of cytokines on rat islets: evidence for
involvement of free radicals and lipid peroxida-
tion. Diabetologia 35:409413
39. Rabinovitch A, Sumoski W, Rajotte RV,
Warnock GL (1990) Cytotoxic effects of cytok-
ines on human pancreatic islet cells in mono-
layer culture. J Clin Endocrinol Metab
71:152156
40. Nerup J, Mandrup-Poulsen T, Helqvist S,
Andersen HU, Pociot F, Reimers JI et al (1994)
On the pathogenesis of IDDM. Diabetologia
37(Suppl 2):S82S89
41. Corbett JA, McDaniel ML (1995) Intraislet
release of interleukin 1 inhibits beta cell func-
tion by inducing beta cell expression of induc-
ible nitric oxide synthase. J Exp Med 181:
559568
42. Rabinovitch A, Suarez-Pinzon WL, Sorensen
O, Bleackley RC (1996) Inducible nitric oxide
synthase (iNOS) in pancreatic islets of nonobese
362 Y.L. Lightfoot et al.
diabetic mice: identification of iNOS- expressing
cells and relationships to cytokines expressed in
the islets. Endocrinology 137:20932099
43. Rabinovitch A, Suarez-Pinzon W, El-Sheikh A,
Sorensen O, Power RF (1996) Cytokine gene
expression in pancreatic islet-infiltrating leuko-
cytes of BB rats: expression of Th1 cytokines
correlates with beta-cell destructive insulitis
and IDDM. Diabetes 45:749754
44. Mandrup-Poulsen T, Corbett JA, McDaniel
ML, Nerup J (1993) What are the types and
cellular sources of free radicals in the pathogen-
esis of type 1 (insulin-dependent) diabetes mel-
litus? Diabetologia 36:470471
45. Corbett JA, Kwon G, Turk J, McDaniel ML
(1993) IL-1 beta induces the coexpression of
both nitric oxide synthase and cyclooxygenase
by islets of Langerhans: activation of cyclooxy-
genase by nitric oxide. Biochemistry 32:
1376713770
46. Takamura T, Kato I, Kimura N, Nakazawa T,
Yonekura H, Takasawa S et al (1998) Transgenic
mice overexpressing type 2 nitric-oxide syn-
thase in pancreatic beta cells develop insulin-
dependent diabetes without insulitis. J Biol
Chem 273:24932496
47. Oliveira HR, Verlengia R, Carvalho CR, Britto
LR, Curi R, Carpinelli AR (2003) Pancreatic
beta-cells express phagocyte-like NAD(P)H
oxidase. Diabetes 52:14571463
48. Morgan D, Oliveira-Emilio HR, Keane D,
Hirata AE, Santos da Rocha M, Bordin S et al
(2007) Glucose, palmitate and pro-
inflammatory cytokines modulate production
and activity of a phagocyte-like NADPH oxi-
dase in rat pancreatic islets and a clonal beta cell
line. Diabetologia 50:359369
49. Newsholme P, Morgan D, Rebelato E, Oliveira-
Emilio HC, Procopio J, Curi R et al (2009)
Insights into the critical role of NADPH
oxidase(s) in the normal and dysregulated pan-
creatic beta cell. Diabetologia 52:24892498
50. Suarez-Pinzon WL, Strynadka K, Rabinovitch
A (1996) Destruction of rat pancreatic islet
beta-cells by cytokines involves the production
of cytotoxic aldehydes. Endocrinology 137:
52905296
51. Shen HM, Lin Y, Choksi S, Tran J, Jin T,
Chang L et al (2004) Essential roles of recep-
tor-interacting protein and TRAF2 in oxidative
stress-induced cell death. Mol Cell Biol
24:59145922
 Chapter 18

Experimental Autoimmune Encephalomyelitis

Praveen Rao and Benjamin M. Segal

Abstract
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the
central nervous system that is induced in laboratory animals by the generation of an immune response
against myelin epitopes. It has been used as a prototype of Th1- and/or Th17-driven, organ-specific auto-
immunity and as a model for the human disease, multiple sclerosis. In this chapter we describe two classic
protocols for EAE induction (active immunization and adoptive transfer of Th1- or Th17-polarized cells)
in Subheadings 3.1 and 3.2, respectively. Subheading 3.3 describes methods for rating clinical disease in
symptomatic animals. Subheading 3.4 includes instructions for the isolation of mononuclear cells from the
inflamed spinal cords of mice with EAE. Subheading 3.5 describes a method for performing the enzyme-
linked immunospot assay.

Key words: Experimental autoimmune encephalomyelitis, Active immunization, Adoptive transfer,

Proteolipid protein, Myelin basic protein, Myelin oligodendrocyte glycoprotein

1. Introduction

Experimental autoimmune encephalomyelitis (EAE) is an

inflammatory disease of the central nervous system (CNS) that is
induced in laboratory animals via the generation of an autoim-
mune response against proteins embedded in the myelin sheath, an
insulating covering around nerve fibers. The typical clinical course
is an ascending paralysis that correlates with inflammation and tis-
sue damage in the thoracolumbar regions of the spinal cord,
although the optic nerves and brain (particularly the subpial white
matter and brain stem) can also be affected (1). The classic patho-
logical features of EAE are (a) perivascular and subpial inflammatory
infiltrates (composed of lymphocytes, macrophages, and activated
microglia) and (b) adjacent areas of demyelination, characterized
by destruction of myelin with relative preservation of axons.
However, there is growing evidence for early axonal transection as

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_18, Springer Science+Business Media New York 2012

363
364 P. Rao and B.M. Segal

well, which might actually correlate more strongly with long-term

disability (2, 3).
EAE is widely used as an animal model of multiple sclerosis
(MS) due to the striking clinical and histopathological similarities
shared by the animal and human diseases. Indeed, many recent
advances in MS therapeutics, including the introduction of glati-
ramer acetate and anti-VLA-4 antibodies, arose from EAE studies
(47). However, beyond its usefulness as a model of MS, EAE is
arguably the best defined experimental model of Th1/Th17-driven
organ-specific autoimmunity. Many principles initially observed in
EAE have been extended to other models of autoimmune disease
with tissue targets as diverse as the joints (collagen-induced arthri-
tis), uvea, thyroid, and bowel. For example, the roles of CD4+
effector T cells and IL-12p40 monokines during the induction
phase, now recognized as critical in all of the above models, were
initially worked out in EAE (817).
In early versions of the EAE model, disease was induced using
spinal cord homogenate, myelin extracts, or whole myelin proteins
[such as myelin basic protein (MBP), proteolipid protein (PLP),
and myelin oligodendrocyte glycoprotein (MOG)] as the immu-
nogen. A wide spectrum of mammals was found to be susceptible,
including mice, rats, pigs, and nonhuman primates (18). However,
as the model has evolved it has become common practice to induce
the disease in well-defined inbred mouse strains by targeting single
MHC Class II-restricted myelin epitopes. A list of the most popu-
lar EAE-susceptible strains and corresponding encephalitogenic
peptides is provided in Tables 1 and 2.
From a practical standpoint, murine EAE holds many advan-
tages. With most of the current protocols, the disease is induced with
a high degree of incidence (80100 %) and reproducibility. Unlike
other models of autoimmune disease that take months to become
manifest, clinical signs begin within 516 days of induction.
Furthermore, different clinical courses simulating various subcatego-
ries of multiple sclerosis can be triggered based upon the particular
strain and autoantigen employed. For example, SJL mice immunized
with a peptide of PLP (PLP139151) or injected with PLP139151-specific
T cells exhibit a relapsing-remitting course, reminiscent of the most
common form of MS. By contrast, C57BL/6 mice actively immu-
nized against a peptide of MOG (MOG3555) develop a progressive
form of EAE, characteristic of later stages of MS.
EAE in the mouse can be triggered either by active immuniza-
tion or by the adoptive transfer of myelin-specific CD4+ T-cell lines.
Both protocols are described in detail in the following sections.
A detailed protocol for generating Th1- and Th17-polarized cells
is also included. This is followed by a description of a clinical scale
used to rate mice with EAE based on the severity of their neuro-
logical deficits. From an immunological standpoint, the encephali-
togenic T cell response has traditionally been measured by
 18 Experimental Autoimmune Encephalomyelitis 365

Table 1
Sequences of encephalitogenic peptides used to induce
EAE in susceptible mouse strains

Peptidea Sequence

PLP4364 EKLIETYFSKNYQDYEYLINVI
PLP103116 YKTTICGKGLSATV
b
PLP 139151
HSLGKWLGHPDKF
PLP185206 SIAFPSKTSASIGSLCADARMY
PLP180199 WTTCQSIAFPSKTSASIGSL
PLP215232 PGKVCGSNLLSICKTAEF
MBPAc111 Ac-ASQKRPSQRHG
MBP89101 VHFFKNIVTPRTP
MBP84104 VHFFKNIVTPRTPPPSQGKGR
MBP3547 TGILDSIGRFFSG
MOG92106 DEGGYTCFFRDHSYQ
MOG3555 MEVGWYRSPFSRVVHLYRNGK
a
References are provided in Table 2
b
In order to increase solubility, the synthetic peptide differs from the native sequence by
the substitution of Serine (S) for Cysteine (C) at position 140

Table 2
Peptides used to induce EAE in susceptible mouse strains

Mouse strain Protein Peptide Reference

SJL PLP 139151 (22)

MBP 89101 (23)
MBP 84104 (24)
C57 BL6 MOG 92106 (25)
MOG 3555 (26)
2D2 Tg MOG 3555 (26)
B10.PL MBP 3547 (27)
PLP 4364 (28)
MBP Ac 111 (29)
SWR PLP 215232 (30)
PLP 103116 (31)
C3H PLP 103116 (32)
BALB/c PLP 185206 (33)
PLP 180199 (33)
366 P. Rao and B.M. Segal

subjecting draining lymph node cells and/or splenocytes to

standard T cell assays, such as thymidine incorporation and IFN/
IL-2 ELISAs or ELISPOT. In the final section we chose to con-
centrate on methods that are more specific to the EAE model,
namely, the isolation and analysis of CNS-infiltrating leukocytes.

2. Materials

2.1. Active 1. Complete Freunds adjuvant (CFA) with killed Mycobacterium

Immunization tuberculosis H37Ra at 5 mg/ml. Supplement standard CFA
(Difco, Detroit, MI) containing 1 mg/ml M. Tuberculosis or
IFA (containing no bacterial products) with desiccated M. tuber-
culosis H37Ra (also available from Difco) to reach the desired
concentration.
2. Synthetic myelin peptides (see Table 2), at 90 % or greater
HPLC purity. Obtain in lyophilized form (Macromolecular
Resources, Fort Collins, CO) and store them in a well-sealed
desiccator at 20 C. For use, dissolve in sterile PBS at high
concentration (8 mg/ml). Sterile filter peptides with low-pro-
tein binding filters from Millipore (Cat # SLGV033RS) and
store at 80 C.
3. Phosphate-buffered saline (PBS).
4. 1, 5, or 10 cc glass syringes (Popper and Sons, Inc, New Hyde
Park, NY).
5. Four way plastic stopcock with swivel male Luer Lock (Smiths
medical ASD inc, Dublin, OH).
6. 1 ml plastic syringes (BD).
7. 25 and 27 gauge needles (BD).
8. Avertin. Stock solution (50) is prepared by dissolving
2,2,2-Tribromoethanol (Sigma, St. Louis, MO) in Tertiary Amyl
alcohol (5 g/6.5 ml). Store at 20 C in 0.51 ml aliquots.
9. Pertussis toxin (salt-free; List Biological Laboratories, Campbell,
CA). It is sold as a lyophilized powder. Reconstitute with PBS
to a final concentration of 2 g/ml and store at 4 C.
10. 812-week-old female mice (the most commonly used strains
are SJL, C57BL/6, and B10.PL) (Jackson Labs, Bar Harbor,
ME; NCI, Frederick, MD; Harlan laboratories). Mice should be
housed under specific pathogen-free conditions with routine test-
ing of sentinels for infections including mouse hepatitis virus.
11. 2 mm ear Punch (Roboz, Gaithersburg, MD).
Peptides should be sterile filtered following resuspension. PBS,
syringes, stopcock, and needles must be sterile.
 18 Experimental Autoimmune Encephalomyelitis 367

2.2. Lymph Node Cell 1. Tissue culture media (TCM) consisting of the following
Culture for Passive ingredients:
Transfer RPMI 1640 with L-glutamine (GIBCO BRL): 1,000 ml.
Fetal Bovine Serum (GIBCO BRL). Heat denature at 58 C
for 45 min and filter sterilize prior to use. Add 100 ml.
2-Mercaptoethanol (Sigma, St. Louis, MO). Prepare stock
at 5 105 M. Add 1 ml.
Sodium pyruvate (100 mM, GIBCO BRL). Add 10 ml.
Non-essential amino acid solution (NEEAS) (10 mM,
GIBCO BRL). Add 10 ml.
Penicillin/streptomycin solution (10,000 U of penicillin
and 10 mg of streptomycin/ml, GIBCO BRL). Add 10 ml.
HEPES (1 M, GIBCO BRL). Add 12.5 ml.
All of the above ingredients should be stored at 4 C except
for Penicillin/Streptomycin and fetal calf serum, both of which
should be stored at 20 C.
2. Sterile Hanks Balanced Salt solution (HBSS).
3. Sterile disposable cell strainers (70 m, Nylon; BD Falcon,
Millville, NJ).
4. Sterile ACK lysing buffer (Invitrogen).
5. 50 ml conical polypropylene centrifuge tubes.
6. Trypan Blue.
7. Hemocytometer.
8. 24-Well plates treated for tissue culture.
9. Synthetic myelin peptides as described above.

2.3. Spinal Cord 1. Peristaltic mini pump with variable flow, medium Flow Rate
Harvest and Isolation (4.085.0 ml/min) (Cat # 54856-075, VWR Scientific, West
of CNS Mononuclear Chester, PA).
Cells 2. Collagenase (Worthington Biochemical corp., Lakewood, NJ).
Prepare stock solution of 8 mg/ml in HBSS or RPMI. Store
aliquots of 5 ml at 80 C.
3. DNAse I (Sigma Chemical Corp, St. Louis, MO). Prepare stock
solution of 20 mg/ml in PBS. Store aliquots of 1 ml at 20 C.
4. Percoll (Amersham, Piscataway, NJ). Store at 4 C.
5. Sterile 15 and 50 ml polypropylene tubes.

2.4. ELISPOT Assay 1. Solutions:

Sterile PBS.
ELISPOT wash buffer (PBS + 0.05 % Tween-20).
PBS + 1 % BSA.
PBS + 0.05 % Tween-20 + 1 % BSA (PBS-TB).
368 P. Rao and B.M. Segal

2. Medium: TCM.
ELISPOT plates (Model MAIP N4550; Millipore, Billerica, MA).
3. Coating antibodies and biotinylated secondary antibodies for
each cytokine (eBioscience, San Diego, CA).
4. Streptavidin Alkaline phosphatase (Southern Biotech,
Birmingham, AL).
5. Vector Blue substrate solution (Vector Laboratories,
Burlingame, CA).
6. 100 mM TrisHCl, pH 8.2.
7. CTL ImmunoSpot Analyzer (Cellular Technology, Cleveland,
OH).

3. Methods

There are two basic approaches for induction of EAE: active immu-
nization and passive transfer. In active immunization, the entire
disease process, from autoreactive T cell priming to CNS infiltration
and demyelination, takes place in the same animal. Depending on
the mouse strain and myelin epitope, it is sometimes necessary to
inject the recipient with pertussis toxin to attain a high rate of inci-
dence and to synchronize the course between experimental sub-
jects. The mechanism of action of pertussis toxin is unknown, but
it is widely believed that it acts by increasing the permeability of the
bloodbrain barrier, thereby expediting migration of effector cells
into the brain and spinal cord (19).
Adoptive transfer allows the separation of the induction and
effector phases, which might be advantageous depending on the
experimental question(s) posed. Myelin peptide-primed lymph
node cells are reactivated with antigen in vitro for 96 h prior to
disease transfer. The in vitro stimulation step is critical for success-
ful disease transfer. Presumably it allows for the selective expansion
of myelin-reactive T cells and/or their terminal differentiation into
encephalitogenic effector cells.

3.1. Induction of EAE 1. Estimate the amount of peptide and CFA you will need:
by Active Irrespective of the specific model, we find that immunization
Immunization of mice with 100 g of myelin peptide in 100 l of an emul-
sion with CFA is sufficient for the reproducible induction of
EAE at high incidence. Therefore the total amount of peptide
you will need in microgram equals 100 n, where n is the
number of mice to be immunized. Dilute the appropriate
amount of peptide (from stock solution) with sterile PBS to a
final concentration of 1 g/l. To prepare the emulsion, CFA
 18 Experimental Autoimmune Encephalomyelitis 369

(5 mg/ml) is mixed with aqueous peptide solution in equal

parts, vol:vol. The final volume (in l) that you will need of the
emulsion is 100n.
2. Prepare the emulsion: For every 1 ml of emulsion, mix 500 l
of CFA, 375 l of PBS, and 125 l of stock peptide solution
(of concentration 8 mg/ml). Mix CFA and peptide solution
by repetitive passage between two glass syringes connected by
a stopcock after removing as much air from the two syringes as
possible. The syringes should be partly submerged in ice. The
luer lock should be adjusted to a position (usually at an angle
of about 45) where one feels sufficient resistance while push-
ing the emulsion from one syringe to another. At periodic
intervals test the emulsion by placing a drop in a Petri dish
containing PBS. If the droplet breaks up and comes apart,
continue mixing. If, on the other hand, the droplet remains
intact, the emulsion is ready for injection. Generally 2025 min
of mixing for 2 ml of emulsion is sufficient. The time needed to
make a good emulsion will increase with an increase in volume
and should be determined for each person making the emulsion.
Use an emulsion as soon as possible without keeping it on ice for
too long.
3. Load plastic syringes with emulsion: Pump all of the emulsion
into one of the glass syringes. Remove the empty glass syringe
from the stopcock and replace it with a plastic 1 cc syringe.
Slowly transfer emulsion to the plastic syringe. Repeat until all
of the emulsion is dispensed into plastic syringes.
4. Anesthetize mice: Dilute stock solution of Avertin in sterile
PBS (200 l in 10 ml). Vortex well. Heat to 56 C prior to use.
Inject i.p. at a dose of 250 mg/kg per mouse (or approxi-
mately 0.33 ml for a mouse weighing 20 g) using a 27 gauge
needle. Periodically assess the mouses level of consciousness
by toe pinch. The animal is properly anesthetized when she
fails to withdraw the limb. Mice should be anesthetized within
several minutes of the injection.
5. Immunize mice: Inject a total of 0.1 cc of emulsion/mouse in
the back s.c. using a 25 gauge needle. Distribute the immuno-
gen equally between four sites over the flanks immunizing as
closely as possible to the axillary nodes on the top and the
inguinal nodes on the bottom flanks.
6. Administer pertussis toxin systemically: Inject pertussis toxin
i.p. or i.v. (300 ng in 0.1 ml PBS per mouse) on days 0 and 2
post immunization using a 27 gauge needle. Note: Pertussis
toxin is required for EAE induction in some models of EAE
and not others. See Table 3 for more details.
7. Identify mice by ear punch.
370 P. Rao and B.M. Segal

Table 3
Requirementa of pertussis toxin in active immunization
models of EAE

Mouse strain/peptide Pertussis required Reference

SJL/PLP139151 Not required (28)

C57 BL6/MOG3555 Yes (20)
B10.PL/MBPAc111 Yes (9)
SWR/PLP103116 Yes (31)
C3H/PLP103116 Yes (34)
BALB/c/PLP185206 Yes (33)
BALB/c/PLP180199 Yes (33)
300 ng of pertussis toxin i.p (2 ng/l) on day 0. Similar dose on day 2
a

8. Place the mice on a paper towel to prevent asphyxiation due to

bedding and monitor the mice till they are fully awake. The
mice typically are fully awake within 40 min.
9. Score mice: Monitor mice on a daily basis for development of
neurological deficits starting on day 7 (see Subheading 3.3 for
details).
10. Please refer to notes 15 for further suggestions.

3.2. Induction of EAE
by Passive Transfer
(Fig. 1)
1. Immunize donor mice as described in Subheading 3.1 but do
not inject pertussis toxin.
2. Sacrifice mice between days 10 and 16 post immunization.
3. Harvest draining lymph nodes: Four axillary (the proper and
accessory axillary on each side) and two inguinal nodes under
aseptic conditions and place in HBSS.
4. Prepare a single-cell suspension by pressing the lymph node
cells through a cell strainer or mesh screen with a plunger from
a sterile 3 or 5 ml syringe. We use disposable nylon cell strain-
ers (70 M) that fit over a 50 ml conical tube. (Alternatively
use a 100-mesh screen from Fisher. Clean and flame sterilize the
screen prior to each use.) During the preparation of the suspen-
sion, periodically douse the strainer with 12 ml aliquots of
sterile HBSS to wash adherent cells through into the 50 ml
conical tube. Remove debris and connective tissue as they
accumulate on the screen with sterile forceps.
5. Once the single-cell suspension is finished, centrifuge the 50 ml
tube at 300 g at 4 C for 7 min.
6. Typically lymph node preparations do not contain significant
number of erythrocytes. In case there is a significant number
oft RBCs, resuspend the pellets in 7.5 ml of ACK lysing buffer
 18 Experimental Autoimmune Encephalomyelitis 371

Adoptive Transfer Protocol

1) Prime with myelin

peptide in CFA s.c. 2) Harvest draining LN Cells on day 10

3) Culture cells with myelin peptide x 96 hours

4) Inject cells into nave syngeneic mice i.p.

Mouse with EAE

Fig. 1. Diagrammatic illustration of the adoptive transfer protocol (described in

Subheading 3.2).

and let tube stand at RT for 12 min. Add 12.5 ml of media,

mix well, and centrifuge at 300 g at 4 C for 7 min.
7. Resuspend the pellet in 20 ml fresh HBSS and spin cells down
again. Repeat for two washes.
8. Count viable cells with a hemocytometer using trypan blue
exclusion.
Expected yield is 5070 106 cells/mouse.
9. Resuspend cells in TCM and dilute to a final concentration of
4 106 cells/ml. Add myelin peptide at a concentration of
50 g/ml. In certain models it is necessary to add recombi-
nant IL-12 or IL-23 to the cultures in order to confer enceph-
alitogenicity. This is not the case for SJL mice immunized with
PLP139151 in CFA. However it is the case for C57BL/6 mice
immunized with MOG3555 in CFA. The addition of polarizing
cytokines is also required in an alternative protocol in which
donor mice are immunized with peptide emulsified in IFA
rather than CFA. More details are provided below under #12.
10. Transfer the cell suspension to 24-well plates in 2 ml aliquots per
well. Incubate at 37 C in a 7.5 % CO2 tissue culture incubator.
11. After 96 h of culture, collect the cells in 50 ml tubes with a
sterile transfer pipette. Centrifuge at 300 g for 12 min. Discard
supernatants and resuspend each pellet in a relatively small
volume of HBSS. Pool cell suspensions together. Centrifuge
again and wash with HBSS 2.
12. If one wishes to polarize myelin-specific T cells to a Th1 or
Th17 phenotype, immunize SJL mice with the PLP139151
372 P. Rao and B.M. Segal

peptide in IFA or the C57BL6 mice with MOG3555 in CFA

without pertussis toxin. Wait 2 weeks for the SJL mice and
10 days for the C57BL6 mice before harvesting the lymph
node cells from these mice.
Use the following conditions for Th1 and Th17 polariza-
tion: For Th1 cells (5 ng/ml rmIL-12, 2 ng/ml rmIFN-,
10 g/ml IL-4 [clone A11B11], and 10 mg/ml IL-23 p19
[CNTO209]). For Th17 cells (5 ng/ml rmIL-23, 10 ng/ml
IL-1, 25 g/ml IFN- [AN18], and 10 g/ml IL-4). For
a control population of Th0 cells, culture with antigen under
neutral conditions (10 g/ml IL-12 p40 [C17.8]). Add
myelin peptide at a concentration of 50 g/ml.
13. Cells can be cultured at 45 106 cells/ml in 75 cm2 tissue
culture flasks in a volume of 3050 ml.
14. Count viable cells by trypan blue exclusion. Cell yield will be
6080 % of the cells at the start of the culture.
15. Spin down cells at 322 g at 4 C for 7 min. Resuspend the
pellet with PBS to a final concentration of 30 106 cells/0.2 cc.
Typically, CD4+ T cells are approximately 2030 % of the total
cell population at this stage.
16. Alternatively, a highly pure CD4+ T cell population can be
obtained at this step by using any CD4 cell purification kits that
are available (Cedarlane, Burlington, NC). Determine the purity
of the T cell preparation by flow cytometry and also keep some
cells aside to determine the phenotype of the polarized T cells
by ELISPOT assay (20). In case of purified T cells,
4 106 cells/0.2 cc can lead to the onset of peak EAE by
57 days. One may wish to titrate the cell numbers to prolong
the onset or severity of the EAE course according to ones needs.
17. Inject 0.2 cc of cell suspension per mouse intraperitoneally or
intravenously using a 1 cc plastic syringe attached to a 25 gauge
needle.
18. Examine mice on a daily basis starting on day 5 and score for
degree of neurological impairment (see Subheading 3.3). Animals
generally develop clinical signs between days 5 and 8 post cell
transfer. Mice that have received Th17-polarized cells typically
develop disease a day or two earlier than Th1 cell recipients.
19. Please refer to notes 610 for further suggestions.

3.3. Clinical 1. Observe the mouse as it ambulates on a smooth surface.

Assessment of Mice 2. If there is no obvious limb paresis, hold the mouse by the scruff
with EAE of the neck and note whether she spontaneously raises her tail.
It the tail does not move or is only lifted transiently, assess its
tone by flicking gently with your index finger. Please refer to
the video files linked with this section depicting mice at various
stages of EAE.
 18 Experimental Autoimmune Encephalomyelitis 373

3. Next, while holding the tail between thumb and index finger,
flip the animal on its back and time how long it takes her to
assume an upright position. A healthy mouse will turn herself
over immediately. A delay suggests hind limb weakness.
4. Place the mouse on a wire cage top or metal grid and observe
as she crosses from one side to the other. Pay particular atten-
tion to whether the hind limbs slip between the bars.
5. Observe for complete paralysis in the limbs. This observation
is critical to distinguish between a score of 3 and 4. Again,
please refer to the video files and observe partial hind limb
activity in the mouse with a score of 3.
Notice complete hind limb paralysis in one or both limbs
in a mouse with a score of 4.
6. Score according to the following 5 point scale:
0: Healthy mouse. No signs of neurological dysfunction.
1: Limp tail only. The tail remains flaccid when the mouse is
picked up.
2: Hind limb paresis, but without frank leg dragging. The
mouse fails the backflip test and has a waddling gait. When
placed on a wire cage top, the mouse frequently slips, with
one or more limbs falling in between the bars.
3: Partial hind limb weakness with one of both hind limbs
dragging, but some movement preserved.
4: Complete hind limb paralysis. (Depicted in Fig. 2.)
5: Moribund. The mouse is paralyzed in both hind limbs and
possibly one forelimb. Inevitably, there is weight loss.
Breathing appears labored.
Occasionally, mice develop EAE displaying signs of an atypical
form of the disease characterized by ataxia. This happens when
lesions develop in the brain stem and cerebellum. Such mice typi-
cally cannot right themselves, are tilted to one side with either
complete or partial paralysis of all limbs, and typically move around
in circles. It is seen sometimes in the active immunization model
but more typically when wild-type (WT) Th17- or Th17-polarized
IFN--deficient cells are used for adoptive transfer to WT recipi-
ents. Please refer to the video displaying symptoms of atypical EAE.
The scoring scale for atypical form of EAE is as follows (21):
0: Healthy.
1: Mouse partly tilted, feet fall into cage fence.
2: Tilted and tumbles.
3: Mouse heavily tilted and moves in circles.
4: Inability to walk, mouse is only rolling.
5. Moribund.
374 P. Rao and B.M. Segal

Fig. 2. Photograph of a mouse with EAE. The arrow points to a female SJL mouse 15 days
after injection with 50 106 PLP139151 reactive cells (according to the protocol described
in Subheading 3.2). At this point, the mouse had reached a clinical score of 4 (see
Subheading 3.3). Neurological signs initially presented as a flaccid tail on day 10 after cell
transfer and then evolved into hind limb paralysis. A nave, healthy littermate is shown for
the sake of comparison.

3.4. Isolation 1. Anesthetize mice with Avertin as described in Subheading 3.1,

of Mononuclear Cells but at double the dose (500 mg/kg).
from Inflamed Spinal 2. Perfuse mice with PBS by the intracardiac route using a peri-
Cords (Please See staltic pump set to low flow and low speed.
Video of This Protocol)
3. Remove the entire spinal column by gross dissection.
4. Fill a 10 cc syringe with PBS and attach an 18 gauge needle to
the tip.
5. Hold the spinal column with a forceps and insert the 18 gauge
needle at the caudal end.
6. Eject the spinal cord into a Petri dish filled with HBSS by
applying steady pressure on the plunger.
7. Prepare an enzyme cocktail composed of collagenase (5 ml
of stock solution), DNAse I (1 ml), and HBSS (14 ml).
8. Transfer the spinal cords to a Petri dish filled with the enzyme
cocktail and mince into small sections with a scalpel.
9. Incubate the dish at 37 C for 4560 min.
10. Prepare a 30 %/70 % Percoll gradient in 15 ml polystyrene
tubes (with 4 ml of each phase per tube) and leave at RT in the
tissue culture hood for the gradient to stabilize. Before preparing
the gradient, prepare the diluting solution by mixing 45 ml of
10 PBS, 3 ml of 0.6 N HCL, and 132 ml of water and adjust
 18 Experimental Autoimmune Encephalomyelitis 375

HBSS
Myelin debris

30 % Percoll
MNCs

70 % Percoll
RBCs/dead cells

Fig. 3. Isolation of mononuclear cells from spinal cords of mice with EAE using a Percoll
gradient (described in Subheading 3.4).

to pH 7.07.2. Filter sterilize before use. For preparing 70 %

Percoll, mix 63 ml of Percoll stock solution (density of 1.13 g/
ml) and 38.7 ml of diluting solution. For 30 % Percoll, mix
43 ml of 70 % Percoll solution with 57 ml of diluting solution.
Prepared Percoll at the required densities can be stored for
reuse at 4 C for short periods of time.
11. Draw the digested spinal cord mix up and down through a
large-bore needle several times in order to further dissociate
the CNS cells into a cell suspension.
12. Carefully overlay the cell suspension over the Percoll (45 ml
of suspension per tube).
13. Centrifuge at 1,085 g for 20 min at room temperature with
the brake disengaged. Red blood cells and dead cells settle at
the bottom of the tube and myelin debris at the 30 %:HBSS
interface. Collect MNCs at the 70 %:30 % interface (Fig. 3).
14. Centrifuge at 575 g, 4 C for 10 min.
15. Resuspend the pellet in 10 ml HBSS and transfer to a 15 ml
tube.
16. Centrifuge at 322 g, 4 C for 7 min.
17. Decant the supernatant and resuspend cells in 12 ml TCM.
18. Count viable cells by trypan blue exclusion. The cell yield
depends on the particular EAE model and the stage of disease
when mice are sacrificed. However, a reasonable estimate is
5 105 cells/cord (range 37 105 cells/cord).
19. Isolated cells can now be used for various studies including
flow cytometric analysis, RNA extraction for RT-PCR, and
ELISPOT assays.
20. Please refer to notes 1618 for further suggestions.
376 P. Rao and B.M. Segal

3.5. ELISPOT Assay 1. Day 1 (under sterile conditions): Dilute coating antibody to
3 g/ml in PBS, add 100 l/well, and leave the plate either at
RT for 2 h or at 4 C overnight.
2. Day 2 (under sterile conditions):
(a) Wash plate 3 with PBS (200 l/well), aspirate.
(b) Block with 200 l of PBS + 1 % BSA, 1 h RT.
(c) Wash plate 3 with PBS.
(d) Prepare 100 g/ml myelin antigen/mitogen in TCM
medium and add 100 l/well. The final concentration of
the peptide will be 50 g/ml.
(e) Resuspend MNC in TCM at 2 106 cells/100 l. Make
serial dilutions as desired.
(f) Add 100 l cell suspension/well to give a total volume
of 200 l. Add the cells with a steady hand in the center
of the well. This ensures a uniform distribution of
ELISPOTs.
(g) Do not mix the cells and peptide. Try to hold the plate as
steady as possible before placing it in the incubator. Incubate
at least for 24 h at 37 C. The incubation times will vary
according to the cytokine being analyzed.
3. Day 3 (can be carried out under non-sterile conditions from
here on):
(a) Wash plate 3 with PBS (200 l/well).
(b) Wash plate 3 with PBS-Tween (200 l/well).
4. Dilute biotinylated secondary antibody to 3 g/ml in PBS-
TBSA. Add 100 l/well and leave the plate either at RT for
2 h or at 4 C overnight.
5. Day 4:
(a) Wash plate 4 with PBS-Tween (200 l/well).
(b) Add StreptavidinAP (1:1,000) 100 l/well diluted in
PBS-TBSA. Leave the plate at RT for 2 h.
(c) Wash plate 3 with PBS (200 l/well).
(d) Add 100 l/well developer (two drops of each solution
per 5 ml 100 mM TrisHCl pH 8.2). Place the plate in a
dark spot and monitor spot formation. Do not let a
significant background develop as counting spots will
become a problem.
(e) Rinse plate with water, and let it air-dry completely before
reading in a counter.
6. Please refer notes 1922 for further suggestions.
 18 Experimental Autoimmune Encephalomyelitis 377

4. Notes

1. Peptides should be sterile filtered following resuspension in

PBS prior to storage. PBS, syringes, stopcock, and needles
must be sterile as well.
2. Prepare 1025 % more emulsion than actually required as some
volume is lost to dead space in the syringe tips and needles. In
addition, material is inevitably lost during the course of mixing
and transferring.
3. Vortex CFA rigorously before adding it to the glass syringe.
Mycobacterial particles settle to the bottom of the tube during
storage.
4. Attempt to remove air bubbles before mixing the emulsion by
tapping on the side of the glass syringe after the CFA and pep-
tide solution has been added to the barrel. Air bubbles can
hamper the emulsification process.
5. In the case of SJL mice immunized against PLP139151, injection
of pertussis toxin is not absolutely necessary for disease induc-
tion. Mice will still succumb to EAE, although at a slightly
lower incidence (7080 % as opposed to 90100 %) and in a
less synchronized manner.
6. Mix cells gently immediately before they are transferred to the
syringe for in vivo injection. This will help insure that a uni-
form number of cells is injected per mouse.
7. Cells should be injected slowly to prevent lysis. Leave the nee-
dle in for several seconds after the cells are dispensed prior to
withdrawal. This will help minimize leakage.
8. When designing experiments, be aware of the fact that you will
lose 2040 % of the cells that you started with by the end of the
96 h culture. This is due to the death of T cells that are not
specific for the myelin antigen as well as the turnover of other
(non-T) cell types.
9. If you want to avoid introducing Mycobacterial products in
the donor mice, you can use the myelin-specific Th1- or Th17-
polarized cells.
10. The pellet obtained after the ACK lysis buffer step typically is
hard to dissolve in TCM due to coagulated protein debris.
Resuspend as much of the pellet as possible keeping in mind
that the entire pellet may not be resuspended.
11. The clinical scale described in Subheading 3.3 is appropriate
for mice that experience the most common course of EAE,
namely, an ascending paralysis. This course correlates with
predominant spinal cord pathology. Occasionally a mouse will
378 P. Rao and B.M. Segal

develop a cerebellar lesion, manifested by a tilt in her posture

and/or gait. We rate an animal with such a deficit as a 3 (since
there is an obvious neurological sign, immediately apparent,
but not severe enough to interfere with essential activities). As
the disease progresses, the mouse may continually lie on its
side, unable to assume an upright stance or might even exhibit
repetitive rolling. Such a mouse is rated a 4 or a 5, based on
whether breathing and/or body weight are affected.
12. Our practice is to sacrifice mice before they progress to a score
of 5.
13. In addition to rating neurological deficits, some investigators
weigh mice on a daily basis and use weight loss as a surrogate
marker of EAE.
14. Typically when the mice reach a score of 3 or 4, they do not
have the ability to reach for food and moisture and may also
appear dehydrated. Take care to provide moist chow and ster-
ile PBS i.p (0.2 cc/mouse) at this stage.
15. It would be ideal to have blinded observers score the mice.
16. The enzyme cocktail can be made slightly in advance and kept
on ice. It should be warmed at 37 C immediately prior to
use.
17. Percoll solutions should be kept at room temperature prior to
use.
18. HBSS without calcium and magnesium is preferred for washing
as this is believed to result in higher cell yields.

19. The concentration of the primary and secondary antibodies for

each cytokine needs to be determined by each investigator.
20. The washing steps need to be done carefully without damag-
ing the membranes in the wells.
21. Ideally aim for a dilution of cells that will yield between 700
and 100 spots/well. Sometimes too many spots cannot be
counted by the ELISPOT counter.
22. Pay careful attention to the background as spots develop in the
plate.

References

1. Raine CS, Barnett LB, Brown A, Behar T,
McFarlin DE (1980) Neuropathology of exper-
imental allergic encephalomyelitis in inbred
strains of mice. Lab Invest 43(2):150157
2. Trapp BD, Peterson J, Ransohoff RM, Rudick
R, Mork S, Bo L (1998) Axonal transection in
the lesions of multiple sclerosis [Comment]. N
Engl J Med 338(5):278285
3. Wujek JR, Bjartmar C, Richer E, Ransohoff
RM, Yu M, Tuohy VK, Trapp BD (2002) Axon
loss in the spinal cord determines permanent
neurological disability in an animal model of
multiple sclerosis. J Neuropathol Exp Neurol
61(1):2332
4. Baron JL, Madri JA, Ruddle NH, Hashim G,
Janeway CA Jr (1993) Surface expression of
 18 Experimental Autoimmune Encephalomyelitis
alpha 4 integrin by CD4 T cells is required for
their entry into brain parenchyma. J Exp Med
177(1):5768
5. Keith AB, Arnon R, Teitelbaum D, Caspary
EA, Wisniewski HM (1979) The effect of Cop
1, a synthetic polypeptide, on chronic relapsing
experimental allergic encephalomyelitis in
guinea pigs. J Neurol Sci 42(2):267274
6. Teitelbaum D, Arnon R, Sela M, Abramsky O
(1989) Clinical trial of copolymer 1 in multiple
sclerosis. Harefuah 116(9):453456
7. Tubridy N, Behan PO, Capildeo R, Chaudhuri
A, Forbes R, Hawkins CP, Hughes RA, Palace
J, Sharrack B, Swingler R, Young C, Moseley
IF, MacManus DG, Donoghue S, Miller DH
(1999) The effect of anti-alpha4 integrin anti-
body on brain lesion activity in MS. The UK
Antegren Study Group.[comment]. Neurology
53(3):466472
8. Segal BM, Dwyer BK, Shevach EM (1998) An
interleukin (IL)-10/IL-12 immunoregulatory
circuit controls susceptibility to autoimmune
disease. J Exp Med 187(4):537546
9. Segal BM, Shevach EM (1996) IL-12 unmasks
latent autoimmune disease in resistant mice.
J Exp Med 184(2):771775
10. Segal BM, Chang JT, Shevach EM (2000) CpG
oligonucleotides are potent adjuvants for the
activation of autoreactive encephalitogenic T
cells in vivo. J Immunol 164(11):56835688
11. Tarrant TK, Silver PB, Chan CC, Wiggert B,
Caspi RR (1998) Endogenous IL-12 is required
for induction and expression of experimental
autoimmune uveitis. J Immunol
161(1):122127
12. Trembleau S, Penna G, Bosi E, Mortara A,
Gately MK, Adorini L (1995) Interleukin 12
administration induces T helper type 1 cells
and accelerates autoimmune diabetes in NOD
mice. J Exp Med 181(2):817821
13. Trembleau S, Germann T, Gately MK, Adorini
L (1995) The role of IL-12 in the induction of
organ-specific autoimmune diseases. Immunol
Today 16(8):383386
14. Neurath MF, Fuss I, Kelsall BL, Stuber E,
Strober W (1995) Antibodies to interleukin 12
abrogate established experimental colitis in
mice. J Exp Med 182(5):12811290
15. Moiola L, Galbiati F, Martino G, Amadio S,
Brambilla E, Comi G, Vincent A, Grimaldi
LM, Adorini L (1998) IL-12 is involved in the
induction of experimental autoimmune myas-
thenia gravis, an antibody-mediated disease.
Eur J Immunol 28(8):24872497
16. Matthys P, Vermeire K, Mitera T, Heremans H,
Huang S, Billiau A (1998) Anti-IL-12 antibody
prevents the development and progression of
379
collagen-induced arthritis in IFN-gamma
receptor-deficient mice. Eur J Immunol
28(7):21432151
17. Malfait AM, Butler DM, Presky DH, Maini
RN, Brennan FM, Feldmann M (1998)
Blockade of IL-12 during the induction of col-
lagen-induced arthritis (CIA) markedly attenu-
ates the severity of the arthritis. Clin Exp
Immunol 111(2):377383
18. Martin R, McFarland HF, McFarlin DE (1992)
Immunological aspects of demyelinating dis-
eases. Annu Rev Immunol 10:153187
19. Linthicum DS (1982) Development of acute
autoimmune encephalomyelitis in mice: factors
regulating the effector phase of the disease.
Immunobiology 162(3):211220
20. Kroenke MA, Carlson TJ, Andjelkovic AV,
Segal BM (2008) IL-12- and IL-23-modulated
T cells induce distinct types of EAE based on
histology, CNS chemokine profile, and response
to cytokine inhibition. J Exp Med
205(7):135141
21. Pollinger B, Krishnamoorthy G, Berer K,
Lassmann H, Bosl MR, Dunn R, Domingues HS,
Holz A, Kurschus FC, Wekerle H (2009)
Spontaneous relapsing-remitting EAE in the
SJL/J mouse: MOG-reactive transgenic T
cells recruit endogenous MOG-specific B cells.
J Exp Med 206(6):13031316
22. McRae BL, Miller SD (1994) Fine specificity of
CD4+ T cell responses to the dominant enceph-
alitogenic PLP 139151 peptide in SJL/J mice.
Neurochem Res 19(8):9971004
23. Sakai K, Zamvil SS, Mitchell DJ, Lim M,
Rothbard JB, Steinman L (1988)
Characterization of a major encephalitogenic T
cell epitope in SJL/J mice with synthetic oligo-
peptides of myelin basic protein. J Neuroimmunol
19(12):2132
24. Tan LJ, Kennedy MK, Miller SD (1992)
Regulation of the effector stages of experimental
autoimmune encephalomyelitis via neuroantigen-
specific tolerance induction. II. Fine specificity
of effector T cell inhibition. J Immunol
148(9):27482755
25. Amor S, Groome N, Linington C, Morris MM,
Dornmair K, Gardinier MV, Matthieu JM,
Baker D (1994) Identification of epitopes of
myelin oligodendrocyte glycoprotein for the
induction of experimental allergic encephalomy-
elitis in SJL and Biozzi AB/H mice. J Immunol
153(10):43494356
26. Rainey-Barger EK, Rumble JM, Lalor SJ, Esen
N, Segal BM, Irani DN (2010) The lymphoid
chemokine, CXCL13, is dispensable for the ini-
tial recruitment of B cells to the acutely inflamed
central nervous system. Brain Behav Immun
(in press)
380 P. Rao and B.M. Segal
27. Zamvil SS, Mitchell DJ, Powell MB, Sakai K,
Rothbard JB, Steinman L (1988) Multiple dis-
crete encephalitogenic epitopes of the autoan-
tigen myelin basic protein include a determinant
for I-E class II-restricted T cells. J Exp Med
168(3):11811186
28. Whitham RH, Bourdette DN, Hashim GA,
Herndon RM, Ilg RC, Vandenbark AA, Offner
H (1991) Lymphocytes from SJL/J mice immu-
nized with spinal cord respond selectively to a
peptide of proteolipid protein and transfer relaps-
ing demyelinating experimental autoimmune
encephalomyelitis. J Immunol 146(1):101107
29. Zamvil SS, Mitchell DJ, Moore AC, Kitamura
K, Steinman L, Rothbard JB (1986) T-cell
epitope of the autoantigen myelin basic protein
that induces encephalomyelitis. Nature
324(6094):258260
30. Endoh M, Rapoport SI, Tabira T (1990) Studies
of experimental allergic encephalomyelitis in old
mice. J Neuroimmunol 29(13):2131
31. Montgomery NI, Rauch HC (1982)
Experimental allergic encephalomyelitis (EAE)
in mice: primary control of EAE susceptibility
is outside the H-2 complex. J Immunol
128(1):421425
32. Tuohy VK, Sobel RA, Lees MB (1988) Myelin
proteolipid protein-induced experimental aller-
gic encephalomyelitis. Variations of disease
expression in different strains of mice. J Immunol
140(6):18681873
33. Lyons JA, Ramsbottom MJ, Trotter JL,
Cross AH (2002) Identification of the
encephalitogenic epitopes of CNS proteo-
lipid protein in BALB/c mice. J Autoimmun
19(4):195201
34. Sobel RA, Tuohy VK, Lees MB (1991) Parental
MHC molecule haplotype expression in
(SJL/J SWR) F1 mice with acute experimen-
tal allergic encephalomyelitis induced with two
different synthetic peptides of myelin proteo-
lipid protein. J Immunol 146(2):543549
 Chapter 19

Mouse Models of Multiple Sclerosis: Experimental

Autoimmune Encephalomyelitis and Theilers
Virus-Induced Demyelinating Disease
Derrick P. McCarthy, Maureen H. Richards, and Stephen D. Miller

Abstract
Experimental autoimmune encephalomyelitis (EAE) and Theilers Murine Encephalitis Virus-Induced
Demyelinating Disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS).
Like MS, both are characterized by mononuclear cell infiltration into the CNS and demyelination. EAE is
induced by either the administration of myelin protein or peptide in adjuvant or by the adoptive transfer
of encephalitogenic T cell blasts into nave recipients. The relative merits of each of these protocols are
compared. Depending on the type of question being asked, different mouse strains and peptides are used.
Different disease courses are observed with different strains and different peptides in active EAE. These
variations are also addressed. Additionally, issues relevant to clinical grading of EAE in mice are discussed.
In addition to EAE induction, useful references for other disease indicators such as DTH, in vitro prolif-
eration, and immunohistochemistry are provided. TMEV-IDD is a useful model for understanding the
possible viral etiology of MS. This section provides detailed information on the preparation of viral stocks
and subsequent intracerebral infection of mice. Additionally, virus plaque assay and clinical disease assess-
ment are discussed. Recently, recombinant TMEV strains have been created for the study of molecular
mimicry which incorporate various 30 amino acid myelin epitopes within the leader region of TMEV.

