Neutrophil Function From Mechanism To Disease

IY30CH19-Zychlinsky
ARI
ANNUAL
REVIEWS
17 February 2012
13:38
Further
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

by Harvard University on 09/10/13. For personal use only.
Click here for quick links to

Annual Reviews content online,
including:
Other articles in this volume
Top cited articles
Top downloaded articles
Our comprehensive search
Neutrophil Function:
From Mechanisms to Disease
Borko Amulic, Christel Cazalet,
Garret L. Hayes, Kathleen D. Metzler,
and Arturo Zychlinsky
Department of Cellular Microbiology, Max Planck Institute for Infection Biology,
Chariteplatz 1, 10117 Berlin, Germany; email: amulic@mpiib-berlin.mpg.de,
cazalet@mpiib-berlin.mpg.de, hayes@mpiib-berlin.mpg.de, metzler@mpiib-berlin.mpg.de,
zychlinsky@mpiib-berlin.mpg.de
Annu. Rev. Immunol. 2012. 30:45989
Keywords
First published online as a Review in Advance on

January 3, 2012
inammation, antimicrobial, granule, phagocytosis, NET
The Annual Review of Immunology is online at

immunol.annualreviews.org
Abstract
This articles doi:

10.1146/annurev-immunol-020711-074942
c 2012 by Annual Reviews.
Copyright
All rights reserved
0732-0582/12/0423-0459$20.00
All authors contributed equally to the work and

are listed alphabetically.
Neutrophils are the most abundant white blood cells in circulation,

and patients with congenital neutrophil deciencies suffer from severe
infections that are often fatal, underscoring the importance of these
cells in immune defense. In spite of neutrophils relevance in immunity,
research on these cells has been hampered by their experimentally intractable nature. Here, we present a survey of basic neutrophil biology,
with an emphasis on examples that highlight the function of neutrophils
not only as professional killers, but also as instructors of the immune
system in the context of infection and inammatory disease. We focus
on emerging issues in the eld of neutrophil biology, address questions
in this area that remain unanswered, and critically examine the experimental basis for common assumptions found in neutrophil literature.
459
IY30CH19-Zychlinsky
ARI
17 February 2012
13:38

INTRODUCTION
In the late nineteenth century, Paul Ehrlich,
dissatised with what he considered an inexcusable disinterest in the white blood cell,
began to utilize newly developed cell-staining
techniques to examine subpopulations of leukocytes. His experimentation led to a new appreciation for the heterogeneity of white blood cells
and to the discovery of several novel leukocyte
subpopulations. Ehrlich named one of these
newly discovered cell types, characterized by a
polymorphous nucleus and a tendency to retain neutral dyes, the neutrophil (1) (see also
the sidebar, A Natural History of Neutrophils).
The function of neutrophils was initially
shrouded in considerable mystery; their conspicuous presence during infections led several
researchers to arrive hastily at a rather ironic
conclusion: They surmised that neutrophils
promote infection, serving as cellular shuttles
for bacteria (2). Their actual function, that of
antimicrobial actors in the immune response,
was eventually demonstrated conclusively by a
contemporary of Ehrlich, Elie Metchnikoff, an
A NATURAL HISTORY OF NEUTROPHILS

