local lymphoid tissues and formaldehyde

C. Frieke Kuper

Introduction - Why local lymphoid tissues?

Epidemiology Association reported between formaldehyde (FA) exposure and haemopoietic tumors, particularly (acute) myeloid leukemia Myeloid leukemia = tumor of haemopoietic precursor cells by oncogenic mutations that impede differentiation. Heterogeneous: thrombocytic, erythrocytic and granulocytic precursors accumulate in bone marrow FA is highly reactive: - Local effects at porte dentree to be expected - Inhalation exposure in animals: nasal tissues - lesions nasal epithelium and nasopharyngeal cancer with strong exposure-response correlation - Effects on local lymphoid tissues?

Induction site of immune response

Ag Soluble antigen Particulate antigen Ag

Effector site of immune response

Mucosal response Secretory Ig

Immune exclusion

NALT
Dendritic cells

Epithelium D

Plasma cells

B C

Lymph node

Lymph node

D
Systemic response or tolerance

Immune Elimination

Local lymphoid tissues in airways of rat and mouse

Effects of FA in rats and mice

- Retrospective examination NALT in chronic Batelle studies in rats and mice: increased % of germinal centre development in NALT of male Fischer rats, exposed to 2 and 6 ppm (6 and 12 months) and 15 ppm (12 months) - Respiratory LLNA (3 days, 3 ppm for 45, 90, 180 or 360 min.) with FA in BalB/c mice was negative: No proliferation in respiratory tract-draining lymph nodes (Arts et al., 2008,
Tox. Sci. 106:423-434. Funded by Dutch Government)

Evaluation of effects difficult, because toxicity NALT rarely reported (NALT rarely examined in inhalation studies)

New study: 28-day inhalation with formaldehyde vapor in male Fischer rats and female B3C3F1 mice
FA vapor conc. ppm (mg/m3) No. of F344 male rats (effects on NALT) No. of B3C3F1 mice (marginally increased incidence of lymphoma in Batelle study) 6 6 6 6 6 6 6

0.5 1 2 6 10 15

(0.6) (1.25) (2.49) (7.5) (12.5) (19)

8 8 8 8 8 8 8

Induction site of immune response

Ag Soluble antigen Particulate antigen Ag

Effector site of immune response

Mucosal response Secretory Ig

Immune exclusion

NALT
Dendritic cells

Epithelium D Adenoid in humans

Plasma cells

B C

SCLN

PCLN

D
Systemic response or tolerance

Immune Elimination

NALT, superficial and posterior cervical LN

NALT mouse
Harrisons fixative allows mild decalcification of bone and easy recognition of lymphoid tissues. H&E-stained section, approx 1 mm
(P.T.C. Harrison, 1984, Lab. Animals 18, 325-331)

NALT rat - overview

Suboptimal examination NALT in nasal cross sections

Rat - isolated NALT, H&E

Approx. 8 mm
Follicle without germinal centre: primary follicle Interfollicular area Follicles with germinal centre

Lympho-epithelium

Microscopy on H&E-stained sections

1. NALT - Histopathology - Enhanced/(semi)quantitative histology: * microphotographs: length x width measured, for automated pathology system translated into size with grades * Decreased cellularity estimated * Germinal centres counted, translated into grades 2. Lymph nodes - Histopathology - Enhanced or (semi)quantitative histology

Results histopathology H&E; NALT of rats (no effects on mice)

ppm Size very small small medium large Decreased cellularity Slight moderate Germinal centre development Simple epithelial hyperplasia 4 5 3 0 (8) 1 2 2 3 0.5 (8) 1 7 1 (8) 2 5 1 2 (8) 1 2 5 6 (8) 3 5 10 (8) 2 3 3 15 (8) 1 6 1

1 3 3 3

1 2

7 **

Results histopathology H&E; Posterior cervical lymph nodes of rats (no effects on mice)

ppm Germinal centre development

0 (8) 5

0.5 (8) 3

1 (8) 3

2 (8) 0*

6 (8) 3

10 (8) 1

15 (8) 0*

Epithelial hyperplasia and decreased cellularity, 15 ppm Rat NALT

control

15 ppm FA

BrdU-stained sections
BrdU pump implanted in animals 3 days before necropsy Counts on two sections/NALT or LN NALT is paired organ Counts on IF 1. interfollicular area 2. follicles (without germinal centres) F 3. Epithelium above interfollicular area 4. Epithelium above follicles

GC

BrdU results rat (no effects mice)

ppm FA Interfollicular epithelium 6.5 3.2 4.9 2.2 5.9 3.4 4.3 2.7 3.3 2.4 5.5 3.5 28.2 11.1 *** Follicular epithelium 12.6 17.5 4.9 3.8 6.4 6.5 4.7 3.2 5.8 5.3 5.7 2.9 23.6 13.6 *

0 0.5 1 2 6 10 15

Principal component analysis of results: correlation between parameters/no effect overlooked?

Scores for D# 1 versus D# 2 0.6 0 1 2 3 4 5 6 115116 113 133 127 126 130 120 136 140 108 117 137139101 121 131 122 114 111 110 129 135 123 107 144 109 124 138 103 134 97 99 143 105 132118 125 128 104 141 98 112 119 142 -0.2 152 145 147 151 146 149 150 148

0.4

All BrdU results NALT and LN Size NALT Germ.cent.developm. NALT

0.2

106 102 -0.4

100 -0.6

A difference is seen between group 6 and the other groups. This difference is seen in PC1*, which explains 14.2% of the total variance in the data set.
* PC1 is epithelial hyperplasia

-0.8 -0.2

-0.1

0.1

0.2

0.3

0.4

0.5

0.6

Summary and Conclusion 28-day study

Enhanced histopathology and proliferation marker (BrdU) to examine NALT and local LNs: - Epithelial hyperplasia of the lymphoepithelium of NALT in rats exposed to 15 ppm FA vapor. (Indirect) effect on lymphocytes in NALT at 15 ppm cannot be excluded. - No effects in mice.

Frieke Kuper, August 2008

17

Discussion
- No effects on local lympohid tissues in mice - Batelle studies: Incidence of lymphoma marginally increased in female mice, exposed to 15 ppm FA by inhalation (Battelle, 1981) Lymphoma in local lymphoid tissues?

Mucosal lymphocytes preferentially home to mucosae

Compounds that affected NALT epithelium upon chronic inhalation exposure

acetaldehyde; rat had nasal squamous cell carcinoma 1-Year inhalation to Propylene oxide: NALT lymphocytic depletion glutaraldehyde (chloride)

Acknowledgements
BASF Lan Ma-Hock Stefan Durrer TNO Lidy van Oostrum Ruud Woutersen

The 28-day inhalation study was financed by Formacare

Mishoming leads to inflammatory diseases

1-year propylene oxide inhalation, Wistar rat NALT

Females, incidence of change

Controls No. of animals examined NALT depletion

Low conc.

Mid conc.

High conc.

10

10

10

10