Key words: Multiple sclerosis, Experimental autoimmune encephalomyelitis, EAE, Emulsion, Active
induction, Adoptive transfer, T cell blasts, Encephalitogenic, Neurodegeneration, Theilers murine
encephalomyelitis virus-induced demyelinating disease, PLP, MOG, Myelin, MBP, VP2, VP3,
Relapsingremitting, Epitope spreading

1. Introduction

Mouse models of demyelinating diseases have been useful in both

the demonstration of T cell-mediated demyelination and in the
characterization of the pathogenesis of immune-mediated demyeli-
nating disease. This chapter describes the methods for inducing and
characterizing two models of demyelinating disease: experimental

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_19, Springer Science+Business Media New York 2012

381
382 D.P. McCarthy et al.

autoimmune encephalomyelitis (EAE) and Theilers murine

encephalomyelitis virus-induced demyelinating disease (TMEV-IDD).
EAE is a CD4+ T cell-mediated, demyelinating autoimmune
disease of the CNS that is characterized by mononuclear cell
infiltration. EAE serves a useful animal model for multiple sclerosis
(MS), since many of the pathologies observed in the CNS of mice
with EAE bear strong similarity to those found in the CNS of MS
patients (19). In both EAE and MS, the white matter of the CNS
presents with demyelinating lesions associated with infiltrating T
cells, macrophages, and B cells (1015). In addition, foam cell-like
macrophages containing phagocytosed hydrophobic myelin debris
have been demonstrated within active lesions (1618). Ascending
hind limb paralysis (described in Subheading 3.1.3, below) is asso-
ciated with inflammation and demyelination of axonal tracks.
Finally, oligoclonal IgG can be found in the CSF of both EAE mice
and MS patients (2, 7, 19).
There are two widely used methods for inducing EAE in mice:
active induction by immunization with myelin antigens and passive
induction by the adoptive transfer of pre-activated myelin-specific
T cells into nave mice. In active EAE, peripheral immunization of
mice with myelin antigen(s) results in the breakdown of peripheral
tolerance and the activation of myelin antigen-specific T cells in the
secondary lymphoid organs. Following activation, myelin-specific
T cells proliferate and differentiate into effector cells, allowing
egress from the secondary lymphoid organs. The expression of
integrins by effector T cells enables them to cross the bloodbrain
barrier (20), where they are reactivated by CNS-resident APCs pre-
senting myelin antigens (21). Reactivation leads to the expression
of pro-inflammatory cytokines by the effector T cells (IFN-, IL-17,
GM-CSF, and TNF-), some of which can directly injure nervous
tissue. In addition, chemokine production by the pathogenic T cells
induces recruitment of nonspecific cellular effectors such as
T cells, monocytes, macrophages, and neutrophils into the CNS
(22, 23). Activation of these inflammatory cells and the bystander
damage they mediate are largely responsible for destruction of the
myelin-sheathed axonal tracts and the formation of lesions.
The effector phase of EAE can be directly induced by the
adoptive transfer of activated, myelin-specific Th1 or Th17 cells
from immunized donors into nave syngeneic recipients (2426).
Although the clinical features of disease induced by passive EAE
are identical to those induced by active EAE, and more reagents
are required, passive EAE has numerous advantages over active
induction since (a) the day of adoptive transfer serves as a definitive
point of introducing encephalitogenic T cells to the recipient mice;
(b) there is no antigen depot to present leading to continuous
de novo activation of nave T cells; and (c) it can be used to track
encephalitogenic T cells in vivo; (d) to study CNS infiltration, and
19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 383

(e) isolate antigen-specific T cells from the CNS (27, 28). Passive
EAE induction is a valuable tool for delineating the relative contri-
butions of T helper subsets in disease, and it is considered to be a
more direct way of characterizing T cell effector function in the
CNS. In the SJL/J mouse, both active induction and adoptive
transfer of disease typically take a relapsingremitting form, while
the C57BL/6 mouse displays chronicprogressive disease following
active or passive EAE induction.
TMEV-IDD has been defined as a mouse model for human
multiple sclerosis (29, 30). TMEV is a natural mouse pathogen
that belongs to the cardiovirus group of the Picornaviridae family
(31, 32), and is composed of a single, positive-strand RNA genome
surrounded by a capsid containing viral proteins, VP1, VP2, and
VP3. TMEV is divided into two subgroups based on the patho-
genesis of the viruses. The first subgroup, which includes GDVII,
is highly virulent and induces fatal encephalitis in infected mice.
The second group, which is defined as the Theilers original subgroup,
includes Daniels (DA) and BeAn 8386 strains that have low viru-
lence and do not induce severe encephalitis, but do establish per-
sistent infections of the CNS associated with immune-mediated
demyelination (33).
TMEV-IDD is an immune-mediated demyelinating disease
dependent on persistent virus infection of the macrophages, micro-
glia, and astrocytes within the CNS (34, 35). TMEV-IDD is asso-
ciated with a mononuclear cell infiltrate consisting predominantly
of CD4+ T cells, macrophages, and B cells. The chronic phase of
TMEV-IDD is mediated by a PLP139151-specific CD4+ Th1 type T
cell response that can be initially detected at approximately
4555 days post-infection (36). As the disease progresses, epitope
spreading leads to autoimmune responses to additional myelin
antigens (37). The inflammation and demyelination observed in
TMEV-IDD are similar to the pathological descriptions in MS
patients (38, 39). Importantly, epidemiological studies suggest a
viral etiology for MS providing additional importance for TMEV-
IDD as a relevant model for multiple sclerosis (40, 41). Infection
of SJL/J mice with the BeAn strain has been directly associated
with the development of a chronicprogressive demyelinating
disease arising approximately 3035 days post-infection characterized
by spastic hind limb paralysis and primary demyelination (42).
In this article, we address the basic methods for inducing both
active and adoptive transfer of EAE and the induction of TMEV-
IDD. While different mouse strains have various susceptibilities to
both models of disease, we focus primarily on the induction in the
most commonly used strains of mice, the SJL/J and C57BL/6.
Additionally, the induction of TMEV-IDD is described using the
BeAn strain of TMEV in the SJL/J mouse. Mouse strain susceptibili-
ties and variation in disease are discussed in the Notes section 5.
384 D.P. McCarthy et al.

2. Materials

2.1. Induction 1. SJL/J or C57BL/6 mice (Harlan Laboratories).

of Active EAE 2. Encephalitogenic protein or peptide or spinal cord
homogenate.
3. Phosphate-Buffered Saline (PBS).
4. Mycobacterium tuberculosis, H37 RA (Difco).
5. Incomplete Freunds Adjuvant (Bacto).
6. 3-Way nylon stopcock (Luer connector) (Kontes Glass Co) or
Sorvall Omni-Mixer (Dupont Instruments).
7. 5-ml snap cap tubes (Falcon).
8. Small animal clippers (Golden A5, Oster).
9. 18 and 25 gauge needles (Becton Dickinson).
10. Pertussis toxin (List Biological Labs).
11. Carbol fuchsin dye.
12. Ear tags (Gey band and tag).

2.2. Induction 1. SJL/J or C57BL/6 mice (Harlan Laboratories).

of Passive EAE 2. Balanced salt solution (BSS).
3. Dulbeccos modified Eagles medium (DMEM).
4. Heat-inactivated fetal calf serum (FCS).
5. 200 mM L-glutamine.
6. 5.5 mM 2-mercaptoethanol.
7. 1,000 U/ml penicillin, 1,000 g/ml streptomycin.
8. Light microscope and hemocytometer.
9. 75 cm2 sterile tissue culture flasks.
10. Myelin antigen or myelin peptide.
11. 10 ml sterile pipette.
12. 100 gauge sterile wire mesh and 90 mm sterile Petri dishes.
13. 3 and 10 ml syringes.
14. 25 gauge needle.

2.3. Induction 1. DMEM (Sigma).

of TMEV-IDD 2. FCS (Sigma).
3. Tryptose phosphate broth (Sigma).
4. Antibioticantimycotic (Gibco BRL).
5. BHK-21 cells (ATCC).
6. 25-, 75-, 162-cm2 tissue culture flasks (Corning).
7. Versene 1:5,000 (Gibco BRL) or TrypsinEDTA (Sigma).
 19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 385

8. Centrifuge tubes.
9. Polyethylene glycol (PEG) (Sigma).
10. Tris base (Sigma).
11. Sodium chloride, NaCl (Sigma).
12. Sodium dodecyl sulfate, SDS (Sigma).
13. Sucrose (Sigma).
14. 21-, 23-, and 27-gauge needles.
15. Cesium sulfate, Cs2SO4 (Sigma).
16. PBS.
17. 60 mm tissue culture dishes (Nunc).
18. Noble agar (Sigma).
19. Penicillin/streptomycin (Life technologies).
20. Crystal violet (Sigma).
21. ClaI restriction enzyme (Promega).
22. DH5 max efficient E. coli (Invitrogen).
23. Sp6/T7 in vitro transcription kit (Roche).
24. Lipofectin reagent (Gibco BRL).
25. SJL/J mice (Harlan Laboratories).

3. Methods

3.1. Induction
of Active
and Passive EAE
3.1.1. Induction
of Active EAE Disease
EAE can be induced in susceptible strains of mice by using proteo-
lipid protein (PLP), myelin basic protein (MBP), myelin oligoden-
drocyte glycoprotein (MOG), or peptides corresponding to the
encephalitogenic portions of these proteins. Peptides (>98 % purity
based on mass spectrophotometry) or spinal cord homogenate to
be used in priming are first dissolved in PBS and irradiated at
6,000 rads for sterilization purposes. For peptides that are insolu-
ble in PBS (pH 7.0), the pH can be raised until the peptide dis-
solves. The pH can then be lowered to physiologic levels; however,
pH has little influence on disease induction with peptide in CFA.
Peptide should be diluted in PBS to a concentration of 1 mg/ml if
inducing disease with PLP139151 in the SJL/J mouse. MOG3555-
induced disease in the C57BL/6 mouse requires a concentration
of 4 mg/ml in PBS as do all other peptides used to induce C57BL/6
or SJL/J disease. An equal volume of peptide/PBS is added to
complete Freunds adjuvant (containing 4 mg/mldesiccated M.
tuberculosis, H37 RA in Incomplete Freunds Adjuvant). This is
then thoroughly mixed to form a thick peptide/CFA emulsion.
For small volumes, the emulsion can be prepared directly between
two 1 ml tuberculin syringes using a 3-way stopcock with a Luer
386 D.P. McCarthy et al.

Table 1
Mouse strain and encephalitogenic peptides in active EAE

Mouse strain H-2 type Peptidea Sequencea Reference

SJL/J H-2s MBP89101 VHFFKNIVTPRTP (57)

MBP84104 VHFFKNIVTPRTPPPSQGKGR (4)
PLP139151b HSLGKWLGHPDKF (55, 58)
PLP104117 KTTICGKGLSATVT (58)
PLP178191 NTWTTCQSIAFPSK (59)
PLP5770 YEYLINVIHAFQYV (60, 61)
MOG92106 DEGGYTCFFRDHSYQ
PL/J, B10.PL H-2u MBPAc111 Ac-ASQKRPQRHG (62)
PLP178191 NTWTTCQSIAFPSK Unpublished (B10.PL)
MBP3547 TGILDSIGRFFSG (62)
PLP4364 EKLIETYFSKNYQDYEYLINVI (63)
(PL/J SJL/J) H-2s/u MBPAc111 Ac-ASQKRPQRHG (62)
F1 PLP4364 EKLIETYFSKNYQDYEYLINVI (63)
PLP139151 HSLGKWLGHPDKF (55)
C57BL/6 H-2b MOG3555 MEVGWYRSPFSRVVHLYRNGK (64)
PLP178191 NTWTTCQSIAFPSK (65)
C3H H-2k PLP103116 YKTTICGKGLSATV (66)
SWR H-2q PLP215232 PGKVCGSNLLSICKTAEF (67)
s/q
(SJL/J B10.PL) H-2 PLP139151 HSLGKWLGHPDKF Unpublished
F1 PLP178191 NTWTTCQSIAFPSK Unpublished
MBPAc111 Ac-ASQKRPQRHG Unpublished
(SJL/J C3H/ H-2s/k PLP190209 SKTSASIGSLCADARMYGVL (68)
HeJ)F1c PLP215232 PGKVCGSNLLSICKTAEFQ (60)
BALB/cPtc H-2d PLP178191 NTWTTCQSIAFPSK (59)
g7
NOD H-2 PLP4870 TYFSKNYQDYEYLINIHAFQYV (69)
MOG3555 MEVGWYRSPFSRVVHLYRNGK (70)
a
Sequences for MBP peptides are based on different species variants of MBP, which have different number-
ing systems; sequences for PLP peptides are based on the mouse sequence. The reader is urged to consult
the indicated references for more detailed information
b
The PLP139151 sequence has a serine (S) for cysteine (C) substitution at position 140 to enhance
solubility
c
The EAE observed in these mice is nonclassical. In (SJL/J C3H/HeJ)F1 mice, the disease causes imbal-
ance and axial rotatory movement (rotatory EAE). In BALB/cPt, mice show lack of balance and forelimb
paralysis in the absence of hindlimb paralysis

connector. For larger volumes, the emulsion can be prepared using

a mechanical mixer with the emulsion on ice. Emulsion should be
removed from the mixer with a standard lab spatula and placed in
a 5-ml snap cap tube. The emulsion is then gently centrifuged. The
emulsion should be loaded into 1 ml tuberculin syringes using an
18 gauge needle taking care not to introduce air bubbles into the
syringe. The 18 gauge needle is replaced with a 27 gauge needle
for immunization. Table 1 provides a comprehensive list of
 19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 387

different peptides of PLP, MBP, and MOG proteins which can be

used to initiate disease in different inbred mouse strains.
The backs of animals to be primed are shaved using small animal
clippers. Ideally, animals receive approximately 100 l of emulsion
subcutaneously divided equally across three sites on the dorsal
flank (on each hip and one along the midline of the back between
the shoulders) using a 27 gauge needle. If inducing EAE in the
C57BL/6 strain or R-EAE in the SJL/J strain with MBP or MOG
antigens, 200 ng of pertussis toxin must be administered intraperi-
toneally (i.p.) on days 0 and 2 relative to immunization. As neces-
sary, animals should be marked for grading purposes. White and
light brown mice can be marked with a red dye (carbol fuchsin).
We routinely mark mice with large spots on the head, midback,
base of tail, and left and right sides. For black mice, one can mark
the tails with permanent markers, e.g., Sharpie, and reapply as nec-
essary (one, two, three stripes, etc.) or use numbered ear tags.

3.1.2. Induction Eight- to twelve-week-old mice are immunized as described above

of Passive EAE for active induction of EAE (24, 25). In some cases, such as the
adoptive transfer of myelin-specific transgenic T cells and encepha-
litogenic T cell lines maintained by in vitro passage, this step is not
necessary (see Notes). Once immunized, the mice are left for
714 days, as described for different models in Notes. The ratio of
donor mice to recipient mice varies according to how many
activated cells are required for adoptive transfer (see Table 2).
Typically, for induction of PLP139151-induced disease in the SJL/J
mouse, one donor mouse for two recipient mice is usually sufficient.
In the C57BL/6 model, typically the ratio is one to one. In cases
where large numbers of cells are to be transferred, higher ratios are
required.
In models where donor mice are immunized (see Notes), ingui-
nal, axillary, and brachial lymph nodes are pooled from primed mice
and placed in BSS containing 5 % FCS. Cell suspensions should be
kept on ice at all times. The pooled lymph nodes are then placed
onto a sterile 100 gauge wire mesh in a 90 mm Petri dish. Using the
plunger of a sterile 10 ml syringe, the lymph nodes are crushed to
produce a single-cell suspension. The cell suspension is centrifuged
at 300 g for 5 min at 4 C in a sterile 50 ml conical centrifuge tube.
The pellet is resuspended in fresh 5 % BSS by vigorous manual agi-
tation or repeated pipetting using a sterile 10 ml pipette. The cells
are washed once more with 5 % BSS and again pelleted as previously
described. After centrifugation, the cells are resuspended in com-
plete culture medium (DMEM containing 10 % FCS, 2 mM L-glu-
tamine, 100 U/ml penicillin and 100 g/ml streptomycin, and
50 M 2-mercaptoethanol). Three milliliter of culture medium are
added for each donor mouse used to obtain lymph node cells.
Alternatively, complete HL-1 (serum free ) can be used as culture
medium in place of DMEM.
388 D.P. McCarthy et al.

Primed LN cells are resuspended at a final concentration of

8 106 cells/ml in complete DMEM medium. Thirty milliliter
(2.4 108 total cells) are placed into a sterile 75 cm2 tissue culture
flask. Myelin protein or peptide antigen is added to the lymph node
suspension at the concentrations indicated in Table 2. Cells are
cultured for 34 days (37 C, 100 % humidity, and 5 % CO2) for
the time periods indicated in Table 2 for the various models. In
transfer systems where T cell lines or nave transgenic T cells (B10.
PLMBPAc111-specific transgenic T cells) are to be reactivated or
activated, respectively, different protocols are followed and can be
obtained from the relevant references (43, 44). In these systems,
addition of recombinant IL-12 to the culture medium is some-
times required (see Table 2) for efficient induction of clinical dis-
ease in nave recipient mice. In some cases, neutralizing antibody
to IL-4 has also been used (43). Culturing lymphocytes in the
presence of IL-23 rather than IL-12 may be desirable if the inves-
tigator is attempting to skew myelin-specific T cells towards a Th17
phenotype (23, 45).
After 34 days of in vitro culture, the cells are resuspended by
repeated pipetting and pelleted in a 50 ml conical tube. Cells are
washed twice with BSS and resuspended in approximately 10 ml
of BSS for each 30 ml tissue culture volume. This blast preparation
consists mainly of CD4+ T cells. If required, CD8+ T cells and any
remaining B cells and macrophages can be depleted using mag-
netic bead separation techniques or other standard depletion
methods. Viable cell counts of the number of T cell blasts are
determined by counting on a hemocytometer. Both the total
numbers of cells and the total numbers of T cell blasts are deter-
mined. Unstimulated T cells appear small, quite regular in shape
with a cytoplasm relatively clear, compared to the large, often
irregular and granular appearance of T cell blasts. Typically, in the
SJL/J transfer system, blasts account for 3040 % of total cells,
although use of complete HL-1 will yield a blast percentage of
2030 % of total cells. In other systems, the percentage may be
lower and in transgenic systems, higher. Cells are resuspended at a
concentration of 2 1061.2 108 T cell blasts/ml in PBS, depend-
ing on the adoptive transfer system and the number of blasts trans-
ferred (see Table 2).
The T cell blasts, derived above, can be injected into nave
syngeneic recipient either intraperitoneally or intravenously (i.v.)
to induce effective clinical disease. However, i.v. injection is typi-
cally more effective, with clinical disease developing faster than
delivery via i.p. injection. Usually the required numbers of cells are
injected in a volume of 0.20.5 ml using a disposable 1 ml tuber-
culin syringe and a 25 or 27 gauge needle. Adoptive transfer of
MOG3555-specific blasts into nave C57BL/6 mice must be accom-
panied by i.p. injection of 200 ng of pertussis toxin on days 0 and
2 relative to adoptive cell transfer.
 Table 2
19

Summary of general parameters for various EAE adoptive transfer modelsa

Donor In vitro antigen In vitro

Mouse Antigen immunization concentration In vitro IL-12 culture Number of blast Disease
strain specificity period (days) (mg/ml) (ng/ml) time (h) transfers (106) Disease type severity

SJL/J PLP 714 50100 7296 510 Relapsingremitting Severe

(27, 7178) PLP139151 714 20 7296 15 Relapsingremitting Severe
MBP 714 50100 7296 4060 Relapsingremitting Moderate
MBP84104 714 50 7296 1020 Relapsingremitting Moderate
C57BL/6 MBP 1014 50100 7296 50 Monophasic/chronic Mild
(64, 7982) MBP84104 1014 50 7296 50 Monophasic/chronic Mild
MOG 1014 50 25 7296 20 Relapsingremitting Moderate
MOG3555 1014 10 25 7296 20 Relapsingremitting Moderate
B10.PLb (43) MBPAc111 50 10 72 1 Monophasic Moderate
B10.S (43) MBP 1011 25 20 96 35 Monophasic Moderate
MBP87106 1011 50 20 96 35 Monophasic Moderate
a
These parameters have been optimized in different laboratories as previously described. *SJL/J protocol parameters from refs. 27, 7178, C57BL/6 proto-
col parameters from refs. 64, 7982, B10.PL protocol parameters from ref. 43, and B10.S protocol parameters from ref. 43
b
Note that the B10.PL system employs T cell receptor transgenic donors which do not require in vivo priming, only in vitro culture with MBPAc111 peptide
and rIL12
Mouse Models of Multiple Sclerosis: Experimental Autoimmune
389
390 D.P. McCarthy et al.

3.1.3. Clinical Grading Following priming, mice should be monitored every other day for
of Active and Passive EAE the development of disease. The appearance of EAE disease induced
by active immunization varies considerably based on mouse strain
and peptide used. For most strains and peptides, disease appears
between the second and fourth week following priming. The disease
is characterized by an ascending hind limb paralysis that begins in
the tail and spreads to involve the hind limbs and forelimbs. The
disease is graded on a 05 scale, though depending on strain and
peptide, mice do not always reach the higher disease grades before
disease resolution or disease plateau. Grade 0: there is no observ-
able difference from nave animals. Grade 1: assigned to mice that
have lost tail tonicity or show hind limb weakness (but not both).
Loss of tail tonicity is judged in mice that when held aloft by the
base of the tail show sagging of the tail and the tail cannot be lifted.
Additionally, the tip of the tail fails to curl. Hind limb weakness is
defined by the objective criterion that when placed on the wire
screen of the cage, the animals legs fall through as it tries to walk.
A waddling gait can also be observed as the animal walks on a flat
surface. The rear limbs are splayed and the rear posture lowered.
Grade 2: assigned to mice that present both a limp tail and show
hind limb weakness as defined above. Grade 3: assigned to mice
that show partial hind limb paralysis defined as the ability of a
mouse to move one or both hind limbs to some extent but not
maintain posture or walk. Grade 4: assigned to mice that cannot
move their hind limbs. The animal moves only by dragging itself
with its front limbs. A spastic paralysis and atrophy of the hind
limbs and lower body are often observed at this point. Mice at this
stage are given food on the cage floor (that can be moistened),
bottles with long sipper tubes, and daily injections of subcutaneous
saline to prevent death by dehydration. Grade 5: assigned to the
most severe end-stage assessment of EAE. These mice show a com-
plete inability to move due to paralysis in all limbs. In addition, any
animals that consistently show high grades and die (death by EAE)
should be given a grade of five. Mice that reach this stage and are
moribund with EAE should be sacrificed for humane reasons.
Histopathologically, the disease can be characterized by CD4+
T cell and F4/80+ (macrophage) inflammatory cell infiltrates that
can be found in both diffuse and focal patterns. In most EAE
models, the pattern of infiltrate tends to concentrate in the tho-
racic section of the spinal cord with less involvement in other
regions of the cord or the brain. However, recent studies have
revealed distinct differences in the histopathological features and
infiltration profile induced by Th1 and Th17 cells (23, 46).

3.1.4. Clinical Disease The first clinical episode is referred to as acute-phase disease which
Course is preceded by pronounced weight loss. Mice will experience this
acute episode for variable times depending on whether the disease
is relapsing and remitting (R/R), monophasic, or chronic/progressive
 19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 391

in nature. The point where disease reaches its highest score is

referred to as the peak of acute disease. After the initial episode or
a subsequent relapse, some strains of mice experience a recovery
(remission). If the recovery lasts for at least 2 days and drops by at
least one grade level, the recovery is deemed an authentic remis-
sion. These recoveries are observed in mice that show relapsing
and remitting (SJL/J) and monophasic disease profiles (B10.PL).
Mice that remit from the initial disease episode and recover fully or
stabilize at a reduced disease score are referred to as monophasic,
e.g., B10.PL primed with MBPAc111. Mice that have an acute dis-
ease that never shows a full grade reduction in disease are said to be
prone to a chronic disease. This chronic disease is characterized by
sustained priming antigen-specific T cell responses, e.g., C57BL/6
following MOG3555 priming.
In adoptive transfer EAE, clinical disease is evaluated using the
same scale as for actively induced EAE. The type of disease, day of
onset, and peak severity of disease depend on the system used (see
Table 2). Onset to peak disease is rapid, necessitating daily evalua-
tion of mouse clinical signs. Results are typically presented as mean
clinical score of mouse groups + standard error of the mean. Other
critical measures include mean day of onset, mean peak score, and
mean day of remission and relapse (the latter two being relevant to
R/R disease). Typically, mice reach peak disease within a day or
two after disease onset and remain at peak disease longer than in
the active EAE induction (average 5 days versus 3 days). Disease
incidence is normally greater than 90 %. Clinical disease may also
be evaluated using a terminal evaluation of histopathology in which
fixed and Epon embedded sections of spinal cord are stained with
Toluidine blue, as described previously (47) (Fig. 1).

Fig. 1. Histopathologic evaluation of 1 m thick Epon embedded spinal cord sections. Panel (a)spinal cord section from
a normal mouse. Note the presence of profuse and evenly distributed ringed structures reflecting myelinated axons, with
no infiltrating immune cells. Panel (b)spinal cord section from an SJL/J mouse with severe EAE. Note the few and
unevenly distributed myelinated axonal ringed structures with large bare areas, along with large numbers of infiltrating
immune cells throughout the section, appearing as dense and dark spots. Magnification: 220.
392 D.P. McCarthy et al.

When choosing the strain of mice and clinical disease course to

be studied, the investigator should take into consideration the
advantages and disadvantages of each model. For example, the
R-EAE disease course exhibited by SJL/J mice may be particularly
useful for studies involving immunoregulation and epitope spread-
ing since the mouse exhibits periods of remission as a result of self-
regulation, and relapses associated with T cell responses spreading
to other myelin epitopes. Although R-EAE recapitulates the most
common clinical manifestation of MS, the chronicprogressive dis-
ease course exhibited by C57BL/6 mice is a popular model for
EAE study due to the availability of transgenics and knockouts on
the H-2b background.

3.2. Induction Virus is produced in BHK-21 cells (ATCC). BHK-21 cells are
of TMEV-IDD grown in DMEM (Sigma) supplemented with 10 % FCS, 0.295 %
tryptose phosphate broth (Sigma), 1.0 % gentamycin (Gibco BRL),
3.2.1. TMEV Infecting
and 1 % antimycoticantibiotic (Gibco BRL). Cells are maintained
Stock
in culture at 37 C and 5 % CO2 and grown to confluence. BHK-
21 cells are removed from the flask by rinsing with versene (1:5,000)
(Gibco BRL) or 1 TrypsinEDTA Solution (Sigma), resuspended
in complete medium (above), and seeded (1:10 split) in a new
flask. These BHK-21 cells are grown to confluence (23 days), and
washed with DMEM without serum or supplements.
Medium is removed from the cells, and TMEV, BeAn 8386
strain, is added at an MOI of 5 in a minimal volume of serum-free
medium ensuring that the cell monolayer is covered with medium.
The infected cells are incubated overnight at 33 C at 5 % CO2 until
BHK-21 cells detach from the flask surface indicating lysis. The
medium is transferred to a conical for centrifugation to pellet the cell
debris. The cell lysate is removed and stored on ice, leaving a small
volume on top of the pelleted cells. The pelleted cells are then soni-
cated using brief pulses to completely lyse the cells, and the resulting
cell debris is again pelleted by centrifugation. The lysate is added to
the stored lysate collected from the first spin and this is aliquoted
into small volumes and stored at 70 C. The titer of the infecting
virus stock is determined by plaque assay (described below).

3.2.2. Purification of TMEV
Virus is produced in large stocks as described in Subheading 3.2.1
until the point of removing the supernatant from the infected cells.
The supernatant is removed from the infected cells and the pH is
adjusted with HCl to a pH below 7.0 and then frozen in bottles at
20 C. The bottles are thawed in 37 C shaking water bath with-
out allowing the supernatant to become too warm. To each 500 ml
of lysate, 14.5 g NaCl and 30 g PEG are added and the lysate is
stirred overnight at 4 C. The precipitated lysate is centrifuged at
7,000g in a Sorvall HB-4 swinging bucket rotor for 45 min at 4 C.
 19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 393

The supernatant is discarded, and the pellet is immediately resus-

pended in 18 ml hypertonic TNE buffer (0.02 M Tris base, 0.5 M
NaCl, 0.002 M EDTA). The resuspended pellets are sonicated to
separate the virus from cellular debris and DNA. The pellets are
pooled, warmed, and incubated with 1 ml 10 % SDS for each 9 ml
of resuspended pellets for 30 min at 37 C. The lysate is then cen-
trifuged to remove any membranous debris. The supernatant is
transferred to clear centrifuge tubes and overlaid onto 22 ml of a
35 % sucrose solution. The virus is pelleted through the sucrose by
centrifuging in an SW28 rotor at 20,000 rpm for 20 h at room
temperature.
Following the centrifugation, the pellet is resuspended in 2 ml
hypertonic TNE buffer, and sonicated to remove clumps. The virus
solution is then incubated with 0.1 ml 10 % SDS for each 1 ml solu-
tion for at least 10 min at 37 C to remove the remaining membra-
nous fractions. The virus solution is then clarified by centrifugation
and overlaid with 23 ml of the resulting supernatant onto 2070 %
sucrose gradients poured in clear centrifuge tubes. The gradients
are centrifuged at 35,000 rpm for 3 h at room temperature.
Following the centrifugation, a blue band containing the virus is
visible by UV or halogen light about 23 cm from the bottom of
the tube. The virus-containing band is collected by puncturing the
side of the tube using a syringe fitted with a 21 gauge needle. The
bands are pooled in new tubes up to a maximum volume of 1.5 ml
per tube. 2.2 ml of 1 g/ml Cs2SO4 solution is then added to each
tube. The tubes are filled with hypotonic TNE buffer, mixed thor-
oughly, and centrifuged at 40,000 rpm for 22 h at 4 C.
The following day, the virus contained in a white band about
1 cm from the bottom of the tube is collected using a syringe fitted
with 23 gauge needle. The virus-containing bands are pooled in a
new centrifuge tube (23 ml per tube) and the tubes are then filled
with hypotonic TNE buffer, mixed, and centrifuged at 35,000 rpm
for 3 h at 5 C. After the centrifugation, the virus pellet is resus-
pended in 0.2 ml PBS and incubated for 24 h at 4 C. To increase
virus yields, sonication of the suspension will help separate virus
particles and remove virus adhering to the tube wall. Quantitate
the virus by measuring the A280 and determine the amount of virus
using the following equation: (average A280 10/35) = mg virus.
Purity is assessed by SDS-PAGE.

3.2.3. TMEV Plaque Assay BHK-21 cells are cultured in 35 mm culture dishes (Thermo
Fischer: Nunc, Rochester, NY) to 90 % confluence as described
above. The BHK-21 cells are washed twice with serum-free
DMEM. Dilutions of the virus stock or tissue homogenate are
made in serum-free DMEM and 0.5 ml of each dilution is added
to the BHK-21 cells in duplicate. The cells are incubated at room
394 D.P. McCarthy et al.

temperature for 1 h with occasional rocking of the dishes.

Meanwhile, a 2 % solution of noble agar (Sigma) is autoclaved and
maintained at 55 C. A 1:1 solution of the 2 % noble agar and 2
DMEM supplemented with 2 % FCS and 2 % penicillin/strepto-
mycin (Life technologies) is then prepared. After the 1-h incuba-
tion, 6 ml of the 1:1 agar:DMEM solution is added to each culture
dish. The cells are then incubated at 33 C and 5 % CO2 for
56 days. The agar is removed from the dish, and the cells fixed
with methanol and stained with a crystal violet solution (0.8 g
crystal violet, 100 ml ethanol, 400 ml H2O) for 5 min. Alternatively
Formalin (Fischer) can be placed on top of the agar for 5 min to fix
cells before the agar is removed and cells are stained with crystal
violet as stated previously. The plate is then rinsed in a dish with
water to remove the excess stain. Plaques are then counted and the
number of plaque-forming units is calculated based on the dilu-
tion, volume of the dilution added to each culture dish, and weight
of tissue used.

3.2.4. Construction The cDNA for BeAn genome has been inserted into pGEM
of TMEV Containing plasmid for molecular manipulations. A restriction enzyme site,
Molecular Mimics of ClaI, was inserted into the leader sequence of the BeAn genome
Myelin Peptides along with a 23 amino acid deletion. Molecular mimic sequences
for myelin epitopes as previously described (48) are inserted into
the ClaI restriction site. PCR mutagenesis was conducted to insert
ClaI sites flanking the sequence to be inserted into the virus
genome. The mimic sequences are 30 amino acids in length to
restore the deletion in the leader protein. The mimic sequence is
ligated into the ClaI site in the BeAn cDNA, and DH5 E. coli are
transformed with the ligated product to produce a BeAn cDNA
containing the mimic sequence in the correct orientation. Next,
in vitro transcription of the BeAn cDNA containing the mimic
sequence is driven by an upstream T7 promoter using an Sp6/T7
in vitro transcription kit (Roche). This produces a single positive-
stranded RNA. The resulting RNA is transfected into BHK-21
cells in a 60 mm culture dish with DMEM supplemented with 2 %
FBS using lipofectin reagent (Gibco BRL) as described by the
manufacturers protocol. The transfected cells are incubated over-
night at 33 C. Following the incubation, the medium is removed
from the cells, replaced with DMEM containing 2 % serum, and
incubated for 23 additional days at 33 C until the cells began to
lyse, indicating virus production. Virus is isolated from the cells
following the procedure described above (Subheading 3.2.1). The
virus is amplified beginning with very small volumes until the virus
titer reaches 104 PFU/ml, and then larger volumes can be used to
produce the recombinant virus for infecting stocks. The viral titer
of the recombinant viruses is determined by plaque assay as
described above (Subheading 3.2.3).
 19
3.2.5. Induction
of TMEV-Induced
Demyelinating Disease
3.2.6. Clinical Assessment
of TMEV-Induced
Demyelinating Disease
Mouse Models of Multiple Sclerosis: Experimental Autoimmune 395
TMEV is a naturally endemic infection in mice spread through the
fecal oral route, which results in a 5060 % incidence of TMEV-
IDD in disease-susceptible animals. Experimentally, disease-suscep-
tible SJL/J mice can be infected intracranially in order to increase
the incidence of TMEV-IDD development to approximately 90 % of
all infected animals. Six- to seven-week-old female SJL/J mice are
anesthetized with aerosolized isoflurane (Abbott Laboratories) and
inoculated with either 5 106 PFU of wild-type TMEV (BeAn 8386
strain) infecting stock (produced as described in Subheading 3.2.1)
or recombinant mimic-expressing viruses (produced as described in
Subheading 3.2.4), in 30 l in the right cerebral hemisphere by free-
hand injection with a 24 gauge needle. The cap of the needle remains
on the needle during injection but is cut down so that approximately
23 mm of the needle is exposed for the injection. This same needle
guard is used in all injections so that the virus is injected at the same
depth in each mouse injected. Mice are marked to allow for indi-
vidual evaluation of clinical and histological disease.
The clinical disease presentation seen in susceptible mouse strains,
such as SJL/J, depends upon the strain of TMEV used for infec-
tion. Following inoculation with the brain-derived DA strain of
virus, mice first develop a flaccid paralysis. Mice recover in approxi-
mately 2 weeks indicating that this phase of disease is self-limiting.
However, 23 weeks after infection, mice then develop a spastic
paresis of the hind limbs, which, in SJL/J mice, has a chronic dis-
ease course resulting in severe spastic paralysis (42). In contrast,
infection of SJL/J mice with the tissue culture-adapted BeAn 8386
strain does not produce clinical evidence of a first-phase disease.
Infected mice begin to show signs of clinical disease between 30
and 40 days post-TMEV infection and develop a chronic, progres-
sive paralysis with no recovery or remitting episodes, similar to
primary progressive MS. Unlike EAE, clinical signs develop slowly,
with no drastic changes in gait from day to day. Mice are moni-
tored for disease progression 23 times per week continuing for
100 days post-infection. Each mouse is assigned a numerical score
between 0 and 5, based on the severity of its impairment: 0, asymp-
tomatic; 1, mild gait abnormalities; 2, severe gait abnormalities; 3,
loss of ability to right itself associated with mild spastic paralysis; 4,
spastic paralysis in both hind limbs combined with urinary inconti-
nence and dehydration; 5, moribund. Infection with recombinant
infection with recombinant TMEV containing different molecular
mimic sequences leads to different disease profiles depending upon
the epitope expressed. Mice infected with the leader deletion
recombinant virus, ClaI-BeAn, do not develop signs of demyeli-
nating autoimmune disease (48). In contrast, mice infected with
the recombinant PLP139-BeAn virus exhibit an earlier onset and
more severe clinical disease, with onset between days 7 and 10
post-infection (48).
396 D.P. McCarthy et al.

3.2.7. Immunological A number of immunological assays can be performed to determine

Aspects of TMEV-Induced the specificity, class, and timing of the immune system in disease
Demyelinating Disease pathology following TMEV infection. Support for a CD4+ T cell-
mediated pathogenesis of TMEV-IDD derives from studies show-
ing that susceptibility strongly correlates with the development of
chronic, high levels of TMEV- and myelin-specific delayed-type
hypersensitivity (DTH) reactions. DTH to TMEV capsid epitopes
in BeAn-infected, susceptible SJL/J mice develops within 510 days
post-infection, preceding the appearance of clinical signs, and
remains at high levels for at least 100 days post-infection (49).
Previous data indicated that VP27486 was the immunodominant
Th1 determinant in TMEV-infected SJL/J mice, as 8090 % of the
DTH response is directed against this virion peptide within the
VP2 protein (50). However, recent work by Jin et al. shows a new
immunodominant peptide within the 3D viral polymerase protein
(3D2136) which a greater number of CD4+ T cells respond to
compared to VP27486 (51). The immunodominant myelin epitope
in SJL/J mice is PLP139151 and responses to this self-antigen are
first observed around 4550 days post-TMEV infection, i.e.,
23 weeks after clinical disease onset (37). As disease progresses,
epitope spreading occurs in a hierarchical order with intermolecu-
lar spreading to PLP178191 and PLP5670 and intramolecular epitope
spreading to MOG92106 and MBP84104, occurring during the
chronic late phase of TMEV-IDD, by day 100 post-infection
(36, 37). These responses can be detected by both DTH and
splenic T cell proliferative responses.
The early detection of PLP139151 responses, between days 10
and 14 post-infection, can be observed following the infection of
mice with PLP139-BeAn (48). This is in contrast to wild-type
TMEV-infected mice where myelin responses arise by day 50
post-infection. In addition, there is evidence for epitope spread-
ing of myelin epitopes to PLP178191 in PLP139-BeAn-infected
mice (48, 52).
In addition, cytokines play an important role in TMEV-IDD.
The release of the pro-inflammatory cytokines (IFN- and LT/
TNF) by both viral and myelin-specific Th1 cells in the CNS leads
to the recruitment and activation of monocytes and macrophages
which cause myelin destruction by a terminal nonspecific bystander
mechanism (36). These cytokines can be quantitated using ELISA,
ELISPOT, and other cytokine measures.
In conclusion, following TMEV infection of the CNS,
bystander damage to myelin is initiated by virus-specific CD4+ Th1
cells, which leads to the release, processing, and presentation of
myelin auto-antigens by CNS APCs. This presentation to autore-
active T cells leads, via epitope spreading, to an autoimmune
response directed against CNS myelin which perpetuates chronic
clinical pathology.
 19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 397

4. Notes

1. These protocols have been developed by numerous laboratories,

with some variation between each protocol. Variations in opti-
mal culture conditions and immunization conditions are appar-
ent. As such, it is important for each laboratory to optimize the
culture system for its environment and reagents. Thus, listed
concentrations are only approximations.
2. Susceptible strains: Varieties of mouse strains are used to study
EAE. The most common are SJL/J, B10.PL, C57BL/6, C3H,
SWR, and the F1 progeny of several of these parental strains.
The importance of pertussis toxin and the type of disease
course (chronic versus relapsing/remitting) for each of these
strains is discussed below. Table 1 describes the haplotype and
reported encephalitogenic peptides for each of these suscepti-
ble strains.
Resistant strains: While many mouse strains are useful in
the study of EAE, not all mouse strains appear to be susceptible
to EAE induction. For instance, A/J, C3H/HeJ, AKR, NZW,
and DBA/2 appear to be resistant to EAE after priming with
known myelin antigens (53).
3. For efficient disease induction in some strains, administration
of pertussis toxin is required on days surrounding peptide
priming. 200 ng (200 l of a 1 g/ml stock in PBS) should be
administered intraperitoneally on the day of priming and again
2 days later. Pertussis should be dissolved at least 24 h prior to
use to prevent death associated with administration of fresh
pertussis. Pertussis toxin is required to initiate EAE in
C57BL/6, B10.PL, and their F1 progeny, as well as (SJL/J
BALB/c)F1 mouse strains. Efficient disease induction in SJL/J
mice requires the use of pertussis toxin only with certain neu-
roantigens, e.g., intact MBP and MPB84104.
4. In addition to induction of disease and clinical disease, several
other techniques have been used to assay the presence and per-
sistence of T cell responses (and importantly antigen-specific
responses) following the development of EAE. These include
DTH (54), proliferation assays (55), immunohistochemistry
(56), and histopathology (Fig. 1). Complete descriptions of
these techniques can be found in the respective citations.
5. The disease course and immune reactivity described in these
methods relate to the BeAn strain of TMEV. The DA strain
also induces a demyelinating disease with some differences in
clinical disease and different immune reactivity. In addition,
the GDVII strain of TMEV induces a lethal encephalitis and
thus does not result in a late-onset demyelinating disease.
398 D.P. McCarthy et al.

The mouse strain described in these methods is the SJL/J

mouse which is susceptible to BeAn strain TMEV-IDD. C57BL/6
are resistant to BeAn strain TMEV-IDD. BALB/c have varying
susceptibility to TMEV-IDD depending on the substrain, where
BALB/cAnNCr are mildly susceptible, and the BALB/cByJ are
resistant to TMEV-IDD. Additionally, the C57BL/6 SJL/J/J
F1 strain of mice has an intermediate frequency of TMEV-IDD
development with approximately 30 % of intracranially infected
animals developing mild to moderate disease symptoms.