Phagocytes are ancient cells that evolved to allow multicellular
organisms to thrive in the face of constant competition with microbes for resources. Metchnikoff s seminal theory of cellular
immunity was based on comparative embryology and observations of phagocytes in various simple organisms, including the microscopic crustacean Daphnia. Remarkably, even the slime mold
Dictyostelium discoideum has phagocytic cells that protect it from
infection (200). The short-lived neutrophil with a lobulated nucleus and granule-packed cytoplasm is a more recent evolutionary
adaptation. In insects, phagocytes are long lived and have round
nuclei. They do, however, produce hydrogen peroxide and carry
distinct classes of granules (201). Bony sh and frogs have bona
de neutrophils that are functionally similar to mammalian ones
(202, 203). In both zebrash and rodents, neutrophils are less
abundant than in humans, comprising only 1520% of immune
cells. In chimpanzees, neutrophils account for more than 50% of
the differential blood count (204).
460
Amulic et al.
early and enthusiastic evolutionary biologist interested in the phagocytic capacity of cells.
Metchnikoff demonstrated that injury of
starsh embryos resulted in recruitment of
phagocytic cells to the site of injury (3). He
theorized (correctly) that these cells migrate to
injured sites and participate in microbe digestion. Remarkably, this prescient view of neutrophil action still aptly summarizes, more than
a century later, the basic role of neutrophils
in immunity. The uniquely lobulated nucleus
of the neutrophil also inspired Metchnikoff to
rename these cells: He called them polymorphonuclear leukocytes (or PMNs), a title that
still enjoys frequent use and that is used interchangeably with neutrophil throughout this review. Together with two other developmentally
related cell types, the eosinophils and basophils
(also discovered by Ehrlich), PMNs form the
granulocyte family of white blood cells, a family whose hallmark is the presence of granules,
unique storage structures important in antimicrobial functions (see section on Granules and
Degranulation, below).
Neutrophils were discovered at the dawn
of the immunological sciences; consequently,
elucidation of their role in the immune response has been an ongoing process stretching
over more than a century. We now know that
they are key components of the innate immune
response and vital in immune function; unfortunately, their importance has often been overshadowed by breakthroughs in the study of the
adaptive immune response (4). Admittedly, this
situation is exacerbated by neutrophils notorious experimental intractability: They exhibit a
short life span and are terminally differentiated,
preventing growth in tissue culture. The standard tools of molecular biology, such as transfection and RNA interference, are of little use
when applied to these cells, and immortalized
neutrophil-like cell lines rarely reect the
functional diversication of neutrophils. Furthermore, neutrophil-like cells studied in the
isolation of a culture dish most certainly do not
mimic the complex biological reality in tissues
or circulation. Conclusions from in vitro studies should, therefore, be carefully interpreted.

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
Unfortunately, in vivo studies of neutrophil

function also raise concerns. Mouse neutrophils, the preferred model for in vivo
studies, differ in important aspects from their
human equivalents. This is perhaps best
exemplied by the differences in the respective
antimicrobial repertoires and the numbers of
PMNs in circulation (30% versus 70% in mice
and humans, respectively).
Despite these difculties, no picture of the
immune response can be complete without
a comprehensive understanding of the neutrophil and its functions. The extensive nature
of neutrophil research, however, precludes a
comprehensive review of the subject matter.
In this review, we intend to provide a survey
of basic neutrophil biology and function, while
emphasizing recent advances in neutrophil research and providing a critical assessment of
some current reports on PMN action.
Our survey of the neutrophil begins in
adult bone marrow where, under the instruction of growth factors and cytokines,
pluripotent hematopoietic cells differentiate
into myeloblasts, a developmental cell type
committed to becoming granulocytes. As these
precursor cells mature to neutrophils, they synthesize proteins that are sorted into different
granules (5). Traditionally, granules have been
subdivided into three different classes based
on their resident cargo molecules: azurophilic,
specic, and gelatinase granules. Although this
subdivision is practical, these designations are
largely articial. Granules are formed through a
continuous process; vesicles bud from the Golgi
apparatus and fuse, producing granular structures. The content of these structures is dictated by the transcriptional program active at
the time of their formation. As the maturing
neutrophil sequentially alters its transcriptional
prole, granule content changes, resulting in a
continuum of granule species with overlapping
cargoes (6).
The release of neutrophils from the bone
marrow is tightly regulated in healthy individuals: Chemokines control the passage
of PMNs into circulation and maintain a
pool of cells ready for release in case of
infection. Indeed, the number of neutrophils