References
1. Brown A, McFarlin DE, Raine CS (1982)
Chronologic neuropathology of relapsing
experimental allergic encephalomyelitis in the
mouse. Lab Invest 46:171185
2. Karcher D, Lassmann H, Lowenthal A, Kitz K,
Wisniewski HM (1982) Antibodies-restricted
heterogeneity in serum and cerebrospinal fluid
of chronic relapsing experimental allergic
encephalomyelitis. J Neuroimmunol 2:93106
3. Lassmann H (1983) Chronic relapsing experi-
mental allergic encephalomyelitis: its value as
an experimental model for multiple sclerosis. J
Neurol 229:207220
4. Tan LJ, Kennedy MK, Miller SD (1992)
Regulation of the effector stages of experimen-
tal autoimmune encephalomyelitis via
neuroantigen-specific tolerance induction. II.
Fine specificity of effector T cell inhibition. J
Immunol 148:27482755
5. Eng LF, Ghirnikar RS, Lee YL (1996)
Inflammation in EAE: role of chemokine/
cytokine expression by resident and infiltrating
cells. Neurochem Res 21:511525
6. Miller SD, Karpus WJ (1994) The immunop-
athogenesis and regulation of T-cell mediated
demyelinating diseases. Immunol Today
15:356361
7. Williams KC, Ulvestad E, Hickey WF (1994)
Immunology of multiple sclerosis [Review)
[300 refs). Clin Neurosci 2:229245
8. Lublin FD (1985) Relapsing experimental
allergic encephalomyelitis. An autoimmune
model of multiple sclerosis. Springer Semin
Immunopathol 8:197208
9. Arnason BG (1983) Relevance of experimental
allergic encephalomyelitis to multiple sclerosis.
Neurol Clin 1:765782
10. Traugott U, Stone SH, Raine CS (1979)
Chronic relapsing experimental allergic enceph-
alomyelitis. Correlation of circulating lympho-
cyte fluctuations with disease activity in
suppressed and unsuppressed animals. J Neurol
Sci 41:1729
11. Traugott U, McFarlin DE, Raine CS (1986)
Immunopathology of the lesion in chronic
relapsing experimental autoimmune encepha-
lomyelitis in the mouse. Cell Immunol
99:395410
12. Boyle EA, McGeer PL (1990) Cellular immune
response in multiple sclerosis plaques. Am J
Pathol 137:575584
13. McCallum K, Esiri MM, Tourtellotte WW,
Booss J (1987) T cell subsets in multiple scle-
rosis. Gradients at plaque borders and differ-
ences in nonplaque regions. Brain
110:12971308
14. Renno T, Krakowski M, Piccirillo C, Lin JY,
Owens T (1995) TNF-alpha expression by resi-
dent microglia and infiltrating leukocytes in the
central nervous system of mice with experimen-
tal allergic encephalomyelitis. Regulation by
Th1 cytokines. J Immunol 154:944953
15. Swanborg RH (1995) Experimental autoim-
mune encephalomyelitis in rodents as a model
for human demyelinating disease [see com-
ments) [Review) [99 refs). Clin Immunol
Immnopathol 77:413
16. Epstein LG, Prineas JW, Raine CS (1983)
Attachment of myelin to coated pits on mac-
rophages in experimental allergic encephalo-
myelitis. J Neurol Sci 61:341348
17. Smith ME (1993) Phagocytosis of myelin by
microglia in vitro. J Neurosci Res 35:480487
18. Sommer MA, Forno LS, Smith ME (1992)
EAE cerebrospinal fluid augments in vitro
phagocytosis and metabolism of CNS myelin
by macrophages. J Neurosci Res 32:384394
19. Colover J (1988) Immunological and cytologi-
cal studies of autoimmune demyelination and
multiple sclerosis. Brain Behav Immun
2:341345
20. Yednock TA, Cannon C, Fritz LC, Sanchez-
Madrid F, Steinman L, Karin N (1992)
Prevention of experimental autoimmune
encephalomyelitis by antibodies against 41
integrin. Nature 356:6366
 19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune
21. Kawakami N, Lassmann S, Li Z, Odoardi F,
Ritter T, Ziemssen T, Klinkert WE, Ellwart JW,
Bradl M, Krivacic K, Lassmann H, Ransohoff
RM, Volk HD, Wekerle H, Linington C, Flugel
A (2004) The activation status of neuroantigen-
specific T cells in the target organ determines
the clinical outcome of autoimmune encepha-
lomyelitis. J Exp Med 199:185197
22. Lees JR, Golumbek PT, Sim J, Dorsey D,
Russell JH (2008) Regional CNS responses to
IFN-gamma determine lesion localization pat-
terns during EAE pathogenesis. J Exp Med
205:26332642
23. Kroenke MA, Carlson TJ, Andjelkovic AV, Segal
BM (2008) IL-12- and IL-23-modulated T cells
induce distinct types of EAE based on histology,
CNS chemokine profile, and response to cytokine
inhibition. J Exp Med 205:15351541
24. Cross AH, Raine CS (1990) Serial adoptive
transfer of murine experimental allergic enceph-
alomyelitis: successful transfer is dependent on
active disease in the donor. J Neuroimmunol
28:2737
25. Raine CS, Mokhtarian F, McFarlin DE (1984)
Adoptively transferred chronic relapsing exper-
imental autoimmune encephalomyelitis in the
mouse. Neuropathologic analysis. Lab Invest
51:534546
26. Zamvil S, Nelson P, Trotter J, Mitchell D,
Knobler R, Fritz R, Steinman L (1985) T-cell
clones specific for myelin basic protein induce
chronic relapsing paralysis and demyelination.
Nature 317:355358
27. Kim C, Tse HY (1993) Adoptive transfer of
murine experimental autoimmune encephalo-
myelitis in SJL.Thy-1 congenic mouse strains.
J Neuroimmunol 46:129136
28. Howard LM, Miller SD (2001) Autoimmune
intervention by CD154 blockade prevents T
cell retention and effector function in the tar-
get organ. J Immunol 166:15471553
29. Dal Canto M, Lipton HL, Miller SD,
Melvold RW, Capen CC, Jones TC, Migaki G
(1986) Theilers murine encephalomyelitis
virus (TMEV) infection in mice as a model for
MS. In: Capen CC, Jones TC, Migaki G (eds.).
In Registry of comparative pathology, hand-
book of animal models of human disease, VIIth
fascicle. AFIP, Washington, D.C.
30. Miller SD (1995) Pathogenesis of Theilers
murine encephalomyelitis virus-induced demy-
elinating disease a model of multiple sclerosis.
ACLAD Newslett 16:46
31. Pevear DC, Borkowski J, Luo M, Lipton H
(1988) Sequence comparison of a highly viru-
lent and a less virulent strain of Theilers virus.
Amino acid differences on a three- dimensional
399
model identify the location of possible immu-
nogenic sites. Ann NY Acad Sci 540:652653
32. Pevear DC, Calenoff M, Rozhon E, Lipton HL
(1987) Analysis of the complete nucleotide
sequence of the picornavirus Theilers murine
encephalomyelitis virus indicates that it is closely
related to cardioviruses. J Virol 61:15071516
33. Lipton HL (1980) Persistent Theilers murine
encephalomyelitis virus infection in mice depends
on plaque size. J Gen Virol 46:169177
34. Clatch RJ, Miller SD, Metzner R, Dal Canto
MC, Lipton HL (1990) Monocytes/mac-
rophages isolated from the mouse central ner-
vous system contain infectious Theilers murine
encephalomyelitis virus (TMEV). Virology
176:244254
35. Peterson JD, Karpus WJ, Clatch RJ, Miller SD
(1993) Split tolerance of Th1 and Th2 cells in
tolerance to Theilers murine encephalomyeli-
tis virus. Eur J Immunol 23:4655
36. Katz-Levy Y, Neville KL, Padilla J, Rahbe SM,
Begolka WS, Girvin AM, Olson JK, Vanderlugt
CL, Miller SD (2000) Temporal development
of autoreactive Th1 responses and endogenous
antigen presentation of self myelin epitopes by
CNS-resident APCs in Theilers virus-infected
mice. J Immunol 165:53045314
37. Miller SD, Vanderlugt CL, Begolka WS, Pao
W, Yauch RL, Neville KL, Katz-Levy Y,
Carrizosa A, Kim BS (1997) Persistent infec-
tion with Theilers virus leads to CNS autoim-
munity via epitope spreading. Nat Med
3:11331136
38. Prineas J (1975) Pathology of the early lesion
in multiple sclerosis. Hum Pathol 6:531554
39. Dal Canto MC, Lipton HL (1975) Primary
demyelination in Theilers virus infection. An
ultrastructural study. Lab Invest 33:626637
40. Nathanson N, Miller A (1978) Epidemiology
of multiple sclerosis: critique of evidence for a
viral etiology. Am J Epidemiol 107:451461
41. Kurtzke JF, Raine CS, McFarlin HF,
Tourtellotte WW (1997) The epidemiology of
multiple sclerosis. In: Multiple sclerosis: clinical
and pathogenetic basis. Chapman and Hall,
London, pp 91139
42. Lipton HL (1975) Theilers virus infection in
mice: an unusual biphasic disease process leading
to demyelination. Infect Immun 11:11471155
43. Walker MR, Mannie MD (2002) Acquisition of
functional MHC class II/peptide complexes by
T cells during thymic development and CNS-
directed pathogenesis. Cell Immunol
218:1325
44. Segal BM, Shevach EM (1996) IL-12 unmasks
latent autoimmune disease in resistant mice.
J Exp Med 184:771775
400 D.P. McCarthy et al.
45. Axtell RC, de Jong BA, Boniface K, van der
Voort LF, Bhat R, De Sarno P, Naves R, Han
M, Zhong F, Castellanos JG, Mair R, Christakos
A, Kolkowitz I, Katz L, Killestein J, Polman
CH, de Waal Malefyt R, Steinman L, Raman C
(2010) T helper type 1 and 17 cells determine
efficacy of interferon-beta in multiple sclerosis
and experimental encephalomyelitis. Nat Med
16:406412
46. Stromnes IM, Cerretti LM, Liggitt D, Harris
RA, Goverman JM (2008) Differential regula-
tion of central nervous system autoimmunity
by T(H)1 and T(H)17 cells. Nat Med
14:337342
47. Dal Canto MC, Melvold RW, Kim BS, Miller
SD (1995) Two models of multiple sclerosis:
experimental allergic encephalomyelitis (EAE)
and Theilers murine encephalomyelitis virus
(TMEV) infection a pathological and immu-
nological comparison. Microsc Res Tech
32:215229
48. Olson JK, Croxford JL, Calenoff M, Dal Canto
MC, Miller SD (2001) A virus-induced molec-
ular mimicry model of multiple sclerosis. J Clin
Invest 108:311318
49. Clatch RJ, Lipton HL, Miller SD (1986)
Characterization of Theilers murine encepha-
lomyelitis virus (TMEV)-specific delayed-type
hypersensitivity responses in TMEV-induced
demyelinating disease: correlation with clinical
signs. J Immunol 136:920927
50. Gerety SJ, Clatch RJ, Lipton HL, Goswami
RG, Rundell MK, Miller SD (1991) Class
II-restricted T cell responses in Theilers murine
encephalomyelitis virus-induced demyelinating
disease. IV. Identification of an immunodomi-
nant T cell determinant on the N-terminal end
of the VP2 capsid protein in susceptible SJL/J
mice. J Immunol 146:24012408
51. Jin YH, Kang B, Kim BS (2009) Theilers virus
infection induces a predominant pathogenic
CD4+ T cell response to RNA polymerase in
susceptible SJL/J mice. J Virol
83:1098110992
52. Miller SD, Olson JK, Croxford JL (2001)
Multiple pathways to induction of virus-
induced autoimmune demyelination: lessons
from Theilers virus infection. J Autoimmun
16:219227
53. Duong TT, Finkelman FD, Singh B, Strejan
GH (1994) Effect of anti-interferon-gamma
monoclonal antibody treatment on the devel-
opment of experimental allergic encephalomy-
elitis in resistant mouse strains. J Neuroimmunol
53:101107
54. Zamvil SS, Steinman L (1990) The T lympho-
cyte in experimental allergic encephalomyelitis.
Annu Rev Immunol 8:579621
55. McRae BL, Vanderlugt CL, Dal Canto MC,
Miller SD (1995) Functional evidence for
epitope spreading in the relapsing pathology of
experimental autoimmune encephalomyelitis. J
Exp Med 182:7585
56. Begolka WS, Vanderlugt CL, Rahbe SM, Miller
SD (1998) Differential expression of
inflammatory cytokines parallels progression of
central nervous system pathology in two clini-
cally distinct models of multiple sclerosis. J
Immunol 161:44374446
57. Sakai K, Zamvil SS, Mitchell DJ, Lim M,
Rothbard JB, Steinman L (1988)
Characterization of a major encephalitogenic T
cell epitope in SJL/J mice with synthetic oligo-
peptides of myelin basic protein. J
Neuroimmunol 19:2132
58. Tuohy VK, Thomas DM (1993) A third
encephalitogenic determinant of myelin prote-
olipid protein (PLP) for SJL/J mice. J Immunol
150:194A
59. Greer JM, Kuchroo VK, Sobel RA, Lees MB
(1992) Identification and characterization of a
second encephalitogenic determinant of myelin
proteolipid protein (residues 178191) for SJL
mice. J Immunol 149:783788
60. Greer JM, Sobel RA, Sette A, Southwood S,
Lees MB, Kuchroo VK (1996) Immunogenic
and encephalitogenic epitope clusters of myelin
proteolipid protein. J Immunol 156:371379
61. Amor S, Groome N, Linington C, Morris MM,
Dornmair K, Gardinier MV, Matthieu JM,
Baker D (1994) Identification of epitopes of
myelin oligodendrocyte glycoprotein for the
induction of experimental allergic encephalo-
myelitis in SJL and Biozzi AB/H mice. J
Immunol 153:43494356
62. Zamvil SS, Mitchell DJ, Moore AC, Kitamura
K, Steinman L, Rothbard JB (1986) T-cell
epitope of the autoantigen myelin basic protein
that induces encephalomyelitis. Nature
324:258260
63. Whitham RH, Jones RE, Hashim GA, Hoy
CM, Wang RY, Vandenbark AA, Offner H
(1991) Location of a new encephalitogenic
epitope (residues 43 to 64) in proteolipid pro-
tein that induces relapsing experimental auto-
immune encephalomyelitis in PL/J and (SJL
PL)F1 mice. J Immunol 147:38033808
64. Mendel I, Kerlero DR, Ben-Nun A (1995) A
myelin oligodendrocyte glycoprotein peptide
induces typical chronic experimental autoim-
mune encephalomyelitis in H-2b mice: fine
specificity and T cell receptor V beta expression
of encephalitogenic T cells. Eur J Immunol
25:19511959
65. Tompkins SM, Padilla J, Dal Canto MC, Ting
JP, Van Kaer L, Miller SD (2002) De novo cen-
 19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune
tral nervous system processing of myelin anti-
gen is required for the initiation of experimental
autoimmune encephalomyelitis. J Immunol
168:41734183
66. Tuohy VK, Lu ZJ, Sobel RA, Laursen RA, Lees
MB (1988) A synthetic peptide from myelin
proteolipid protein induces experimental aller-
gic encephalomyelitis. J Immunol
141:11261130
67. Endoh M, Kunishita T, Nihei J, Nishizawa M,
Tabira T (1990) Susceptibility to proteolipid
apoprotein and its encephalitogenic determi-
nants in mice. Int Arch Allergy Appl Immunol
92:433438
68. Muller DM, Pender MP, Greer JM (2000) A
neuropathological analysis of experimental
autoimmune encephalomyelitis with predomi-
nant brain stem and cerebellar involvement and
differences between active and passive induc-
tion. Acta Neuropathol (Berl) 100:174182
69. Maron R, Hancock WW, Slavin A, Hattori M,
Kuchroo V, Weiner HL (1999) Genetic suscep-
tibility or resistance to autoimmune encephalo-
myelitis in MHC congenic mice is associated
with differential production of pro- and anti-
inflammatory cytokines. Int Immunol
11:15731580
70. Slavin A, Ewing C, Liu J, Ichikawa M, Slavin J,
Bernard CC (1998) Induction of a multiple
sclerosis-like disease in mice with an immu-
nodominant epitope of myelin oligodendrocyte
glycoprotein. Autoimmunity 28:109120
71. McRae BL, Kennedy MK, Tan LJ, Dal Canto
MC, Miller SD (1992) Induction of active and
adoptive chronic-relapsing experimental auto-
immune encephalomyelitis (EAE) using an
encephalitogenic epitope of proteolipid pro-
tein. J Neuroimmunol 38:229240
72. Miller SD, Tan LJ, Kennedy MK, Dal Canto
MC (1991) Specific immunoregulation of the
induction and effector stages of relapsing EAE
via neuroantigen-specific tolerance induction.
Ann NY Acad Sci 636:7994
73. Tuohy VK, Thomas DM (1995) Sequence
104-117 of myelin proteolipid protein is a
401
cryptic encephalitogenic T cell determinant for
SJL/J mice. J Neuroimmunol 56:161170
74. Skundric DS, Kim C, Tse HY, Raine CS (1993)
Homing of T cells to the central nervous system
throughout the course of relapsing experimen-
tal autoimmune encephalomyelitis in Thy-1
congenic mice. J Neuroimmunol 46:113121
75. Fritz RB, Zhao ML (1994) Encephalitogenicity
of myelin basic protein exon-2 peptide in mice.
J Neuroimmunol 51:16
76. Segal BM, Raine CS, McFarlin DE, Voskuhl
RR, McFarland HF (1994) Experimental aller-
gic encephalomyelitis induced by the peptide
encoded by exon 2 of the MBP gene, a peptide
implicated in remyelination. J Neuroimmunol
51:719
77. Pettinelli CB, McFarlin DE (1981) Adoptive
transfer of experimental allergic encephalomy-
elitis in SJL/J mice after in vitro activation of
lymph node cells by myelin basic protein:
requirement for Lyt 1+ 2 T lymphocytes. J
Immunol 127:14201423
78. Pettinelli CB, Fritz RB, Chou CHJ, McFarlin
DE (1982) Encephalitogenic activity of guinea
pig myelin basic protein in the SJL mouse. J
Immunol 129:12091211
79. Shaw MK, Kim C, Hao HW, Chen F, Tse HY
(1996) Induction of myelin basic protein-
specific experimental autoimmune encephalo-
myelitis in C57BL/6 mice: mapping of T cell
epitopes and T cell receptor V beta gene seg-
ment usage. J Neurosci Res 45:690699
80. Clark RB, Grunnet M, Lingenheld EG (1997)
Adoptively transferred EAE in mice bearing the
lpr mutation. Clin Immunol Immnopathol
85:315319
81. Mendel I, Shevach EM (2002) Differentiated
Th1 autoreactive effector cells can induce
experimental autoimmune encephalomyelitis in
the absence of IL-12 and CD40/CD40L inter-
actions. J Neuroimmunol 122:6573
82. Segal BM, Dwyer BK, Shevach EM (1998) An
Interleukin (IL)-10/IL-12 immunoregulatory
circuit controls susceptibility to autoimmune
disease. J Exp Med 187:537546
 Chapter 20

Pathogenesis of Multiple Sclerosis: What Can We Learn

from the Cuprizone Model
Peter Acs and Bernadette Kalman

Abstract
Multiple sclerosis is an inflammatory demyelinating and neurodegenerative disorder of the central nervous
system (CNS). The primary cause of the disease remains unknown, but an altered immune regulation with
features of autoimmunity has generally been considered to play a critical role in the pathogenesis.
Historically, lesion development has been attributed to activation of CD4 and CD8 T lymphocytes, B
lymphocytes, and monocytes in the peripheral circulation and the migration of these cells through the
bloodbrain barrier to exert direct or indirect cytotoxic effects on myelin, oligodendrocytes and neuronal
processes in the CNS. This broadly accepted concept was significantly influenced by the experimental
autoimmune encephalitis (EAE) model, in which either immunization with myelin antigens or injection of
a myelin antigen-specific T cell line into a recipient results in inflammatory demyelination in the CNS.
More recent studies reveal that the loss of oligodendrocytes and neurons begins in the earliest stages of the
disease and may not always be associated with blood-derived inflammatory cells. The pathology affects
both the white and the gray matters and the clinical disability best correlates with the overall neurodegen-
erative process. These newer observations prompted several revisions of the classical concept of MS and
facilitated a shift from using EAE to using other model systems. This chapter summarizes the classical and
more contemporary concepts of MS, and provides methodologies for employing the cuprizone model for
further explorations of the pathogenesis and treatment of the disease.

Key words: Multiple sclerosis, Autoimmunity, Oligodendrocytopathy, Demyelination, Cuprizone

model, Mitochondrion, Apoptosis, Histological methods, Gene expression quantitation

1. Inflammation,
Demyelination,
and Neuro-
degeneration in Multiple sclerosis is an inflammatory process associated with demy-
Multiple Sclerosis elination and neurodegeneration in the central nervous system
(CNS). The etiology remains unknown, but the development of
1.1. Immune-Mediated the disease has been linked to an altered immune response qualified
Mechanisms of MS by many measures for autoimmunity (1).

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_20, Springer Science+Business Media New York 2012

403
404 P. Acs and B. Kalman

Box 1
The EAE Model

The efferent arm of inflammatory demyelination has been comprehensively

studied in the experimental autoimmune or allergic encephalitis (EAE)
model. In active EAE, rodents (most commonly mice, rats, or guinea
pigs) or nonhuman primates are injected subcutaneously with a myelin-
related antigen or peptide homogenized in complete Freunds adju-
vant. Intravenous injection of Bordetella Pertussis toxin on
post-immunization days 0 and 2 may also be required (2). Activated
myelin antigen-specific CD4+ T cell clones express an array of adhesion
molecules, enzymes, cytokines, and chemokines and their receptors,
which facilitate the breakdown of bloodbrain barrier (BBB) and the
migration of these lymphocytes into the CNS. Myelin antigen-specific
T cells recognize their antigenic determinants presented by antigen-
presenting cells (monocytes, macrophages, dendritic cells, B cells,
microglia, astrocytes), undergo continuous activation, and exert cyto-
toxic effects in the CNS. A paralytic disease affecting predominantly the
tail and hind limbs, but sometimes also the forelimbs, will typically
develop on post-immunization days 515. Acute monophasic as well as
chronic relapsing forms of the model were generated by subtle
modifications of the immunization protocol. In passive transfer experi-
ments, first draining lymph nodes of animals immunized previously
with a myelin antigen are removed and restimulated with the same anti-
gen in vitro. The cell line or clone is then injected into a nave recipient,
and the disease develops in a few days after the injection of pathogenic
cells (3). Both the active immunization and the passive transfer models
have been extensively used to better understand immune-mediated
mechanisms of demyelination and neurodegeneration in MS. New drug
development for MS usually also involves a preclinical testing in the
EAE model.

Influenced by the EAE model (Box 1), lesion development in MS

has been attributed to activated CD4 T lymphocytes, particularly
to the T helper (TH)-1 and TH-17 subpopulations, that cross the
BBB, activate residential immune cells (microglia, astroglia), and
exert direct cytotoxic effects on oligodendrocytes (OLs) and myelin
in the CNS. Edema, demyelination, loss of remyelinating oligo-
dendrocytes, astrogliosis, and neuroaxonal degeneration develop
in association with inflammation over time (1). Among effector
cells, TH-1 cells are characterized by the expression of interferon
(IFN)-g, interleukine (IL)-2, tumor necrosis factor (TNF)-b, and
nitric oxide (NO). These immune mediators control the expression
 20 Pathogenesis of Multiple Sclerosis 405

of major histocompatibility (MHC) Class I/II and adhesion

molecules (IFN-g), maintain T cell differentiation and activation
(IL-2), contribute to oligodendrocyte and myelin injury (TNF-b,
NO), and modulate the balance among effector and regulatory
immune cell populations (1, 4). Activation of TH-1 cells is associ-
ated with the expression of numerous cell surface molecules (e.g.,
very-late-activation antigen VLA-4 or integrin a4b1) and enzymes
(e.g., members of the matrix metalloproteaseMMPfamily),
which allow these cells to bind to their corresponding receptors
(e.g., VLA4 on TH-1 cells binds to vascular cell adhesion molecule
or VCAM-1 on endothelial cells) and degrade matrix (e.g., MMP-9
and MMP-2) and breakthrough of the BBB. Recognition of TH-17
cells as major effector cells in EAE prompted intense analyses of
this subpopulation in MS (1, 4, 5). These cells produce IL-17 and
their survival depends on IL-23 produced by dendritic cells and
macrophages. In contrast to the TH-1 and TH-17 subpopulations,
TH-2 cells are primarily viewed as regulatory cells, which produce
IL-4, IL-5, IL-6, IL-10, and IL-13 cytokines. TH-2 cells down-
regulate TH-1 cells and thus limit the inflammatory process in MS.
TH-2 cells, however, support B cell differentiation and promote
immunoglobulin production. Another important regulatory T cell
population is defined by the CD4+CD25+FOXp3+ phenotype, and
is known to control the polarization, expansion, and effector func-
tion of TH-1, TH-2, and TH-17 cell types (6).
A direct pathogenic role of CD8 lymphocytes was recently
suggested in MS. CD8 T cells are present in lesions, show signs of
clonal expansion, and have cytotoxic properties. Both blood and
cerebrospinal fluid (CSF)-derived CD8 lymphocytes of MS patients
are enriched for memory and activated CD8 phenotypes compared
to normal controls (1). The proportion of CD8 cells may greatly
vary depending on the age of a plaque. CD8 T cells have been
implicated in MHC I-dependent cytotoxicity directed against
demyelinated axons (1). Regulatory CD8+ CD25+ FOXp3+ T cells
also are present in the blood and CSF of MS patients, and influence
pro-inflammatory properties of effector-cytotoxic and antigen-
presenting cells (7).
Much evidence support the involvement of B cells in MS. The
functionally most important components of this lineage include B
lymphocytes (CD19+,CD138), plasma blasts (CD19+,CD138+),
and plasma cells (CD19,CD138+). While CD19 plasma cells are
rare in the CSF of MS patients, CD19+ cells compose 1012 % of
CSF cells (8). CNS antigen-specific B cells with signs of clonal
expansion can be detected in the brain and CSF of MS patients
(911). The high rate of somatic mutations preferentially affecting
the complementarity-determining regions in the rearranged immu-
noglobulin genes contributes to intraclonal diversity of these cells,
406 P. Acs and B. Kalman

and to the occurrence of unique VH sequences in plaques (911).

A physiological mechanism to prevent B cell autoimmunity is
molecular editing, a process replacing elements in the rearranged
immunoglobulin genes after the re-expression of RAG1/RAG2
genes. This editing appears inefficient in MS as autoreactive B cells
with unsuccessfully edited receptors are present in the CSF (12).
In addition, there are meningeal and parenchymal B cell aggre-
gates in MS brains, which resemble B cell aggregates found in the
target organs of other autoimmune disorders (13, 14). While these
observations indirectly suggest the contributions of autoreactive B
cells to the development of MS lesions, evidence supports the
involvement of antibody and complement-mediated phagocytosis
in demyelination (15).
In addition, B lymphocytes, monocytes, macrophages, den-
dritic cells, and residential glia (microglia, astrocytes) play impor-
tant roles in antigen presentation and thus, contribute to induction
and maintenance of autoimmunity. Antigen-presenting cells con-
trol T cell activation and modulate T subset differentiation (5, 16).
Macrophages are also scavengers that phagocytose and clean up
degenerating myelin in the CNS.
Manipulating several of the pro-inflammatory cytokines (e.g.,
by specific monoclonal antibodies, small competitive antagonists,
expression regulation in transgenic and knockout models) and cell
surface molecules (e.g., VLA-4, CD20) can beneficially control
EAE, and some of these strategies have been successfully extended
to treat MS (e.g., anti-VLA4 or Natalizumab, anti-CD20 or
Rituximab) (1719). However, modifying some of the conditions,
such as the timing or route of intervention while using the same
molecule (e.g., anti-IFN-g, anti-TNF-b), can lead to either
enhancement or inhibition of EAE, and interventions that inhibit
EAE may cause exacerbations of MS (e.g., anti-TNF-b) (4). There
are a number of potential reasons of the inconsistent treatment
effects in EAE and MS: (1) While EAE is induced by T cells acti-
vated in vitro or generated in vivo in the peripheral immune sys-
tem, MS may be an autoimmune condition ignited inside the CNS;
(2) the adaptive immune response is involved in the development
of MS, but it is not clear whether or not these cells have primary
pathogenic role or only contribute to the perpetuation of lesion
evolution; (3) the tested therapeutic antibodies may or may not
reach the appropriate cells within MS lesions; and (4) the charac-
teristics of the immune system differ in mice and men (e.g., the
dichotomy of murine TH-1 and TH-2 cytokine profiles does not
exist in humans; individual human T cell clones may produce mixed
TH-1/TH-2 cytokines) (1, 4, 6).

1.2. The Histo- Since most histopathology studies have to rely on sampling at a
pathology of MS single time point, the temporal sequence of events and the exact
role of blood-derived mononuclear cells (MNCs) in lesion development
 20 Pathogenesis of Multiple Sclerosis 407

remain somewhat ambiguous in MS. However, it is unequivocally

established that MS is the disease of the whole brain affecting not
only oligodendrocytes, myelin, and axons in the white matter, but
also neurons and their processes in the cortical and deep gray mat-
ter (2022). Acute relapsingremitting (RR)-MS starts as discrete
inflammatory demyelinating foci in the white and gray matter (23).
Over time, the process spreads to diffuse inflammation and micro-
glial activation in the whole brain associated with axonal transec-
tions, white and gray matter demyelination, and neuronal loss.
Reaching a functional threshold of neuroaxonal loss may correlate
with the conversion of RR-MS to secondary progressive (SP)-MS
(20, 21). The temporal evolution of pathology results in heteroge-
neous lesions in regard to age and immune histological characteris-
tics in cross-sectional studies. The kinetics of lesion development
involves a rise in the number of activated immune cells with MHC
Class II molecules in early active lesions, but these cells gradually
diminish in chronic active lesions. Inactive lesions are characterized
by hypocellularity and gliosis (21).

1.2.1. Heterogeneity of MS Using biopsied and postmortem MS tissues with early lesions
Lesions and Loss of Lucchinetti et al. (24) proposed four intra-individually consistent
Oligodendrocytes: The subtypes of acute demyelinating lesions. Type I and II lesions express
Concept of Histological signs of autoimmunity with perivenular inflammation and demyeli-
Heterogeneity of MS nation. Type I lesions are only characterized by MNC infiltration.
Evolved in Two Directions Type II lesions also have MNC infiltration, but with signs of immu-
noglobulin and complement deposition. In contrast to type I and II
Interindividual
lesions, type III and IV lesions have sparse inflammatory cell
Heterogeneity and
infiltration and instead of autoimmunity, they express signs of oligo-
Intra-individual
dendrocytopathy. The loss of oligodendrocytes is best characterized
Homogeneity of MS
in type III lesions. Type IV lesions with oligodendrocyte loss are the
Lesions
least frequent among all MS lesions (Box 2a).

Sequence of Lesion A more uniform mechanism was proposed for early plaque forma-
Development and Temporal tion with heterogeneity over time by Barnett and Prineas (25)
Heterogeneity (Box 2b). Studying new symptomatic lesions in patients who died
shortly after the onset of a relapse, these authors observed exten-
sive oligodendrocyte apoptosis with nuclear condensation but
without caspase 3 activation. The loss of oligodendrocytes in dis-
crete, still myelinated tissue regions was associated with microglial
activation in the absence of MNC infiltration. Myelin edema and
tissue vacuolization appeared to follow the loss of oligodendro-
cytes, and to trigger the immigration of activated lymphocytes.
A subsequent analysis by the same group confirmed that oligo-
dendrocyte apoptosis is the earliest event in prephagocytic lesions
with still intact myelin and without T and B cell infiltration in tis-
sue bordering rapidly expanding MS lesions. Phagocytic lesions
include macrophages associated with myelin fragmentation, where
the role of innate immune response is to scavenge injured myelin.
408 P. Acs and B. Kalman

Box 2
Loss of Oligodendrocytes

(a) Model 1: oligodendrocytopathy in type III and IV lesions (24,

4951).
In type III lesions, oligodendrocytes undergo an apoptosis-like
cell death which begins with a dying back mechanism. An ini-
tial loss of myelin-associated glycoprotein (MAG) can be
detected in the distal periaxonal oligodendrocyte processes while
myelin basic protein (MBP) and proteolipid lipoprotein are still
intact on the outer surface of the myelin sheet. Subsequently,
nuclear condensation and apoptosis-like loss of oligodendro-
cytes occur without caspase-3 activation. The expression of
hypoxia inducible factor (HIF)-1a and HIF-1b resembles
hypoxic deconditioning. After translocated to the nucleus, the
heterodimer of the HIF-1a (upregulated) and b (constitutively
expressed) binds to HIF responsive elements (HREs) in pro-
moters of neuroprotective molecules. The signs of hypoxic his-
totoxicity with HIF-1a upregulation in type III lesions are
indicators of a mitochondrial energy failure similar to that noted
in acute stroke. In areas surrounding sublethal hypoxic injury,
stress proteins are upregulated. Heat-shock protein (HSP)-70 is
a molecular chaperon that mediates resistance to subsequent
injuries, and facilitates the regeneration of proteins in type III
and Balo type of lesions.
The rarely observed type IV lesions are also characterized by
oligodendrocytopathy, but the loss of cells occurs by a non-apop-
totic mechanism.
(b) Model 2: early loss of oligodendrocytes (25, 26).
The earliest event is the apoptosis of oligodendrocytes in
regions with intact myelin.
As demyelination develops, lesions are infiltrated by scaveng-
ing macrophages (innate immune response), which phagocy-
tose and clean up degraded myelin.
Recently demyelinated lesions have infiltration by blood-
derived T and B cells (adaptive immune response) which may
contribute to perpetuation of pathology or tissue repair.
(c) Possible mechanisms of oligodendrocyte loss (2527, 32). Both
caspase-dependent and -independent mechanisms were described.
Activated microglia and other immune cells or their soluble
inflammatory products may induce oligodendrocyte apoptosis by a
death-ligand engagement (TNF-TRAIL, FasL-Fas), which triggers
activation of the caspase cascade. Involvement of a mitochondrial
mechanism is supported by the upregulation of HIF-1a in type III
lesions. Inflammation-induced oxidative stress (NO, ROS) causes
mitochondrial damage, defect in oxidative phosphorylation, drop
in the mitochondrial transmembrane potential, and altered Ca
homeostasis, which is further worsened by glutamate and ultimately

(continued)
 20 Pathogenesis of Multiple Sclerosis 409

Box 2
(continued)

leads to a mitochondrial mechanism of apoptosis. Epigenetic effects

of exogenous factors (viruses or toxins) are also possible, but
have not been tested. The above-reviewed mechanisms causing
oligodendrocyte apoptosis may also play a role in neuronal injury
and loss.

T and B cells are mainly seen in recently demyelinated areas and

this adaptive immune response may be associated with
oligodendrocyte regeneration (26). These observations chal-
lenge the generally accepted view of a T lymphocyte-initiated
demyelination in MS and point out that oligodendrocyte apop-
tosis may not only occur in type III lesions (24), but rather rep-
resents the earliest stage of lesions underlying MS exacerbation
(25, 26). Oligodendrocyte and myelin damage exposes CNS
antigens that subsequently trigger the immigration of blood-
borne MNCs into the CNS. Interactions between MHC Class
II-positive antigen-presenting cells and TH1/TH17 lympho-
cytes as well as immunoglobulins, complement, and soluble
products of activated MNCs may contribute to the amplification
of tissue injury (27). Alternatively, the appearance of adaptive
immune response may be associated with oligodendrocyte repair
and regeneration (26). In the line of these observations, Breij
et al. (28) acknowledge the temporal heterogeneity of early active
and developing lesions, but suggest a homogeneous presentation
of established demyelinating lesions.

1.2.2. Neuroaxonal Oligodendrocytes are not the only cell types undergoing apoptosis
Degeneration in MS in MS brains. Trapp et al. (20) demonstrated axonal transection in
association with inflammation in the white matter by using immu-
nohistochemistry and confocal microscopy. In contrast, apoptotic
neurons and transection of axonal and dendritic processes were
described in areas of demyelination but in the absence of notable
MNC infiltration in cortical MS lesions (29). Only activated
microglia surrounded injured dendrites, neuritis, and neuronal
pericaria, suggesting that the neuroaxonal loss may be related to or
followed by microglial activation (22). Demyelinating lesions and
neuronal loss were observed not only in cortical but also in deep
gray matter regions (30). Observations suggest that the loss of
410 P. Acs and B. Kalman
1.2.3. Possible Causes
of Cell Injury in MS Brains
neurons likely follow a caspase-independent pathway. Consistent
with the above data, neocortical thinning with loss of neurons,
glia, and synapses was also reported (31).
Thus far, no primary endogenous or exogenous causes of oligoden-
drocyte and neuronal apoptosis have been identified. Some of the
factors contributing to cell and tissue injury developing secondary
to inflammation are reviewed in Box 2c. Direct cytotoxic mecha-
nisms mediated by cytokines and their receptors have been compre-
hensively explored in experimental systems but direct evidence is
sparse in MS (32, 33). Our team proposed that inflammation ignites
a mitochondrion-driven mechanism that contributes to tissue
degeneration in MS (3437). Activated monocytes and microglia
in MS express inducible nitric oxide synthase (iNOS) and produce
increased amounts of NO, which damages proteins by generating
nitration adducts (e.g., nitrotyrosine). NO can also react with O2
(a component of reactive oxygen speciesROS) resulting in a toxic
intermediate called peroxinitrite (38, 39). ROS are produced in
increased amounts by activated inflammatory cells in MS and give a
ring-like appearance on MRI representing macrophages around
acute plaques (40, 41). NO and ROS cause oxidative damage to
macromolecules in the site of inflammation (34). While evidence
suggests that scavengers of NO and physiologic antioxidants can
provide significant therapeutic effects in EAE (42), upregulation of
such molecules was not detected in MS lesions. In contrast, a
significant accumulation of oxidative damage to DNA and a
decreased activity of Complex I were found in chronic active plaques
(34). Despite the accumulated oxidative damage to mitochondrial
(mt)DNA, this decreased Complex I activity was not related to an
accelerated accumulation of somatic mtDNA deletions in chronic
active plaques compared to other brain regions or to age-matched
controls (43). Altered mRNA and protein expressions of mito-
chondrial molecules and impaired activity of other mitochondrial
enzymes were also observed in MS cortex and plaques (4446).
Using histochemistry, Mahad et al. (47) found impaired function
of Complex IV (cytochrome c oxidaseCOX) in Balos type of
concentric sclerosis and in pattern III but not in pattern II MS
lesions (24). This defect in enzyme activity was accompanied by a
decreased expression of the catalytic component (COX-I) of
Complex IV in oligodendrocytes, hypertrophied astrocytes, and
axons (46, 47). These observations suggest that Complex IV func-
tional defect may contribute to the injury of mature oligodendro-
cytes in subtypes of demyelinating MS lesions. In addition to
inflammation, demyelination may directly contribute to a mito-
chondrial mechanism facilitating axonal loss. This mechanism
results from the increased expression and redistribution of the
voltage-gaited Na-channels from the node of Ranvier to the entire
 20 Pathogenesis of Multiple Sclerosis 411

length of demyelinated axons. The higher numbers of Na+/

K+-channels are associated with higher energy demand, but at a
time when the ATP synthesis is compromised by inflammation.
This relative energy deficiency in chronically depolarized naked
axons ultimately leads to an excessive axoplasmic Ca2+ accumula-
tion via the Na+/Ca++ exchanger, impaired structural integrity,
and axonal degeneration (45, 46). In addition, both the energy-
depleted axons and oligodendrocytes are highly sensitive to toxic
effects of glutamate mediated by distinct glutamate receptors (48).
These observations suggest that using a model system that involves
a mitochondrial mechanism of glial or neuronal injury may facili-
tate a better understanding of important features of inflammation-
related tissue degeneration in MS.

2. Cuprizone-
Induced
Oligodendrocyte
Apoptosis and In the past few decades, the cuprizone-induced demyelination
Demyelination model attracted prominent interest, since contrary to other models
of MS, this one provides a highly reproducible system of primary
2.1. The Utility of the oligodendrocyte (OL) apoptosis and secondary demyelination.
Cuprizone Model in The administration of the copper chelating agent cuprizone
(bis-cyclohexanone oxaldihydrazone) to mice induces spatially and
Studying MS
temporally well-defined histopathological alterations in the CNS.
The earliest event is the appearance of megamitochondria (52),
followed by oligodendrocyte apoptosis. The peak of the apoptotic
events is between the third and tenth days (53) of the cuprizone
challenge, but apoptotic oligodendrocytes can be detected during
the entire administration, and even during the recovery period
12 weeks posttreatment (54). The exact mechanism of oligoden-
drocyte apoptosis is not fully understood, and is often debated.
However, it is generally accepted that cuprizone induces metabolic
disturbances in oligodendrocytes which leads to caspase-independent,
apoptosis inducing factor (AIF)-mediated cell death, involving a
mitochondrial mechanism (36, 55).
The massive OL apoptosis is followed by extensive demyelina-
tion. The loss of myelin is preceded and accompanied by a down-
regulation of myelin-related proteins with varying kinetics. For
example, a down-regulation of MAG expression can be seen in a
few days after the initiation of cuprizone administration, while a
complete demyelination of the corpus callosum is usually observed
after 6 weeks of treatment. While demyelination was thought to
affect only particular white matter tracts (i.e., corpus callosum,
superior cerebellar peduncle) (56), recent studies reveal that other
regions including the hippocampus, putamen, cerebellum, and
even distinct gray matter areas in the cortex also undergo demyeli-
nation (57).
412 P. Acs and B. Kalman

Another prominent pathological feature associated with oligo-

dendrocyte apoptosis is the invasion of the demyelinated areas by
activated microglial cells. These cells originate from residential
microglia, but macrophages immigrating from the blood (58) also
contribute to the marked numbers of phagocytic cells seen most
abundantly around the third week of cuprizone treatment. Beyond
the phagocytosis of disrupted myelin sheets, the role of activated
macrophages and microglia in the cuprizone model is controver-
sial. These cells may further amplify the cuprizone-initiated oligo-
dendroglial cell death by the production and secretion of
pro-inflammatory cytokines (59). Alternatively, microglia may have
a beneficial role by stimulating oligodendrocyte precursor cells and
promoting remyelination (56, 60).
If mice return to normal diet after 6 weeks of cuprizone expo-
sure, demyelination is followed by a spontaneous and complete
remyelination driven by the repopulation and maturation of oligo-
dendrocyte progenitor cells (61). If the cuprizone challenge is pro-
longed for 12 weeks, the degree of remyelination may be limited
or remyelination may even fail to occur (54). If it occurs, this spon-
taneous but incomplete remyelination begins between the fourth
and sixth weeks of the ongoing cuprizone administration (62).
Histopathological features of the cuprizone-induced demyeli-
nation closely resemble those of the Lucchinetti and coworkers
defined type III MS lesions (Box 2a, refs. 24, 55). The most
significant similarities include the following: a prominent OL apop-
tosis and microglial activation in the actively demyelinating lesions,
the lesions are not perivenous and their borders are ill defined, and
there is an early and profound down-regulation of the MAG mRNA
level (Box 2a) (63). In addition, the cuprizone model shares com-
mon features with the earliest phases of MS lesion development as
described by Barnett and Prineas (25) and Henderson et al. (26)
(Box 2b), where first, apoptosis of oligodendrocytes occurs in
regions with intact myelin. As the pathology further evolves, early
demyelinating lesions get loaded with scavenging macrophages
(innate immune response), which phagocytose and clean up
degraded myelin. However, in contrast to either the type III lesions
(ref. 24, Box 2a) or to the acute (earliest) MS lesions (refs. 25, 26,
Box 2b), there are no signs of the involvement of the adaptive
immune response in lesions seen in the cuprizone model.
Considering the above-mentioned histopathological features,
the cuprizone model is highly suitable for studying basic mecha-
nisms of acute and chronic demyelination and remyelination,
exploring the pathophysiology of oligodendrocyte apoptosis, and
testing preclinically new interventions for promoting remyelina-
tion and repair in MS lesions.
 20 Pathogenesis of Multiple Sclerosis 413

3. Materials

Mice and cuprizone

1. C57BL/6 mice (Charles River Laboratories, USA).
2. Cuprizone (bis-cyclohexanone oxaldihydrazone) (Sigma-
Aldrich, USA).
Drugs
3. Low molecular weight heparin, 9500 NE 1.0 ml injection
(Glaxo Smith Kline, UK).
4. Diazepam, 2 ml injection (Richter Gedeon Rt., Hungary).
5. Ketamine, 50 mg/ml injection (Richter Gedeon Rt., Hungary).
Fixation, embedding
6. Paraformaldehyde (Sigma-Aldrich, USA).
7. Glutaraldehyde solution (Sigma-Aldrich, USA).
8. Ethanol absolute (Reanal Zrt, Hungary).
9. Osmium tetroxide solution (Sigma-Aldrich, USA).
10. Uranyl acetate dihydrate (Sigma-Aldrich, USA).
11. Propylene oxide (Sigma-Aldrich, USA).
12. Durcupan A/M epoxy resin (Sigma-Aldrich, USA).
13. Lead(II) citrate tribasic trihydrate (Sigma-Aldrich, USA).
Histology
14. Luxol fast blue: Solvent blue 38 (Sigma-Aldrich, USA).
15. Cresyl Violet acetate (Sigma-Aldrich, USA).
16. Hematoxylin (Sigma-Aldrich, USA).
17. (3-Aminopropyl)triethoxysilane (Sigma-Aldrich, USA).
18. Glass slides (Spektrum-3D Kft, Hungary).
19. Lithium carbonate (Sigma-Aldrich, USA).
20. Xylenes histological grade (Sigma-Aldrich, USA).
21. Vectastain Universal ABC Kit (Vector Laboratories Ltd, UK).
22. Hydrogen peroxide solution (Sigma-Aldrich, USA).
23. Diaminobenzidine (Sigma-Aldrich, USA).
Antibodies
24. Anti-MBP antibody (Novocastra Laboratories, UK).
25. Anti-glial fibrillary acidic protein antibody (GFAP, DAKO,
USA).
26. Anti-APC (Ab-7) monoclonal antibody (CC-1) (Calbiochem,
USA).
27. Anti-Iba1 antibody (Wako, USA).
414 P. Acs and B. Kalman

28. Biotin-XX F(ab)2 fragment of goat anti-rabbit IgG (Molecular

Probes, Invitrogen, USA).
General buffers
29. Phosphate-buffered saline (PBS): 8 g NaCl, 0.2 g of KCl,
1.44 g Na2HPO4, 0.24 g KH2PO4 dissolved in 1 l of H2O with
pH adjusted to 7.4.
30. Tris(hydroxymethyl)aminomethane TRIS buffer (1 M):
121.1 g Tris dissolved in 800 ml of ultrapure water. pH adjusted
with HCl.
Western blotting
31. Tissue lysis buffer for Western blotting: 10 mM TRIS buffer
pH 7.4, 0.5 mM sodium metavanadate, 1 mM EDTA, and
protease inhibitor cocktail (1:200) (all ingredients from Sigma-
Aldrich, Hungary).
32. Laemmli buffer: 4 % SDS, 10 % 2-mercaptethanol, 20 % glyc-
erol, 0.004 % bromophenol blue, 0.125 M TrisHCl.
33. Standard 1 Tris-glycine migration buffer: 25 mM Tris base,
190 mM glycine, 0.1 % SDS.
34. 5 % BSA blocking buffer: 5 g BSA in Tris Buffer Saline
Tween20-TBST.
35. Anti-rabbit, horseradish peroxidase-conjugated secondary
antibody (Sigma-Aldrich, USA).
36. Amersham ECL Plus Western Blotting Detection Reagents
(GE Healthcare, USA).
qRT PCR
37. RNA isolation: NucleoSpin kit (Macherey-Nagel, Germany).
38. Reverse transcription: M-MLV Reverse Transcriptase kit
(Invitrogen, USA).
39. iQ SYBR Green Supermix (Bio-Rad, Germany).
40. qRT PCR detection system: MyIQ rtPCR Detection System
(BioRad, Germany).
Instruments
41. MRI equipment: Spectrometer Varian INOVA 400 WB NMR
(Varian, Palo Alto, CA, USA); 9.4 T, 89-mm vertical bore
magnet (Oxford Instruments, UK); Litz-type transmit/receive
volume RF coil and built-in self-shielded water-cooled gradi-
ent system (Doty Scientific, Columbia, SC); VNMR 6.1C and
Image Browser (Varian, Palo Alto, CA) software; Sun Ultra 30
workstation (Sun Microsystems, Mountain View, CA).
42. Cryostat, Leica CM3050 S (Leica Microsystems, Germany).
 20 Pathogenesis of Multiple Sclerosis 415

43. Ultramicrotome: Leica EM UC6 (Leica Microsystems,

Germany).
44. Electron microscope: JEOL 1200EX-II (JEOL Ltd, Japan).
45. Light microscope: Olympus BX-50 microscope with a SPOT
RT color digital camera.
46. Mouse brain atlas (http://www.hms.harvard.edu/research/
brain/atlas).

3.1. Methods As the cuprizone model is suitable for asking a wide variety of
scientific questions, here we only aim to describe the basic method-
ology applicable to different experimental systems. Using these
methods, investigators will be able to investigate general patho-
logical features of oligodendrocyte apoptosis, demyelination, and
remyelination, to test and evaluate the effectiveness of cuprizone
treatment, and to learn a general scenario that occurs in most of
the cuprizone experiments. Before starting a cuprizone experi-
ment, we recommend performing pilot experiments and determin-
ing the kinetics of pathological events in order to have a baseline
for subsequent studies.

3.1.1. Mice The most commonly used strain is the C57BL/6J, and the most
suitable region for the evaluation of demyelination is the corpus
callosum. Therefore, the description below is focused on the alter-
ations found in the corpus callosum of 8-week-old, C57BL/6J,
male mice. It has to be noticed that the cuprizone treatment
induces highly reliable and reproducible demyelination in the CNS
(Fig. 1), although strain, gender, and age differences influence the
distribution and extent of demyelination in mice (Fig. 2; for details
see Subheading 2.3.5) (59, 60).