drastically increases during infection and some
diseases. Interestingly, neutrophils circulate
for only approximately 68 h and are among
the shortest-lived cells in the human body.
Although the reason for this short life is unclear,
it may ensure neutrophil integrity; this hypothesis is bolstered by observations that apoptosis
prevents the release of noxious molecules.
Still, the question of why evolution opted for
eliminating neutrophils quickly as opposed
to reducing leakage of their dangerous cargo
remains an unanswered and intriguing mystery.
Mature neutrophils emerge from the bone
marrow intent on pursuing one simple, yet
essential, question: Has host integrity been
compromised by potentially harmful invaders?
Should the answer prove to be yes, the
neutrophil must swiftly enact a carefully
choreographed process to locate, attack, and
destroy the potential threat. At its disposal is
an impressive arsenal of antimicrobial weapons
that are deadly, indiscriminate, and brutish in
their application. Although effective in their
destructive capacity, these weapons can prove
to be just as dangerous to the host cells as to
their intended targets, the microbial invaders.
Therefore, their deployment must be executed
with exquisite precision and timing, at locations
where they are both contained and effective.
How then does the neutrophil locate and
identify infections? How does it transition
at the correct time and place from an inactive cellular bystander to a fully activated
microbial killing machine? This transition
process, during which the neutrophil integrates a complex barrage of environmental
cues and translates them into specic actions,
is known as neutrophil activation. As it
pursues microbes, the neutrophil will enact an
impressive multitude of cellular mechanisms:
It will mobilize secretory vesicles and granules,
identify chemotactic gradients and traverse
them through destruction and reorganization
of the actin skeleton, penetrate the endothelial
barrier and navigate a course through the
basement membrane, and begin transcription
of cytokines for recruitment of new immune
www.annualreviews.org Neutrophil Functions
461
IY30CH19-Zychlinsky
ARI

Selectins:
transmembrane
glycoproteins that
mediate cell adhesion
via binding to sugar
moieties
Integrins:
transmembrane
receptors that mediate
attachment to the
extracellular matrix, as
well as direct cell-cell
interaction and
signaling
Oxidative/respiratory
burst: a rapid increase
in oxygen
consumption upon
neutrophil activation
due to production of
ROS by the NADPH
oxidase
462
17 February 2012
13:38
cells. Ultimately, upon arriving at the infection

site, it will seek the insulting pathogens and
unleash its extensive arsenal of antimicrobial
weapons. The initiation of these processes occurs in the bloodstream, where the neutrophil
acts as a monitor for host distress, patrolling
vessels and vigilantly seeking out indications of
an incipient inammatory response.
NEUTROPHIL ACTIVATION
At inammatory sites, bacterial-derived and
host-produced inammatory signals are
abundant; these compounds stimulate the
endothelial cells near the inammatory site.
These stimulants, such as the bacterial-derived
lipopolysaccharide (LPS) and fMLP, as well
as the classical chemoattractants and cytokines
tumor necrosis factor (TNF)-, interleukin
(IL)-1, and IL-17, prompt endothelial cells to
produce adhesion molecules on their luminal
side: the P-selectins, E-selectins, and several
members of the integrin superfamily, the
ICAMs (5). As neutrophils traverse the circulatory system, they continuously and randomly
probe the vessel wall; the postcapillary venules,
where ow dynamics and the constricted space
are particularly amenable to increased random
probing, are often the best-suited location
for neutrophils to encounter the stimulated
endothelial cells (7, 8).
On the surface of neutrophils, two constitutively expressed proteins are critical for recognition of the endothelial inammatory signals:
the glycoprotein P-selectin glycoprotein
ligand-1 (PSGL-1) and L-selectin (9, 10). Upon
random contact with the endothelium, these
molecules engage the P- and E-selectins of
endothelial cells, resulting in selectin-mediated
tethering of neutrophils to the vessel wall.
This is followed by a characteristic rolling of
neutrophils along the endothelium. It is here
that the complex activation cascade begins
and the neutrophil commitment to microbial
killing commences. What changes occur in the
neutrophil at this early time point? The engagement of PSGL-1 and L-selectin on neutrophils
activates a variety of kinases, including Src
Amulic et al.
family kinases, Syk, phosphoinositide 3-kinase