Fig. 1. Cuprizone-induced demyelination demonstrated by Luxol fast blue staining. A rep-

resentative image of corpus callosum from an untreated male mouse (left panel ) and a
male mouse treated with cuprizone for 5 weeks (right panel ). Intact myelin sheets appear
blue. Profound demyelination can be seen in the corpus callosum of cuprizone-treated
mouse. Arrowheads indicate corpus callosum. 4 Magnification.
416 P. Acs and B. Kalman

Fig. 2. Gender differences in cuprizone-induced demyelination. Luxol fast blue staining of

a female (left panel ) and a male (right panel ) mouse after 5 weeks of cuprizone treatment.
Myelinated fibers appear blue. The images demonstrate that cuprizone induces more
profound demyelination in male mice (corpus callosum is almost entirely demyelinated in
male mouse, while only the peripheral part of the corpus callosum is demyelinated in
female mouse). Arrowheads indicate the peripheral part of corpus callosum, arrows indi-
cate the medial part of corpus callosum. 4 Magnification.

3.1.2. Cuprizone 1. Add 2 g of cuprizone to 1 kg of milled mouse chow (0.2 %).

Administration Mix it thoroughly. (Avoid using any metal instrument while
handling cuprizone.)
2. Feed mice ad libitum with 0.2 % cuprizone.
Note: The dosage of cuprizone can be different. Starting at 8 weeks
of age, 0.2 % cuprizone induces profound demyelination. Lower
dose of cuprizone might be ineffective in this age group. A higher
dose can be used for more profound demyelination, but giving
cuprizone at 0.3 % dose may cause death in approximately 10 % of
the animals in the first 2 weeks of the experiment.

3.1.3. In Vivo Monitoring Cuprizone experiments commonly last for several weeks, during
of the Cuprizone Effect which the pathological effects of the toxin are monitored without
sacrificing animals.

Measuring the Weight The optimal weight range to start the cuprizone administration is
of the Mice between 20 and 24 g in case of male mice. A loss of 34 g in the
first 2 weeks of treatment is characteristic of cuprizone challenge.
However, the weight loss is not an essential element of a successful
cuprizone experiment. After the omission of cuprizone, a rapid
weight gain is expected.
Note: If the weight of mice is to be precisely recorded, it is useful
to change the standard pellet chow to milled chow a couple of days
before the beginning of the experiment, as the switch to the milled
chow itself may induce a minor weight loss (12 g).
 20 Pathogenesis of Multiple Sclerosis 417

MRI Neuroimaging MRI neuroimaging is an established technique for the in vivo

assessment and possible quantitation of evolving demyelination
during cuprizone treatment.
1. Mice are anesthetized by intraperitoneal injection of diazepam
(5 mg/kg) and ketamine (80 mg/kg).
2. Animals are fixed into an epoxy resin animal holder tube
modified to accommodate the tip of teeth and position the
eyes of each animal to the same location above the isocenter of
the magnet.
3. MR images are obtained using a Varian INOVA 400 WB NMR
spectrometer (Varian, Palo Alto, CA) with an 89-mm vertical
bore magnet of 9.4 T (Oxford Instruments, UK) using a
35-mm inner diameter hollow microimaging probe with a
Litz-type transmit/receive volume RF coil and built-in self-
shielded water-cooled gradient system with 600 mT/m gradient
strength and <100 microseconds (s) rise time (Doty Scientific,
Columbia, SC).
4. Coronal cross-sectional images are recorded in a T2-weighted
multi-slice spin echo experiment obtaining 21 contiguous
slices, each 1.0 mm thick.
5. Post-processing is performed by VNMR 6.1C and Image
Browser (Varian, Palo Alto, CA) software on a Sun Ultra 30
workstation (Sun Microsystems, Mountain View, CA).
6. Characteristic images of a cuprizone-treated and an untreated
control mouse are shown in Fig. 3.

3.1.4. Tissue Harvesting The choice of tissue harvesting techniques may depend on the aim
of the experiment. Generally, transcardial perfusion is recom-
mended for histochemistry, but this method of fixation is not suit-
able for protein or RNA studies. The detailed description of tissue
harvesting processes for protein and RNA assays is shown in the
corresponding Subheadings 2.3.8 and 2.3.9.

Fig. 3. Cuprizone-induced demyelination in the corpus callosum demonstrated by in vivo

MRI. Representative T2-weighted spin echo coronal MR image of brain from an untreated
mouse (left panel ) and a mouse treated for 4 weeks with cuprizone (right panel ).
Arrowheads indicate hyperintensities (suggesting demyelination) or hypointensity (intact
myelin status) in the corpus callosum.
418 P. Acs and B. Kalman

Transcardial perfusion
1. Mice are pretreated with 0.1 ml low-molecular-weight heparin
30 min prior to the perfusion.
2. Mice are anesthetized by intraperitoneal injection of diazepam
(5 mg/kg) and ketamine (80 mg/kg).
3. Mice are pinned to the procedure table, the chest is opened,
and the left ventricle is punctured with a green butterfly nee-
dle, while the right atrium is opened.
4. The fixative is introduced into the main circulation via the
green butterfly needle placed into the left ventricle.
5. 4 % Paraformaldehyde solution in PBS is used for fixation.
6. The perfusion is followed by a careful dissection of the brain
and an overnight postfixation in the same fixative.
7. For frozen sections, the brains are placed first into a 10 %, and
then into a 15 % sucrose solution for 1-1 h and subsequently
soaked in a 20 % sucrose solution overnight.
8. For paraffin sections, the brains are routinely dehydrated and
embedded into paraffin.
Note: If low-molecular-weight heparin is unavailable, the perfu-
sion may be started with PBS or physiological saline to wash the
blood out of the vessels. The exact duration of this initial PBS/
saline wash may vary, but as a general rule, the perfusion with the
fixative can be started when the fluid leaking out of the dissected
right atrium becomes water clear.

3.1.5. Evaluation Demyelination is one of the most prominent neuropathological

of Demyelination by LFB features of the cuprizone model. Luxol Fast Blue (LFB) staining
provides a reliable, easy, and inexpensive way to evaluate demyeli-
nation. The application of standard Hematoxylin and Eosin (HE)
staining may provide information on damaged white matter tracts
and hypercellularity. However, HE does not add information to
that revealed by LFB staining if it is combined with nuclear coun-
ter staining, which we prefer to use routinely. If a more in-depth
analysis of the cuprizone-induced cellular and molecular pathology
is needed, we choose using immunohistochemistry and protein or
RNA analyses, as detailed below. However, HE staining may be
useful for an initial identification of anatomical landmarks for sec-
tioning preferentially affected or other selected brain regions.
The LFB staining allows a quick determination of whether or
not the cuprizone challenge is successful, and serves as a useful
pilot method to compare differences in demyelination in drug
administration studies (Fig. 1). The demyelination process becomes
well detectable by LFB after 3 weeks of cuprizone treatment, and
reaches its maximum at 5 weeks.
 20 Pathogenesis of Multiple Sclerosis 419

The modifying effect of gender on the cuprizone-induced

pathology is still a controversial issue. An elegant, recently reported
study (64) suggests that patterns of demyelination and remyelina-
tion are similar between C56BL/6J male and female mice. In con-
trast, our group found that female mice are less susceptible to
cuprizone challenge (unpublished data, Fig. 2) and female sexual
steroids may have a beneficial role by mitigating the developing
pathology (58).
1. LFB dye is used in 0.1 % solution. (1 g substrate is dissolved in
1,000 ml 96 % ethanol.) Five milliliter 10 % acetic acid is added
to this solution. The dye is filtered before usage.
2. Eight-micrometer thick, paraffin-embedded, coronal sections
on silan-coated slides are used. (Frozen sections and the rou-
tinely used gelatin-coated slides are not suitable for LFB
staining.)
3. Sections are cut at the levels of 161, 181, 209, and 221 accord-
ing to mouse brain atlas by Sidman et al. (http://www.hms.
harvard.edu/research/brain/atlas).
4. Sections are routinely deparaffinized. Fifty percent ethanol is
used in the final step; it is recommended to use freshly made
ethanol solution. The sections are incubated in LFB for 1624 h
at 56 C. (Staining jars should be closed tightly to prevent
evaporation of the dye. Although the best quality of staining is
gained after the above-mentioned incubation time, when fast
result is needed, incubation time can be reduced to 5 h.)
5. After incubation, let the staining dishes cool to room
temperature.
6. Sections are rinsed in 96 % ethanol and then washed in distilled
water.
7. Sections are differentiated in 0.05 % lithium carbonate solu-
tion, followed by three washing steps in 70 % ethanol.
8. The degree of differentiation is controlled by visual monitor-
ing under the microscope. If the fibers of the cingular cortex
are distinct, the degree of differentiation is appropriate. If the
differentiation is incomplete, the sections are further differen-
tiated in lithium carbonate solution after a quick rinse in dis-
tilled water.
9. Cresyl violet is used for nuclear counterstaining in 0.1 % solu-
tion with 0.8 ml of 10 % acetic acid added to 100 ml of dye.
Before usage, the dye is heated to 65 C and filtered.
10. The sections are dipped to the dye for 2 min.
11. Sections are washed three times in 96 % ethanol. (Two drops
of glacial acetic acid are added to the ethanol used for the first
wash.)
12. Sections are washed three times in xylol and coverslipped.
420 P. Acs and B. Kalman

13. As a result of the above staining processes, myelinated fibers

will appear blue and nuclei will appear purple (Figs. 1 and 2).
14. Demyelination is evaluated by a semiquantitative scoring
method. Scoring on a scale of 03 is performed by three inde-
pendent experts in a double-blinded manner. A score of 3 is
equivalent to the myelin status of a mouse not treated with
cuprizone, whereas 0 is equivalent to totally demyelinated cor-
pus callosum. A score of 1 and 2 indicates that one-third and
two-thirds of the fibers of the corpus callosum were myeli-
nated, respectively (65).
15. Figure 1 shows typical findings in the corpus callosum after
5 weeks of cuprizone treatment.
16. Figure 2 shows gender differences in susceptibility to cupri-
zone treatment as reflected by the different degrees of demy-
elination in the corpus callosum of female and male mice.
Note: The best way to make intergroup comparisons of demyelina-
tion (e.g., that in control vs. cuprizone or cuprizone vs. cupri-
zone + drug groups) is to apply sections from precisely the same level
of the brains of subjects in each group onto one slide. The level of the
bregma is a suitable region for these studies. Although this method
requires advanced technical skills, the results are more credible, as the
staining conditions are the same for each experimental group.
The dynamics of the disappearance and reappearance of par-
ticular myelin-related proteins (MBP, myelin-oligodendrocyte gly-
coprotein, CGP) underlying the changes reflected by LFB staining
may greatly differ (63). It is well established that mRNA levels of
the myelin proteins are downregulated after a couple of days of
cuprizone treatment, and spontaneously upregulated during the
administration period as well. Therefore, methods complementing
the LFB staining are recommended for more precise analyses of
the neuropathological features of demyelination, as detailed in
Subheadings 2.3.62.3.9.

3.1.6. Detection Apoptosis of oligodendrocytes is one of the most characteristic

of Apoptosis by Electron neuropathological features of cuprizone-induced demyelination.
Microscopic Techniques While apoptotic oligodendrocytes can be found in the brain during
the entire cuprizone administration period, apoptotic cells are most
abundant between the third and tenth day of cuprizone challenge
(53). Following the manufacturers protocols of most commer-
cially available TUNEL kits provides a suitable technique for the
detection of apoptosis. However, our methodological experience
suggests that apoptosis can most reliably be detected on frozen
sections. For ultrastructural characterization of oligodendrocyte
apoptosis, electron microscopic techniques may be used.
1. Mice are transcardially perfused with phosphate buffer (PB
0.1 M, pH 7.4) followed by 4 % paraformaldehyde with 2.5 %
glutaraldehyde in phosphate buffer.
 20 Pathogenesis of Multiple Sclerosis 421

Note: The sine qua non of obtaining reasonable results by

electron microscopy is the rigorous fixation of the brain!
2. Brains are removed from the skulls; approximately 1 mm3 large
blocks are cut from the corpus callosum at the level of the ante-
rior comissure. Blocks are postfixed overnight at 4 C in the
same fixative used for perfusion.
Note: The brains have to be handled with extreme carefulness,
as even harder mechanical manipulations can induce artifacts.
3. After overnight postfixation at 4 C in the fixative used for the
perfusion, blocks are fixed in 1 % osmium tetroxide in 0.1 M
PB for 35 min.
4. Blocks are dehydrated in an ascending ethanol series. To
increase the contrast, 1 % of uranyl acetate is added into the
70 % ethanol.
5. Blocks are transferred to propylene oxide, then placed into
aluminum-foil boats containing Durcupan resin, and then
embedded in gelatin capsules containing the same resin.
6. Semithin (1 mm) and serial ultrathin (100 nm) sections are cut
with a Leica ultramicrotome.
7. Sections are mounted either on mesh or on Collodion-coated
single-slot, copper grids.
8. Additional contrast is provided by post-staining with 5 % uranyl
acetate solution (3 min) followed by lead citrate solution (2 min).
Precise timing of post-staining has to be determined individually.
It is highly important that grids be carefully dried before going
from uranyl acetate solution to lead citrate solution.
9. Sections are examined in JEOL 1200EX-II electron
microscope.
10. A characteristic electron microscopic image of oligodendro-
cyte apoptosis is shown in Fig. 4.

3.1.7. Detection of the The loss of oligodendrocytes, demyelination, accumulation of

Histopathological Features macrophages, and astrogliosis may be visualized by immunohis-
of Cuprizone Model by tochemistry. For immunohistochemical studies, Vectastain Universal
Immunohistochemistry ABC Kit is used according to the manufacturers protocol.
1. After intracardial perfusion with 4 % paraformaldehyde, brains
are embedded in paraffin. (Frozen sections may also be used for
immunohistochemistry, but with different incubation times.)
2. After routine deparaffinization, 8 mm thick, coronal sections are
cut at the levels of 161, 181, 209, and 221 according to mouse
brain atlas by Sidman et al. (http://www.hms.harvard.edu/
research/brain/atlas) and placed onto gelatin-coated slides.
3. For a 10-min-long blocking of the endogenous peroxidase,
3 % H2O2 solution can be used. (This step is only necessary if
DAB reaction is used for visualization.)
422 P. Acs and B. Kalman

Fig. 4. Electron microscopic image of an apoptotic oligodendrocyte in the corpus callosum.

The condensation of chromatin in the nucleus and the disappearance of cytoplasmic
organelles indicate an early phase of programmed cell death. The image was taken after
3 days of cuprizone administration. The arrowhead indicates a giant mitochondrion.
10,000 Magnification.

4. Sections are washed 3 for 10 min in PBS.

5. For unmasking antigenic determinants, sections are placed into
a pH 6 citric acid buffer and heated in microwave oven for 2
for 5 min.
6. Sections are washed 3 for 10 min in PBS.
7. Normal horse serum is used from the Vectastain Universal
ABC Kit for an hour-long background blocking.
8. The following primary antibodies are used: for visualization of
MBP and astrocytes, anti-MBP antibody (1:75, Novocastra
Laboratories, UK) and anti-GFAP antibody (1:500, DAKO,
USA) are used, respectively. Mature oligodendrocytes are
immunostained using anti-APC (Ab-7) monoclonal antibody
(CC-1) (1:200; Calbiochem, USA), whereas anti-Iba1 anti-
body (1:250, Wako, USA) is used as a pan-macrophage marker.
Sections are incubated with primary antibodies in a humidity
chamber overnight at 4 C, followed by a 1-h-long incubation
at room temperature.
9. Sections are washed 3 for 10 min in PBS.
10. Appropriate biotinylated secondary antibodies are applied
(1:200, Molecular Probes, USA) for 1 h.
11. Sections are washed 3 for 10 min in PBS.
12. Sections are incubated with Vectastain avidinbiotin complex
for 2 h.
 20 Pathogenesis of Multiple Sclerosis 423

13. Sections are washed 3 for 10 min in PBS.

14. The immunostaining is visualized by adding the diamino benzi-
dine (DAB) substrate. Ten milligram of DAB is dissolved in
TRIS buffer (pH 7.4). Immediately before usage, 25 ml of 3 %
H2O2 is added to the solution. The DAB solution is applied
onto the sections. A dark brown precipitate indicates positive
immunostaining. The development of DAB reaction should
always be controlled by visual monitoring under a microscope.
15. The sections are rinsed in PBS and distilled water.
16. Nissl or hematoxylin nuclear counterstain may be used.
17. Results of immunohistochemistry studies are shown in Fig. 5.
Note: The LFB staining may be combined with immunohis-
tochemistry (described by Samuel Komoly). In this case, the
immunohistochemistry is followed by the LFB staining.
Although not detailed here, we recommend using frozen sec-
tions and immunofluorescence labeling for testing multiple
antigens (colocalization studies).

3.1.8. Immunoblot Analysis Immunohistochemistry is a reliable method for investigating dis-

of Myelin Basic Protein tribution of different proteins in whole brain. However, unless
there are significant differences, immunostaining does not allow a
precise quantitative comparison among protein levels. The immu-
noblot technique allows us to perform a comparative assessment of
the expression levels of different proteins. With careful dissection
of the brain, it is possible to intra-individually compare the expres-
sion of proteins in different brain regions (for example corpus cal-
losum compared to cortex). As MBP is one of the most frequently
investigated proteins in cuprizone studies, a detailed protocol of
MBP immunoblotting is provided below.
1. Mice are sacrificed with quick decapitation.
2. Brain is quickly removed from skull, the corpus callosum is
dissected, and the samples are placed in a round-bottom
microcentrifuge tube and immersed in liquid nitrogen. (These
steps have to be performed as quickly as possible to prevent
any degradation by proteases. Samples can be stored at 80 C
for later use.)
3. Add 300 ml of lysis buffer to each 5 mg of samples. Lysis buffer:
10 mM TRIS buffer pH 7.4, 0.5 mM sodium metavanadate,
1 mM EDTA, and protease inhibitor cocktail (1:200). The
samples are homogenized with electronic homogenizer and
then constantly agitated on an orbital shaker at 4 C for 2 h.
4. The samples are centrifuged for 20 min at 8,000 g at 4 C in
a microcentrifuge. The tubes are gently removed from the
centrifuge and immediately placed on ice. The supernatant is
aspirated and placed in a fresh tube previously kept on ice.
424 P. Acs and B. Kalman

Fig. 5. Characteristic neuropathological features of cuprizone administration. (a, b) MBP

immunohistochemistry. Demyelination of the peripheral part of the corpus callosum after
4 weeks of cuprizone treatment (b). The myelin sheets are intact in an untreated mouse
(a). Arrowheads indicate corpus callosum. (c, d): GFAP immunohistochemistry. Massive
astrocytosis in the midline of the corpus callosum after 4 weeks of cuprizone administra-
tion (d) compared to untreated mouse (c). (e, f): APC immunohistochemistry. Almost total
loss of matured oligodendrocytes in the corpus callosum of a cuprizone-treated mouse (f).
(e) Typical number and arrangement of matured oligodendrocytes in an untreated mouse.
(g, h) Iba1 immunohistochemistry. Massive macrophage accumulation in the peripheral
corpus callosum after 4 weeks of cuprizone challenge (h). (g) The corpus callosum of an
untreated mouse. 12 Magnification.
 20 Pathogenesis of Multiple Sclerosis 425

5. The concentration of the lysates is determined by Bradford

assay. Bovine serum albumin is used for protein standard. After
determining the protein concentration, samples can be stored
at 80 C or can be prepared for loading onto an SDS poly-
acrylamide gel.
6. Standard 2 Laemmli buffer is used for loading buffer. The
Laemmli buffer contains 4 % SDS, 10 % 2-mercaptoethanol,
20 % glycerol, 0.004 % bromophenol blue, and 0.125 M Tris
HCl. Samples are mixed with the 2 Laemmli buffer in a 1:1
ratio and boiled at 95100 C for 5 min. Samples are mixed by
vortexing before and after the heating step.
7. Twenty microgram of total protein is loaded per well of an
SDS polyacrylamide gel. (Using of special gel loading tips is
recommended. Take care not to poke the bottom of the well
with the tip, and not to overfill the well.)
8. The gel is submerged in migration buffer. Standard 1 Tris
glycine migration buffer is used (25 mM Tris base, 190 mM
glycine, 0.1 % SDS).
9. The gel is run for 1 h at constant 200 V, and then the samples
are transferred to a nitrocellulose membrane. The transfer lasts
for 2 h at constant 100 mA.
10. The membrane is blocked with a 5 % BSA containing blocking
buffer (5 g BSA in Tris Buffer Saline, Tween20 (TBST)) for
1 h, at 4 C under constant agitation. The membrane is rinsed
for 5 s in TBST after incubation.
11. The membrane is incubated with an anti-MBP antibody
(Novocastra Laboratories, Germany) which is diluted to
1:1,000 in blocking buffer. The membrane is incubated over-
night, at 4 C, with constant agitation.
12. The membrane is washed 4 for 5 min in TBST.
13. Anti-rabbit, horseradish peroxidase-conjugated secondary
antibody (Sigma-Aldrich, Germany) is used in 1:5,000 dilu-
tion in TBST. The membrane is incubated for 1 h at room
temperature under constant agitation.
14. For the visualization of MBP detection, a commercially avail-
able ECL+ (GE Healthcare) kit is used according to the manu-
facturers protocol.
15. The film is developed using an automated X-ray film developer.
16. The expected weight size of MBP isoform is 21 kDa. If a semi-
quantitative measurement is needed, the films are scanned and
the pixel volumes of the bands are determined by using the
National Institutes of Health Image J software (Bethesda,
MD, USA).
17. MBP immunoblotting reveals decreased MBP expression in the
corpus callosum after 5 weeks of cuprizone feeding (Fig. 6).
426 P. Acs and B. Kalman

Fig. 6. MBP immunoblot from the dissected corpus callosum after 4 weeks of cuprizone
administration. MBP expression is almost absent in the sample from the cuprizone-treated
mouse. Even protein loadings were confirmed by an anti-Akt antibody and immunoblotting.

3.1.9. Gene Expression The selective regional vulnerability is one of the major characteris-
Analyses of Myelin Basic tic features of cuprizone model. The physiological and pathologi-
Protein by Real-Time PCR cal mechanisms causing this phenomenon are still unknown. The
combination of micropunch technique with qRT PCR allows us to
investigate anatomically closely localized but functionally different
brain regions.
1. Mice are rapidly decapitated and brains are quickly removed
and placed on dry ice.
2. Samples are obtained by micropunching technique (66). While
this method may require higher numbers of animals (since the
amounts of the obtained samples are small), it provides the best
quality of RNA and allows the most precise localization of the
acquired punched samples. Samples obtained by this technique
will exclusively contain tissue from a highly selected brain region.
3. Two-hundred micrometer thick serial sections are made in a
cryostat (Leica CM3050 S).
4. The slides are immediately placed on dry ice for transfer, and
kept at 80 C until needed.
5. The micropunching is performed with a punching needle
(200 mm in diameter) on dry ice, under stereomicroscopic
control. Samples can be obtained from the corpus callosum or
other selected parts of the brain, and kept at 80 C until
further experiments.
6. Total RNA is isolated using the NucleoSpin kit (Macherey-
Nagel, Germany) according to the manufacturers protocol.
7. The concentration of RNA is extrapolated from the OD260
spectrophotometric measurement. The quality of RNA may be
assessed based on the OD260/280 ratio and the integrity of
 20 Pathogenesis of Multiple Sclerosis 427

the 18S/28S/5S bands in the RNA minigel. If available, more

advanced RNA quality assessment may be performed by using
the Nanodrop ND-1000 and the Agilents 2100 Bioanalyzer.
8. Reverse transcription is performed by using the Invitrogen
M-MLV RT-kit with random hexanucleotide primers accord-
ing to the manufacturers protocol.
9. The RT PCR master mix (pro probe) is made in the following
order: 5 ml iQ SYBR Green Supermix, 2 ml ultrapure H2O,
0.5 ml antisense primer (10 mM), 0.5 ml sense primer (10 mM).
The master mix is mixed carefully. (It is recommended to pre-
pare 10 % more master mix than needed.) The following primer
sequences are used for MBP: sense: 5-cca tcc aag aag acc cca
ca-3, antisense: 5-ccc ctg tca ccg cta aag aa-3 (expected PCR
product: 191 bp). For housekeeping gene, the hypoxanthine-
guanine phosphoribosyltransferase (HPRT) gene is used with
the following primer sequences: sense: 5-gct ggt gaa aaggac
ctc t-3, antisense: 5-cac agg act aga aca cct gc-3 (expected
PCR product: 248 bp).
10. Eight microliter of the master mix is pipetted into the wells of
a 96-well plate and then 2 ml of each sample is added into the
wells. The plate is sealed.
11. The qPCR reactions are conducted using the MyIQ rtPCR
Detection System (BioRad, Germany) under the following con-
ditions: 10-min enzyme activation at 95 C, 45 cycles of 15-s
denaturation at 95 C, 30-s annealing at 60 C, 30-s extension
at 72 C, and 5-s fluorescence measurement at 80 C.
12. Relative quantification of the target gene mRNA is performed
using the delta cycle threshold (dCT) method which defines the
ratio of target and the housekeeping reference mRNA (HPRT).
In each run, external standard curves are generated by using
logarithmic dilutions of the standard cDNA. The concentra-
tion of the target mRNA is determined by comparing the cross-
ing time values of the target and standard amplicons.
13. Melting curves and gel electrophoresis of the amplicons are
routinely analyzed to confirm the specificity of the RT-PCR
reaction.
14. In our hands, 5 weeks of cuprizone treatment caused a highly
significant (fourfold) down-regulation of MBP mRNA level
(data not shown).

4. Conclusion

Data suggest that the tissue loss is a complex and multifactorial

process in MS. There is a phenotypic and genetic heterogeneity of
the disease, accounting for, at least in part, the great variability of
428 P. Acs and B. Kalman

observations. Apoptotic loss of oligodendrocytes may be present in

the earliest lesions, but depletion of oligodendrocytes, transection
of axons, and loss of neurons progressively occur during lesion
evolution. MNCs usually co-localize with demyelinated lesions and
axonal transections, but the loss of oligodendrocytes in the earliest
stages of acute lesions and the apoptosis of neurons in chronic gray
matter lesions appear to be predominantly driven by unknown
intrinsic or extrinsic factors and are accompanied by activated
microglia in the absence of MNC infiltration. The cytotoxic effects
of inflammatory cells contributing to demyelination and neurode-
generation either by immune celltissue interactions or by soluble
inflammatory products (NO, ROS, glutamate, cytokines, immu-
noglobulins with or without complement) also remain to be fur-
ther evaluated. Interpretation of data from cross-sectional
histological observations is a complex process and may not always
accurately reflect the temporal sequence of immune pathological
events in the CNS. The potential intrinsic (soluble inflammatory
cell products) or extrinsic (viral products, toxic factors) causes of
oligodendroglial and neuronal apoptosis remain to be better under-
stood. Recent observations raise the possibility that the loss of oli-
godendrocytes and neurons develops prior to or in association with
microglial activation. The innate immune response (macrophages)
clears the damaged myelin by phagocytosis in both the gray and
white matter, while the adaptive immune response involving CD4
and CD8 T cells and B lymphocytes may appear secondary to the
developing CNS pathology and perpetuates the tissue injury or
facilitates repair. The old as well as the newer paradigms of lesion
development require further clarifications and an identification of
the primary trigger of the pathological process. The cuprizone
model, while offering analyses of causes and consequences of oli-
godendrocyte injury by a mitochondrial mechanism, greatly facili-
tates a better understanding of MS pathogenesis, and supports the
development of tissue repairpromoting treatment modalities.

References
1. Bennett JL, Stve O (2009) Update on
inflammation, neurodegeneration, and immuno-
regulation in multiple sclerosis: therapeutic impli-
cations. Clin Neuropharmacol 32(3):121132
2. Linthicum DS, Munoz JJ, Blaskett A (1982)
Acute experimental autoimmune encephalomy-
elitis in mice. I. Adjuvant action of Bordetella
pertussis is due to vasoactive amine sensitiza-
tion and increased vascular permeability of the
central nervous system. Cell Immunol
73(2):299310
3. Westarp ME, Wekerle H, Ben-Nun A et al
(1987) T lymphocyte line-mediated experimen-
tal allergic encephalomyelitis a pharmacologic
model for testing of immunosuppressive agents
for the treatment of autoimmune central ner-
vous system disease. J Pharmacol Exp Ther
242(2):614620
4. Steinman L (2010) Mixed results with modula-
tion of TH-17 cells in human autoimmune dis-
eases. Nat Immunol 11(1):4144
5. Crome SQ, Wang AY, Levings MK (2010)
Translational mini-review series on TH17 cells:
function and regulation of human T helper 17
cells in health and disease. Clin Exp Immunol
159(2):109119
6. OConnor RA, Taams LS, Anderton SM (2010)
Translational mini-review series on Th17 cells:

CD4 T helper cells: functional plasticity and
differential sensitivity to regulatory T cell-
mediated regulation. Clin Exp Immunol
159(2):137147
7. Correale J, Villa A (2010) Role of regulatory
CD8+CD25+FoxP3+ T cells in multiple scle-
rosis. Ann Neurol 67(5):625638
8. Cepok S, Rosche B, Grummel V et al (2005)
Short-lived plasma blasts are the main B cell
effector subset during the course of multiple
sclerosis. Brain 128:16671676
9. Qin Y, Duquette P, Zhang Y et al (1998) Clonal
expansion and somatic hypermutation of Vh
genes of B cells from the cerebrospinal fluid in
multiple sclerosis. J Clin Invest 102:10451050
10. Owens GP, Burgoon MP, Anthony J et al (2001)
The immunoglobulin G heavy chain repertoire
in multiple sclerosis plaques is distinct from the
heavy chain repertoire in peripheral blood lym-
phocytes. Clin Immunol 98:258263
11. Monson NL, Brezinschek HP, Brezinschek RI
et al (2005) Receptor revision and atypical
mutational characteristics in clonally expanded
B cells from the cerebrospinal fluid of recently
diagnosed multiple sclerosis patients. J
Neuroimmunol 158(12):170181
12. Lambracht-Washington D, OConnor KC,
Cameron EM et al (2007) Antigen specificity
of clonally expanded and receptor edited cere-
brospinal fluid B cells from patients with relaps-
ing remitting MS. J Neuroimmunol
186(12):164176
13. Serafini B, Rosicarelli B, Magliozzi R et al
(2004) Detection of ectopic B-cell follicles with
germinal centers in the meninges of patients
with secondary progressive multiple sclerosis.
Brain Pathol 14(2):164174
14. Willis SN, Stadelmann C, Rodig SJ et al (2009)
EpsteinBarr virus infection is not a character-
istic feature of multiple sclerosis brain. Brain
132(Pt 12):33183328
15. Lucchinetti C, Brck W, Noseworthy J (2001)
Multiple sclerosis: recent developments in neu-
ropathology, pathogenesis, magnetic resonance
imaging studies and treatment. Curr Opin
Neurol 14(3):259269
16. Stoeckle C, Tolosa E (2009) Antigen processing
and presentation in multiple sclerosis. Results
Probl Cell Differ 2010;51:149172
17. Goodin DS, Cohen BA, OConnor P et al
(2008) Therapeutics and Technology Assessment
Subcommittee of the American Academy of
Neurology. Assessment: the use of natalizumab
(Tysabri) for the treatment of multiple sclerosis
(an evidence-based review): report of the
Therapeutics and Technology Assessment
Subcommittee of the American Academy of
Neurology. Neurology 71(10):766773
20 Pathogenesis of Multiple Sclerosis 429
18. Hauser SL, Waubant E, Arnold DL et al (2008)
HERMES Trial Group. B-cell depletion with
rituximab in relapsing-remitting multiple scle-
rosis. N Engl J Med 358(7):676688
19. Bar-Or A, Calabresi PA, Arnold D et al (2008)
Rituximab in relapsing-remitting multiple scle-
rosis: a 72-week, open-label, phase I trial. Ann
Neurol 63(3):395400
20. Trapp BD, Peterson J, Ransohoff RM et al
(1998) Axonal transection in the lesions of
multiple sclerosis. N Engl J Med 338:278285
21. Trapp BD, Bo L, Mork S, Chang A (1999)
Pathogenesis of tissue injury in MS lesions. J
Neuroimmunol 98:4956
22. Peterson JW, Bo L, Mork S, Chang A et al
(2002) VCAM-1-positive microglia target oligo-
dendrocytes at the border of multiple sclerosis
lesions. J Neuropathol Exp Neurol 61:539546
23. Kutzelnigg A, Lucchinetti CF, Stadelmann C
(2005) Cortical demyelination and diffuse
white matter injury in multiple sclerosis. Brain
128(Pt 11):27052712
24. Lucchinetti C, Brck W, Parisi J et al (2000)
Heterogeneity of multiple sclerosis lesions:
implications for the pathogenesis of demyelina-
tion. Ann Neurol 47(6):707717
25. Barnett MH, Prineas JW (2004) Relapsing and
remitting multiple sclerosis: pathology of the
newly forming lesion. Ann Neurol 55:
458468
26. Henderson AP, Barnett MH, Parratt JD,
Prineas JW et al (2009) Multiple sclerosis: dis-
tribution of inflammatory cells in newly form-
ing lesions. Ann Neurol 66:739753
27. Barnett MH, Sutton I (2006) The pathology of
multiple sclerosis: a paradigm shift. Curr Opin
Neurol 19:242247
28. Breij EC, Brink BP, Veerhuis R et al (2008)
Homogeneity of active demyelinating lesions in
established multiple sclerosis. Ann Neurol
63(1):1625
29. Peterson JW, Bo L, Mork S et al (2001)
Transected neurites, apoptotic neurons, and
reduced inflammation in cortical multiple scle-
rosis lesions. Ann Neurol 50:389400
30. Vercellino M, Plano F, Votta B et al (2005)
Grey matter pathology in multiple sclerosis.
J Neuropathol Exp Neurol 64:11011107
31. Wegener C, Esiri MM, Chance SA et al (2006)
Neocortical neuronal, synaptic, and glial loss in
multiple sclerosis. Neurology 67:960967
32. Zipp F (2000) Apoptosis in multiple sclerosis.
Cell Tissue Res 301(1):163171
33. Hestvik AL, Skorstad G, Vartdal F et al (2009)
Idiotype-specific CD4+ T cell induced apoptosis
of human oligodendrocytes. J Autoimmun
32(2):125132
430 P. Acs and B. Kalman
34. Lu F, Selak M, OConnor J et al (2000)
Oxidative damage to mitochondrial DNA and
activity of mitochondrial enzymes in lesions of
multiple sclerosis. J Neurol Sci 177:95103
35. Kalman B, Albert RH, Leist TP (2002) Genetics
of multiple sclerosis: determinants of autoim-
munity and neurodegeneration. Autoimmunity
35(4):225234
36. Kalman B, Laitinen K, Komoly S (2007) The
involvement of mitochondria in the pathogen-
esis of multiple sclerosis. J Neuroimmunol
188(12):112
37. Kalman B (2006) Role of Mitochondria in MS.
Curr Neurol Neurosci Rep 6:244252
38. Bo L, Dawson TM, Wesselingh S et al (1994)
Induction of nitric oxide synthase in demyeli-
nating regions of multiple sclerosis brains. Ann
Neurol 36:778786
39. Cross AH, Manning PT, Stern MK et al (1997)
Evidence for the production of peroxynitrite in
inflammatory CNS demyelination. J nervous system. Brain Pathol 11:107116
Neuroimmunol 80:121130
40. Vladimirova O, OConnor J, Cahill A et al
(1998) Oxidative damage to DNA in plaques
of MS brains. Mult Scler 4:413418
41. Powell T, Sussman JG, Davies-Jones GA (1992)
MR imaging in acute multiple sclerosis: ringlike
appearance in plaques suggesting the presence
of paramagnetic free radicals. Am J Neurorad
13:15441546
42. Hooper DC, Bagasra O, Marini JC et al (1997)
Prevention of experimental allergic encephalo-
myelitis by targeting nitric oxide and peroxini-
trite: implications for the treatment of multiple
sclerosis. Proc Natl Acad Sci 94:25282533
43. Blokhin A, Vyshkina T, Komoly S et al (2008)
Lack of mitochondrial DNA deletions in lesion
of multiple sclerosis. Neuromolecular Med
10:187194
44. Dutta R, McDonough J, Yin X et al (2006)
Mitochondrial dysfunction as a cause of axonal
degeneration in multiple sclerosis patients. Ann
Neurol 59:478489
45. Mahad D, Lassmann H, Turnbull D (2008)
Review: mitochondria and disease progression
in multiple sclerosis. Neuropathol Appl
Neurobiol 34(6):577589
46. Mahad DJ, Ziabreva I, Campbell G et al (2009)
Mitochondrial changes within axons in multiple
sclerosis. Brain 132(Pt 5):11611174
47. Mahad D, Ziabreva I, Lassmann H et al (2008)
Mitochondrial defects in acute multiple sclero-
sis lesions. Brain 131(Pt 7):17221735
48. Trapp BD, Stys PK (2009) Virtual hypoxia and
chronic necrosis of demyelinated axons in
multiple sclerosis. Review. Lancet Neurol 8:
280291
49. Aboul-Enein F, Lassmann H (2005)
Mitochondrial damage and histotoxic hypoxia:
a pathway of tissue injury in inflammatory brain
disease? Acta Neuropathol (Berl) 109:4955
50. Stadelmann C, Ludwin S, Tabira T et al (2005)
Tissue preconditioning may explain concentric
lesions in Balos type of multiple sclerosis. Brain
128:979987
51. Graumann U, Reynolds R, Steck AJ et al (2003)
Molecular changes in normal appearing white
matter in multiple sclerosis are characteristic of
neuroprotective mechanisms against hypoxic
insult. Brain Pathol 13:554573
52. Ludwin SK (1978) Central nervous system
demyelination and remyelination in the mouse:
an ultrastructural study of cuprizone toxicity.
Lab Invest 39:597612
53. Matsushima GK, Morell P (2001) The neuro-
toxicant, cuprizone, as a model to study demy-
elination and remyelination in the central
54. Lindner M, Fokuhl J, Linsmeier F et al (2009)
Chronic toxic demyelination in the central ner-
vous system leads to axonal damage despite
remyelination. Neurosci Lett 453:120125
55. Veto S, Acs P, Bauer J et al (2010) Inhibiting
poly(ADP-ribose) polymerase: a potential ther-
apy against oligodendrocyte death. Brain
133:822834
56. Komoly S (2005) Experimental demyelination
caused by primary oligodendrocyte dystrophy.
Regional distribution of the lesions in the ner-
vous system of mice [corrected]. Ideggyogy Sz
58:4043
57. Kipp M, Clarner T, Dang J (2009) The cupri-
zone animal model: new insights into an old
story. Acta Neuropathol 118:723736
58. Remington LT, Babcock AA, Zehntner SP et al
(2007) Microglial recruitment, activation, and
proliferation in response to primary demyelina-
tion. Am J Pathol 170(5):17131724
59. Pasquini LA, Calatayud CA, Bertone Una AL
et al (2007) The neurotoxic effect of cuprizone
on oligodendrocytes depends on the presence
of pro-inflammatory cytokines secreted by
microglia. Neurochem Res 32:279292
60. Acs P, Kipp M, Norkute A et al (2009)
17b-Estradiol and progesterone prevent cupri-
zone provoked demyelination of corpus callo-
sum in male mice. Glia 57:807814
61. Lindner M, Heine S, Haastert K et al (2008)
Sequential myelin protein expression during
remyelination reveals fast and efficient repair
after central nervous system demyelination.
Neuropathol Appl Neurobiol 34:105114
62. Armstrong RC (2007) Growth factor regulation
of remyelination: behind the growing interest

in endogenous cell repair of the CNS. Future
Neurol 2:689697
63. Morell P, Barrett CV, Mason JL et al (1998)
Gene expression in brain during cuprizone-
induced demyelination and remyelination. Mol
Cell Neurosci 12:220227
64. Taylor LC, Gilmore W, Matsushima GK (2009)
SJL mice exposed to cuprizone intoxication
20 Pathogenesis of Multiple Sclerosis 431
reveal strain and gender pattern differences in
demyelination. Brain Pathol 19:467479
65. Hiremath MM, Saito Y, Knapp GW et al (1998)
Microglial/macrophage accumulation during
cuprizone-induced demyelination in C57BL/6
mice. J Neuroimmunol 92:3849
66. Palkovits M (1983) Punch sampling biopsy
technique. Methods Enzymol 103:368376
 Chapter 21

Assessing Inflammatory Disease at Mucosal

Surfaces in Murine Genetic Models
R.W. Engelman and William G. Kerr

Abstract
Inflammatory diseases of the mucosal surfaces are rising worldwide and particularly in the Western world
that is witnessing unprecedented increases in the number and incidence of both asthma and inflammatory
bowel disease. The laboratory mouse allows the application of the full panoply of modern genetic, immu-
nological and biochemical tools to increase our understanding of how inflammation arises and how it
might be controlled at mucosal surfaces. Here we provide a detailed description of how to systematically
assess inflammatory disease in the lung and intestines of the laboratory mouse. We provide histopathology
examples from SHIP mutant mice that are the only known genetic mutant to suffer from pulmonary con-
solidation, asthma, and Crohns disease. The intent of this chapter is to facilitate increased surveillance of
mucosal inflammation in studies where the laboratory mouse is utilized so that we can better understand
the cell types, genes, and microorganisms that contribute to mucosal inflammatory disease and thereby
develop more effective therapies and preventive strategies.

Key words: Pneumonia, Asthma, IBD, Mouse models, T cells

Crohns disease (CD) and ulcerative colitis (UC) are chronic

inflammatory diseases of the alimentary tract with multiple etiologies,
distinguishable by unique histopathological phenotypes and patho-
genic mechanisms (15). CD classically occurs in the ileum, begins
with the formation of multiple aphthoid ulcers infiltrated by neu-
trophils, which progress and coalesce into a segmental transmural
ileitis with granuloma, strictures, fissures, and fistulas. In contrast,
UC is classically confined to the colon and comprises a diffuse,
superficial, mucosalsubmucosal colitis. Histopathological hetero-
geneity exists within each disorder.
Etiologically, this spectrum of enteric autoimmune inflammatory
diseases results from complex polygenic and environmental inter-
actions. Although genome-wide association studies have identified
more than 30 susceptibility loci for IBD that disturb either intesti-
nal epithelial cell (IEC) barrier homeostasis or immune effector or
regulatory cell functions and interactions with luminal commensal

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_21, Springer Science+Business Media New York 2012

433
434 R.W. Engelman and W.G. Kerr

microflora, many susceptibility genes possess differing functions in

IEC and hematopoietic cells, and the specific IEC or immunologic
defects in a majority of genetically susceptible individuals remain
enigmatic (69).
Mouse models representative of the major IBD phenotypes
have contributed to an understanding of the pathogenic mecha-
nisms of IBD, and more are needed to identify potential opportu-
nities for therapeutic approaches. Models include those mice that
spontaneously develop IBD-like enteritis/colitis as a result of a
naturally occurring mutation or via gene targeting or transgene
introduction, or mice that are induced to develop IBD-like enteri-
tis/colitis by exposure to an exogenous (e.g., haptenating) agent.
Mouse models have contributed to identifying many of the inter-
acting factors that contribute to IBD pathogenesis, including IEC
integrity and function, alterations in the mucusantimicrobial
secreted barrier, and the presence of defective regulatory or exces-
sive effector T-cell function (1022).
Mouse models have revealed an etiologic interplay between
immunologic dysregulation and commensal microbiota. Model
strain-specific immunoregulatory defects that result in an IBD-
spectrum phenotype include targeted deletion of receptors for either
transforming growth factor (TGF)- or interleukin (IL)-10, defects
in regulatory T cell (Treg) modulation of effector functions via
TGF-- or IL-10-dependent mechanisms, aberrant B-cell specificity,
serum autoreactivity, or myeloid cell dysfunction. Pro-inflammatory
cytokines increase in IBD, including tumor necrosis factor (TNF)-,
the Th1-polarizing cytokines IL-12 and IFN-, and IL-23, the latter
of which sustains a Th17-associated immune response (1016).
That primary defects in immune cell function contribute to CD in
some human patients is supported by reports of disease remission
following allogeneic bone marrow transplantation (17).
Mouse models have also revealed that epithelial barrier func-
tions are critical to enteric homeostasis. Model strain-specific
defects in epithelia proposed to result in enteritis include altered
IEC cytokine expressions, increased IEC paracellular permeability
due to inadequate IEC tight junctions, mucosal leakiness caused
by a dominant negative epithelial N-cadherin, the absence of an
intestinal mucus component in Muc2/ mice which contributes to
an endoplasmic reticulum (ER) stress response in goblet cells caus-
ing reduced mucus secretion and an increase in pro-inflammatory
cytokines, an enterocyte-specific ablation of nuclear factor kappa B
(NF-B) activation which permits microflora to invade beyond the
epithelial barrier and results in an exuberant inflammation, the
inability of a Paneth cell-derived NOD2 variant to expel luminal
bacteria, diminished IEC autophagy and antimicrobial granule
exocytosis, and altered IEC toll-like receptor (TLR) expression
with potentially dysregulated responses to common bacterial
motifs (1824).
21 Assessing Inflammatory Disease at Mucosal Surfaces in Murine Genetic Models 435

Luminal commensal microbiota is normally host protective,

but in the presence of mucosal immunologic dysregulation, con-
tribute to IBD progression. Normally, TLR signaling activated by
commensal microbiota provides cytoprotective signals to epithelia.
In the absence of microflora in germ-free mice, immune dysregula-
tion does not result in IBD. Indeed, dysregulated, exaggerated
immune responses in IBD may be directed against microflora ini-
tially and later evolve into an autoimmune disease, to include
autoantibody formation. CD and UC phenotypes appear to have
unique pathogenic mechanisms with respect to host interactions
with commensal microflora. NOD2 (CARD15), the intracellular
sensor of bacterial peptidoglycan, and autophagy factors ATG16L1
and IRGM, are genetic factors for CD but not for UC.
Understanding how hostmicrobiota interactions are disrupted in
IBD phenotypes also requires an understanding of the composi-
tion of normal luminal microflora.
Mouse models of IBD are often induced rather than spontane-
ously developing models and frequently involve the colon (5, 7,
25). Three CD mouse models that spontaneously develop chronic
inflammation of the ileum are the SAMP1/Yit mouse, the
TNFARE mouse, and the Src homology 2 (SH2)-containing
inositol-5-phosphatase (SHIP)/ mouse (2628). SAMP1/Yit
mice spontaneously develop terminal ileitis by 10 weeks of age
driven by Th1- and Th2-type immune responses, the severity of
which can be decreased by anti-TNF antibodies (25, 29, 30).
Although SAMP1/Yit mesenteric lymph node CD4+ T cells are
capable of adoptively transferring ileitis to SCID mice, increased
IEC paracellular permeability may be a principle susceptibility fac-
tor in this model (22). The TNFARE mouse model was gener-
ated by deletion of 69 bp within the AU-rich region (ARE) of the
gene that encodes TNF, resulting in elevated systemic TNF levels
associated with expansion of CD8+ effector cell subpopulations
that mediate chronic ileitis and arthritis in both heterozygous and
homozygous mice (27, 31). Hematopoietic cells of neither the
SAMP1/Yit nor the TNFARE mouse are capable of adoptively
transferring CD-like disease to an immunocompetent host. SHIP/
mice develop segmental, transmural pyogranulomatous ileitis that
recapitulates many features of CD enteric pathology by 8 weeks of
age (28). Reconstitution of SHIP/ mice with SHIP-competent
hematopoietic cells prevents CD-like ileitis, while transfer of
SHIP/ spleen cells to an immunocompetent host elicits CD-like
ileitis. SHIP signaling limits the number and function of immuno-
regulatory cells either by limiting the survival of FoxP3+ Treg cells
or by preventing the inappropriate acquisition of FoxP3 expression
by nave CD4 T cells (3241). FoxP3+ Treg cells play an important
anti-inflammatory role. SHIP-deficiency also promotes the inap-
propriate expansion of myeloid immunoregulatory (MIR) cells,
and limits PI3K activation of key downstream immune effector
436 R.W. Engelman and W.G. Kerr

pathways such as Akt and NF-B (35, 36). SHIP/ granulocytes

are less susceptible to apoptotic signals, and granulocytemonocyte
infiltrations are found in multiple organs in SHIP/ mice. A role of
SHIP1 in human IBD is suggested by the demonstration that a
chromosome 2 polymorphism located at 2q37 is associated with a
significantly increased and early onset of IBD, and that the SHIP1
locus is found in the 2q37 region (42).
Mucosal homeostasis appears to result from various coopera-
tive host contributions that reinforce the IEC structural barrier
including its tight junctions, relies on an appropriately orchestrated
mucosal immune response, all interacting with commensal micro-
biota at the luminal surface. These host contributions include gob-
let cell secretion of mucus, Paneth cell secretion of antimicrobial
peptides, IEC NF-B activation by bacterial products, IEC trans-
port of dimeric immunoglobulin A (IgA) produced by plasma cells
in the lamina propria, and the IEC release of intra-luminal secre-
tory IgA. Diminution of this structural and functional mucosal
barrier may permit commensal microbiota to aberrantly or exces-
sively stimulate the mucosal-based immune system and elicit a pro-
inflammatory T cell-mediated immune response, acutely manifest
as a granulocytemonocyte infiltration. Primary defects in immu-
nologic regulation may also initiate IBD in the absence of an IEC
breach, as commensal microflora may routinely trigger mucosal-
based antigen-presenting cells (APC) to produce pro-inflammatory
cytokines, and encourage a mixed Th1/Th17-driven inflammation,
which in the absence of adequate immunologic regulation mani-
fests as an IBD spectrum disease.
Evaluations of future IBD mouse models require a systematic
and comprehensive approach. Since the spectrum of enteric auto-
immune inflammatory diseases that may model IBD are histo-
pathologically heterogeneous, the entire alimentary tract, including
the esophagus, stomach, duodenum, jejunum, ileum, cecum, and
colon and associated mesentery and mesenteric lymph nodes may
need to be evaluated morphologically. A comprehensive evaluation
of all organs will assist with determining whether other inflammatory
lesions in organs outside the alimentary tract develop concordant
with the IBD phenotype. For example, TNFARE mice develop
both ileitis and arthritis and SHIP/ mice develop both ileitis and
eosinophilic crystalline pneumonia (4345).
During necropsy, collection and preparation of the gastrointes-
tinal tract should be accomplished first to minimize autolysis. The
small intestine should be separated from the cecum at the rotund
sac and gently lifted from the abdominal cavity, removing the
attached mesenteric tissue, and the pancreas at the duodenojejunal
flexure, and then separated from the stomach at the pars pylorica.
After removal of the reproductive organs, the cecum is lifted and
the colon removed by freeing it from attached mesentery. The
intestines must then be insufflated with a 10 ml syringe of fixative
21 Assessing Inflammatory Disease at Mucosal Surfaces in Murine Genetic Models 437

Fig. 1. (a) Insufflated small intestine rolled to fit as a Swiss roll into a cassette for
histological processing. (b) Full-length small intestine thin section preparation for microscopic
assessment from an SHIP/ mouse with regional thickening of the ileum. (c) Insufflated
lungs ready for cassette placement and histological processing.

by introducing a 1722 gauge sharp needle along the length and

at the ends of the bowel, and holding the intestine and penetrating
needle firmly with forceps, or by using a 20 gauge ball-tip gavage
needle introduced at the ends of the bowel. Long intestinal seg-
ments (e.g., small intestine, large intestine) should be rolled to fit
as Swiss rolls into cassettes for histological processing (Fig. 1a).
438 R.W. Engelman and W.G. Kerr

Fig. 2. Eosinophilic crystalline pneumonia of SHIP/ mice with (a) large crystals in bronchiolar airways, focal mixed
leukocyte infiltration, bronchiolar subepithelial fibrosis, hypertrophy, and mucous metaplasia of bronchiolar epithelium,
numerous alveolar macrophages, and multinucleate cells, which result in patchy (b) to lobar (c) pulmonary consolidation.

This will create a full-length intestinal preparation for microscopic

assessment (Fig. 1b). The insufflated intestine is rolled in concen-
tric circles, orienting the proximal end within the center of the roll.
Similarly, the lungs should be insufflated with fixative to ensure
adequate presentation for microscopic morphologic evaluation
(Fig. 2). During necropsy, the soft tissues of the ventral neck as
well as the ventral thoracic ribcage are removed exposing the epi-
glottis, larynx, trachea, esophagus, thymus, heart, and lungs. Using
a similar needle and syringe as with the intestine, the needle is
placed in the glottis and the lungs are insufflated to a size normal
achieved during inhalation.
Histological sections of each segment of the alimentary tract
should be masked, assessed, and assigned an inflammatory grade
relevant to the IBD phenotype that presents. Grading schemes
used may vary depending on the IBD phenotype that manifests,
but should reflect disease progression and represent the most
advanced lesion identified in each section. As an example, in the
SHIP/ mouse model of CD, grades of 06 were assigned to sec-
tions of ileum, with grade 6 representing the most progressed,
severe lesion (Fig. 3). Inflammatory grades assigned reflected pro-
gressively more severe disease with an inflammatory grade 0 repre-
senting no significant abnormalities; an inflammatory grade 1
representing mild predominantly polymorphonuclear (PMN) leu-
kocyte infiltrations (<25 PMN/hpf) of the lamina propria and/or
enteric lymph nodule; an inflammatory grade 2 representing mod-
erate predominantly PMN leukocyte infiltrations (>25 cells/hpf)
of the lamina propria and/or lymph nodule; an inflammatory grade
3 representing marked inflammatory cell infiltrations with extension
below the muscularis mucosa causing architectural distortion of
the mucosa and submucosa with attendant crypt hyperplasia; an
inflammatory grade 4 representing marked infiltrations with exten-
sion into the tunica muscularis; an inflammatory grade 5 represent-
ing marked transmural leukocyte infiltrations; and an inflammatory
grade 6 representing marked transmural leukocyte infiltrations
 21 Assessing Inflammatory Disease at Mucosal Surfaces in Murine Genetic Models 439

Fig. 3. Histopathological appearance of the small intestine of SHIP/ mice with CD-like disease assessed as inflammatory
(a) grade 1 with mild polymorphonuclear (PMN) leukocyte infiltrations of the lamina propria and enteric lymph nodule
(arrow); (b) grade 2 with moderate PMN leukocyte infiltrations; (c) grade 3 with marked PMN infiltrations extending below
the muscularis mucosa; (d) grade 4 with marked infiltrations extending into the tunica muscularis; (e) grade 5 with marked
transmural leukocyte infiltrations; (f) grade 6 with transmural leukocyte infiltrations extending into the mesentery.

with extension into the mesentery and/or other organs. In each

section the presence of granuloma, crypt abscess, stricture, or
fissure can be noted.

Acknowledgements

This work was supported in part by grants from the NIH (RO1
HL72523, R01 HL085580, R01 HL107127) and the Paige
Arnold Butterfly Run. WGK is the Murphy Family Professor of
Childrens Oncology Research and an Empire Scholar of the State
University of NY. The staffs of the Weiskotten (SUNY) and Moffitt
(USF) vivariums are thanked for their very capable assistance with
tissue harvesting and processing.

References

1. Sartor RB (2006) Mechanisms of disease: mutations, and immunoregulatory defects in

pathogenesis of Crohns disease and ulcerative
colitis. Nat Clin Pract Gastroenterol Hepatol
3:390407
2. Xavier RJ, Podolsky DK (2007) Unravelling
the pathogenesis of inflammatory bowel dis-
ease. Nature 448:427434
3. Packey CD, Sartor RB (2008) Interplay of
commensal and pathogenic bacteria, genetic
the pathogenesis of inflammatory bowel dis-
eases. J Intern Med 263:597606
4. Cho JH (2008) The genetics and immunop-
athogenesis of inflammatory bowel disease. Nat
Rev Immunol 8:458466
5. Wirtz S, Neurath MF (2007) Mouse models of
inflammatory bowel disease. Adv Drug Deliv
Rev 59:10731083
440 R.W. Engelman and W.G. Kerr
6. Barrett JC, Hansoul S, Nicolae DL et al (2008)
Genome-wide association defines more than 30
distinct susceptibility loci for Crohns disease.
Nat Genet 40:955962
7. Sollid LM, Johansen FE (2008) Animal models
of inflammatory bowel disease at the dawn of
the new genetics era. PLoS Med 5:e198
8. Bouma G, Strober W (2003) The immunologi-
cal and genetic basis of inflammatory bowel
disease. Nat Rev Immunol 3:521533
9. Shanahan F (2002) Crohns disease. Lancet
359:62
10. Monteleone G, Biancone L, Marasco R et al
(1997) Interleukin 12 is expressed and actively
released by Crohns disease intestinal lamina
propria mononuclear cells. Gastroenterology
112:11691178
11. Fuss IJ, Neurath M, Boirivant M et al (1996)
Disparate CD4+ lamina propria (LP) lym-
phokine secretion profiles in inflammatory
bowel disease. Crohns disease LP cells mani-
fest increased secretion of IFN-gamma, whereas
ulcerative colitis LP cells manifest increased
secretion of IL-5. J Immunol 157:12611270
12. Duerr RH, Taylor KD, Brant SR et al (2006) A
genome-wide association study identifies
IL23R as an inflammatory bowel disease gene.
Science 314:14611463
13. Yen D, Cheung J, Scheerens H et al (2006)
IL-23 is essential for T cell-mediated colitis and
promotes in fl ammation via IL-17 and IL-6.
J Clin Invest 116:13101316
14. Singh B, Read S, Asseman C et al (2001)
Control of intestinal inflammation by regula-
tory T cells. Immunol Rev 182:190200
15. Kazemi-Shirazi L, Gasche CH, Natter S et al
(2002) IgA autoreactivity: a feature common
to inflammatory bowel and connective tissue
diseases. Clin Exp Immunol 128:102109
16. Dieckgraefe BK, Korzenik JR (2002) Treatment
of active Crohns disease with recombinant
human granulocyte-macrophage colony-stimu-
lating factor. Lancet 360:14781480
17. Lopez-Cubero SO, Sullivan KM, McDonald
GB (1998) Course of Crohns disease after
allogeneic marrow transplantation.
Gastroenterology 114:433440
18. Hisamatsu T, Suzuki M, Reinecker HC et al
(2003) CARD15/NOD2 functions as an anti-
bacterial factor in human intestinal epithelial
cells. Gastroenterology 124:9931000
19. Cadwell K, Liu JY, Brown SL et al (2008) A
key role for autophagy and the autophagy gene
Atg16l1 in mouse and human intestinal Paneth
cells. Nature 456:259263
20. Cario E, Podolsky DK (2000) Differential
alteration in intestinal epithelial cell expression
of toll-like receptor 3 (TLR3) and TLR4 in
inflammatory bowel disease. Infect Immun
68:70107017
21. Pizarro TT, Michie MH, Bentz M et al (1999)
IL-18, a novel immunoregulatory cytokine, is
up-regulated in Crohns disease: expression
and localization in intestinal mucosal cells.
J Immunol 162:68296835
22. Olson TS, Reuter BK, Scott KG et al (2006)
The primary defect in experimental ileitis origi-
nates from a nonhematopoietic source. J Exp
Med 203:541552
23. Heazlewood CK, Cook MC, Eri R, Price GR,
Tauro SB et al (2008) Aberrant mucin assem-
bly in mice causes endoplasmic reticulum stress
and spontaneous inflammation resembling
ulcerative colitis. PLoS Med 5(3):e54
24. Van der Sluis M, De Koning BA, De Bruijn
AC, Velcich A, Meijerink JP et al (2006) Muc2-
deficient mice spontaneously develop colitis,
indicating that MUC2 is critical for colonic
protection. Gastroenterology 131:117129
25. Strober W, Fuss IJ, Blumberg RS (2002) The
immunology of mucosal models of
inflammation. Annu Rev Immunol 20:
495549
26. Matsumoto S, Okabe Y, Setoyama H et al
(1998) Inflammatory bowel disease-like enteri-
tis and caecitis in a senescence accelerated
mouse P1/Yit strain. Gut 43:7178
27. Kontoyiannis D, Pasparakis M, Pizarro TT et al
(1999) Impaired on/off regulation of TNF
biosynthesis in mice lacking TNF AU-rich ele-
ments: implications for joint and gut-associated
immunopathologies. Immunity 10:387398
28. Kerr WG, Park MY, Maubert M, Engelman
RW (2010) SHIP deficiency causes Crohns
disease-like ileitis. Gut 10:11361148
29. Kosiewicz MM, Nast CC, Krishnan A et al
(2001) Th1-type responses mediate spontane-
ous ileitis in a novel murine model of Crohns
disease. J Clin Invest 107:695702
30. Marini M, Bamias G, Rivera-Nieves J et al
(2003) TNF-alpha neutralization ameliorates
the severity of murine Crohns-like ileitis by
abrogation of intestinal epithelial cell apopto-
sis. Proc Natl Acad Sci U S A 100:83668371
31. Ho J, Kurtz CC, Naganuma M et al (2008) A
CD8+/CD103high T cell subset regulates
TNF-mediated chronic murine ileitis. J Immunol
180:25732580
32. Liu L, Damen JE, Ware M et al (1997) A new
player in cytokine-induced signalling. Leukemia
11:181184
33. Kerr WG (2008) A role for SHIP in stem cell
biology and transplantation. Curr Stem Cell
Res Ther 3:99106
 21 Assessing Inflammatory Disease at Mucosal Surfaces in Murine Genetic Models
34. Wang JW, Howson JM, Ghansah T et al (2002)
Influence of SHIP on the NK repertoire and
allogeneic bone marrow transplantation.
Science 295:20942097
35. Ghansah T, Paraiso KH, Highfill S et al (2004)
Expansion of myeloid suppressor cells in SHIP-
deficient mice represses allogeneic T cell
responses. J Immunol 173:73247330
36. Paraiso KH, Ghansah T, Costello A et al (2007)
Induced SHIP deficiency expands myeloid reg-
ulatory cells and abrogates graft-versus-host
disease. J Immunol 178:28932900
37. Rauh MJ, Ho V, Pereira C et al (2005) SHIP
represses the generation of alternatively acti-
vated macrophages. Immunity 23:361374
38. Collazo MM, Wood D, Paraiso KH et al (2009)
SHIP limits immunoregulatory capacity in the
T-cell compartment. Blood 113:29342944
39. Locke NR, Patterson SJ, Hamilton MJ et al
(2009) SHIP regulates the reciprocal develop-
ment of T regulatory and Th17 cells. J Immunol
183:975983
40. Kashiwada M, Cattoretti G, McKeag L et al
(2006) Downstream of tyrosine kinases-1
441
and Src homology 2-containing inositol
5-phosphatase -are required for regulation
of CD4+CD25+ T cell development. J Immunol
176:39583965
41. Kerr WG (2011) Inhibitor and activator: dual
functions for SHIP in immunity and cancer.
Ann N Y Acad Sci 1217:117
42. Imielinski M, Baldassano RN, Griffiths A et al
(2009) Common variants at five new loci asso-
ciated with early-onset inflammatory bowel dis-
ease. Nat Genet 41:13351340
43. Helgason CD, Damen JE, Rosten P et al
(1998) Targeted disruption of SHIP leads to
hemopoietic perturbations, lung pathology,
and a shortened life span. Genes Dev 12:
16101620
44. Guo L, Johnson RS, Schuh JC (2000)
Biochemical characterization of endogenously
formed eosinophilic crystals in the lungs of
mice. J Biol Chem 275:80328037
45. Hoenerhoff MJ, Starost MF, Ward JM (2006)
Eosinophilic crystalline pneumonia as a major
cause of death in 129S4/SvJae mice. Vet Pathol
43:682688
 Chapter 22

Rodent Models of Experimental Autoimmune Uveitis

Rajeev K. Agarwal, Phyllis B. Silver, and Rachel R. Caspi

Abstract
The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets
immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well
as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and reproduc-
ible model that is easily followed and quantitated. It is inducible with synthetic peptides derived from reti-
nal autoantigens in commonly available strains of rats and mice. The ability to induce EAU in various
gene-manipulated, including HLA-transgenic, mouse strains makes the EAU model suitable for the study
of basic mechanisms as well as in clinically relevant interventions.

Key words: Uveitis, Uveoretinitis, EAU, Autoimmunity, T cells, Tolerance, Th1, Th2, IRBP, S-Ag

1. Introduction

Experimental autoimmune uveoretinitis (EAU) is an organ-specific,

T-cell mediated autoimmune disease that targets the neural retina
and related tissues that is induced by immunization with retinal
antigens (13). The pathology of EAU closely resembles human
uveitic diseases of a putative autoimmune nature in which patients
display immunological responses to retinal antigens. Examples of
such diseases, which have similar pathology to EAU and in which
patients frequently have circulating lymphocytes that respond to
retinal proteins, are sympathetic ophthalmia, birdshot retinochor-
oidopathy, Behcets disease, and others (4, 5). In the United States
alone, there are approximately 70,000 cases of uveitis per year, and
autoimmune uveitis is estimated to account for approximately 10 %
of severe vision loss. Although none of the animal models mimics
all the features of human disease, each has distinguishing character-
istics that are reminiscent of different aspects of clinical uveitis.
Although the retinal antigens that might be involved in human
uveitis have not been definitively identified, many uveitis patients

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_22, Springer Science+Business Media New York 2012

443
444 R.K. Agarwal et al.

are seen to respond to the retinal soluble antigen (S-Ag = arrestin)

and to a lesser extent to other retinal antigens. The EAU model
has served as an invaluable tool to evaluate novel immunothera-
peutic and conventional therapeutic strategies. The EAU model is
also useful to study basic mechanisms of tolerance and autoimmu-
nity to organ-specific antigens in immunologically privileged sites
(5). Thus, EAU is useful as a tool for clinical as well as for basic
studies of ocular and organ-specific autoimmunity.
EAU can be induced in susceptible animals by peripheral
immunization with a number of evolutionarily well-conserved
uveitogens (purified protein antigens extracted from the retina, or
their peptides) in adjuvant or by adoptive transfer of lymphocytes
specific to these antigens (2, 5, 6). In many cases the sequence of
these proteins is known and the pathogenic fragments have been
identified. The majority of the studies have been performed using
heterologous bovine antigens because autologous rat or mouse
retinal proteins cannot be obtained in sufficient quantities.
Uveitogenic retinal proteins are molecules whose homologues can
be found as far down the phylogenetic scale as the invertebrates
(79). The uveitogenic retinal proteins identified so far are:
1. The retinal soluble antigen (S-Ag, arrestin): This 48 kDa intra-
cellular photoreceptor protein is involved in the phototrans-
duction cascade. It binds to photoactivated-phosphorylated
rhodopsin, thereby apparently preventing the transducin-
mediated activation of phosphodiesterase (10).
2. Interphotoreceptor retinoid-binding protein (IRBP): This
148 kDa protein is found in the interphotoreceptor matrix,
which helps in transporting Vitamin A derivatives between the
photoreceptor and the retinal pigment epithelium (RPE).
IRBP is composed of four evolutionary conserved homolo-
gous domains, which are thought to have arisen by gene dupli-
cation (8).
3. Rhodopsin, and its illuminated form, opsin: This 40 kDa mem-
brane protein is the rod visual pigment (7): Pathogenicity of
this protein appears to be conformation-dependent, as rho-
dopsin is more pathogenic than opsin (11).
4. Recoverin: This is 23 kDa calcium-binding protein.
5. Phosducin: This is 33 kDa soluble cytosolic photoreceptor
protein.
Susceptibility to EAU is genetically controlled. It has been observed
that different species, and strains within species, vary in their sus-
ceptibility. Thus, rats develop EAU after immunization with either
S-Ag or IRBP. Guinea pigs are susceptible to S-Ag, but not to
IRBP; and mice develop severe disease with IRBP, but not with
S-Ag. Within each species there are susceptible and resistant strains.
In mice and in rats both MHC and non-MHC gene control has
 22 Rodent Models of Experimental Autoimmune Uveitis 445

been implicated (12, 13). MHC control is likely to be connected

to the ability to bind and present uveitogenic epitopes. Non-MHC
control is more complex and controlled by multiple genetic path-
ways that are not all defined. One important factor is the type of
effector response that a given strain is genetically programmed to
mount. Uveitis can be either Th17 or Th1 driven, and each of
these effector T cell types is pathogenic when polarized in culture
and infused into nave mice (14). Strains that are naturally high
Th1 and/or Th17 responders such as C57BL/6 and B10.RIII
tend to be susceptible, whereas strains that are overt Th2 respond-
ers (e.g., BALB/c mouse) tend to be resistant (15, 16).
Furthermore, skewing of the response towards a Th2-like pheno-
type, e.g., by treatment with the regulatory cytokines IL-4 and
IL-10, can ameliorate EAU (17). Another factor is different level
of expression of retinal antigens in the thymus, provoking efficient
central tolerance to antigens expressed in adequate amounts
(18). Other factors, including the hypothalamus pituitary adrenal
(HPA) axis control, the number of mast cells in the eye, have also
been implicated (19, 20). Genetic control of clinical and experi-
mental uveitis has been reviewed (21).
Although in many clinical diseases and in some autoimmunity
models there is a gender bias of susceptibility, in EAU there does
not seem to be an obvious difference in susceptibility between
males and females. However, reduced susceptibility is seen in preg-
nant females (22) and in animals harboring an active infection
(Caspi et al., unpublished). Both these phenomena may be con-
nected to the cytokine milieu elicited by the physiological state of
the individual. Pregnant mice were found to have elevated TGF-b
levels (22) and infection elicits the production of IFN-g, which has
a protective role in tissue-specific autoimmunity, including EAU
(23). There are no controlled studies of age-dependency of suscep-
tibility. We have used animals between 6 weeks and 8 months of
age, without any noticeable differences in disease development
(Caspi et al., unpublished observations).
EAU in many poorly susceptible strains can be enhanced by
the treatment with Bordetella pertussis in the form of heat-killed
bacteria or better yet, as purified pertussis toxin (PT), concurrently
with immunization. This is similar to other autoimmune disease
models, such as experimental autoimmune encephalomyelitis
(EAE). Administration of PT concurrently with uveitogenic immu-
nization permits expression of disease in resistant strains, and
enhances it in susceptible strains (15, 24, 25). The mechanism has
for a long time been thought to involve the opening of the blood
organ barrier by PT (26, 27); however, recently we showed that a
dominant effect of PT in EAU is to enhance the Th1 response and
the Th17 response, both pathogenic in EAU (15, 24, 28). This
effect is at least in part due to maturation of dendritic cells by PT
(16) and is mediated by the B subunit of PT (29). It is important
446 R.K. Agarwal et al.

to point out that PT can have strong inhibitory effects on disease

as well if it is present at the time of cell migration to the target
organ, due to its inhibitory effects on chemokine receptor signaling
through G-protein inhibition (30). Therefore, not only the timing
of PT administration, but also the amount administered, is impor-
tant because excess PT administered at the time of immunization
will persist into the effector stage of the disease and inhibit its
expression (31).
EAU in mice and in rats induced with the different uveitogenic
proteins or their peptides appears to share essentially the same
immunological mechanisms and histological features; however,
there are species-specific and strain-specific differences in the course
of disease (1, 3, 5, 32, 33). The type, number, and size of lesions
serve as a basis for a semiquantitative grading system used to score
disease severity. A degree of familiarity with ocular histology is
needed to grade the disease in a specific fashion. In practice, dis-
ease scores assigned by different observers will not always be iden-
tical; however, when performed by the same person, grading should
be consistent.
Although originally developed in the guinea pig, the two major
EAU models in use today are the mouse and the rat. Each has its
unique advantages. The mouse is immunologically well character-
ized and many congenic and gene-manipulated strains are available
that permit sophisticated studies of basic immunological mecha-
nisms. The rats larger size permits therapeutic and surgical manip-
ulations that are more difficult to do in the mouse and traditionally
is the preferred model for endocrine and physiological studies.
More specific attributes of EAU in the rat and mouse models are
described ahead, under Subheading 3. EAU models in the guinea
pig, the rabbit, and the monkey have been reviewed elsewhere
(1, 5, 34). The choice of model will therefore depend on the
specific needs of the study.
The methods and descriptions ahead deal with the rat and the
mouse models of EAU induced by defined synthetic peptides of
retinal antigens. Synthetic peptides were chosen because they can
be synthesized in every laboratory and do not require access to
native retinal antigens from natural or recombinant sources.

2. Materials

1. Mice (B10.RIII and C57.BL/6) (Jackson Laboratories, Bar

Harbor, ME).
2. Lewis rats (Charles River Laboratories, Wilmington, MA).
3. Pertussis Toxin (Catalog #179A; List Biologicals, Campbell, CA).
4. IRBP and S-Ag peptides.
 22 Rodent Models of Experimental Autoimmune Uveitis 447

5. DMEM, RPMI-1640, HBSS (Hyclone Laboratories, Logan,

UT).
6. L-glutamine, sodium pyruvate, nonessential amino acids, gen-
tamycin (Invitrogen, Carlsbad, CA) as medium additives.
7. Fetal bovine serum (FBS) (Hyclone).
8. Complete Freunds adjuvant (CFA) and Mycobacterium tuber-
culosis (Difco, Detroit, MI).
9. Purified Flt-3 ligand plasmid (pUMCV-mFLex containing the
extracellular domain of murine fms-like tyrosine kinase 3
ligand; secreted form; National Gene Vector Laboratory,
University of Michigan, Ann Arbor, MI).
10. Collagenase D (from Clostridium histolyticum) and DNase I
(Roche, Nutley, NJ).
11. EDTA (Quality Biological, Inc., Gaithersburg, MD).
12. CD16/32, anti-CD40 monoclonal antibodies (BD Biosciences,
San Jose, CA).
13. CD11c-conjugate magnetic beads (Miltenyi Biotec, Auburn,
CA).
14. GM-CSF (Peprotech, Rocky Hill, NJ).
15. 12-Well tissue culture plates.
16. LPS (Escherichia coli 0111:B4, Sigma-Aldrich).
17. 4 % Glutaraldehyde (Fisher Scientific, Fair Lawn, NJ) solution
was prepared in phosphate buffered saline (PBS).
18. 10 % neutral buffered formalin (Sigma-Aldrich).
19. Ophthalmic dilating solutions: 1 % Tropicamide (Alcon
Laboratories, Inc., Fort Worth, Tx) and phenylephrine hydro-
chloride; 2.5 % (Bausch & Lomb) for fundus examination
procedure.
20. Sterile eye irrigating physiological solution (Ciba Vision
Ophthalmics, Atlanta, GA).

3. Methods

3.1. EAU in Rats The model of EAU, originally established in the guinea pig using
homologous uveal tissue (3537), was adapted to the rat in 1973
by Wacker and Kalsow (38) using whole retinal extracts, and was
subsequently refined by de Kozak et al. using the retinal S-Ag (39).
IRBP was shown to be uveitogenic in rats (40).
Susceptibility of EAU varies among different rat strains. The
strain most commonly used for EAU studies is the Lewis rat. In the
Lewis rat, the disease is of monophasic course, which develops
448 R.K. Agarwal et al.

Table 1
Susceptibility to EAU of some inbred rat strainsa

Strain MHC Susceptibility Antigen Reference

Lewis RT1l High S-Ag, IRBP (3, 19)

F344 RT1lv1 Low S-Ag, IRBP peptide R16 (3, 19, 31)
CARb RT1l High S-Ag (3, 19)
n c
BN RT1 Low S-Ag (5)
PVG RT1c High S-Ag (39)
a
Please see other references for more detailed list (41, 42)
b
Resistance of this strain may vary in different colonies (19)
c
Resistance is not overcome by PTX treatment

characteristically severe uveitis and has served as a standard

against which responses of other strains are compared. It appears
that both MHC and non-MHC genes play a role (12), however,
due to the limited availability of congenic and MHC-recombinant
rat strains, their relative effects have not been well separated.
Table 1 summarizes the susceptibility of some common inbred rat
strains to EAU induced with the native S-Ag or IRBP. A more
detailed list has been published elsewhere (41, 42).
The retinal proteins known as uveitogenic have historically
been defined as such in the Lewis rat model, the most highly sus-
ceptible rat strain known. The normal dose is 3050 mg of S-Ag or
IRBP, or 50100 mg of rhodopsin emulsified in CFA. Lewis rats do
not require PT as part of the immunization protocol to develop
disease, but if used, PT will cause earlier onset and enhanced dis-
ease scores. The immunizing protocol of 30 mg of peptide R16 of
bovine IRBP (Table 2) in CFA normally results in disease onset on
day 9 or 10. In contrast, immunization with 30 mg of S-Ag in CFA
usually results in onset between days 12 and 14. Other strains may
require PT as an additional adjuvant. Subcutaneous (s.c.) immuni-
zation in the thighs and base of tail with an emulsion of S-Ag or
IRBP in CFA was as good or better for induction of disease as the
footpad route (43).
Table 2 shows the commonly used epitopes that were found to
be consistently pathogenic in the Lewis strain. The peptides which
are pathogenic at low doses are considered to contain a major
pathogenic epitope.
Induction of EAU in the Lewis rat by active immunization. As men-
tioned above, Lewis rat strain is highly susceptible to EAU. (In
strains that are less susceptible, such as the F344, one must use PT
as additional adjuvant and a higher dose of antigen is recom-
mended.) Two peptides are recommended as being strongly and
consistently uveitogenic in the Lewis: peptide R16 of bovine IRBP
 Table 2
Retinal protein-derived peptides that are pathogenic for Lewis ratsa

Source Nickname (if any) Positionb a.a. sequencec Minimal dosed Reference

Bovine S-Ag peptide N 281302 (287297) VPLLANNRERRGIALDGKIKHE 50 mg (59, 60)

22

peptide M 303320 (303317) DTNLASSTIIKEGIDKTV 50 mg (61)

Bovine IRBP R23 1,0911,115 PNNSVSELWTLSQLEGERYGSKKSM 100 nM (280 mg) (62)
R4 1,1581,180 HVDDTDLYLTIPTARSVGAADGS 67 mg (63)
R14 1,1691,191 PTARSVGAADGSSWEGVGVVPDV 0.1 nM (0.2 mg) (6, 64)
(1,1821,190)
R16 1,1771,191 ADGSSWEGVGVVPDV 0.1 nM (0.2 mg) (6)
(1,1821,190)
Human S-Ag peptide 19 181200 VQHAPLEMGPQPRAEATWQF 25 mg (65)
peptide 35 341360 (343356) GFLGELTSSEVATEVPFRLM 5 mg (65, 66)
peptide 36 351370 (356366) VATEVPFRLMHPQPEDPAKZE 50 mg (65, 67)
Human IRBP H-IRBP 715 521540 (527534) YLLTSHRTATAAEEFAFLMQe 0.1 mg (68)
a
More detailed list of other peptides pathogenic to Lewis rats can be found elsewhere (41, 42)
b
In parenthesis: position of minimal sequence (if known)
c
The minimal pathogenic sequence (if known) is underlined
d
Pathogenicity was tested in most cases using PTX as an additional adjuvant (12 1010 heat-killed organisms per rat)
e
An additional epitope may be encoded by the N terminus (seq. YLLTSHRTATAA)
Rodent Models of Experimental Autoimmune Uveitis
449
450 R.K. Agarwal et al.

(residues 1,1771,191, sequence ADGSSWEGVGVVPDV) and

peptide S35 of human S-Ag (residues 341360, sequence
GFLGELTSSEVATEVPFRLM). S35 tends to cause stronger disease, but
onset is a day or two later than R16.
1. Use 68 weeks old female Lewis rats, preferably housed in
specific pathogen-free environment with food and water ad
libitum. If rats are procured from an outside vendor, it is best
to acclimate them for a few days before immunization.
2. Prepare an emulsion of the chosen peptide: 30 mg of R16 or o
f S35, in CFA (1:1 v/v) (Support Protocol 3.3.3) by sonication
to provide 30 mg of peptide in 200 ml per rat. Spin at ~400 g
to remove any air bubbles embedded in the emulsion. A well-
prepared emulsion should have the consistency of thick cream.
3. A 16-G blunt-ended needle is used to draw the emulsion into a
1 ml glass syringe preferable with a Luer Lock tip (rubber plung-
ers in plastic syringes tend to soften and stick due to the oil in
CFA). Carefully remove any residual air bubbles trapped in the
syringe. Change the needle to a 23-G needle and inject subcu-
taneously 100 ml at the base of the tail and 50 ml in each thigh.
4. 79 days after immunization, start inspecting the eyes with a
flashlight for loss of red reflex (Fig. 1). Grade the disease on a
scale of 0 (no disease) to 4 (severe disease) based on the scor-
ing method in Table 3.
5. Approximately 16 days after immunization (or at least 7 days
after onset of the disease) euthanize the rats. Remove the eyes
and process them for histopathology (Support Protocol 3.3.2).
6. Examine the hematoxylin and eosin-stained sections under a
microscope and grade the disease histopathologically following
the guidelines listed in Table 4.

Fig. 1. Clinical appearance of EAU in the Lewis rat by anterior chamber examination. (a) Normal eye; translucent appear-
ance; pupil and iris blood vessels are clearly visible and the vessels are not congested. (b) Uveitic eye; the eye appears
larger due to swelling and proptosis; red reflex is absent and pupil is obscured.
 22 Rodent Models of Experimental Autoimmune Uveitis 451

Table 3
Clinical grading of EAU in the rat

Gradea Criteria

0 No disease; eye is translucent and reflects light (red reflex)

0.5 (trace) Dilated blood vessels in the iris
1 Engorged blood vessels in iris; abnormal pupil
contraction
2 Hazy anterior chamber; decreased red reflex
3 Moderately opaque anterior chamber, but pupil still
visible; dull red reflex
4 Opaque anterior chamber and obscured pupil; red reflex
absent; proptosis
a
Each higher grade includes the criteria of the preceding one

Table 4
Scoring EAU histopathologically in the rat

Area of retinal
Grade section affected Criteria

0 None No disease, normal retinal architecture

0.5 <1/4
(trace)
1 1/4
2 1/4
3 1/4
4 1/4
Mild inflammatory cell infiltration of the
retina with or without photoreceptor
damage
Mild inflammation and/or photoreceptor
outer segment damage
Mild to moderate inflammation and/or
lesion extending to the outer nuclear layer
Moderate to marked inflammation and/or
lesion extending to the inner nuclear layer
Severe inflammation and/or full-thickness
retinal damage

Induction of EAU in the Lewis rat by adoptive transfer. EAU is a

CD4+ T-cell mediated disease. The full histopathological picture
can be obtained by adoptive transfer of immune lymph node or
spleen cells or long-term CD4+, MHC class II-restricted T cell
lines in the absence of detectable titers of serum antibodies (2, 44).
The cells must be activated with antigen or mitogen just prior to
transfer in order to efficiently mediate disease, suggesting that
activation-dependent functions (lymphokine production, expression
of adhesion molecules, etc.) are important. The minimal number
of cells required to transfer the disease depends on their source and
452 R.K. Agarwal et al.

specificity (2, 44, 45). The following protocol describes induction

of disease using primary cultures of lymph node cells and peptide
antigen.
1. Rats to be used as cell donors are immunized as mentioned in
the active immunization protocol, above. The donor rats are
sacrificed 1012 days post-immunization and draining lymph
nodes (inguinal and iliac) are harvested and cultured with the
immunizing peptide as follows.
2. Keep isolated lymph nodes in RPMI 1640 media containing
1 % FBS or rat serum. Prepare single cell suspension by crush-
ing the nodes using a plunger from a disposable plastic syringe
on a sterile mesh in a Petri dish, with some sterile media to
release the cells.
3. Transfer the cells to a 50 ml centrifuge tube and wash the Petri
dish with additional media. Spin the cells at 300 g and discard
the supernatant. Resuspend the pellet in fresh media and repeat
the washing procedure one to two times. Resuspend the final
cell pellet in a small volume of RPMI 1640 media containing
1 % FBS.
4. Count viable cells using an exclusion dye such as Trypan Blue.
5. Adjust the cell suspension to 5 106 cells/ml by adding com-
plete RPMI media (Support Protocol 3.3.4). Add the peptide
used for immunization of the cell donors (R16 or S35) to a
final concentration of 5 mg/ml.
6. Distribute 2 ml aliquots into 12-well tissue culture plates and
incubate the culture at 37 C for 3 days in 5 % CO2 tissue
incubator.
7. After 72 h collect, wash, and count the cells (as described
above). All procedures are preferably done using RPMI 1640
media with 1 % serum for maximal cell viability.
8. Inject 3050 106 cells (in 0.30.5 ml) intraperitoneally into
recipient Lewis rats.
9. Starting on day 3 after adoptive transfer, inspect the recipients
for disease by examining the eyes with a flashlight for loss of
red reflex (Fig. 1) and grade the disease as described in
Table 3.
10. Between 11 and 14 days after adoptive transfer (around 7 days
after disease onset), euthanize the rats, collect the eyes (Support
Protocol 3.3.2) and process them for histopathological grad-
ing of EAU according to Table 4.

3.1.1. Expected Course The course of disease in the Lewis rat is typically acute and of short
of Disease duration. The time of onset will vary depending on severity of the
developing disease, the antigen and the antigen dose. S-Ag and its
peptides tend to give more severe disease but a later onset than
 22 Rodent Models of Experimental Autoimmune Uveitis 453

IRBP and its peptides. For example, 30 mg of S35 peptide in CFA

without PT results in onset around day 1214, while a similar dose
of R16 in CFA without PT results in onset around day 1012. If
pertussis is used as additional adjuvant, the time of onset will be
shortened by 2 days, and will usually be more uniform than with-
out pertussis. The amount of antigen used to elicit disease can be
reduced several-fold if PT is used. Onset of EAU induced by adop-
tive transfer is usually on day 47, i.e., about a week earlier than for
active immunization. The active EAU in the Lewis rats lasts
12 weeks, and the disease does not relapse. The rapid onset and
acute course of EAU in the Lewis rat makes it difficult to evaluate
therapeutic intervention during active disease. Alternatively, to
look at efferent-stage disease, begin intervention 7 days after
immunization, when immune lymphocytes are already present, or
to use an adoptive transfer system.

Quantitation Clinical. Onset of disease in the albino Lewis rat can be recognized
by inspecting the eyes with the aid of a good flashlight (Fig. 1).
The normal eye appears translucent and reflects the light (red
reflex). The first sign of uveitis is engorgement of blood vessels in
the iris and an irregular pupil that cannot contract in response to
light (caused by the iris adhering to the lens). Leukocyte infiltration
and deposition of fibrin is first seen as dulling of the red reflex,
progressing to complete opacification of the anterior chamber. The
eye swells and can protrude from its socket (proptosis). In very
severe cases hemorrhages in the anterior chamber and even perfo-
ration of the cornea can occur. In the latter case the animal should
be euthanized. We grade clinical EAU on a scale of 0 (no disease)
to 4 (severe disease), using the criteria listed in Table 3.
Histopathology. EAU is defined primarily as a posterior segment
disease, because the target antigens reside in the retina. Lewis rats
can develop very severe anterior chamber inflammation, which can
lead to corneal perforation. Therefore, although clinical follow-up
by anterior chamber inflammation is important and yields valuable
information, the final readout should be recorded by histopathol-
ogy. We grade EAU by histopathology based on number and extent
of lesions on an arbitrary scale of 0 (none) to 4 (maximum sever-
ity), in half-point increments, as shown in Table 4 (42). Typical
EAU vs. normal histology is shown in Fig. 2. Although the Figure
shows EAU histopathology in the mouse, it is representative of
what is seen in rat.

3.2. EAU in Mice Mice are highly resistant to EAU induction with S-Ag, but some
strains can develop severe EAU after immunization with IRBP
(33). As in rats, age and sex do not appear to have a major influence
on susceptibility to disease. Table 5 lists some common EAU sus-
ceptible mouse strains and IRBP epitopes that have been found to
induce disease in each. The epitopes of the IRBP molecule
454 R.K. Agarwal et al.

Fig. 2. Clinical appearance of EAU in the B10.RIII mouse by fundoscopic exam. Eyes were photographed with a fundus
camera during the acute phase of disease (day 1421). The range of disease severity scores parallel the pathological
scores in Fig. 3. (Figure adapted from reference 69).
 22 Rodent Models of Experimental Autoimmune Uveitis 455

Table 5
Susceptibility of different mouse strains to IRBP-EAUa

Expected
Strain H-2 disease score Epitope, position, and reference

B10.RIII r Very high SGIPYIISYLHPGNTILHVD (161180b) (48)

Medium HPGNTILHVDTIYNRPSNTT (171190) (49)
Medium SLGWATLVGEITAGNLLHTR (541560) (49)
B10.A a (I-Ak) High ADKDVVVLTSSRTGGV (201216) (47)
B10.BR k High Same as B10.A
A/J a (I-Ak) Medium Same as B10.A
C57BL/6 b Moderate GPTHLFQPSLVLDMAKVLLD (120) (46)
Moderate LRHNPGGPSSAVPLLLSYFQ (461480) (49)
Moderate LAQGAYRTAVDLESLASQLT (651670) (49)
C57BL/10 b Medium Same as C57BL/6
a
More detailed list of other mouse strains can be found elsewhere (41)
b
Human sequence. PT is not required

pathogenic for the H-2b, H-2r, and H-2k haplotypes have been
identified (4648). In the B10.A mouse (Kk, Ak, Ek, Dd) MHC
control of susceptibility in mice has been tentatively mapped to the
I-A region with modifying influences from the I-E (13). In addi-
tion to a susceptible H-2 haplotype that can bind and present the
uveitogenic epitopes, the strain must also have a permissive
background to express disease. Studies with MHC-congenic mice
showed that a nonpermissive background can completely prevent
expression of disease in mice having a susceptible H-2. Known
susceptible H-2 haplotypes include H2r, H-2k, H-2a (shares class II
subregion with H-2k), H-2b, H-2q, and H-2d. The last two, initially
thought to be resistant, can in fact be shown to be EAU-susceptible
in situations where IFN-g is neutralized or knocked out (ref. 23
and Grajewski and Caspi, unpublished).
The most susceptible mouse strain currently known is B10.
RIII (H-2r) (Table 5). Unlike other mouse strains, this strain does
not require PT to develop disease by active immunization either
with IRBP or with its major pathogenic epitope, residues 161180
of human IRBP. Human epitopes 171190 and 541560 require
PT, as does the murine sequence of peptide 161180. This is likely
due to thymic elimination of high-affinity T cells reacting with the
endogenous version of peptide 161180 (46). In less susceptible
mouse strains, such as B10.A or C57BL/6, a higher dose of IRBP
(50150 mg) or of the peptide appropriate for the haplotype in
CFA is injected s.c., concurrently with PT (0.30.4 mg) given i.p.
456 R.K. Agarwal et al.

One of the strengths of the mouse model of EAU is the ready

availability of many gene-manipulated mouse strains. Knockouts
and transgenics for various immunologically relevant genes have
been, and continue to be, instrumental in unraveling the basic
mechanisms in uveitis. EAU can be induced with human IRBP
residues 120 in mice of the H-2b haplotype, which is expressed by
the C57BL/6 and 129 strains that typically serve for the produc-
tion of transgenics and knockouts (46). More recently, additional
epitopes that induce EAU in the H-2b haplotype, IRBP residues
171190 and 541560 have been identified (49). It is also impor-
tant to mention here one newly established model, the human-
ized EAU model in HLA class II transgenic mice. Uveitic diseases
in humans show strong associations with specific HLA class I or
class II alleles, that vary depending on the disease and the popula-
tion studied. The genetic associations in uveitis have recently been
reviewed (21). MHC association strongly supports a role for anti-
gen presentation by HLA molecules in the etiology of uveitis. HLA
class I and class II transgenic mice afford a model to study these
effects. Both HLA class I (A29) and class II transgenic mice (DR3,
DR4, DR2, DQ6, DQ8) have been used to study uveitis. HLA
A29 transgenics develop a spontaneous uveitis (50) whereas the
various class II transgenic mice develop EAU after immunization
with IRBP (51). Importantly, HLA-DR3 transgenic mice develop
severe uveitis after immunization with S-Ag, which is thought to
be involved in human uveitis, but which is not uveitogenic in wild
type mice. These humanized models support an etiological role
for retinal antigens, which are uveitogenic in animals, in human
uveitis and validate use of the EAU model for the study of human
disease.
Induction of EAU in the B10.RIII mouse by immunization. The
following protocol is given for B10.RIII mice, the most susceptible
mouse strain currently known. Human IRBP peptide 161180 is
used. This peptide does not require PT as part of the immuniza-
tion protocol, although PT will promote more severe disease and
an earlier onset (24). See ahead for a variation of the protocol for
use in C57BL/6 mice.
1. Use 68 weeks old female B10.RIII mice (H2r), preferably
housed under specific pathogen-free conditions with food and
water available ad libitum. If purchased from an outside ven-
dor, let them acclimate to the animal facility for a few days
before immunization.
2. Emulsify IRBP peptide 161180 (see sequence in Table 5) in
CFA (1:1 v/v) by sonication to provide 1025 mg peptide in
0.2 ml emulsion per mouse. Severity of disease obtained will
depend on the amount of peptide used (24). Spin at 2,000 rpm
to remove air bubbles trapped in the emulsion. A well-prepared
emulsion has the consistency of thick cream.
 22 Rodent Models of Experimental Autoimmune Uveitis 457

Table 6
Clinical scoring of EAU in the mouse

Grade Criteria

0 No change
0.5 (trace) Few (12) very small, peripheral focal lesion;
minimal vasculitis/vitritis
1 Mild vasculitis; <5 small focal lesions; 1 linear lesion
2 Multiple (>5) chorioretinal lesions and/or
infiltrations; severe vasculitis (large size, thick wall,
infiltrations); few linear lesions (<5)
3 Pattern of linear lesions; large confluent lesions;
subretinal neovascularization; retinal hemorrhages;
papilledema
4 Large retinal detachment; retinal atrophy

3. Use a 16-G blunt ended needle to draw the emulsion into a

1 ml glass syringe with a Luer Lock tip (rubber plungers in
plastic syringes tend to soften and stick due to the oil in CFA).
Carefully remove any air bubbles trapped in the syringe.
Change the needle to a 23-G needle and inject each mouse
subcutaneously with 0.2 ml emulsion, dividing the dose among
two thighs (50 ml each) and base of the tail (100 ml).
4. 1012 days post-immunization, monitor the eyes of these mice
for disease induction by inspecting the fundus under a binocu-
lar microscope (Support Protocol 3.3.1). Grade the animals on
a scale of 0 (no disease) to 4 (severe disease) using the criteria
described in Table 6 and in Fig. 2.
5. Approximately 21 days after immunization (or 7 days after the
disease onset), euthanize the mice and remove eyes for histo-
pathology (Support Protocol 3.3.2).
6. Examine the hematoxylin and eosin-stained sections under a
microscope and grade the disease histopathologically following
the guidelines in Table 7 and in Fig. 3.
Alternative protocol: Induction of EAU in the C57BL/6 mouse by
immunization
1. Use 68 weeks old female C57BL/6 mice (H2b), preferably
housed under specific pathogen-free conditions with food and
water available ad libitum. If purchased from an outside ven-
dor, let them acclimate to the animal facility for a few days
before immunization.
2. Emulsify human IRBP peptide 120 (see sequence in Table 5)
in CFA (1:1 v/v) by sonication to provide 200300 mg peptide
458 R.K. Agarwal et al.

Table 7
Grading EAU histopathologically in the mouse

Grade Criteria

0 No change
0.5 (trace) Mild inflammatory cell infiltration. No tissue damage
1 Infiltration; retinal folds and focal retinal detachments; few
small granulomas in choroid and retina, perivasculitis
2 Moderate infiltration; retinal folds, detachments and focal
photoreceptor cell damage; small to medium sized
granulomas, perivasculitis and vasculitis
3 Medium to heavy infiltration; extensive retinal folding with
detachments, moderate photoreceptor cell damage;
medium sized granulomatous lesions; subretinal
neovascularization
4 Heavy infiltration; diffuse retinal detachment with serous
exudate and subretinal bleeding; extensive photoreceptor
cell damage; large granulomatous lesions; subretinal
neovascularization

in 0.2 ml emulsion per mouse. Severity of disease obtained will

depend on the amount of peptide used (46). Spin at 2,000 rpm
to remove air bubbles trapped in the emulsion. A well-prepared
emulsion has the consistency of thick cream.
3. Prepare pertussis toxin as adjuvant. Pertussis bacteria are not as
potent as the purified toxin. Dilute stock to 3 mg/ml just before
use in PBS or RPMI media with 1 % syngeneic serum. Inject
0.1 ml i.p. concurrently with the antigen emulsion. Alternatively,
inject 0.2 mg of PT on day 0 and 0.2 mg again on day 2 (see
Notes).
4. Proceed as described above in the B10.RIII protocol starting
from step 3.
Induction of EAU in B10.RIII or C57BL/6 mice by adoptive trans-
fer. Adoptive transfer of primed uveitogenic effector cells, just like
in rats, can induce EAU in mice. Primary cultures from immunized
donors, as described in the protocol ahead, can be used as a source
of effector cells. If desired, the cells may be polarized to Th1 or
Th17 phenotype using published methods before adoptive transfer
(52, 53). Alternatively, long-term antigen-specific T cell lines,
which are typically CD4+ cells of the Th1 phenotype, can be
derived from draining lymph-node cells of IRBP- or peptide-
immunized mice (48).
1. Immunize donor mice as described above, using peptide 161
180 of human IRBP for B10.RIII mice and peptide 120 of
human IRBP for C57BL/6 mice.
 22 Rodent Models of Experimental Autoimmune Uveitis 459

2. After 1014 days, harvest draining lymph nodes (inguinal and

iliac) as well as spleens from donor mice.
3. Place isolated lymph nodes and spleens in DMEM media con-
taining 1 % FBS or mouse serum. Prepare a single cell suspen-
sion (lymph nodes and spleen) by disrupting the spleens and
the lymph nodes using a sterile rubber plunger from a dispos-
able plastic syringe on a sterile mesh in a Petri dish with some
sterile media to release the cells.
4. Pool the suspensions of lymph node cells and splenocytes and
transfer to 50 ml centrifuge tubes. Wash the cells by centrifug-
ing at 300 g. Discard the supernatant and resuspend the pellet
in fresh media. Repeat the washing one to two times. Resuspend
the final cell pellet in a small volume of DMEM media contain-
ing 1 % serum to maintain maximal viability of the cells.
5. Take a small aliquot and count the viable cells using a vital dye
such as Trypan Blue.
6. Adjust the cell concentration to 10 106 live cells/ml in complete
DMEM media (Support Protocol 3.3.4) containing 2025 mg/ml
of the immunizing peptide. Optional: including 5 ng/ml of
IL-12 in the culture will result in a more highly Th1-polarized
population that will transfer disease with fewer cells (46).
7. Distribute 150 ml aliquots into 175-cm2 tissue culture flasks
and incubate for 3 days in a humidified 37 C tissue incubator
with 10 % CO2.
8. Important: After 24 h and again after 48 h of culture, bring the
nonadherent cells into suspension by gently rocking the flasks
and transfer the entire suspension to a new flask of the same
size. This gets rid of excess (adherent) macrophages that pro-
duce inhibitory factors.
9. After 72 h of culture, collect, wash, and count the viable cells.
There usually are many dead cells in the culture at that point.
Purifying the cells by centrifugation over Ficoll (Lympholyte
M, Accurate Biochemicals) can help obtain a more pure popu-
lation of live cells and facilitate counting. Suspend the counted
cells in DMEM media with 1 % normal mouse serum.
10. Inject cells intraperitoneally into syngeneic recipients in a vol-
ume of 0.5 ml or less. For B10.RIII mice, inject 3050 106
cells. For recipient C57BL/6 mice, inject 50100 106 cells. If
IL-12 is used during culture, this number may be reduced.
11. Starting 4 days after adoptive transfer, inspect the recipients
for disease induction by fundus examination (see Support
Protocol 3.3.1 and Fig. 2).
12. Between 12 and 14 days after adoptive transfer (at least 7 days
after disease onset), euthanize the mice, collect the eyes, and
process them for histopathology. Score the eyes according to
the criteria listed in Table 7 and Fig. 3.
460 R.K. Agarwal et al.

Induction of EAU in B10.RIII mice by antigen-pulsed dendritic cells

The following is a recently described protocol to induce EAU
in B10.RIII mice by the injection of matured dendritic cells loaded
with peptide 161180 (DC-EAU) (54):
1. Use 68-week-old female B10.RIII mice, preferably housed
under specific pathogen-free conditions with food and water
available ad libitum. If purchased from an outside vendor, let
acclimatize to the animal facility for a few days before
immunization.
2. To increase the number of functional DCs in vivo, a single
injection of 50 mg of Flt-3 ligand DNA was delivered by the
hydrodynamic protocol described by Knapp and Liu (55).
3. Harvest spleen 7 days later and prepare single-cell suspension
following Support Protocol 3.3.5.
4. Block cells with Fc receptor Ab, CD16/CD32. Add anti-
CD11c-conjugated magnetic beads and purify the CD11c+
population according to the manufacturers directions. Run
the possel separation program. Check the purity of the
CD11c+ cell population by flow cytometry.
5. Suspend the purified CD11c+ cells in complete medium for cell
culture (see Support Protocol 3.3.4) substituting 1 % fresh-
frozen mouse serum for the FBS, and adding 10 ng/ml
GM-CSF. Deliver 2 106 cells in 1 ml aliquots into 12-well
plates. Allow to adhere for 1 h at 37 C.
6. Rinse wells 2 with media in order to remove nonadherent
cells.
7. To mature the DC deliver complete medium (step 5) addition-
ally supplemented with 100 mg/ml peptide 161180, 1 mg/ml
LPS, and 5 mg/ml anti-CD40. Incubate 4.5 h at 37 C.
8. Harvest DC and wash twice with HBSS containing 1 % normal
mouse serum.
9. Inject one to two million DC s.c. into one footpad of recipient
mice (day 0). Inject 0.30.4 mg PT i.p. on day 2. Repeat injec-
tion with fresh in vitro matured DC on day 4 (See induction
regimen, Fig. 3).
10. EAU evaluation by fundus exam or histology is performed on
day 18.

3.2.1. Expected Course The severity and clinical course of EAU induced by active immuni-
of Disease zation (CFA-EAU) with peptide 161180 in B10.RIII mice is
typically monophasic, resembling the Lewis rat, and lasts for
23 weeks. High intensity immunization results in an acute form
of disease with onset as early as day 8 or 9 and widespread photo-
receptor damage, whereas lower intensity of immunization will
result in milder disease with progressively later onset (24). In other
 22 Rodent Models of Experimental Autoimmune Uveitis 461

Fig. 3. Histopathology of EAU in the B10.RIII mouse. Eyes were collected from B10.RIII mice 21 days after uveitogenic
immunization with IRBP, representing a range of disease scores. (Figure adapted from references 49, 69). EAU in the rat
shows essentially the same type of histopathology.

strains (e.g., B10.A) it is possible to observe relapsing disease after

a low to intermediate intensity protocol (56). As in the Lewis, the
rapid onset and acute course of EAU induced with peptide 161
180 in B10.RIII mice makes it difficult to evaluate therapeutic
intervention during active disease. Alternatively, to look at efferent-
stage disease, begin intervention 7 days after immunization, when
immune lymphocytes are already present, or to use an adoptive
transfer system.
Interestingly, the DC-EAU model exhibits unique clinical
manifestations and inflammatory characteristics when compared to
traditional CFA-induced EAU. DC-EAU is less severe, of shorter
duration, and is characterized by focal infiltrates and short linear
lesions. While the cellular infiltrate of CFA-EAU is composed of
462 R.K. Agarwal et al.

monocytes and lymphocytes, DC-EAU is largely granulocytic with

a small population of eosinophils. Intraocularly, IFNg predomi-
nates in DC-EAU with little or no detectable IL-17, i.e., a domi-
nant Th1 response. In contrast, the eye milieu of CFA-EAU
consists of a combination of IFNg, IL-17, and IL-18, i.e., a mixed
Th1/Th17 response. The differences observed in these two EAU
models highlights the variability of clinical and immunological out-
comes when the same antigen is processed in the context of dis-
tinct innate stimuli. The DC-EAU induction system can offer the
investigator an opportunity to choose a disease model that is devoid
of IL-17 influence, has a milder outcome, and is free of prolonged
innate stimulation generated by the Ag/CFA depot.

Quantitation Clinical. Unlike rats, in susceptible strains of mice (B10 back-

ground), black pigmentation of the eyes and an often mild involve-
ment of the anterior chamber preclude detection of disease by
anterior chamber examination. However, in pigmented strains it is
possible to observe changes in the fundus of the eye under a bin-
ocular microscope after dilating the pupil (Support Protocol 3.3.1)
(4). Visualization of the fundus is possible if the anterior chamber
does not become infiltrated with cells, or after the infiltrate clears
(usually within a few days after onset). The nature, number, and
severity of the lesions are used as criteria for clinical scoring, on a
scale of 0 (no change) to 4 (severe disease) (Table 6).
Histopathology. Histological grading is the final readout of the dis-
ease and is performed on methacrylate-embedded tissue sections
(Support Protocol 3.3.2). Disease is scored on a scale of 0 (no
disease) to 4 (maximum disease) in half-point increments, accord-
ing to a semiquantitative system described earlier (33). Examples
of various grades of pathology are shown in Fig. 2.

3.3. Support Protocols Fundoscopic examination can be used to detect and evaluate EAU
in pigmented animals, provided that the anterior segment of the
3.3.1. Fundoscopic
eye remains clear. In this procedure, retina of the eye is examined
Examination
through the dilated pupil under a microscope. Fundoscopy is a
good tool for determining the onset and clinical grading of disease
in pigmented animals but not in albinos.
Materials
Ophthalmic dilating solutions: 1 % Tropicamide and
Phenylephrine.
Sterile physiological solution (either normal saline or PBS or
artificial tears).
Binocular microscope with coaxial illumination.
Microscope coverslip
Anesthetize the animals and dilate the pupil with one to two
drops of dilating solution. It takes several minutes for the

Fig. 4. Protocol for induction of autoimmune uveitis in B10.RIII mice with antigen-pulsed DCs (54).
3.3.2. Handling of Eyes
for Histopathology
22 Rodent Models of Experimental Autoimmune Uveitis 463
medication to take effect. Place a drop of sterile physiological
solution and a microscope coverslip on the cornea to equalize
refraction. Manipulate the head of the mouse under the micro-
scope to inspect as far up the sides of the retina as possible. Look
for engorged blood vessels, constricted blood vessels (cuffing),
white linear lesions, subretinal hemorrhages, and retinal detach-
ment. Typical appearance of a uveitic fundus is shown in Fig. 4.
Eyes should be collected within 15 min of euthanasia; otherwise,
autolysis may preclude correct evaluation of the results. Enucleation
in rats should be performed by carefully dissecting the globe from
the periocular tissues and the optic nerve without applying pres-
sure on the globe, to avoid maceration of delicate ocular tissues
that become even more fragile when inflamed. In mice for enucle-
ation, the eye should be made to protrude by applying pressure on
the skull, and plucked free of the tissue with a curved forceps.
Freshly enucleated eyes are fixed in 4 % phosphate-buffered glu-
taraldehyde for 1 h and then transferred into 10 % phosphate-buffered
formalin at least overnight or until processing. The brief fixation in
4 % glutaraldehyde prevents artifactual detachment of the retina
from the choroid. However, leaving the eyes in glutaraldehyde for
too long will cause excessive hardening of the lens, which will make
sectioning difficult. The grading is conveniently done on methacry-
late or paraffin-embedded tissue sections cut up to 8 mm in thick-
ness, stained with hematoxylin and eosin. To arrive at the final
grading, several sections cut through the pupillary-optic nerve axis
should be examined for each eye. It should be remembered that in
this type of visual scoring, there is always an element of subjectivity.
Therefore, it is important that the results be read in a masked fash-
ion, preferably always by the same person. Whenever possible, both
eyes should be evaluated for the disease, as disease may be unilat-
eral. If it becomes experimentally necessary to take only one eye,
always collect the same eye to average out this random variation.
464 R.K. Agarwal et al.
3.3.3. Complete Freunds 100 mg heat killed M. tuberculosis (strain H37Ra; Difco) is crushed
Adjuvant
3.3.4. Complete Medium
for Cell Culture (RPMI 1640
or DMEM)
3.3.5. Preparation
of a Single-Cell
Suspension of Splenocytes
4. Notes
into a fine powder using a porcelain mortar and pestle. Mix with
70 ml of CFA (1.0 mg/ml of M. tuberculosis). Store at 4 C until
used. The suspension must be thoroughly mixed before each use as
the mycobacterial particles settle quickly to the bottom.
500 ml bottle of RPMI 1640 or DMEM.
50 ml FBS (10 % final).
5 ml of 200 mM L-glutamine (2 mM final).
5 ml of 100 mM Sodium pyruvate (1 mM final).
5 ml of 10 mM Nonessential amino acids (0.1 mM final).
0.5 ml of 50 mg/ml Gentamycin (50 mg/ml final).
0.05 ml of 0.5 M 2-mercaptoethanol (5 mM final).
Filter through 0.2 mm and store up to 4 weeks at 4 C.
Single-cell suspension preparation of splenocytes follows the pro-
tocols of Bjorck (57) and Vremec et al. (58) with modifications.
Briefly, mince the spleen into small fragments and digest in 10 ml
complete media (Support Protocol 3.3.4) supplemented with
1.0 mg/ml collagenase D and DNase I for 45 min at 37 C with
frequent shaking. One milliliter of 0.1 M EDTA is then added to
break the DC-T cell complexes. Continue shaking for 5 min. Pass
tissue through a 70-mM nylon cell strainer (BD Falcon) and centri-
fuge. Lyse red blood cells, wash, and then suspend in culture
media.
Although the EAU model is very robust, problems can arise with
the technique at a number of levels. If no disease is obtained, the
following questions should be considered (some may seem trivial
or obvious, but we have encountered all!):
Has the antigen been uveitogenic in previous experiments? If
this is a new synthesis, a synthesis error might have changed
the pathogenic epitope.
Has a sufficient dose of antigen been used?
Has the adjuvant been prepared correctly: enough mycobacte-
ria, mixed before sampling (mycobacteria may have settled),
well-prepared (thick) emulsion?
Has the correct strain and substrain of mouse or rat been used?
Substrains of the same strain, and even the same strain from a
different vendor, may vary in their susceptibility.
 22 Rodent Models of Experimental Autoimmune Uveitis 465

Are the animals in poor health? stressed? harboring an infection?

Mice that are unhealthy, chronically stressed (have elevated
corticosteroid levels) or actively infected (high levels of circu-
lating interferon) will frequently fail to develop disease.
Have the mice developed abscesses at the site of immunization
(reaction to the mycobacteria in CFA) and were promptly
treated by the facility vet with a nonsteroidal anti-inflammatory
agent (NSAID) such as ibuprofen? NSAIDs may inhibit induc-
tion of disease.
Susceptibility varies with strain. For some commonly used
mouse strains immunized with IRBP, hierarchy of susceptibility is
B10.RIII > B10.A = B10.BR > C57BL/10 > C57BL/6 129.
Chronic exposure to strong light can damage photoreceptor
cells and cause retinal degeneration. Albino animals are especially
sensitive to this due to lack of pigment in their eyes. For this rea-
son, it is important to protect animals that will be used in EAU
experiments from strong light, as the resulting retinal damage may
confound correct EAU assessment. This includes frequent and
prolonged fundoscopies.
Some common laboratory mouse strains carry the retinal
degeneration1 mutation (rd1) and congenitally lack photoreceptor
cells. These strains are of course not appropriate for EAU studies,
as they do not possess the target tissue. Strains carrying the rd1
gene include FVB/N, CBA, SLJ/J, PL/J, and most C3H sub-
strains. However, F1 hybrids of these strains with a sighted strain
such as B10.RIII or C57BL/6 will be sighted. As determined by
crosses between sighted strains, a hybrids susceptibility is usually
intermediate between the two parental strains. Of note, recently it
was reported that the C57BL/6N mouse substrain (but not
C57BL/6J), carry the rd8 mutation (70). This retinal degenera-
tion phenotype is more subtle and does not completely destroy the
retina, but its impact on EAU susceptibility has not been
determined.
If a strain other than B10.RIII, or a B10.RIII hybrid with a less
susceptible strain is being used, pertussis toxin may be needed.
Because the activity and toxicity of PT varies among vendors and
product lots, it is advisable to perform a dose response trial with the
reagent before proceeding to the actual experiment. Animal deaths
after immunization are usually associated with PT administration.
Immunize groups of mice with the IRBP peptides and inject sev-
eral doses of PT in a range between 0.2 and 0.5 mg/mouse. Choose
a dose that results in moderate to high disease without toxicity.
Splitting the PT administration into two low doses on day 0 and
day 2 is an alternative protocol. To alleviate stress to the animals
after immunization, it is helpful to add DietGel (PharmaServ,
Framingham, MA) to the bottom of the cages to provide them
with easy access to hydration and nutritional support. The PT dose
for rats can be approximately doubled that for mice.
466 R.K. Agarwal et al.

When anesthetizing animals for any reason, including

fundoscopy, it is important to keep in mind that anesthetized
animals sleep with their eyes open and do not blink. Therefore, if
animals are going to be asleep for more than just a few minutes, it
is necessary to place an ointment on the eyes to prevent drying of
the cornea. Drying of the eyes will inevitably result in exposure
keratitis, which will cause corneal opacification and will make fol-
low-up of clinical disease difficult or impossible.
Notes on fundoscopy: fundoscopy is best done under general
anesthesia. With some practice, fundoscopy can be performed on
non-anesthetized animals, but if disease is borderline or severity
scores are to be assigned, it advisable to lightly anesthetize the
mouse prior to fundoscopy to facilitate a more thorough inspec-
tion. Note: the dilating drops cause a temporary opacification of
the lens within 510 min after application, so it is important to
complete the fundoscopy within that time frame.
Notes on histopathology: When cutting the embedded eye tis-
sue, it is important to make sure that the cut is made through the
pupillary-optic nerve plane. If the inflammation is mild, pathology
is often most apparent around the optic nerve head. Therefore, if
sections are cut more laterally, it can be missed. Especially in mild
cases, specimens positive on fundoscopy may appear to be negative
on histology due to the fact that pathology is focal and the section-
ing may have missed it. Therefore, it is important to prepare and
examine several nonconsecutive sections.

References

1. Caspi RR (1989) Basic mechanisms in immune- 5. Gery I, Mochizuki M, Nussenblatt RB (1986)

mediated uveitic disease. In: Lightman SL (ed)
Immunology of eye disease, Vol. Ch. 5. Kluwer
Academic Publishers, Lancaster, UK, pp 6186
2. Caspi RR, Roberge FG, McAllister CG, el Saied
M, Kuwabara T, Gery I, Hanna E, Nussenblatt
RB (1986) T cell lines mediating experimental
autoimmune uveoretinitis (EAU) in the rat.
J Immunol 136:928933
3. Gery I, Robinson WG Jr, Shichi H, El-Saied
M, Mochizuki M, Nussenblatt RB, Williams
RM (1985) Differences in susceptibility to
experimental autoimmune uveitis among rats
of various strains. In: Chandler JW, OConner
GR (eds) Advances in immunology and immu-
nopathology of the eye (Proceedings of the
third international symposium on immunology
and immunopathology of the eye), Vol. Ch.
59. Masson Publishing, NY, pp 242245
4. Nussenblatt RB, Whitcup SM, Palestine AG
(1996) Uveitis: fundamentals and clinical prac-
tice, 2nd edn. Mosby - Year Book, Inc., St.
Louis, MO
Retinal specific antigens and immunopatho-
genic processes they provoke. Prog Retinal Res
5:75109
6. Sanui H, Redmond TM, Kotake S, Wiggert B,
Hu LH, Margalit H, Berzofsky JA, Chader GJ,
Gery I (1989) Identification of an immu-
nodominant and highly immunopathogenic
determinant in the retinal interphotoreceptor
retinoid-binding protein (IRBP). J Exp Med
169:19471960
7. Applebury ML, Hargrave PA (1986) Molecular
biology of the visual pigments. Vision Res
26:18811895
8. Borst DE, Redmond TM, Elser JE, Gonda
MA, Wiggert B, Chader GJ, Nickerson JM
(1989) Interphotoreceptor retinoid-binding
protein. Gene characterization, protein repeat
structure, and its evolution. J Biol Chem
264:11151123
9. Mirshahi M, Boucheix C, Collenot G, Thillaye
B, Faure JP (1985) Retinal S-antigen epitopes
 22 Rodent Models of Experimental Autoimmune Uveitis
in vertebrate and invertebrate photoreceptors.
Invest Ophthalmol Vis Sci 26:10161021
10. Pfister C, Chabre M, Plouet J, Van Tuyen V,
De Kozak Y, Faure JP, Khn H (1985) Retinal
S antigen identified as the 48 K protein regulat-
ing light-dependent phosphodiesterase in rods.
Science 228:891893
11. Schalken JJ, Winkens HJ, van Vugt AH, Bove-
Geurts PH, de Grip WJ, Broekhuyse RM
(1988) Rhodopsin-induced experimental auto-
immune uveoretinitis: dose-dependent clinico-
pathological features. Exp Eye Res
47:135145
12. Hirose S, Ogasawara K, Natori T, Sasamoto Y,
Ohno S, Matsuda H, Onoe K (1991) Regulation
of experimental autoimmune uveitis in rats
separation of MHC and non-MHC gene
effects. Clin Exp Immunol 86:419425
13. Caspi RR, Grubbs BG, Chan CC, Chader GJ,
Wiggert B (1992) Genetic control of suscepti-
bility to experimental autoimmune uveoretini-
tis in the mouse model: Concomitant regulation
by MHC and non-MHC genes. J Immunol
148:23842389
14. Luger D, Silver PB, Tang J, Cua D, Chen Z,
Iwakura Y, Bowman EP, Sgambellone NM,
Chan CC, Caspi RR (2008) Either a Th17 or a
Th1 effector response can drive autoimmunity:
conditions of disease induction affect dominant
effector category. J Exp Med 205:799810
15. Caspi RR, Silver PB, Chan CC, Sun B, Agarwal
RK, Wells J, Oddo S, Fujino Y, Najafian F,
Wilder RL (1996) Genetic susceptibility to
experimental autoimmune uveoretinitis in the
rat is associated with an elevated Th1 response.
J Immunol 157:26682675
16. Sun B, Rizzo LV, Sun SH, Chan CC, Wiggert
B, Wilder RL, Caspi RR (1997) Genetic sus-
ceptibility to experimental autoimmune uveitis
involves more than a predisposition to generate
a T helper-1-like or a T helper-2-like response.
J Immunol 159:10041011
17. Rizzo LV, Xu H, Chan CC, Wiggert B, Caspi
RR (1998) IL-10 has a protective role in exper-
imental autoimmune uveoretinitis. Int
Immunol 10:807814
18. Egwuagu CE, Charukamnoetkanok P, Gery I
(1997) Thymic expression of autoantigens cor-
relates with resistance to autoimmune disease.
J Immunol 159:31093112
19. Caspi RR, Chan C-C, Fujino Y, Oddo S, Najafian
F, Bahmanyar S, Heremans H, Wilder RL,
Wiggert B (1992) Genetic factors in susceptibility
and resistance to experimental autoimmune uve-
oretinitis. Curr Eye Res 11(suppl):8186
20. Mochizuki M, Kuwabara T, Chan CC,
Nussenblatt RB, Metcalfe DD, Gery I (1984)
467
An association between susceptibility to experi-
mental autoimmune uveitis and choroidal mast
cell numbers. J Immunol 133:16991701
21. Pennesi G, Caspi RR (2002) Genetic control of
susceptibility in clinical and experimental
uveitis. Int Rev Immunol 21:6788
22. Agarwal RK, Chan CC, Wiggert B, Caspi RR
(1999) Pregnancy ameliorates induction and
expression of experimental autoimmune uveitis.
J Immunol 162:26482654
23. Caspi RR, Chan CC, Grubbs BG, Silver PB,
Wiggert B, Parsa CF, Bahmanyar S, Billiau A,
Heremans H (1994) Endogenous systemic
IFN-gamma has a protective role against ocular
autoimmunity in mice. J Immunol 152:
890899
24. Silver PB, Chan CC, Wiggert B, Caspi RR
(1999) The requirement for pertussis to induce
EAU is strain-dependent: B10.RIII, but not
B10.A mice, develop EAU and Th1 responses
to IRBP without pertussis treatment. Invest
Ophthalmol Vis Sci 40:28982905
25. Arimoto H, Tanuma N, Jee Y, Miyazawa T,
Shima K, Matsumoto Y (2000) Analysis of
experimental autoimmune encephalomyelitis
induced in F344 rats by pertussis toxin admin-
istration. J Neuroimmunol 104:1521
26. Ma RZ, Gao J, Meeker ND, Fillmore PD,
Tung KS, Watanabe T, Zachary JF, Offner H,
Blankenhorn EP, Teuscher C (2002)
Identification of Bphs, an autoimmune disease
locus, as histamine receptor H1. Science 297:
620623
27. Sudweeks JD, Todd JA, Blankenhorn EP,
Wardell BB, Woodward SR, Meeker ND, Estes
SS, Teuscher C (1993) Locus controlling
Bordetella pertussis-induced histamine sensiti-
zation (Bphs), an autoimmune disease-suscep-
tibility gene, maps distal to T-cell receptor
beta-chain gene on mouse chromosome 6.
Proc Natl Acad Sci U S A 90:37003704
28. Chen X, Howard OM, Oppenheim JJ (2007)
Pertussis toxin by inducing IL-6 promotes the
generation of IL-17-producing CD4 cells.
J Immunol 178:61236129
29. Su SB, Silver PB, Wang P, Chan CC, Caspi RR
(2003) Dissociating the enhancing and inhibi-
tory effects of pertussis toxin on autoimmune
disease. J Immunol 171:23142319
30. Su SB, Silver PB, Zhang M, Chan CC, Caspi
RR (2001) Pertussis toxin inhibits induction of
tissue-specific autoimmune disease by disrupting
G protein-coupled signals. J Immunol 167:
250256
31. Agarwal RK, Sun SH, Su SB, Chan CC, Caspi
RR (2002) Pertussis toxin alters the innate and
the adaptive immune responses in a pertussis-
468 R.K. Agarwal et al.
dependent model of autoimmunity. J
Neuroimmunol 129:133140
32. Caspi RR (1993) Immunogenetic aspects of
clinical and experimental uveitis. Reg Immunol
4:321330
33. Caspi RR, Roberge FG, Chan CC, Wiggert B,
Chader GJ, Rozenszajn LA, Lando Z,
Nussenblatt RB (1988) A new model of auto-
immune disease. Experimental autoimmune
uveoretinitis induced in mice with two different
retinal antigens. J Immunol 140:14901495
34. Faure JP (1980) Autoimmunity and the retina.
Curr Top Eye Res 2:215301
35. Aronson SB, Hogan MJ, Zweigart P (1963)
Homoimmune uveitis in the guinea-pig. I.
General concepts of auto- and homoimmunity,
methods and manifestations. Arch Ophthalmol
69:105109
36. Aronson SB, Hogan MJ, Zweigart P (1963)
Homoimmune uveitis in the guinea-pig. III.
Histopathologic manifestations of the disease.
Arch Ophthalmol 69:208219
37. Aronson SB, Hogan MJ, Zweigart P (1963)
Homoimmune uveitis in the guinea-pig. II.
Clinical manifestations. Arch Ophthalmol 69:
203207
38. Wacker WB, Kalsow CM (1973) Autoimmune
uveo-retinitis in the rat sensitized with retina
photoreceptor cell antigen. Int Arch Allergy
Appl Immunol 45:582592
39. de Kozak Y, Sakai J, Thillaye B, Faure JP (1981)
S antigen-induced experimental autoimmune
uveo-retinitis in rats. Curr Eye Res 1:327337
40. Gery I, Wiggert B, Redmond TM, Kuwabara
T, Crawford MA, Vistica BP, Chader GJ (1986)
Uveoretinitis and pinealitis induced by immu-
nization with interphotoreceptor retinoid-
binding protein. Invest Ophthalmol Vis Sci 27:
12961300
41. Caspi R (1996) Experimental autoimmune
uveoretinitis (EAU) mouse and rat. In:
Kruisbeek A, Margulies D, Shevach E, Strober
W (eds) Current protocols in immunology,
Vol. January. Wiley, New York, NY, USA, pp
Unit 15.16.11Unit 15.16.18
42. Caspi RR (1994) Experimental autoimmune
uveoretinitis: rat and mouse. In: Cohen I,
Miller A (eds) Autoimmune disease models: a
guidebook. Academic, New York
43. Mozayeni RM, Chan CC, Grubbs BE, Stober
DI, Wiggert B, Chader GJ, Nussenblatt RB,
Caspi RR (1995) Alternative methods of immu-
nization for the induction of experimental auto-
immune uveoretinitis (EAU) in rodent models:
a comparison. Ocul Immunol Inflamm 3:8187
44. Mochizuki M, Kuwabara T, McAllister C,
Nussenblatt RB, Gery I (1985) Adoptive transfer
of experimental autoimmune uveoretinitis in rats.
Immunopathogenic mechanisms and histologic
features. Invest Ophthalmol Vis Sci 26:19
45. Beraud E, Kotake S, Caspi RR, Oddo SM,
Chan CC, Gery I, Nussenblatt RB (1992)
Control of experimental autoimmune uveore-
tinitis by low dose T cell vaccination. Cell
Immunol 140:112122
46. Avichezer D, Silver PB, Chan CC, Wiggert B,
Caspi RR (2000) Identification of a new
epitope of human IRBP that induces autoim-
mune uveoretinitis in mice of the H-2b haplo-
type. Invest Ophthalmol Vis Sci 41:127131
47. Namba K, Ogasawara K, Kitaichi N, Matsuki N,
Takahashi A, Sasamoto Y, Kotake S, Matsuda H,
Iwabuchi K, Ohno S, Onoe K (1998)
Identification of a peptide inducing experimental
autoimmune uveoretinitis (EAU) in H-2Ak-
carrying mice. Clin Exp Immunol 111:442449
48. Silver PB, Rizzo LV, Chan CC, Donoso LA,
Wiggert B, Caspi RR (1995) Identification of a
major pathogenic epitope in the human IRBP
molecule recognized by mice of the H-2r hap-
lotype. Invest Ophthalmol Vis Sci 36:946954
49. Cortes LM, Avichezer D, Silver PB, Luger D,
Mattapallil MJ, Chan CC, Caspi RR (2008)
Inhibitory peptide analogs derived from a
major uveitogenic epitope protect from anti-
retinal autoimmunity by inducing type 2 and
regulatory T cells. J Leukoc Biol 84:577585
50. Szpak Y, Vieville JC, Tabary T, Naud MC,
Chopin M, Edelson C, Cohen JH, Dausset J,
de Kozak Y, Pla M (2001) Spontaneous retin-
opathy in HLA-A29 transgenic mice. Proc Natl
Acad Sci U S A 98:25722576
51. Pennesi G, Mattapallil MJ, Sun SH, Avichezer
D, Silver PB, Karabekian Z, David CS, Hargrave
PA, McDowell JH, Smith WC, Wiggert B,
Donoso LA, Chan CC, Caspi RR (2003) A
humanized model of experimental autoim-
mune uveitis in HLA class II transgenic mice.
J Clin Invest 111:11711180
52. Axtell RC, de Jong BA, Boniface K, van der
Voort LF, Bhat R, De Sarno P, Naves R, Han M,
Zhong F, Castellanos JG, Mair R, Christakos A,
Kolkowitz I, Katz L, Killestein J, Polman CH, de
Waal MR, Steinman L, Raman C (2010) T helper
type 1 and 17 cells determine efficacy of inter-
feron-beta in multiple sclerosis and experimental
encephalomyelitis. Nat Med 16:406412
53. Domingues HS, Mues M, Lassmann H,
Wekerle H, Krishnamoorthy G (2010)
Functional and pathogenic differences of Th1
and Th17 cells in experimental autoimmune
encephalomyelitis. PLoS One 5:e15531
54. Tang J, Zhu W, Silver PB, Su SB, Chan CC,
Caspi RR (2007) Autoimmune uveitis elicited
with antigen-pulsed dendritic cells has a dis-
 22 Rodent Models of Experimental Autoimmune Uveitis
tinct clinical signature and is driven by unique
effector mechanisms: initial encounter with
autoantigen defines disease phenotype. J
Immunol 178:55785587
55. Knapp JE, Liu D (2004) Hydrodynamic deliv-
ery of DNA. Methods Mol Biol 245:245250
56. Chan CC, Caspi RR, Ni M, Leake WC, Wiggert
B, Chader GJ, Nussenblatt RB (1990)
Pathology of experimental autoimmune uveo-
retinitis in mice. J Autoimmun 3:247255
57. Bjorck P (2001) Isolation and characterization
of plasmacytoid dendritic cells from Flt3 ligand
and granulocyte-macrophage colony-stimulat-
ing factor-treated mice. Blood 98:35203526
58. Vremec D, Pooley J, Hochrein H, Wu L,
Shortman K (2000) CD4 and CD8 expression
by dendritic cell subtypes in mouse thymus and
spleen. J Immunol 164:29782986
59. Donoso LA, Yamaki K, Merryman CF,
Shinohara T, Yue S, Sery TW (1988) Human
S-antigen: characterization of uveitopathogenic
sites. Curr Eye Res 7:10771085
60. Singh VK, Nussenblatt RB, Donoso LA,
Yamaki K, Chan CC, Shinohara T (1988)
Identification of a uveitopathogenic and lym-
phocyte proliferation site in bovine S-antigen.
Cell Immunol 115:413419
61. Donoso LA, Merryman CF, Sery TW, Shinohara
T, Dietzschold B, Smith A, Kalsow CM (1987)
S-antigen: characterization of a pathogenic
epitope which mediates experimental autoim-
mune uveitis and pinealitis in Lewis rats. Curr
Eye Res 6:11511159
62. Kotake S, Redmond TM, Wiggert B, Vistica B,
Sanui H, Chader GJ, Gery I (1991) Unusual
immunologic properties of the uveitogenic
interphotoreceptor retinoid-binding protein-
derived peptide R23. Invest Ophthalmol Vis
Sci 32:20582064
63. Sanui H, Redmond TM, Hu LH, Kuwabara T,
Margalit H, Cornette JL, Wiggert B, Chader
469
GJ, Gery I (1988) Synthetic peptides derived
from IRBP induce EAU and EAP in Lewis rats.
Curr Eye Res 7:727735
64. Kotake S, de Smet MD, Wiggert B, Redmond
TM, Chader GJ, Gery I (1991) Analysis of the
pivotal residues of the immunodominant and
highly uveitogenic determinant of interphoto-
receptor retinoid-binding protein. J Immunol
146:29953001
65. de Smet MD, Bitar G, Roberge FG, Gery I,
Nussenblatt RB (1993) Human S-Antigen:
Presence of multiple immunogenic and
immunopathogenic sites in the Lewis rat.
J Autoimmun 6:587599
66. Merryman CF, Donoso LA, Zhang XM,
Heber-Katz E, Gregerson DS (1991)
Characterization of a new, potent, immunop-
athogenic epitope in S-antigen that elicits T
cells expressing V beta 8 and V alpha 2-like
genes. J Immunol 146:7580
67. Gregerson DS, Merryman CF, Obritsch WF,
Donoso LA (1990) Identification of a potent
new pathogenic site in human retinal S-antigen
which induces experimental autoimmune uveo-
retinitis in LEW rats. Cell Immunol 128:
209219
68. Donoso LA, Merryman CF, Sery T, Sanders R,
Vrabec T, Fong SL (1989) Human interstitial
retinoid binding protein. A potent uveitopatho-
genic agent for the induction of experimental
autoimmune uveitis. J Immunol 143:7983
69. Horai R, Caspi RR (eds) (2010) Retinal
inflammation: uveitis/uveoretinitis. Animal
models for retinal diseases, Neuromethods, vol
46. Humana, New York, NY
70. Mattapallil MJ, Wawrousek EF, Chan CC,
Zhao H, Roychoudhury J, Ferguson TA,
Caspi RR (2012) The rd8 mutation of the
Crb1 gene is present in vendor lines of
C57BL/6N mice and embryonic stem cells,
and confounds ocular induced mutant pheno-
types. Invest Ophthalmol Vis Sci. (in press)
 Chapter 23

Tolerance Induction via B-Cell Delivered Gene Therapy

Robert J. Rossi, Belinda M. Jackson, Ai-Hong Zhang, and David W. Scott

Abstract
A master control of both the innate and adaptive immune system is the bodys ability to distinguish
between self and foreign entities. This is accomplished by the elimination of autoreactive leukocytes
through a series of checkpoints both in the thymus (central deletion) and in the circulating periphery
(peripheral tolerance), thus establishing tolerance to self-antigens. When one or more of these controls is
disrupted, there is the potential for the development of autoimmune disease. Current available therapies
for these diseases often rely on global immune suppression or expensive treatments that are not affordable
to all. Herein, we describe a novel therapeutic technique in which tolerance to self can be re-established
via B-cell delivered antigen-specific tolerogenic gene constructs. Furthermore, this technique shows prom-
ise in the gene therapeutic treatment of monogenic disorders and the acceptance of tissue transplants.

Key words: Autoimmunity, Immune tolerance, B cells, Gene therapy, Immunoglobulin fusion protein

1. Introduction

1.1. Self Tolerance and
Autoimmune Disease
It has been well established that the immune system is designed to
recognize and eliminate foreign invaders from the host and at the
same time largely maintain the homeostasis of the body. One of the
many challenges the immune system faces is recognition of self
from non-self. It has evolved the ability to discriminate selfness
from foreignness; thus, self-tolerance is considered the basic tenet
of immune function. This tolerance to self is maintained in at least
two levels. The first mechanism, known as central tolerance,
involves autoreactive T cells being deleted in the thymus before
reaching the periphery. Similar events for clonal deletion or recep-
tor editing of immature autoreactive B cells occur in the bone mar-
row. Central deletion is not foolproof, however, and some
autoreactive cells escape deletion in the thymus/bone marrow, and
must be controlled in the periphery; otherwise, autoimmunity is
likely to occur. Some of the safety mechanisms designed to

Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_23, Springer Science+Business Media New York 2012

471
472 R.J. Rossi et al.
1.2 .Antigen-Specific
Tolerance Induction
1.3. B-Cell Delivered
Gene Therapy for
Tolerance Induction
suppress autoreactivity in the periphery include regulatory cells,
peripheral anergy, antigen segregation, and clonal exhaustion.
However, when one or more of these mechanisms fail, the poten-
tial for self-tolerance breakdown becomes possible with the end
result being an increased susceptibility to autoimmune disease.
Some common autoimmune diseases include type I diabetes, mul-
tiple sclerosis, rheumatoid arthritis, and systemic lupus erythema-
tosus. Additional circumstances in which induced tolerance is
desirable include gene or protein replacement therapies. Such is
the case for hemophilia A patients requiring replacement Factor
VIII (FVIII), an essential blood clotting protein. A significant per-
centage of people who suffer from hemophilia A form antibodies
(inhibitors) to FVIII replacement therapy because their bodies rec-
ognize the replacement FVIII as foreign, and thus eliminate it
from the body. In addition, there are occasions when tolerance to
foreign tissue is desirable, such as tissue transplantation.
Unfortunately, there are no cures available for any of these condi-
tions as of yet. Currently, the first line of treatment is global immune
suppression, which lacks specificity and can cause severe side effects
like secondary infection and cancer (1). Among new treatment
strategies being developed, an antigen-specific tolerance induction
approach is very promising, especially for those autoimmune dis-
eases in which the disease causing auto-antigen(s) have been or can
be identified.
Antigen-specific tolerance can be defined as a specific unresponsive
or hypo-responsive immunologic state induced by prior exposure
to antigenic epitopes (2). It has long been established that antigen-
specific tolerance can be deliberately induced. In 1953, Medawar
and his colleagues clearly demonstrated, for the first time, that
active acquired tolerance to skin grafts from donor mice of a dif-
ferent strain could be achieved by fetal or neonatal exposure to
tissue antigens from the donor strain (3). This seminal finding was
extended by many other studies using a variety of antigens. It is of
note that in the 1970s, Borel and colleagues (4, 5) demonstrated
in adult animals that hapten-carrier conjugates were highly tolero-
genic when some serum proteins were used as carriers. Of all serum
proteins tested, immunoglobulin G (IgG) was the most tolero-
genic. In addition, the exclusive usage of a certain type of antigen-
presenting cells (APC) may dictate the outcome of an immune
response. In contrast to mature dendritic cells, B cells, both resting
or activated, were shown to be excellent tolerogenic APC in a vari-
ety of systems (68).
Based on the ability of B cells to function as tolerogenic APCs as
well as the excellent tolerogenicity of IgG as protein carrier, our lab
developed a novel B-cell gene therapy platform for antigen-specific
tolerance induction (9, 10). Thus, the target antigen (peptide or
 23 Tolerance Induction via B-Cell Delivered Gene Therapy 473

Table 1
Common autoimmune diseases and their corresponding antigenic targets
for gene therapy

Disease Antigen(s) Effects

Multiple Myelin basic protein (MBP) Inflammation from lymphocytes

sclerosis (MS) Myelin oligodendrocyte glycoprotein infiltrating the central nervous
(MOG) system (CNS) causes nerve damage
Proteolipid protein (PLP) leading to loss of muscular strength
and control.
Type I diabetes Glutamic acid decarboxylase Insulin producing cells (b-cells) in the
(GAD-65) pancreas are destroyed. Without
Chromogranin A insulin, glucose cannot be absorbed
Islet-specific glucose-6-phosphatase into the bodys cells where it would
catalytic subunit-related protein be used for energy. The build-up of
(IGRP) glucose in the bloodstream can
Insulin cause life-threatening complications.
Myasthenia Acetylcholine receptor (nAChR) Muscular weakness caused by destruc-
gravis tion or impairment of the receptor
for neurotransmitter acetylcholine
at the neuromuscular junction.
Reduced number of receptors leads
to loss of muscular strength
and control.
Systemic lupus dsDNA Immune complex deposits cause
erythematosis Sm glomerulonephitis, vasculitis,
(SLE) arthritis, and/or skin rash.
Uveitis Interphotoreceptor retinoid binding T cell-mediated inflammation in
protein (IRBP) S-antigen anterior chamber of the eye and/or
(arrestin) iris which can lead to sensitivity to
light, blurry vision, and blindness.

protein domain-) is engineered in frame into the N-terminal of the

murine IgG1 heavy chain. The fusion protein can then be expressed
in syngeneic B cells via retroviral vector-mediated transduction
in vitro. Upon adoptive transfer, such genetically engineered B
cells are highly tolerogenic. Recipient animals become hypo-
responsive against re-challenge with the target antigen, even in the
presence of complete Freunds adjuvant. Over the last 15 years this
approach has been successfully applied to at least a dozen different
antigens, including targets in five autoimmune disease models and
FVIII domains in hemophilia A (1118). As an example, tolerance
induction in experimental autoimmune encephalomyelitis (EAE),
the animal model for multiple sclerosis, will be used here in describ-
ing the detailed protocol for the B-cell delivered gene therapy
(Table 1).
474 R.J. Rossi et al.

2. Materials

1. MBAE retroviral vector (MBAE = murine Moloney leukemia

virus with the b-actin promoter and enhancer) or murine stem
cell virus (MSCV).
2. Restriction endonucleases: NotI, XhoI, SalI, BamHI, HinDIII.
3. DNA electrophoresis system.
4. BSSK-Ig (pBluescript + SK with murine heavy chain IgG1).
5. Viral packaging cell line such as GP + E-86.
6. 1.22 % agarose gel.
7. QIAquick gel extraction kit (Qiagen, Valencia, CA).
8. Calf intestinal alkaline phosphatase (New England BioLabs,
Ipswich, MA).
9. Rapid DNA ligation kit (Roche, Indianapolis, IN).
10. JM109 Competent cells (Promega, Madison, WI).
11. NIH 3T3 cell line.
12. 60 and 100 mm tissue culture (TC) plates.
13. 2.5 M CaCl2: 183.8 g of CaCl2 dihydrate (CaCl2H2O), 500 mL
LPS-free water, sterile filter, and freeze aliquots at 20 C.
14. HEPES buffered saline (2): 16.4 g NaCl (final concentration
0.283 M), 11.9 g HEPES acid (final concentration 0.023 M),
0.21 g Na2HPO4 (final concentration 1.5 mM), add dH2O to
1 L, adjust pH to 7.05 with NaOH, filter sterilize, and freeze
at 20 C.
15. Polystyrene tubes.
16. Trypsin-Versene (Lonza, Walkersville, MD).
17. 24-Well tissue culture plates.
18. Complete DMEM medium: 500 mL DMEM with media sup-
plement and 10 % FCS.
19. Geneticin G418 (Invitrogen, Carlsbad, CA).
20. Sterile cloning disks (Bel-Art Products, Pequannock, NJ).
21. Forceps.
22. Ethanol.
23. Freezing media: 35 mL RPMI, 10 mL dimethyl sulfoxide
(DMSO), 3 mL 1 M HEPES, 2 mL 100 mM sodium pyru-
vate. Filter and store at 4 C. Add 50 mL of fetal calf serum
(FCS) to freezing media before using to freeze cells.
24. Coomassie Blue stain: 1 g/L Coomassie Blue powder, 40 %
ethanol, 10 % acetic acid.
25. Polybrene (Hexadimethrine Bromide) (Sigma-Aldrich, St.
Louis, MO).
 23 Tolerance Induction via B-Cell Delivered Gene Therapy 475

26. 96-Well flat bottom cell culture plates (Corning Inc., Corning
NY).
27. Complete Freunds adjuvant (Sigma-Aldrich, St. Louis, MO).
28. Anti-Thy 1 supernatant (clone 30H12, Rat IgG2b).
29. Anti-CD4 supernatant (clone GK1.5, Rat IgG2b).
30. Anti-CD8 supernatant (clone 3.155, Rat IgM).
31. Low-Tox M rabbit complement (Cedarlane Labs, Burlington,
NC).
32. Lympholyte M (Cedarlane Labs, Burlington, NC).
33. Lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma-
Aldrich, St. Louis, MO).
34. Red blood cell lysis buffer (buffered ammonium chloride):
4.15 g NH4Cl, 0.5 g KHCO3 dissolved in 500 mL H2O with
pH adjusted to 7.2 by gassing with 10 % CO2.
35. Media supplement for complete RPMI and DMEM: Stock 1:
0.1 M b-mercaptoethanol; combine 6.5 mL PBS with 50 ml
b-mercaptoethanol. Stock 2: 100 mL of 100 mM sodium
pyruvate, 100 mL of 100 solution MEM nonessential amino
acids, 100 mL of 1 M HEPES buffer, 100 mL of
200 mM L-glutamine, 100 mL of 100 Penicillin-Streptomycin
(Cellgro by Mediatech, Manassas, VA). Add 5 mL of stock 1 to
stock 2 and stir to combine. Sterile filter, aliquot, and freeze at
20 C.
36. Complete RPMI-1640 medium: 500 mL RPMI with 25 mL
media supplement from above and 5 % FCS (Atlanta Biologicals,
Lawrenceville, GA).
37. b-Mercaptoethanol (Sigma-Aldrich, St. Louis, MO).
38. Phosphate-buffered saline (PBS): 8.5 g NaCl, 0.57 g Na2HPO4,
0.14 g KH2PO4 dissolved in 1 L of H2O with pH adjusted to
7.4.
39. Anti-B220 (clone RA36B2, Rat IgG2c k).
40. Biomag goat a-rat IgG (Polysciences Inc., Warrington, PA).
41. Magnetic separator.
42. Centrifuge.
43. Rotating mixer.
44. Falcon cell strainer (Fisher Scientific, Pittsburgh, PA).
45. Conical tubes (Fisher Scientific, Pittsburgh, PA).
46. [3H] thymidine (Perkin Elmer, Waltham, MA).
47. Glass fiber filtermats.
48. Sample bag for filtermats.
49. Heat sealer.
50. Scintillation fluid.
476 R.J. Rossi et al.

51. MicroBeta FilterMate 96-well Cell Harvester (Perkin Elmer,

Waltham, MA).
52. MicroBeta2 Scintillation Counter (Perkin Elmer, Waltham, MA).
53. Nunc Immulon 96-well flat bottom plates for ELISA (Fisher
Scientific, Pittsburgh, PA).
54. Microplate reader capable of measuring absorbance at 450 nm.
55. ELISA coating buffer.
56. ELISA assay diluent: PBS with 10 % FCS.
57. ELISA wash buffer: PBS with 0.05 % Tween-20.
58. ELISA substrate developing solution: Tetramethylbenzidine
(TMB) and hydrogen peroxide (BD Pharmingen, San Diego,
CA).
59. 1 M H3PO4 or 2 N H2SO4.
60. ELISPOT plates.
61. ELISPOT blocking solution: Complete DMEM or RPMI with
10 % FCS.
62. ELISPOT wash buffer I: 1 PBS with 0.05 % Tween-20.
63. ELISPOT wash buffer II: 1 PBS.
64. ELISPOT dilution buffer: 1 PBS with 10 % FCS.
65. ELISPOT Substrate Solution: AEC substrate reagent set (BD
Pharmingen, San Diego, CA).
66. ELISPOT plate reader.
67. Fluorescence-activated cell sorting (FACS) buffer: 1 PBS
with 3 % FCS and 0.1 % sodium azide.
68. 5 mL polypropylene round bottom tubes (BD Falcon,
Bedford, MA).
69. Fluorescently conjugated antibodies.
70. Propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) cell
viability stain.
71. Fluorescence-activated cell sorter.

3. Methods

The methods described here outline (a) insertion of gene of inter-

est (GOI) into the N terminus of murine IgG1 heavy chain in
pBluescript + SK vector, constructed with murine heavy chain IgG1
(BSSK-Ig); (b) isolation and sub-cloning of GOI into MBAE ret-
roviral vector; (c) transduction of MBAE vector containing GOI
into packaging cell line; (d) B-cell isolation, activation, and trans-
duction; (e) adoptive transfer of transduced B cells into experimen-
tal host and immunization of appropriate disease model antigen
 23 Tolerance Induction via B-Cell Delivered Gene Therapy 477

(or challenge with target model antigen such as ovalbumin); and

(f) sample collection and analysis by ELISA, ELISPOT, clinical
disease scoring (EAE), T cell proliferation assay, and/or FACS
analysis. It is also possible to use MSCV as the retroviral vector,
however, it is not discussed in detail here.

3.1. Insertion of Gene 3.1.1. Using the NotI and Xho1 restriction sites, insert the PCR
of Interest into product of gene of interest into BSSK cassette which
BSSK-Ig contains a murine IgG1 heavy chain cDNA.
1. PCR amplify your GOI such that restriction sites NotI
and XhoI are at the 5 and 3 ends, respectively. In design-
ing the 5 primer ensure that translation of the fusion
protein will occur in frame by adding one additional
nucleotide between the NotI site and the start of your
GOI (see vector sequence below).

2. Digest both the GOI PCR amplicon and the BSSK-IgG

vector with restriction endonucleases NotI and XhoI.
3. Gel extract the digested fragments.
4. Ligate the DNA fragments and transform JM109 com-
petent bacterial cells. Plate transformed cells onto LB
agar/ampicillin media.

3.2. Isolation 3.2.1. Digest the BSSK-Ig-gene of interest construct with Sal I.
and Sub-cloning Run digestion product on a 1.22 % agarose gel to isolate
of Ig-Gene of Interest the GOI away from pBluescript. It is necessary to deter-
into MBAE or MSCV mine the length of the fusion product, so that you will know
where the target fragment will be separated in the gel. The
pBluescript is approximately 2,800 bp. If the GOI is less
than 800 bp or more than 1,500 bp, it is likely that a 1.2 %
gel will work. Otherwise, it may be necessary to digest the
pBluescript with an additional enzyme not found in the
GOI. This will allow for better separation of pBluescript
from the gene insert. Purify the Ig-gene of interest with
a suitable gel purification kit (QIAquick from Qiagen).
3.2.2. Linearize the MBAE vector by digesting with Sal I. In order
to prevent self-ligation of the vector, it is necessary to
treat with calf intestinal alkaline phosphatase (CIAP) for
478 R.J. Rossi et al.

60 min at 37 C to remove the phosphoryl groups from the

5 termini of the DNA fragment (protocol can be found on
the New England BioLabs Web site). Purify the DNA by
gel purification or spin-column purification to remove any
residual phosphatase.
3.2.3. Using the Rapid DNA ligation kit from Roche, ligate the
Ig-gene of interest to the linear MBAE vector which has
been gel purified (protocol available on Roche Web site).
3.2.4. Transform JM109 competent cells (Promega, Madison, WI).
Plate 100 ml of undiluted cells and one to two dilutions of
cells on LB/ampicillin plates. Incubate for 1214 h at 37 C.
3.2.5. Pickup colonies from LB-plate and prepare small amount of
plasmid DNAs by Miniprep. The transformation and ori-
entation of ligation can be tested by digesting the plasmid
with BamHI and HindIII or by PCR.
3.2.6. Sequence the positive colonies and prepare a large amount
of DNA by Maxiprep for packaging cell transfection.

3.3. Stable Day 1: Add 1 106 GP + E-86 fibroblast cells in complete DMEM
Transfection with 10 % FCS to two 100 mm tissue culture plates for every
of GP + E-86 construct being transfected.
Fibroblasts
Day 2: Introduce DNA into plated GP + E-86 cells by calcium
phosphate precipitation. (It is also possible to follow another
transfection method such as using FuGENE kit).
1. Add 1050 mg DNA to a polystyrene tube.
2. Add enough sterile water to bring the volume to 450 ml.
3. Add 50 ml of 2.5 M CaCl2 and gently vortex the mix.
4. To a clean polystyrene tube, add 500 ml of 2 HEPES buffered
saline.
5. While gently vortexing, add the DNA in a dropwise fashion to
the 2 HEPES buffered saline.
6. Let stand at room temperature (RT) for 45 min to 1 h to allow
precipitate to form.
7. Add DNA-HEPES mixture to plated GP + E-86 cells, again in
a dropwise fashion.
8. Incubate overnight at 37 C + 7 % CO2.
Day 3: Remove complete DMEM and add media containing G418
selection with a starting concentration of 0.5 mg/mL and increase
the concentration of G418 to 1 mg/mL on day 4 or day 5.

3.4. Isolation 3.4.1. Approximately 1014 days post retroviral DNA transfection,
of GP + E-86 Viral colonies should be visible on the tissue culture plates. While
Producing Colonies there are several different ways to pick colonies from the plate,
our lab utilizes sterile cloning disks from Bel-Art Products.
 23 Tolerance Induction via B-Cell Delivered Gene Therapy 479

1. Set up one 50 mL conical tube with 70 % ethanol and one with

1 PBS. Place forceps in 70 % ethanol.
2. Carefully remove all media from the 100 mm tissue culture
dish containing clones.
3. Gently rinse TC dish with 10 mL PBS. Pipette out PBS.
4. Shake a few cloning disks out onto the surface of a clean 60 mm
petri dish. Add 12 mL of trypsin-versene solution to same
60 mm petri dish.
5. Carefully remove the forceps from the ethanol and dip in PBS
several times to remove excess ethanol.
6. Using forceps, remove a single cloning disk from trypsin-
versene and touch the edge to a dry part of the petri dish to
wick away excess trypsin-versene. Then place the disk gently,
but firmly on a colony. Continue adding additional disks to
other colonies on the same plate (should be able to get 20
disks on a plate).
7. Incubate for 5 min at room temperature.
8. Without tilting the plate and making sure that the forceps only
touch the back of the disks, pick up each disk and place it in a
single well of a 24-well plate.
9. Add complete DMEM with 1 mg/mL G418 to each well con-
taining a cloning disk.
10. Allow 714 days for colonies to grow.
11. When growth has become confluent, transfer cells to 25 cm2
Flasks. Also, freeze an aliquot from the first passage of this flask.

3.5. Viral Titers Day 1: Harvest supernatant and freeze in 2 mL aliquots until
needed. Thaw each vial one time only. As an alternative, you
3.5.1. Harvest Viral
can change media on sub-confluent packaging cells and use
Supernatant from
fresh media on day 2. Be sure there is no selection component
Packaging Cell Line
such as G418 in the media.

3.5.2. Titer of Viral Day 1: Split confluent NIH 3T3 cells and plate 5 105 cells in
Supernatant 60 mm dishes in complete DMEM. You will need to set up
three plates for every viral cell line checked.
Day 2:
1. Label plates with construct name and dilutions. Discard media
and add 1 mL fresh complete DMEM.
2. Perform serial dilutions of viral supernatant by adding 100 ml
to plate 1. This will be a 1:10 dilution or 0.1 mL virus. Gently
mix supernatant into plated media until it has been well
distributed.
3. Remove 100 ml media from plate 1 and add it to plate 2. Mix
following the same procedure as above. This is a 1:100
480 R.J. Rossi et al.
Day 3:
Day 1013 Count colonies
3.6. B-Cell Isolation
and Activation
dilution or 0.01 mL virus. Repeat the procedure on a third
plate for a 1:1,000 dilution or 0.001 mL virus.
4. Add 6 mg of polybrene to each plate and mix to combine.
5. Incubate at 37 C for 2 h. Gently mix plates one more time
and return to incubator for an additional 2 h.
6. Once again, remove plates from incubator and add 3 mL com-
plete DMEM to each plate. Incubate overnight at 37 C + 7 %
CO2.
1. Wash plates with PBS and add 1 mL of trypsin to each plate.
Incubate at 37 C + 7 % CO2 until cells detach from plate.
2. Break up cell clumps by gently pipetting up and down several
times. Split cells 1:10 after adding 100 ml of trypsin/cell solu-
tion to fresh 100 mm TC dishes.
3. Add 1215 mL of complete DMEM supplemented with 1 mg/
mL G418.
1. Remove media from 100 mm TC plates.
2. Wash each plate with 2 mL of PBS.
3. Add 12 mL Coomassie Blue stain and wait 12 min.
4. Wash off stain with water.
5. Count the colonies and record the number (colonies should be
visible to the naked eye).
6. Calculate the viral titer using the formula below:
Titer of colony forming units / mL (CFU )
# of colonies the inverse of the dilution of plated cells
=
mL of virus added
1. Aseptically harvest spleens from mice and make into single cell
suspension by preferred method.
2. Wash one time with RPMI and centrifuge at 250 g for 5 min.
3. Pour off supernatant and re-suspend cell pellet.
4. Lyse red blood cells by adding 5 mL of buffered ammonium
chloride per spleen. Mix well and allow to incubate at room
temperature for approximately 5 min.
5. Following 5 min incubation, add 1015 mL RPMI media and
centrifuge for 5 min at 250 g.
6. Prepare anti-T cell cocktail using a 1:1:1 ratio of anti-Thy 1
(clone 30H12), anti-CD4 (clone GK1.5), and anti-CD8 (clone
3.155). You will need 1 mL of cocktail for each spleen.
7. Harvest pelleted spleen cells with gentle pipetting and filter
through a cell strainer. Centrifuge for 5 min at 250 g.
 23 Tolerance Induction via B-Cell Delivered Gene Therapy 481

8. Discard supernatant, re-suspend pellet, and add appropriate

amount of anti-T cell cocktail.
9. Incubate on ice for 30 min.
10. Wash two times with cold RPMI.
11. Reconstitute Low-Tox M rabbit complement according to
manufacturers instructions and use within 12 h. Briefly, add
1 mL cold distilled water and 4 mL plain RPMI and mix. This
will be enough for five spleens.
12. Add 1 mL rabbit complement per spleen and incubate for
30 min at 37 C.
13. Wash cells two times with cold RPMI. Spin for 5 min at
250 g.
14. After incubation, to eliminate dead cells, re-suspend the pellet
in 10 mL of complete RPMI. Layer cell suspension over 5 mL
of Lympholyte-M and centrifuge for 20 min at 1,000 g at
room temperature.
15. Carefully collect the interface layer and wash with RPMI one
time.
16. Count cells and re-suspend at a concentration of 25 106 cells/
mL.
17. To activate B cells, add 525 mg/mL LPS. Prior to adding
LPS, it is important to heat it to 100 C for 5 min and then
vortex vigorously. CD40 ligand (CD40L) or a-IgM can also
be used to activate B cells.
18. After adding LPS, incubate cells at 37 C + 7 % CO2 for 24 h.

3.7. Transduction 1. On day 0, prepare activated B cells as described above.

of Activated B Cells 2. A few days before B-cell activation, thaw viral producing pack-
with Virally Produced aging cells and expand them in DMEM media with 10 % FCS.
Tolerogenic Construct On day 0, the same day that B cells are stimulated with LPS,
irradiate packaging cells with 1,500 rad. Wash cells one time.
3. Prepare irradiated viral producing packaging cells to a concen-
tration of 12 105 cells/mL and seed 10 mL of cells per
100 mm cell culture dish. Incubate packaging cells at
37 C + 7 % CO2 overnight to let cells recover and adhere to
culture dishes.
4. On day 1, to set up co-culture, bring activated B cells to a con-
centration of 1 106 cells/mL in complete RPMI with 5 %
FCS.
5. Add 10 mL of activated B cells (10 106) to a 100 mm TC
dish. If you prefer to use 150 mm TC dish, add 35 106 irra-
diated packaging cells in 25 mL culture media per dish on day
0 and add 25 mL activated B cells (25.0 106) to the dish on
day 1.
482 R.J. Rossi et al.

6. Add 36 mg/mL of polybrene and mix well.

7. Co-culture for 24 h at 37 C + 7 % CO2.
8. Day 2, harvest suspension cells (transduced B cells) and wash
two times with PBS.
9. Count live cells and re-suspend cells at a concentration of
2 107 cells/mL in PBS.
10. Inject 0.5 mL/mouse through intraperitoneal (ip) route.
11. After 710 days, challenge mice with antigen emulsified in
Complete Freunds adjuvant (CFA) via subcutaneous (sc)
injection on one footpad and the base of tail. The dose of
immunized antigen depends on which animal model is used.
For example, when inducing tolerance to ovalbumin protein,
we use a typical dose of 25 mg of ovalbumin protein or 323
339 peptide in 50 ml volume.
12. Fourteen days later, collect blood for serum and aseptically
harvest draining lymph nodes (popliteal and inguinal).
13. Process lymph nodes into a single cell suspension and set up
proliferation assay and/or FACS analysis.
14. Set up ELISA to test serum for antibody production.
15. Set up ELISPOT plate for detecting cytokine production (Fig. 1).

3.8. Proliferation 1. Immunize animals in one hind footpad and the base of tail
Assay with peptide or antigen emulsified in CFA.
2. After 1014 days, euthanize mice and remove popliteal and
inguinal lymph nodes on the side of the body that the immu-
nization was given. Working sterilely in a biological safety cabi-
net, mash lymph nodes into single cell suspension, and suspend
in complete RPMI with 2.5 % FCS or X-VIVO serum-free
medium.
3. Wash cells two times. Centrifuge at 250 g for 5 min.
4. Re-suspend cells at a concentration of 5 106 cells/mL.
5. Prepare proliferation plates by adding 100 ml medium per well
in 96-well flat bottom cell culture plate. Each well contains no
or different concentrations of stimulation antigen. Typically an
antigen titration is set up in the range of 100 ng to 30 mg,
6. Add 100 ml cells (5 106 cells/mL) per well, so that final cul-
ture volume is 200 mL and final cell number is 0. 5 106.
Incubate plates at 37 C + 7 % CO2.
7. After 48 h, add 0.5 mCi/well [3H] thymidine to each well and
incubate an additional 1418 h.
8. Harvest cells from plate onto glass fiber filters using a cell
harvester.
9. Add scintillation fluid and read filter using scintillation counter.
 23 Tolerance Induction via B-Cell Delivered Gene Therapy 483

B cell delivered gene therapy protocol

Purify B cells from spleen
Activate B cells with LPS,
-IgM, or CD40L for 24 hours
Co-culture activated B cells
with virus producing packaging
cells
Packaging cells produce tolerogenic
construct consisting of IgG heavy chain
backbone containing in frame target antigen
Harvest spleen
Collect blood serum for
cytokine analysis/antibody
response using ELISA
14 Days Harvest draining lymph nodes
for T cell proliferation assay, FACS,
and/or ELISPOT
Inject mice sub cutaneaously Clinical scoring of paralysis in
with antigen/CFA in footpad Experimental Autoimmune
Encephalomyelitis (EAE)
7-10 Days Measure blood glucose levels in
type 1 diabetes (T1D)

Inject mice i.p. with B cells

transduced with tolerogenic
construct

Fig. 1. Schematic representation of protocol for B-cell delivered gene therapy. Splenic B cells from donor mice are purified
and activated with either LPS, a-IgM, or CD40 ligand (CD40L) for 24 h. Irradiated viral packaging cells transfected with
tolerogenic construct are then co-cultured with activated B cells which allows transduction of tolerogenic construct into B
cells. Transduced B cells can then be injected intravenously or intraperitoneally into recipient mice who are subsequently
challenged with immunogenic form of a disease-specific antigen emulsified in Complete Freunds adjuvant. Blood collec-
tion for serum analysis, EAE clinical disease scoring, and blood glucose levels (T1D) can be monitored. Approximately
2 weeks post footpad injection, draining lymph nodes can be removed for proliferation assay, ELISA, and FACS.

3.9. Blood Collection Circulating cytokines and/or antibodies can be detected in blood
and ELISA serum by enzyme linked immunosorbent assay (ELISA).

3.9.1. Serum Collection
1. Collect blood into microcentrifuge tubes via retro-orbital eye
bleed utilizing glass capillary tubes that DO NOT contain hep-
arin or other anticoagulants.
2. Place tubes containing blood on ice for 30 min to allow blood
to clot. Centrifuge for 1015 min at 1,000 g to separate
serum from other blood components.
3. After centrifugation, pipette clear top layer (serum) into a clean
microcentrifuge tube and assay immediately or freeze at 20 C.

3.9.2. ELISA 1. Dilute capture antibody or protein antigen to proper concen-

tration in coating buffer and add 100 ml/well into a 96-well
ELISA plate.
2. Incubate at 4 C overnight.
3. Remove plate from 4 C and allow to come to room temperature
before proceeding. Aspirate capture antibody and wash plate
484 R.J. Rossi et al.

three to four times with 280300 ml of PBS-Tween (see Note 1).

Blot plate on clean paper towel to remove residual moisture.
4. Block nonspecific binding by adding 200 ml of assay diluent
and incubating plate for 1 h at room temperature.
5. Aspirate plate and wash three to four times with PBS-Tween as
above.
6. Add 100 ml of diluted standards or serum samples to plate
(see Note 2). Standards should be added in duplicate while
samples should be added in duplicate or triplicate. Incubate
plate at room temperature for 2 h, and cover to prevent any
dust from settling into wells.
7. Aspirate and wash plate three to four times as above.
8. Add 100 ml of diluted detection antibody to each well and
incubate for 1 h at room temperature.
9. Aspirate and wash plate three to four times as above.
10. Add 100 ml of TMB substrate solution to each well. Incubate
for 1530 min in the dark at room temperature.
11. Once color development has occurred, add 50 ml of 1 M
H3PO4 or 2 N H2SO4 to stop reaction.
12. Read absorbance at 450 nm on plate reader.

3.10. ELISPOT 1. Use coating buffer to dilute capture antibody and add 100 ml
to each well of an ELISPOT plate.
2. Incubate at 4 C overnight.
3. Aspirate coating antibody and wash wells 12 with blocking
solution (see Note 1).
4. Block any nonspecific binding by adding 200 ml of blocking
solution/well. Incubate at room temperature for 2 h.
5. Aspirate blocking solution and wash plate.
6. Dilute capture antibody in complete medium and add 100 ml/
well. Add 100 ml lymph node single cell suspension(s) to plate.
Incubate plate at 37 C + 5 % CO2 1824 h.
7. After incubation, remove cells and wash plate with 280 ml
deionized water 2, soaking wells for 5 min between washes.
Wash wells three times with 280 ml wash buffer I.
8. Add 100 ml diluted detection (e.g., biotinylated) antibody to
each well and incubate for 2 h at room temperature.
9. After incubation with detection antibody, aspirate wells and
wash plate with 280 ml/well wash buffer I, allowing wells to
soak for 2 min between washes.
10. Dilute streptavidin-HRP enzyme conjugate in dilution buffer and
add 100 ml/well. Incubate plate for 1 h at room temperature.
11. Aspirate plate and wash 4 with 280 ml/well wash buffer I,
allowing plate to soak for 2 min between washes.
 23 Tolerance Induction via B-Cell Delivered Gene Therapy 485

12. Wash an additional 2 with wash buffer II.

13. Add 100 ml AEC substrate solution to each well and develop.
Once development has occurred, stop reaction with deionized
water. Air dry plate, and then read spot development using
ELISPOT reader.

3.11. Clinical Scoring 3.11.1. Grading of paresis/paralysis in EAE studies is conducted

of Paralysis in EAE using a scale from 0 to 5 with 0 being typical of an animal
showing no abnormality and 5 being a moribund animal
that must be euthanized immediately.
Grade 0Animal(s) show no abnormality. Animals should
be monitored on a daily basis.
Grade 1First signs of disease become visible. Animals will
exhibit flaccid tails, but no outward signs of parapare-
sis. Investigators should record weight of study ani-
mals, add extra bedding and enrichment to the cage.
Assessment and scoring of animals should be done at
least every other day. Monitor animals on a daily basis.
Grade 2Paraparesis evident with weakness in hind limbs
causing difficulty walking. Investigators should place
gel nourishment on cage floor. Continue to monitor
mice daily and record weight weekly.
Grade 3Complete hind limb paralysis and possible loss
of bladder control. Investigators may need to express
bladders of affected mice and supply fluids via subcu-
taneous (sc) injection.
Grade 4Paralysis of all four limbs. Depending on inves-
tigators approved protocol, animals may need to be
euthanized.
Grade 5Animals are moribund and must be euthanized
immediately.
It is also acceptable for investigators to use half numbers when
animals exhibit some, but not all characteristics of each grade listed
above.

3.12. Fluorescence- 1. Euthanize animals and remove draining lymph node as in pro-
Activated Cell Sorting liferation assay.
Analysis 2. Place a cell strainer atop a 50 mL conical tube. Wet cell strainer
with 12 mL of RPMI media and place lymph nodes inside.
Crush lymph nodes using a syringe plunger and wash cells
through cell strainer using RPMI, collecting eluate in bottom
of 50 mL conical tube (see Note 3).
3. Centrifuge single cell suspension for 5 min at 250 g.
4. Pour off supernatant and vortex pellet.
5. Re-suspend cells in 1020 mL of FACS buffer and centrifuge
one more time as above.
486 R.J. Rossi et al.

6. Pour off supernatant and vortex pellet.

7. Re-suspend cells in FACS buffer and count by your preferred
method (e.g., hemocytometer or automated cell counter).
After counting, bring cells to a concentration of 5 106 cells/
mL in FACS buffer.
8. Add 200 ml of cells to a 5 mL polypropylene tube and add
appropriate fluorescently conjugated antibodies.
9. Incubate on ice in the dark for 3040 min.
10. Wash cells with FACS buffer 2 to remove unbound antibody
and re-suspend in 200400 ml FACS buffer.
11. Samples are now ready to be run on a flow cytometer.

4. Notes

1. In both ELISA and ELISPOT procedures, be sure not to touch

bottom of wells with pipette tips to avoid disturbing bound
antibodies.
2. Use at least two dilutions of serum when performing ELISA,
which will minimize the chances of samples being too concen-
trated or not concentrated enough to fall within the range of
the standard curve.
3. After crushing lymph node cells through cells strainer, it is a
good idea when pouring media through strainer to tip strainer
up so that there is an air space between strainer and 50 mL
conical collecting tube. This will prevent surface tension on the
cell strainer, which will cause spillage of media and cells.

References
1. Caspi RR (2008) Immunotherapy of autoim-
munity and cancer: the penalty for success. Nat
Rev Immunol 8:970976
2. Scott DW (1994) The Nature of Immunologic
Tolerance. R.G. Landes, Austin, TX
3. Billingham RE, Brent L, Medawar PB (1953)
Actively acquired tolerance of foreign cells.
Nature 172:603606
4. Borel Y, Lewis RM, Stollar BD (1973)
Prevention of murine lupus nephritis by carrier-
dependent induction of immunologic tolerance
to denatured DNA. Science 182:7678
5. Borel Y (1980) Haptens bound to self IgG
induce immunologic tolerance, while when cou-
pled to syngeneic spleen cells they induce immune
suppression. Immunol Rev 50:71104
6. Lassila O, Vainio O, Matzinger P (1988) Can B
cells turn on virgin T cells? Nature 334:253255
7. Eynon EE, Parker DC (1992) Small B cells as
antigen-presenting cells in the induction of tol-
erance to soluble protein antigens. J Exp Med
175:131138
8. Fuchs EJ, Matzinger P (1992) B cells turn off
virgin but not memory T cells. Science 258:
11561159
9. Zambidis ET, Barth RK, Scott DW (1997)
Both resting and activated B lymphocytes
expressing engineered peptide-Ig molecules
serve as highly efficient tolerogenic vehicles in
immunocompetent adult recipients. J Immunol
158:21742182
10. Kang Y, Melo M, Deng E, Tisch R, El-Amine
M, Scott DW (1999) Induction of hypore-
sponsiveness to intact foreign protein via
retroviral-mediated gene expression: the
IgG scaffold is important for induction and
 23 Tolerance Induction via B-Cell Delivered Gene Therapy
maintenance of immune hyporesponsive-
ness. Proc Natl Acad Sci U S A 96:
86098614
11. Agarwal RK, Kang Y, Zambidis E, Scott DW,
Chan CC, Caspi RR (2000) Retroviral gene
therapy with an immunoglobulin-antigen
fusion construct protects from experimental
autoimmune uveitis. J Clin Invest 106:
245252
12. Liang W, Karabekian Z, Mattapallil M, Xu Q,
Viley AM, Caspi R et al (2006) B-cell delivered
gene transfer of human S-Ag-Ig fusion protein
protects from experimental autoimmune
uveitis. Clin Immunol 118:3541
13. Xu B, Scott DW (2004) A novel retroviral gene
therapy approach to inhibit specific antibody
production and suppress experimental autoim-
mune encephalomyelitis induced by MOG and
MBP. Clin Immunol 111:4752
14. Xu B, Haviernik P, Wolfraim LA, Bunting KD,
Scott DW (2006) Bone marrow transplanta-
tion combined with gene therapy to induce
487
antigen-specific tolerance and ameliorate EAE.
Mol Ther 13:4248
15. Melo ME, Qian J, El-Amine M, Agarwal RK,
Soukhareva N, Kang Y et al (2002) Gene trans-
fer of Ig-fusion proteins into B cells prevents
and treats autoimmune diseases. J Immunol
168:47884795
16. Soukhareva N, Jiang Y, Scott DW (2006)
Treatment of diabetes in NOD mice by gene
transfer of Ig-fusion proteins into B cells: role
of T regulatory cells. Cell Immunol 240:
4146
17. Satpute SR, Soukhareva N, Scott DW, Moudgil
KD (2007) Mycobacterial Hsp65-IgG-
expressing tolerogenic B cells confer protection
against adjuvant-induced arthritis in Lewis rats.
Arthritis Rheum 56:14901496
18. Lei TC, Scott DW (2005) Induction of toler-
ance to factor VIII inhibitors by gene therapy
with immunodominant A2 and C2 domains
presented by B cells as Ig fusion proteins. Blood
105:48654870
 INDEX
A stain cell-surface ........................................................ 265
Active immunization ....................................... 366, 368370
Active induction, EAE
clinical grades ............................................................ 390
disease ................................................................ 385387
materials .................................................................... 384
mouse strain and encephalitogenic peptides .............. 386
Adoptive transfer
EAE
and active ............................................................. 383
clinical disease...................................................... 391
models ................................................................. 389
MOG ........................................................................ 388
myelin-specific
CD4+ T-cell lines ................................................ 364
T cells .................................................................. 382
Th1/Th17 cells .................................................... 382
protocol...................................................................... 371
separation, induction and effector phases .................. 368
Adoptive transfer models..................................173, 176, 389
AMAs. See Antimitochondrial antibodies (AMAs)
7-Amino-4-trifluoromethyl-coumarin (AFC) .................. 85
ANA. See Antinuclear antibody (ANA)
Antibody-secreting cell (ASC) ................................ 117118
Antigen
ASC................................................................... 117118
autoreactive CTLs ..................................................... 350
beta cell ...................................................................... 348
clonal expansion......................................................... 114
D10 and AE7 cells .................................................... 178
DNA methylation...................................................... 171
EAE model ............................................................... 404
exogenous and endogenous factors ................................ 2
Freunds Adjuvant ...................................................... 189
9G4 ........................................................................... 110
Ig DNA rearrangements ............................................ 110
immunization ............................................................ 184
MHC ........................................................................ 329
presentation ....................................................... 329, 330
pulsed dendritic cells.......................................... 460, 463
retrieval solution ........................................................ 302
S-Ag (see Soluble antigen (S-Ag))
specific tolerance induction........................................ 472
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4, Springer Science+Business Media New York 2012
489
stock .......................................................................... 306
targets, gene therapy .................................................. 473
types .............................................................................. 5
Antimitochondrial antibodies (AMAs)
and cytokine detection ............................................... 312
dominant PDC-E2 ................................................... 293
levels .......................................................................... 297
profile......................................................................... 292
serological Ig reactivity .............................................. 306
Antinuclear antibody (ANA)
and anti-dsDNA antibodies .............................. 273274
anti-dsDNA, anti-nucleosome antibody
production ........................................................... 275
FANA test (see Fluorescent antinuclear
antibody (FANA) test)
GN ............................................................................ 273
testing .................................................................... 1213
Apoptosis
detection ............................................................ 420421
exogenous and endogenous factors ................................ 2
flow cytometry ........................................................... 259
mitochondrial
assessment, flow cytometry .................................... 72
ATP synthesis ........................................................ 62
caspase enzyme assays ............................................ 80
intracellular ATP and ADP levels ......................... 75
MHP ..................................................................... 63
monitoring ....................................................... 7172
NO and H2O2.................................................. 6263
signaling abnormalities, T cell death...................... 64
transcription factors ............................................... 64
mouse lupus model
Faslpr (lpr) ..................................................... 138139
targeted genetic mutations ................................... 141
neurons ...................................................................... 428
OL (see Oligodendrocyte (OL))
T cell.......................................................................... 258
Arthritis
mouse lupus models
BDX2 .................................................................. 139
characteristics and uses ........................................ 137
rheumatoid ................................................................ 328
 AUTOIMMUNITY: METHODS AND PROTOCOLS
490 Index
Autoantibody
anti-ssDNA ............................................................... 256
BXSB......................................................................... 139
high-titer, SLE .................................................. 110111
hydrocarbon oil .......................................................... 140
lupus-specific ............................................................. 254
mediated pathogenicity.............................................. 181
MRL and CD95 mutants .......................................... 138
production ......................................................... 271273
profiles ....................................................................... 272
SLE ............................................................................. 91
Autoantigen (IgG)
clinical diagnosis ............................................................ 5
exposures ........................................................... 331, 332
Autoimmune diseases
antigenic targets ......................................................... 473
fibrocytes contributions ..................................... 331, 332
organ-specific................................................................. 4
system ............................................................................ 3
Autoimmunity
B cell .......................................................................... 406
EAU .......................................................................... 445
endocytic recycling role (see Endocytic
recycling, SLE)
mitochondrial dysfunction assessment
(see Mitochondrial dysfunction assessment,
SLE patients)
mouse lupus models (see Mouse lupus models)
pathogenesis and spectrum
antigens.................................................................... 5
causes, diseases ..................................................... 45
description ........................................................... 1, 3
diseases, organ-specific autoimmune ....................... 4
exogenous and endogenous factors ...................... 2, 3
gene expression profiling.......................................... 7
organ systems ........................................................... 3
theory....................................................................... 5
TNF....................................................................56
SLE (see Systemic lupus erythematosus (SLE))
and T cell DNA hypomethylation ..................... 171172
T cell signaling abnormalities (see T cell
signaling abnormalities, human SLE)
type I and II lesions ................................................... 407
B GC............................................................... 114115
Bacillus Calmette-Guerin (BCG) ................................... 140
BBB. See Blood brain barrier (BBB)
B-cell delivered gene therapy, tolerance induction
adoptive transfer ........................................................ 473
antigen-specific .......................................................... 472
autoimmune diseases and antigenic
targets .................................................................. 473
functions ............................................................ 472473
materials ............................................................ 474476
methods
blood collection and ELISA ........................ 483484
BSSK ................................................................... 477
description ................................................... 476477
EAE .................................................................... 485
ELISPOT.................................................... 484485
FACS analysis .............................................. 485486
GP + E-86 fibroblasts, stable
transfection and isolation ....................... 478479
isolation and activation ................................ 480481
MBAE/MSCV ............................................ 477478
proliferation assay ................................................ 482
transduction ......................................... 481482, 483
viral titers ..................................................... 479480
self tolerance and autoimmune
disease .......................................................... 471472
B cell receptor (BCR)
antigen engagement ................................................... 114
CD10 marker ............................................................ 111
IgD/IgM ................................................................... 115
immunoglobulins (Ig) ................................................ 110
transitional stage ..................................................... 111
B cells
APC .................................................................. 255, 257
CD4 T cell................................................................. 254
deficient mice............................................................. 296
depletion .................................................................... 298
epitopes...................................................................... 292
flow cytometric analyses ............................ 236, 242243
GVHD assessment .................................................... 258
hyperactivity
BXSB................................................................... 139
MRL mutants ...................................................... 138
NZB mice .................................................... 136, 138
immunity ........................................................... 293294
investigating mutant .................................................. 256
loss, tolerance ............................................................. 296
role, antigen presentation............................................. 13
SLE
antibody-secreting cells (ASC) .................... 117118
BCR (see B cell receptor (BCR))
CD27+ memory ........................................... 115116
characterization, memory B cells ................. 111113
double-negative (DN) ................................. 116117
high-titer autoantibody ................................ 110111
immunoglobulins (Ig) .......................................... 110
signature ...................................................... 118119
transitional and mature-nave ...................... 111, 114
BCG. See Bacillus Calmette-Guerin (BCG)
BCR. See B cell receptor (BCR)
Beta cell dysfunction. See Oxidative stress
and beta cell dysfunction
Beta cells.................................................................. 348350

Blood brain barrier (BBB)
BALB/c mice............................................................. 184
epinephrine ................................................................ 190
LPS ........................................................................... 190
NPSLE mouse model ................................................ 184
B lymphocyte.................15, 61, 109, 121, 127, 405, 406, 428
Bovine serum albumin (BSA)
alpha glyceramide immunization ....................... 293294
2-OA-BSA, immunized mice (see 2-Octynoic
acid-BSA-immunized mice)
BSA. See Bovine serum albumin (BSA)
C GM-CSF................................................................... 332
Calcium response................................................... 26, 3436
Calf intestinal alkaline phosphatase (CIAP) ........... 477478
Caspases .................................................62, 8182, 350, 359
CD. See Crohns disease (CD)
CD3 .................................................................... 50, 93, 95
Cell activation
and lysis ................................................................. 36, 40
mitochondrial checkpoints, T cell.......................... 6263
pathogenesis, SLE nephritis ...................................... 208
Cellular immunology, SLE T cell signaling molecules
confocal microscopy............................................... 5556
flow cytometry
cell surface staining .......................................... 5152
intracellular staining ........................................ 5253
fluorescence microscopy......................................... 5355
Central nervous system (CNS) lupus
antibodies .................................................................. 183
BALB/c ..................................................................... 183
BBB (see Blood brain barrier (BBB))
cardiac perfusion
description ........................................................... 186
materials .............................................................. 186
methods ....................................................... 192193
description ......................................................... 181182
detecting antibody deposition .................................... 195
detecting preapoptotic neurons .................................. 196
ELISA assays
description ........................................................... 184
methods ....................................................... 189190
testing serum antibody titer ................................. 184
epinephrine injection ................................................. 182
FluoroJade, staining ................................................... 196
free-floating section staining ..................................... 195
immunization
description ........................................................... 183
induction...................................................... 183184
methods ....................................................... 188189
injured/apoptotic neurons and
immunoglobulin deposition ......................... 186187
LPS ........................................................................... 182
membrane-enriched brain fractions
preparation........................................................... 187
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
491
WB analysis ................................................. 197198
NMDAR ........................................................... 186, 192
passive transfer
antibody purification............................................ 191
description ........................................................... 185
materials ...................................................... 185186
monoclonal antibodies production............... 190191
pathogenicity, mouse model ......................... 191192
physical barrier........................................................... 182
staining brain sections ............................................... 194
WB analysis, NMDARs .................................... 187188
Cerebrospinal fluid (CSF)
MS patients
CD8 lymphocytes ................................................ 405
CD19-plasma cells .............................................. 405
CFA. See Complete Freunds adjuvant (CFA)
Chemokine
C-C motif.................................................................. 331
CXCR4, 331, 332
and liver cytokine............................................... 303, 312
receptors ............................................................ 329, 330
ChIP assay. See Chromatin immunoprecipitation
(ChIP) assay
Chromatin immunoprecipitation (ChIP) assay
cross-linking and preparation, cell extract.................... 48
description ................................................................... 47
phosphorylated transcription factors ........................... 49
procedure ............................................................... 4849
salmon sperm DNA-blocked
sepharose protein A/G........................................... 48
CIAP. See Calf intestinal alkaline
phosphatase (CIAP)
ClaI restriction enzyme ................................................... 394
CNS. See Central nervous system (CNS)
CNS lupus. See Central nervous system (CNS) lupus
Collagen
CD45 and procollagen-1 expression.................. 335336
collagen-1 .................................................................328
collagen V .................................................................. 330
extracellular matrix components ................................ 328
I/III/IV, vimentin and tenascin.................................. 330
procollagen-I staining ........................................ 338, 339
Complement
components ................................................................. 12
Low-Tox M rabbit ............................................. 475, 481
proteins and toll-like receptors .................................... 13
serum ......................................................................... 188
targeted genetic mutations ......................................... 141
Complete Freunds adjuvant (CFA)
antigen emulsified ...................................................... 482
EAE
emulsion .............................................................. 369
estimation ............................................................ 368
Mycobacterium tuberculosis .....................................366
 AUTOIMMUNITY: METHODS AND PROTOCOLS
492 Index
Crohns disease (CD)
human patients .......................................................... 434
mouse models .................................................... 435436
occurrence .................................................................. 433
phenotypes................................................................. 435
CSF. See Cerebrospinal fluid (CSF)
Cuprizone
administration ............................................416, 424, 426
effect .......................................................................... 416
Cytokines
and chemokines ......................................................... 299
cocktails ..................................................................... 238
development, autoimmunity .......................................... 6
ELISAs...................................................................... 242
exogenous and endogenous factors ................................ 2
expression .................................................................... 43
gene expression, intracellular ..................................... 258
genes .......................................................................... 142
IFN ............................................................................333
IL-4 and IL-13.......................................................... 332
inflammatory ..................................................... 297, 298
intracellular staining .................................. 258260, 269
pro-inflammatory ...................................................... 406
tested agents .............................................................. 255
TH1 ............................................................................ 329
TH2 ............................................................................ 329
TH-2 ......................................................................... 406
D
Delayed-type hypersensitivity (DTH)............................. 396
Demyelination
corpus callosum ......................................................... 417
cuprizone induced.............................................. 411412
EAE model ............................................................... 404
evaluation, LFB ................................................. 418420
gender differences ...................................................... 416
lesions and neuronal loss.................................... 409410
Luxol fast blue staining.............................................. 415
oligodendrocyte loss .................................................. 408
Dendritic cells (DCs)
antibodies use, flow cytometry ........................... 220221
enrichment, magnetic beads use ........................ 222223
functional studies, renal
arginase assay ............................................... 225226
BM-derived macrophages............................ 224225
cathepsin and metalloproteinase activity.............. 224
i-NOS activity, assay ............................................ 225
renal ................................................................... 210211
subgroups................................................................... 210
Diabetes mellitus (DM)
IDDM ................................................................... 5, 292
organ-specific diseases ................................................... 4
DMEM. See Dulbeccos modified Eagles medium
(DMEM)
DNA methylation
description ......................................................... 169170
importance ................................................................. 170
and T cell function..................................................... 170
Dominant negative TGF- receptor II (dnTGFRII)
serum samples............................................................ 306
TGF- receptor II Mice (see TGF- receptor)
Drug-induced lupus ........................................................ 172
DTH. See Delayed-type hypersensitivity (DTH)
Dulbeccos modified Eagles medium (DMEM)
BHK-21 cells..............................................392, 393, 394
contains...................................................................... 387
GP + E-86 fibroblast cells ......................................... 478
induction
passive EAE ........................................................ 384
TMEV-IDD ............................................... 384385
lymph nodes and spleens ........................................... 459
and RPMI 1640 ........................................................ 464
E
EAE. See Experimental autoimmune
encephalomyelitis (EAE)
Early endosome ........................................................... 9294
EAU. See Experimental autoimmune
uveoretinitis (EAU)
Electron microscopic techniques ............................. 420421
Electron transport chain (ETC)
complexes I-IV, testing ................................................ 83
digitonin purification ............................................. 8384
permeabilizing PBL..................................................... 83
reagents........................................................................ 82
ELISAs. See Enzyme linked immunosorbent
assays (ELISAs)
Emulsion ...........189, 368369, 377, 385386, 450, 456458
Encephalitogenic peptides ................................365, 386, 397
Endocytic recycling, SLE
description ............................................................. 9192
human lymphocytes preparation
PBL separation ...................................................... 99
PBMC separation .................................................. 98
T lymphocytes isolation ................................. 99100
materials ................................................................ 9698
metabolic control
HRES-1/Rab4 expression ............................... 93, 94
mTOR ................................................................... 92
NO production ...................................................... 92
mouse splenocytes separation .................................... 100
proximal TCR signaling control .................................. 95
receptor internalization .............................................. 104
recycling assay
human PBLs and isolated T cells ................ 100101
mouse splenocytes................................................ 102
TCR signal transduction, dysregulation ...................... 93
western blotting ................................................. 102103

End-stage renal disease (ESRD) ..................................... 272
Environmental factors ............................................. 157, 272
Enzyme linked immunosorbent assays (ELISAs) .......... 242,
260261, 483484
Eosinophilic crystalline pneumonia, SHIP-/-mice ......... 438
Epigenetics ...................................................................... 409
Epitope
dominant PDC-E2 ................................................... 293
IRBP ................................................................. 453, 455
Lewis strain ....................................................... 448, 449
mouse strains, IRBP-EAU ........................................ 455
retrieval, tissue ........................................................... 309
Epitope spreading.................................................... 383, 396
ESRD. See End-stage renal disease (ESRD)
E2 subunits of pyruvate dehydrogenase (PDC-E2)
apoptosis .................................................................... 299
dominant ................................................................... 293
PAGE ........................................................................ 301
QSARs ...................................................................... 298
self-tolerance, PBC .................................................... 298
serological Ig reactivity .............................................. 306
ETC. See Electron transport chain (ETC)
Experimental autoimmune encephalomyelitis (EAE)
active and passive induction
clinical disease course................................... 390392
clinical grades ...................................................... 390
disease .......................................................... 385389
materials .............................................................. 384
bloodbrain barrier .................................................... 382
classic pathological features ............................... 363364
clinical scale ....................................................... 377378
CNS, MS patients ..................................................... 382
disease, induce ........................................................... 364
materials
active immunization ............................................ 366
CNS mononuclear cells ....................................... 367
ELISPOT assay ........................................... 367368
lymph node cell culture, passive transfer .............. 367
methods
active immunization .................................... 368370
clinical assessment, mice .............................. 372374
ELISPOT assay ................................................... 376
mononuclear cells, isolation ......................... 374375
passive transfer ............................................. 370372
MS............................................................................. 404
multiple sclerosis (MS) .............................................. 364
paresis/paralysis ......................................................... 485
sections .............................................................. 364, 366
SJL mice .................................................................... 377
susceptible mouse strains ................................... 364, 365
T cells ........................................................................ 382
tolerance induction .................................................... 473
Experimental autoimmune uveoretinitis (EAU)
activity and toxicity, PT ............................................. 465
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
493
clinical uveitis .................................................... 443444
factors ........................................................................ 445
fundoscopy and histopathology ................................. 466
guinea pig .................................................................. 446
induction ................................................................... 444
laboratory mouse strains ............................................ 465
materials ............................................................ 446447
mouse, induction
adoptive transfer, B10.RIII/C57BL/6 ......... 458459
antigen-pulsed dendritic cells, B10.RIII .............. 460
clinical course, disease .................................. 460462
HLA class I and II .............................................. 456
immunization, B10.RIII .............................. 456457
immunization, C57BL/6 ............................. 457458
mouse strains and IRBP epitopes ................ 453454
quantitation ......................................................... 462
pathology ................................................................... 443
pertussis toxin (PT) ........................................... 445446
rat, induction
active immunization, Lewis rat .................... 448451
adoptive transfer, Lewis rat .......................... 451452
clinical courses, disease ................................ 452453
immunizing protocol ........................................... 448
quantitation ......................................................... 453
susceptibility ................................................ 447448
strains and species .............................................. 444445
support protocols
complete Freunds adjuvant.................................. 464
fundoscopic examination ............................. 462463
histopathology, eyes ............................................. 463
RPMI 1640/DMEM .......................................... 464
single-cell suspension preparation, splenocytes .... 464
susceptibility, males and females ................................ 445
treatments, Bordetella pertussis .....................................445
uveitogenic proteins/peptides .................................... 446
uveitogenic retinal proteins ........................................ 444
F
FANA test. See Fluorescent antinuclear antibody
(FANA) test
Fibroblast ................................................................ 318, 478
Fibrocytes identification, scleroderma
circulation .......................................................... 328330
description ................................................................. 328
differentiation and homing
CD14+ monocyte fraction ................................... 329
CXCL12 .............................................................. 330
murine fibrocytes ................................................. 329
SSc-ILD .............................................................. 331
disease associations .................................................... 328
functions
autoimmune pathogenesis ........................... 331, 332
IFN and TNF .................................................... 333
PDGF and TGF-1 ............................................ 332
 AUTOIMMUNITY: METHODS AND PROTOCOLS
494 Index
Fibrocytes identification, scleroderma (cont.)
phenotype ............................................................ 331
-SMA ................................................................ 331
T cell responses .................................................... 333
materials ............................................................ 333334
methods
human peripheral blood mononuclear cell
(see Human peripheral blood)
isolation of blood and lung cells, mouse
(see Mouse blood and lung cells isolation)
SSc ............................................................................. 327
Fibrotic skin disease. See Innate immunity, murine skin
Flow cytometry. See also Multiparameter flow
cytometry and bioanalytics, SLEanalyses,
T and B cell ......................................... 236, 242243
analysis, splenic lymphocyte populations ........... 257258
apoptosis .................................................................... 259
assessment, apoptosis and necrosis............................... 72
CFSE staining ........................................................... 260
equipment and software............................................. 260
fibrocytes (see Fibrocytes identification, scleroderma)
identification, fibrocytes..................................... 336, 337
intracellular staining .......................................... 259260
materials
cell preparation ............................................ 302303
liver and spleen cell populations .......................... 303
methods
cell preparation ............................................ 310311
liver and spleen cell populations .................. 311312
miscellaneous materials.............................................. 259
mitochondrial transmembrane potential (ym) ...... 7273
preparation, blood ...................................................... 341
renal mononuclear phagocytes ................... 219221, 229
SLE T cell signaling molecules
cell surface staining .......................................... 5152
intracellular staining ........................................ 5253
splenocyte
annexin V staining protocol ......................... 265266
Foxp3 and Ki67 ................................................... 265
intracellular staining protocol ...................... 264265
surface staining protocol ...................................... 264
stained samples .......................................................... 342
surface staining antibodies ......................................... 259
Fluorescent antinuclear antibody (FANA)
test ....................................................... 146, 153154
G Glomerulonephritis (GN), mouse lupus models
Gene expression, SLE T cell signaling molecules
ChIP assay (see Chromatin immunoprecipitation
(ChIP) assay)
mRNA stability assay .................................................. 46
nucleofection ............................................................... 47
oligonucleotide pulldown assay.............................. 4951
real-time PCR ............................................................. 46
reverse transcriptase polymerase chain reaction
agarose gel electrophoresis ..................................... 45
estimation, total mRNA amount ........................... 44
mRNA isolation .............................................. 4344
polymerase chain reaction ...................................... 45
reverse transcription ............................................... 44
TA cloning ............................................................. 45
transfection ............................................................ 4647
Genes
Bcl6 ........................................................................... 116
exogenous and endogenous factors ................................ 2
expression analyses, myelin basic protein ........... 426427
genetic contributions, SLE ........................................ 272
immunoglobulins (Ig) V region ................................. 110
interactions, lupus alleles ........................................... 282
methylation sensitive
CD40LG .............................................................. 173
Itgal (CD11a) and Tnfsf7 (CD70) ........................172
precision mapping, SLE .................................... 284285
QTL effect ........................................................ 284285
SLE-gene mapping ................................................... 282
SLE-gene precision ................................................... 272
somatic mutations ...................................................... 114
susceptibility (see Systemic lupus
erythematosus (SLE))
Gene therapy
autoimmune diseases and antigenic targets................ 473
B-cell delivered, tolerance induction.................. 472473
Genetic analysis and SLE trait mapping
extraction, mouse tail genomic dna.................... 277278
generation, NZM2328 backcross mice ...................... 277
PCR genotyping ................................................ 278279
size analysis, PCR-amplified SSLP markers
(see Polymerase chain reaction (PCR))
SLE QTL .................................................................. 281
SSLP and SNP .......................................................... 278
Genetic association
GWAS (see Genome-wide genetic association
studies (GWAS))
uveitis ........................................................................ 456
Genome-wide genetic association studies (GWAS)
description ................................................................... 15
functional alleles .......................................................... 15
genes ............................................................................ 16
SLEGEN .................................................................... 15
Germinal center (GC) ............................................. 114115
BCG injection ........................................................... 140
BDX2 ........................................................................ 139
BXSB......................................................................... 139
CD95 mutants ................................................... 138139
description ................................................................. 136
diagnosis .................................................................... 156
hydrocarbon oil .......................................................... 140

MRL mutants............................................................ 138
NZ group........................................................... 136, 138
Palmerston North (PN) ............................................. 139
SCG/Kj ..................................................................... 139
selection ..................................................................... 143
Glutathione
agarose ....................................................................... 301
GCH-Glo assay ........................................................ 352
HPLC assay........................................................... 7980
islet cells..................................................................... 357
peroxidase .................................................................. 349
preswollen .................................................................. 304
TrisHCl ................................................................... 304
9G4, monoclonal antibody
antibodies .................................................................. 128
biotinylated ................................................................ 120
description ................................................................. 110
memory B cell panel, staining cells .................... 123124
non-self-reactive B cells ..................................... 114115
Goodpasture syndrome.................................................... 3, 5
Graft-vs.-host disease (GVHD)
assessment
ELISA ..........................................260261, 266267
flow cytometry ............................................. 259260
real-time polymerase chain reaction .... 261, 267269
renal histology ..................................................... 266
renal studies ......................................................... 260
splenocyte flow cytometry............................ 264266
splenocyte isolation .............................................. 264
induction
CFSE staining, donor cells .................................. 263
donor cell preparation and transfer ...................... 258
donor T cell purification, negative isolation ......... 262
donor T cell purification, positive isolation .......... 263
intravenous injection ............................................ 259
IV injection .................................................. 263264
negative T cell isolation ............................... 258259
positive T cell isolation ........................................ 259
splenocyte preparation ......................................... 262
Granulomatosis with polyangiitis ........................................ 3
GVHD. See Graft-vs.-host disease (GVHD)
GWAS. See Genome-wide genetic association
studies (GWAS)
H
Hemophilia ............................................................. 472, 473
Histological methods............................................... 407409
Histopathology, MS
cell injury, brain ................................................. 410411
heterogeneity ..................................................... 407409
neuroaxonal degeneration .................................. 409410
HPA. See Hypothalamus pituitary adrenal (HPA)
HPRT. See Hypoxanthineguanine
phosphoribosyltransferase (HPRT)
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
495
Human peripheral blood
lung cell isolation and staining .......................... 336338
separation, mononuclear cells .................................... 335
staining, CD45 and procollagen-1..................... 335336
Hypothalamus pituitary adrenal (HPA) .......................... 445
Hypoxanthineguanine phosphoribosyltransferase
(HPRT) ............................................................... 427
I
IBD. See Inflammatory bowel disease (IBD)
IDDM. See Insulin-dependent diabetes
mellitus (IDDM)
IEC. See Intestinal epithelial cell (IEC)
IFN. See Interferon-gamma (IFN)
IgA. See Immunoglobulin A (IgA)
ILD. See Interstitial lung disease (ILD)
Immunoblot analysis, myelin basic
protein ................................................. 423, 425426
Immunoblotting
MBP ...........................................................423, 425, 426
T cell signaling .................................................29, 38, 39
Immunoglobulin A (IgA) ................................................ 436
Immunohistochemistry, cuprizone model........ 421423, 424
Inflammation
chamber ..................................................................... 453
histopathology, EAU ......................................... 451, 453
MS (see MS, pathogenesis)
Inflammatory bowel disease (IBD)
CD mouse models ............................................. 435436
epithelial barrier functions ......................................... 434
evaluations ................................................................. 436
histological sections ........................................... 438439
IEC............................................................ 433434, 436
immunoregulatory defects ......................................... 434
luminal commensal microbiota .................................. 435
necropsy ............................................................. 436438
phenotypes................................................................. 434
pro-inflammatory cytokines....................................... 434
UC and CD occurrence ............................................. 433
Innate immunity, murine skin
inflammation and fibrosis .......................................... 318
ligand stability ........................................................... 324
materials .................................................................... 319
methods
gene expression, skin ............................................ 323
induction, anesthesia and pump insertion .... 321322
selection and loading, osmotic pumps.................. 320
skin, pump outlet ......................................... 322323
mouse weights and PBS ............................................ 324
poly(I:C), osmotic pumps .................................. 318319
pump insertion and osmotic pumps........................... 325
subcutaneous osmotic pumps and utility,
modeling skin disease .................................. 317318
wound dehiscence and mouse skin ............................ 325
 AUTOIMMUNITY: METHODS AND PROTOCOLS
496 Index
Insulin
organ-specific autoimmune diseases .............................. 4
secretion and content
arginine-stimulated .............................................. 355
DNA quantification assay .................................... 356
ELISA ......................................................... 355356
glucose-stimulated ............................................... 355
islet cells............................................................... 355
KCl-stimulated .................................................... 355
syringe VWR ............................................................. 259
Insulin-dependent diabetes mellitus (IDDM).............4, 5, 7,
292, 295
Interferon
cellular signaling/regulation ....................................... 142
DNA methylation...................................................... 170
IFN- ..................................................................404405
signaling pathway ........................................................ 15
Interferon-gamma (IFN) ............................................... 333
Interferon regulatory factor (IRF)
association, IRF5 ......................................................... 15
cellular signaling/regulation ....................................... 142
Interphotoreceptor retinoid-binding protein (IRBP)
bovine and human ............................................. 448, 449
and EAU ................................................................... 455
epitopes.............................................................. 453454
148 kDa protein......................................................... 444
Lewis rat ............................................................ 448, 449
peptide ....................................................... 456, 457458
residues ...................................................................... 456
Interstitial lung disease (ILD) ......................... 327328, 331
Intestinal epithelial cell (IEC) ......................... 433435, 436
IRBP. See Interphotoreceptor retinoid-binding
protein (IRBP)
IRF. See Interferon regulatory factor (IRF)
L cuprizone-induced demyelination ............................. 415
Large Lupus Association Study 2 (LLAS2)
approaches ................................................................... 18
collaborators and role............................................. 1618
description ................................................................... 16
genotyping ................................................................... 18
papers published .......................................................... 19
project .................................................................... 1819
Lewis rat, EAU induction
active immunization .......................................... 448451
adoptive transfer ................................................ 451452
clinical appearance ..................................................... 450
pathogenic ................................................................. 449
LFB staining. See Luxol Fast Blue (LFB) staining
Linkage disequilibrium ................................................ 15, 20
Lipopolysaccharide (LPS) ....................................... 182, 190
LLAS2. See Large Lupus Association Study 2 (LLAS2)
LPS. See Lipopolysaccharide (LPS)
Lupus
ANA/anti-dsDNA, anti-nucleosome antibody
production and GN ............................................. 275
ANA staining ............................................................ 237
Cgnz1 and Agnz1...................................................... 275
C57L/J, F1 progeny ................................................... 287
CNS (see Central nervous system (CNS) lupus)
cytokines ........................................................................ 6
directional dominance................................................ 287
exogenous and endogenous factors ................................ 2
flow cytometry and bioanalytics
(see Multiparameter flow cytometry
and bioanalytics, SLE)
genetic contributions, human SLE ............................ 272
genome-wide screening ............................................. 288
immunofluorescence staining..................................... 237
materials .................................................................... 276
methods
generation, Non-SLE NZM2328 (see Non-SLE
NZM2328 generation)
genetic analysis and SLE trait mapping, NZM2328
(see Genetic analysis)
murine (see Murine)
precision mapping, chromosome 1............... 284285
mouse models (see Mouse lupus models)
mouse models, nephritis .................................... 272273
murine lupus models (see T cell DNA hypomethylation,
murine lupus models)
nephritis loci, NZM2328 .......................................... 274
NZM2328 females .................................................... 287
NZM2328, SLE ................................................ 273274
serologic assessment........................................... 245246
serum levels, anti-dsDNA antibody................... 237, 245
SLE ....................................................271272, 472, 473
Luxol Fast Blue (LFB) staining
evaluation, demyelination .................................. 418420
gender differences, mouse .......................................... 416
Lymphokine .......................................................64, 350, 451
M
Macrophages (M)
antibodies use, flow cytometry ........................... 220221
enrichment, magnetic beads use ........................ 222223
and fibroblasts ............................................................ 329
functional studies, and renal DCs (see Dendritic
cells (DCs))
granulocytemacrophage ........................................... 332
inflammatory ............................................................. 209
inflammatory protein 1 .............................................331
regulatory................................................................... 210
renal ................................................................... 210211
RNS........................................................................... 348

T cells ........................................................................ 258
xanthine oxidase system ............................................. 348
Major histocompatibility complex (MHC)
DNA methylation...................................................... 170
non MHC control ..................................... 444445, 448
Mammalian target of rapamycin (mTOR)
endocytic pathway, SLE T cells ................................... 92
MHP ........................................................................... 63
Matrix metalloprotease (MMP) ..............................224, 229,
233, 332, 405
Mature-Nave B cell ................................................ 111, 114
MBP. See Myelin basic protein (MBP)
Memory B cell
CD27+ ............................................................... 115116
gating strategy ........................................................... 124
9G4, staining ..................................................... 123124
MHC. See Major histocompatibility complex (MHC)
MHP. See Mitochondrial hyperpolarization (MHP)
Mitochondria
ABCB1 transporter ................................................... 114
AMA ......................................................................... 292
ATP production......................................................... 349
autoantigens............................................................... 292
autoantigens, immunoreactivity determination
ELISA ......................................................... 306307
recombinant PDC-E2 ................................. 304305
western blot ......................................................... 305
dysfunction ................................................................ 348
MHP ........................................................................... 92
PDC-E2 .................................................................... 298
ROS production ........................................................ 350
Mitochondrial dysfunction assessment, SLE patients
AFC calibration curve ................................................. 85
ATP assay
and ADP, assessment ............................................. 79
cell collection ......................................................... 75
precautions............................................................. 77
protocol............................................................ 7778
reagents .................................................................. 77
caspase enzyme assays............................................ 8082
description ............................................................. 6162
ETC activity (see Electron transport chain (ETC))
flow cytometric analysis, ym ................................. 7273
fluorochromes .............................................................. 84
HPLC assay, glutathione levels.............................. 7980
intracellular pH measurements .............................. 7475
Lowry assay ................................................................. 76
luciferase reaction ........................................................ 75
lymphocyte
CD3/CD28 co-stimulation, PBL .......................... 70
description ............................................................. 70
Fas-mediated cell death, PBL................................ 71
H2O2 treatment ..................................................... 71
monitoring, cell death ...................................... 7172
monocytes and PBL separation ............................. 70
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
497
PBMC separation ............................................ 6970
materials
caspase substrate peptides ...................................... 68
cytokines ................................................................ 66
flow cytometer ....................................................... 67
luminometer .......................................................... 67
monoclonal antibody ............................................. 69
pharmacological targeting
GSH/GSSG ratios ................................................ 66
NO production ...................................................... 66
oxidative stress, transcription factors................ 64, 66
propidium iodide (PI)............................................ 8485
ROI production, measurement .............................. 7374
T cell activation and apoptosis (see T cells)
Mitochondrial hyperpolarization (MHP)
elevation, ym............................................................... 63
mTOR, activation........................................................ 63
pharmacological targeting...................................... 64, 66
ROI production ........................................................... 62
Mitochondrial transmembrane potential
elevation................................................................. 6263
flow cytometric analysis ......................................... 7273
hyperpolarization ......................................................... 63
MHP ........................................................................... 63
mTOR activity ............................................................ 92
ROI production ........................................................... 62
signaling abnormalities, T cell death............................ 64
Mitochondrion ...................................................62, 410, 422
MMP. See Matrix metalloprotease (MMP)
MOG. See Myelin oligodendrocyte glycoprotein (MOG)
Mouse blood and lung cells isolation
analysis, flow cytometry data (see Flow cytometry)
bronchoalveolar lavage and perfusion ................ 338340
flow cytometry
stained samples .................................................... 342
preparation of blood, flow cytometry ......................... 341
removal and enzymatic digestion, lungs..................... 340
sacrifice ...................................................................... 338
staining, murine ................................................. 341342
Mouse lupus models
blood collection, alternatives .............................. 158159
description ......................................................... 135136
diagnosis ............................................................ 156157
ELISA systems .................................................. 160161
experimentally induced models
BCG injection ..................................................... 140
chronic GVHD ................................................... 140
genetically targeted animals ......................... 140143
hydrocarbon oil .................................................... 140
16/6 idiotype........................................................ 140
IgG2a reagents .......................................................... 161
implementation
chronic GVHD ........................................... 148149
hydrocarbon oil ............................................ 147148
materials .............................................................. 144
 AUTOIMMUNITY: METHODS AND PROTOCOLS
498 Index
Mouse lupus models (cont.)
inbred and spontaneous
BDX2 .................................................................. 139
BXSB........................................................... 138139
Flaky skin (fsn) .................................................... 140
MRL and CD95 mutants ............................ 138139
New Zealand (NZ) group............................ 136, 138
NZW x BSXB F1................................................ 139
NZW x SB/Le F1 ............................................... 139
Palmerston North (PN) ....................................... 139
SCG/Kj ............................................................... 139
necropsy
materials .............................................................. 145
tissue harvest................................................ 149152
tissue processing................................................... 152
renal disease assessment
immunofluorescence .................................... 147, 156
light microscopy................................... 147, 155156
surrogate assays .................................................... 156
retroorbital (RO) approach ................................ 157158
selection
end-organ disease manifestations ........................ 143
non-lupus-like traits ............................................ 143
reagents/knowledge ..................................... 143144
timeframe ............................................................ 143
serum analysis
anti-dsDNA, Crithidia ................................ 146, 154
FANA test ........................................... 146, 153154
immunoglobulin isotype ...................... 145146, 153
total rheumatoid factor .................146147, 154155
serum collection method.................................... 159160
types and characteristics .................................... 135136
Mouse models, IBD
CD mouse models ............................................. 435436
epithelial barrier functions ......................................... 434
etiologic interplay ...................................................... 434
evaluations ................................................................. 436
pathogenic mechanisms ............................................. 434
Mouse strain ............................ 136, 139140, 198, 365, 368,
370, 383, 386, 389, 395, 397, 398, 453, 455, 456, 465
MS. See Multiple sclerosis (MS)
MSCV. See Murine stem cell virus (MSCV)
MS, pathogenesis
apoptotic loss, oligodendrocytes ................................ 428
inflammation, demyelination and neuro degeneration
histopathology ............................................. 406411
immune-mediated mechanisms ................... 403406
materials ............................................................ 413415
methods
cuprizone administration ..................................... 416
detection, apoptosis...................................... 420421
evaluation, demyelination ............................ 418420
gene expression analyses, myelin basic
protein ................................................... 426427
histopathological features,
cuprizone model .................................... 421423
in vivo monitor, cuprizone effect ................. 416417
immunoblot analysis, myelin
basic protein ........................................... 423426
mice ............................................................. 415416
tissue harvesting........................................... 417418
oligodendrocyte apoptosis
and demyelination ....................................... 411412
tissue loss ........................................................... 427428
mTOR. See Mammalian target of
rapamycin (mTOR)
Multiparameter flow cytometry and bioanalytics, SLE
B cells (see B cells, SLE)
cryopreservation......................................................... 127
description ......................................................... 109110
fluorescence-minus-one (FMO) controls .......... 127128
materials ............................................................ 119121
phenotypic B cell profiling......................................... 121
primary analysis ......................................................... 124
principal-component analysis (PCA) ................ 129130
profiling-based visualization ...................................... 129
quality and consistency ...................................... 128129
sample preparation
freezing and thawing ................................... 122123
PBMC isolation................................................... 122
stain blood-cell samples ............................... 123124
stain compensation controls ................................. 123
secondary analysis
cell-number calculations ...................................... 125
clinical blood count (CBC).......................... 124125
clustering and principal-component ............ 125126
global profiling approach ..................................... 125
Multiple sclerosis (MS)
demyelinating diseases ....................................... 381382
disease course and immune reactivity ........................ 397
EAE
active and passive ......................................... 382383
bloodbrain barrier .............................................. 382
CNS, patients ...................................................... 382
T cells .................................................................. 382
efficient disease induction .......................................... 397
laboratory protocols ................................................... 397
materials
active and passive EAE, induction....................... 384
TMEV-IDD, induction............................... 384385
methods
active and passive EAE, induction............... 385392
TMEV-IDD, induction............................... 392396
mouse strain............................................................... 398
resistant and susceptible strains ................................. 397
T cell responses.......................................................... 397
TMEV-IDD
definition ............................................................. 383

demyelinating disease .......................................... 383
induction.............................................................. 383
subgroups ............................................................. 383
Murine
glomerulonephritis
acute GN ............................................................. 285
chronic GN.................................................. 285, 286
SLE ........................................................................... 275
Murine autoimmune model, RA and SLE
arthritis assessment
materials .............................................................. 236
score ..................................................................... 244
serum levels.................................................. 243244
assessing histologic severity ............................... 246247
ChIP assays ............................................................... 248
chromatin immunoprecipitation assays
materials .............................................................. 236
methods ....................................................... 239240
Def6........................................................................... 234
DO11.10 mice ................................................... 234235
flow cytometric analyses T and B cell
materials .............................................................. 236
methods ....................................................... 242243
IL-17 and IL-21................................................ 233234
immune complex-mediated glomeru
lonephritis .................................................... 246247
immunofluorescence Staining .................................... 246
IRF4 .......................................................................... 234
lupus assessment
ANA staining ...................................................... 237
immunofluorescence staining,
immunecomplex deposition ........................... 237
serum levels, anti-dsDNA antibody ............. 237, 245
PBS ........................................................................... 249
retroviral infection, CD4+ T cells
DNA, transfection ............................................... 241
formation, calcium phosphate
and DNA precipitate ..................................... 241
isolation and activation ........................................ 241
materials .............................................................. 236
mouse Nave ................................................ 241242
restimulation ........................................................ 242
resting .................................................................. 242
293T cells, transfection ................................ 240, 241
RORt ....................................................................... 235
serologic assessment........................................... 245246
TGF- and IL-6 ....................................................... 234
T-helper cell 17 differentiation
detection, IL-17 ........................................... 238239
FACS sorting ....................................................... 238
isolation and purification ..................................... 237
materials ...................................................... 235236
mouse Nave CD4+ T cells ................................... 238
Murine genetic models. See Inflammatory
bowel disease (IBD)
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
499
Murine models, SLE nephritis
chronic models................................................... 211213
description ................................................................. 211
Murine stem cell virus (MSCV) ............................. 477478
Myelin basic protein (MBP)
EAE
active .................................................................... 386
adoptive transfer models ...................................... 389
induce, susceptible mouse strains ......................... 365
gene expression analyses .................................... 426427
immunoblot analysis .................................. 423, 425426
Myelin oligodendrocyte glycoprotein (MOG)
active EAE ................................................................ 386
EAE
active immunization models ................................ 370
adoptive transfer models ...................................... 389
induce, susceptible mouse strains ......................... 365
Myelin, peptides .............................................................. 394
N
Natural killer T cells (NKT)
CD1d restricted ......................................................... 299
cell activator ............................................................... 299
N. aromaticivorans .......................................................300
proinflammatory phenotype ...................................... 297
Th1 and Th2 cytokines and chemokines ................... 299
Necrosis
assessment, flow cytometry .......................................... 72
DNA fragmentation assay ..................................... 7172
endogenous NO production ...................................... 349
insulin secretion ......................................................... 348
ROI production ........................................................... 62
T cell death, signaling abnormalities ........................... 64
TNF .......................................................................... 333
TNF..........................................................................56
TNF- ................................................................404405
Nephritis. See also Lupusmouse lupus models
characteristics and uses ........................................ 137
glomerulonephritis (see Glomerulonephritis (GN))
renal mononuclear phagocytes (see Renal
mononuclear phagocytes, SLE)
Neuroaxonal degeneration, MS ............................... 409410
Neurodegeneration .................................................. 403, 404
Next-generation sequencing, SLE ............................... 1920
Nitric oxide (NO)
beta cells .................................................................... 350
iNOS ......................................................................... 350
production, SLE T cells ............................................... 92
and reactive oxygen species production...................... 358
NKT. See Natural killer T cells (NKT)
NMDAR. See N-methyl-D-aspartate receptor (NMDAR)
N-methyl-D-aspartate receptor (NMDAR)
blocking, mediated pathogenicity in vivo ...........186, 192
WB analysis ........................................187188, 197198
NO. See Nitric oxide (NO)
 AUTOIMMUNITY: METHODS AND PROTOCOLS
500 Index
NOD. See Non-obese diabetic (NOD)
Non-obese diabetic (NOD)
BCG injection ........................................................... 140
NOD.ABD Mouse............................................ 292295
NOD.1101 and NOD.B6 Idd10 Idd18r2 mice......... 298
NOD.c3c4 mouse mode ............................................ 296
Non-SLE NZM2328 generation
selection, congenic strains .......................................... 282
speed congenic protocol
Lc1 congenic strain ...................................... 283284
MASP ......................................................... 282283
Nonsteroidal anti-inflammatory agent (NSAID) ............ 465
NSAID. See Nonsteroidal anti-inflammatory
agent (NSAID)
NZM2328
female mice ........................................................ 285, 286
genetic analysis and SLE trait mapping
(see Genetic analysis)lupus nephritis loci ............. 274
lupus-prone strain ...................................................... 273
males/females, C57L ................................................. 287
non-SLE (see Non-SLE NZM2328 generation)
SLE model ........................................................ 273274
O P
2-Octynoic acid-BSA-immunized mice
MHC restriction ....................................................... 299
mitochondrial autoantigen......................................... 298
N. aromaticivorans ...............................................299300
NKT .................................................................. 298299
PDC-E2 .................................................................... 300
xenobiotic .......................................................... 298299
OL. See Oligodendrocyte (OL)
Oligodendrocyte (OL)
apoptosis
cuprizone induced........................................ 411412
electron microscopic image .................................. 422
neuropathological features ................................... 420
pathological features ............................................ 415
direct cytotoxic effects ............................................... 404
early loss .................................................................... 408
mechanisms, loss................................................ 408409
and myelin damage .................................................... 409
type III and IV lesions ............................................... 408
Osmotic pump..................................................... 6, 317325
Oxidative stress
and beta cell dysfunction (see Oxidative stress
and beta cell dysfunction)
inflammation-induced ................................. 408409
pharmacological targeting, mitochondrial ................... 64
Oxidative stress and beta cell dysfunction
CTLs and MHC ....................................................... 350
cytokines .................................................................... 350
cytosolic Fe2+ .............................................................. 349
death .................................................................. 348, 349
FPIR.......................................................................... 348
hypotheses ................................................................. 351
islet-inflitrating CD4+ and CD8+ T cells ................... 350
materials ............................................................ 350353
methods
cytomix and inhibitors treatment......................... 354
glutathione quantification, islet cells .................... 357
isolation and culture, mouse pancreatic islets ....... 353
measurement, intracellular ATP
and ADP ..................................................... 356357
nitric oxide and reactive oxygen species
production (see Nitric oxide (NO))
stimulated insulin secretion measurement
(see Insulin)
treatment, alloxan and inhibitors ................. 354355
treatment, peroxide and inhibitors ....................... 354
viability and caspase enzyme
activity assays ......................................... 358360
p47 phox subunit .......................................................... 350
ROS and STZ ........................................................... 348
TID ........................................................................... 347
2-Oxo acid dehydrogenase (BCOADC-E2) ................... 306
2-Oxo-glutarate dehydrogenase (OGDC-E2) ................ 306
Parent-into-F1 murine model
analysis, biological agents .......................................... 255
B6 F1 mice ........................................................... 254
DBA F1 mice ....................................................... 254
description ......................................................... 253254
GVHD (see Graft-vs.-host disease (GVHD))
intracellular staining, cytokine production ................. 269
investigating mutant T cell ................................ 256257
mutations, host cells................................................... 257
splenocyte isolation.................................................... 262
tail vein injections ...................................................... 269
testing, T cell mutations .................................... 255256
tissue harvesting ........................................................ 261
Pathogenesis and spectrum
description ................................................................. 1, 3
exogenous and endogenous factors ............................ 2, 3
PBC. See Primary biliary cirrhosis (PBC)
PBLs. See Peripheral blood lymphocytes (PBLs)
PBMC. See Peripheral blood mononuclear cells (PBMC)
PBS. See Phosphate-buffered saline (PBS)
PCA. See Principal component analysis (PCA)
PCR. See Polymerase chain reaction (PCR)
Peripheral blood lymphocytes (PBLs)
human lymphocytes preparation.................................. 99
recycling assay .................................................... 100101
Peripheral blood mononuclear cells (PBMC)............ 99, 100
Phosphate-buffered saline (PBS) .................................... 249
PLP. See Proteo lipid protein (PLP)
Polymerase chain reaction (PCR)
buffer ......................................................................... 276
genotyping mouse tail DNA, SSLP .................. 278279

marker ....................................................................... 278
size analysis, amplified SSLP
capillary polymer-based ............................... 279280
metaphor agarose gel electrophoresis ........... 280281
Primary biliary cirrhosis (PBC)
AMAs........................................................................ 292
eppendorf................................................................... 320
materials
ELISA ................................................................. 301
flow cytometry analysis ................................ 302303
flow cytometry, liver and spleen ........................... 303
frozen tissue preparation ...................................... 301
H&E staining ...................................................... 302
immunohisto chemical staining, CD4
and CD8, 302
liver cytokine and chemokine analysis ................. 303
paraffin tissue preparation.................................... 301
recombinant PDC-E2, PAGE ............................ 301
recombinant proteins preparation ................ 300301
serum cytokine analysis........................................ 303
western blot ......................................................... 301
methods
flow cytometry analysis ................................ 310311
flow cytometry, liver and spleen ................... 311312
frozen tissue preparation .............................. 307308
H&E staining ...................................................... 308
immunohistochemical staining, CD4
and CD8 ................................................ 308310
immunoreactivity, mitochondrial autoantigens
(see Mitochondria)
liver cytokine and chemokine analysis ................. 312
paraffin tissue preparation.................................... 307
recombinant proteins preparation ................ 303304
serum cytokine analysis........................................ 312
mice treated ............................................................... 324
mouse models
dominant negative TGF- receptor II mice
(see TGF- receptor)
IL-2R-/-Mice ............................................ 297298
NOD.ABD.................................................. 292, 295
Novosphingobium aromaticivorans ................................292
2-OA-BSA-immunized mice (see 2-Octynoic
acid-BSA-immunized mice)
organ-specific autoimmune diseases .............................. 4
osmotic pumps........................................................... 323
sterile solution ........................................................... 320
Principal component analysis (PCA)
dimension-reduction technique ......................... 129130
generic workflow................................................ 125126
Proteo lipid protein (PLP)
active EAE ................................................................ 386
EAE
active immunization models ................................ 370
induce, susceptible mouse strains ......................... 365
EAE adoptive transfer models................................... 389
myelin epitope ........................................................... 396
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
501
Q
QTL. See Quantitative trait loci (QTL)
Quantitative trait loci (QTL)
acute and chronic GN ............................................... 274
allele effects ............................................................... 282
autoimmune phenotype ............................................. 273
genetic mapping, SLE ............................................... 281
mapping software ...................................................... 276
validity ....................................................................... 281
X-linked .................................................................... 287
R
Reactive oxygen intermediates (ROI) ....................6264, 66,
6971, 7374, 84, 92, 94
Real-time PCR........................................................ 426427
Receptor recycling ..................................................... 94, 104
Relapsing-remitting................................................. 389, 407
Renal mononuclear phagocytes, SLE
arginase and iNOS assays .......................................... 229
cDNA synthesis ......................................................... 229
cell
purification .......................................................... 229
sorting .................................................................. 221
cellular turn over studies .................................... 223224
cytospin ..................................................................... 229
damage assessment
albumin estimation, urine .................................... 218
blood urea nitrogen (BUN) ................................. 218
description ........................................................... 217
DCs (see Dendritic cells (DCs))
flow cytometry ................................................... 219221
functional studies ............................................... 224226
gene expression studies
cDNA synthesis ................................................... 227
real-time PCR ..................................................... 227
RNA isolation, Trizol method ............................. 226
harvest ............................................................... 218219
immunohistologic studies .................................. 227228
macrophages (see Macrophages (M))
magnetic beads .................................................. 222223
materials
buffers .................................................................. 216
chemical reagents ................................................. 216
equipment ............................................................ 215
kits.. ............................................................. 215216
plasticware ................................................... 216217
morphological studies ................................................ 223
murine models, nephritis (see Murine models,
SLE nephritis)
nephritis............................................................. 207208
pathogenesis, nephritis
autoantibody deposition....................................... 208
mechanisms, renal damage........................... 208, 209
remission induction ............................................. 208
protein quantification and analysis ............................ 228
 AUTOIMMUNITY: METHODS AND PROTOCOLS
502 Index
Renal mononuclear phagocytes, SLE (cont.)
proteinuria, measurement .......................................... 228
remission induction
F4/80hi cells.................................................. 214215
flow cytometric analysis ....................................... 214
microarray analysis ............................................... 215
real-time PCR ..................................................... 213
triple therapy treatment ....................................... 213
Retinal pigment epithelium (RPE) ................................. 444
ROI. See Reactive oxygen intermediates (ROI)
RPE. See Retinal pigment epithelium (RPE)
S dysfunction assessment, SLE patients)
Scleroderma. See also Fibrocytes identification,
scleroderma
mouse lupus models ................................................... 137
organ system .................................................................. 3
Self antigens .......................................................1, 5, 65, 143
Signal transduction
TCR ............................................................................ 93
TGF- ........................................................................296
Single nucleotide polymorphism (SNP)
GWAS................................................................... 1516
LLAS2................................................................... 16, 18
and SSLP................................................................... 278
SLE. See Systemic lupus erythematosus (SLE)
Small intestine
histopathological appearance ..................................... 439
section preparation..................................................... 437
separation, cecum....................................................... 436
Swiss roll, ................................................................ 437
SNP. See Single nucleotide polymorphism (SNP)
Soluble antigen (S-Ag)
EAU induction .................................................. 453, 455
48 kDa intracellular photoreceptor protein................ 444
Lewis rat ............................................................ 448, 449
rat strains, EAU ......................................................... 448
Src homology 2 (SH2)-containing inositol-
5-phosphatase (SHIP)
eosinophilic crystalline pneumonia ............................ 438
histopathological appearance, small intestine ............ 439
mouse models, IBD ........................................... 435436
regional thickness, ileum............................................ 437
SSc. See Systemic sclerosis (SSc)
Swiss roll....................................................................... 437
Systemic lupus erythematosus (SLE). See also Murine
autoimmune model, RA and SLE
autoimmune disease ................................................... 271
endocytic recycling (see Endocytic recycling, SLE)
flow cytometry and bioanalytics (see Multiparameter
flow cytometry and bioanalytics, SLE)
gene precision mapping ............................................. 275
mapping susceptibility gene
ANA testing .................................................... 1213
causes ..................................................................... 12
examination ........................................................... 13
GWAS............................................................. 1516
LFRR .............................................................. 1314
LLAS2............................................................. 1619
next-generation sequencing ............................. 1920
pathogenesis .......................................................... 13
prevalence and risk................................................. 12
single-gene human diseases ................................... 11
mitochondrial dysfunction assessment (see Mitochondrial
murine models, renal mononuclear phagocytes
(see Renal mononuclear phagocytes, SLE)
NZM2328 ......................................................... 273274
pathogenesis .............................................................. 272
precision mapping, chromosome 1 .................... 284285
Sle1, Sle2, and Sle3 ..................................................... 273
T cell signaling abnormalities (see T cell
signaling abnormalities, human SLE)
trait analysis ............................................................... 274
trait mapping, NZM2328 (see Genetic analysis)
Systemic Lupus Erythematosus
Genetics (SLEGEN) ....................................... 15, 16
Systemic sclerosis (SSc)
disease progression..................................................... 328
vs. FSC ...................................................................... 337
SSc-ILD ............................................................ 327328
T
T cell blasts ...................................................................... 388
T cell DNA hypomethylation,
murine lupus models
adoptive transfer models ............................................ 176
AE7 cells ................................................................... 178
and autoimmunity
5-azaC induced autoreactivity ............................. 171
dominant negative MEK (dnMEK) ............ 171173
inhibitors ............................................................. 171
autoreactivity assays
cytotoxicity .......................................................... 176
proliferation ......................................................... 176
5-azacytidine ............................................................. 177
cells and 5-azacytidine treatment
AE7 ..................................................................... 175
D10.G4.1 (D10) .................................................. 175
polyclonal CD4+.................................................. 175
variations ............................................................. 176
D10 cells .................................................................... 178
DNA methylation (see DNA methylation)
IgG, IgM, and anti-DNA antibody assays ........ 176177

materials
5-azacytidine ....................................................... 174
cell lines ............................................................... 174
IL-2 ..................................................................... 174
media ................................................................... 174
mice ..................................................................... 174
polyclonal cell lines .................................................... 177
relevance, human lupus .............................................. 173
T cells
activation and apoptosis, mitochondrial
ATP synthesis ........................................................ 62
MHP ..................................................................... 63
mitochondrial redox and metabolic
checkpoints ................................................ 63, 65
ROI production ............................................... 6263
signaling abnormalities, T cell death................ 63, 64
anti-T cell cocktail ............................................. 480, 481
bile duct destruction .................................................. 298
CD4+................................................................. 296, 435
CD8+................................................................. 296, 405
exogenous and endogenous factors ................................ 2
investigating mutant .......................................... 256257
mouse lupus models
MRL and CD95 mutants .................................... 138
NZ group............................................................. 138
targeted genetic mutations ........................... 141142
mutations ........................................................... 255256
myelin antigen-specific .............................................. 404
negative.............................................................. 258259
NKT (see Natural killer T cells (NKT))
pathogenesis, ABD .................................................... 295
positive....................................................................... 259
purification
negative isolation ................................................. 262
positive isolation .................................................. 263
SLE (see Endocytic recycling, SLE)
T cell signaling abnormalities, human SLE
antibiotics .................................................................... 28
antibodies
activating ............................................................... 28
cross-linking .......................................................... 28
fluorescent-labeled ................................................. 29
isotype controls ...................................................... 28
staining .................................................................. 28
applications .................................................................. 26
cellular immunology, signaling molecules
confocal microscopy ......................................... 5556
flow cytometry ................................................. 5153
fluorescence microscopy ................................... 5355
density gradient centrifugation and protein
concentration ......................................................... 30
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
503
gene expression (see Gene expression,
SLE T cell signaling molecules)
general materials and supplies................................ 2728
instruments ............................................................ 3031
reagents.................................................................. 2930
RT-PCR, gene expression analysis .............................. 30
study design ........................................................... 2627
TCR
CD3-mediated signaling analysis
(see TCR/CD3-mediated signaling analysis,
SLE T cells)
signaling........................................................... 2526
T lymphocytes
cell culture........................................................ 3334
magnetic separation, MicroBeads .................... 3233
PBMC separation ............................................ 3132
RosetteSep ............................................................. 33
TCR/CD3-mediated signaling analysis, SLE T cells
cell proliferation assay ............................................ 4243
cytokine expression ...................................................... 43
immunoprecipitation ............................................. 3940
intracellular calcium response, measurement ......... 3436
kinetics, tyrosine phosphorylation ............................... 39
lipid raft-associated TCRz-chain .......................... 4041
lipid raft isolation .................................................. 4142
tyrosine phosphorylation, cellular protein
substrates ......................................................... 3639
western blotting ........................................................... 39
TGF- receptor II mice
AMAs........................................................................ 296
CD8/CD4 T cells ...................................................... 296
CD1d expression ....................................................... 297
dnTGFRII mice ...................................................... 296
IL-12p40 deletion ..................................................... 297
NOD.c3c4 mouse model ........................................... 296
treatment, young mice ............................................... 297
Theilers murine encephalomyelitis virus-induced
demyelinating disease (TMEV-IDD)
definition ................................................................... 383
demyelinating disease ................................................ 383
induction
clinical assessment ............................................... 395
immunological aspects ......................................... 396
infecting stock...................................................... 392
materials .............................................................. 384
molecular mimics, myelin peptides ...................... 394
plaque assay ................................................. 393394
purification .................................................. 392393
SJL/J mice ........................................................... 395
subgroups................................................................... 383
T-helper cell 1 (Th1) ........................................445, 458, 462
 AUTOIMMUNITY: METHODS AND PROTOCOLS
504 Index
T-helper cell 2 (Th2) ....................................................... 445
Tissue, harvesting .................................................... 417418
TNF. See Tumor necrosis factor (TNF)
TNFDARE mouse model ............................................... 436
Tolerance
autoimmune cholangitis ............................................ 296
PBC ........................................................................... 298
PDC-E2 ............................................................ 294, 299
T cell.......................................................................... 297
Transcription factors
CD3 deficiency, SLE T cells ...................................... 93
E2F transcription factor 2 (E2F2) ............................. 141
Elf-1 ...................................................................... 47, 48
exogenous and endogenous factors ................................ 2
JunB........................................................................... 142
oxidative stress ............................................................. 64
phosphorylated ............................................................ 49
Tumor necrosis factor (TNF)
cytokines .................................................................... 333
fibrocyte differentiation ............................................. 329
genetic association ....................................................... 19
liver cytokine and chemokine analysis ....................... 303
mAb against .............................................................. 260
MHP ........................................................................... 63
ROI production ........................................................... 62
serum cytokine analysis ............................................. 303
soluble factors ............................................................ 329
splenocyte flow cytometry ................................. 264265
TNF antagonists ..................................................... 56
TNF- ................................................................404405
Tyrosine phosphorylation, cellular protein substrates
cell activation and lysis ................................................ 36
detection ................................................................ 3839
immunoblotting ........................................................... 38
protein assay ................................................................ 36
protein separation ........................................................ 37
transfer................................................................... 3738
U
UC. See Ulcerative colitis (UC)
Ulcerative colitis (UC)
occurrence .................................................................. 433
phenotypes................................................................. 435
Uveitis
EAU (see Experimental autoimmune uveoretinitis
(EAU))
organ-specific autoimmune diseases .............................. 4
rodent models (see Experimental autoimmune
encephalomyelitis (EAE))
V
Viability and caspase enzyme activity
annexin V and PI staining, islet cells ......................... 359
BCA protein assay ..................................................... 359
caspase activity assay .................................................. 360
caspase-8 and-3 fluorometric assays .......................... 359
MTT assay ................................................................ 358
Viral proteins
VP2.................................................................... 383, 396
VP3............................................................................ 383
Virus.... .......... 2, 117, 241, 242, 381398, 409, 474, 479, 480
W
WB analysis, NMDARs .......................................... 187188
X
Xanthine oxidase system.................................................. 348