(PI3K), and p38 mitogen-activated protein
kinase (1113). This cascade initiates a number
of changes in neutrophil biology and sets the
stage for integrin activation and rm adhesion.
After selectin-mediated rolling, neutrophils
enter a rm adhesion state mediated by the
2 integrin family of proteins (LFA-1 and
Mac-1 proteins on the neutrophil); rm adhesion is characterized by the arrest of neutrophil
rolling in preparation for transendothelial
migration (13, 14). As the neutrophil rolls
along the endothelium, interaction with
selectins, chemoattractants, cytokines, and
bacterial products results in activation and
clustering of the 2 integrins on the surface of
the neutrophil (15, 16). The 2 integrins then
engage their endothelial ligands, members of
the ICAM-1 immunoglobulin superfamily,
resulting in arrest of neutrophil rolling and
rm adhesion. This integrin engagement, as
well as continuing input from inammatory
chemoattractants and cytokines, prepares the
neutrophil for its nal chemotactic pursuit: The
cell spreads, producing a leading-edge lamellipodium where chemokine and phagocytic
receptors are concentrated, the cytoskeleton is
rebuilt and targeted toward movement along
chemotactic gradients, and initiation of the
neutrophil oxidative burst begins (17, 18).
Now rmly adhered, the neutrophil must
negotiate a path through the endothelium into
the underlying tissue. In a process dependent
on 2 integrins and ICAMs, neutrophils
crawl along the vessel wall until a preferred
site of transmigration is reached (1921).
Upon arrival at an endothelial cell junction, a
complex interaction between (a) the neutrophil
integrins and their endothelial partners and
(b) neutrophil surface proteins and various
endothelial junction molecules results in transmigration through the endothelial junction
(13). Once through the endothelial lining,
the neutrophil must navigate the basement
membrane, a protein mesh consisting largely
of laminins and collagen type IV. Speculation
abounds that granule proteases assist in this
migration by digesting the protein mesh

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
subsequent to degranulation; however, conclusive experimental evidence for this is lacking.

Once the endothelial barrier has been
traversed, the neutrophil nds itself in a
much different inammatory milieu: Here, the
environment is awash in a soup of chemoattractants and inammatory stimulants, both
host derived and of pathogenic origin. These
compounds will now be the primary dictators
of neutrophil behavior and assume responsibility for initiating the concluding steps of
neutrophil activation. In the interstitial space,
the neutrophil follows chemotactic gradients
toward the invading microbes, pursuing hostproduced cytokines (e.g., IL-8) and, in parallel,
pathogen-derived chemoattractants (e.g.,
fMLP). During this process, these chemoattractants bind to their respective neutrophil
receptors (often G proteincoupled receptors,
as is the case with the fMLP receptor FPR1 or
the chemokine receptors), which initiate a signaling cascade dominated by the MAPK/ERK
pathway (22, 23). Downstream molecules
prompt assembly of the oxidative burst machinery, a hallmark of neutrophil activation.
Furthermore, the stimulation of FPR1 triggers
the release of ATP, whose autocrine action
through activation of purinergic receptors is
critical for the initiation of effective functional
responses in neutrophils (24). Concomitantly,
a family of molecules, the pattern-recognition
receptors, is activated through recognition of
specic nonself patterns present on many microbes (25). Perhaps the best-known example
of this family is the Toll-like receptors (TLRs);
they are responsible for recognizing a number
of pathogen-derived compounds, collectively
called pathogen-associated molecular patterns
(PAMPs), including LPS (TLR4), bacterial
lipopeptides (TLR2), agellin (TLR5), and
DNA (TLR9). In neutrophils, all but one
of these receptors (TLR3) are constitutively
expressed, and their stimulation contributes
to further activation, e.g., induction of the
oxidative burst (25, 26). As the neutrophil nears
its target, continued activation by chemoattractants further stimulates the oxidative response
and degranulation. Upon nally reaching a
point of high chemoattractant concentration,

where no discernible gradient exists, the
neutrophil halts and begins the nal release of
its antimicrobial arsenal; the neutrophil is now
fully in an antimicrobial attack state.
The complex signaling cascade leading to
nal neutrophil activation has several facets
worthy of note. The movement to ever-higher
concentrations of chemoattractant is key in
this process, as individual chemoattractants
may have very different effects on neutrophil
physiology at different concentrations, a
phenomenon exemplied by one of the key
neutrophil-recruiting chemokines and activators, IL-8. At low concentrations, IL-8
stimulates L-selectin shedding and increased
expression of 2 integrins; slightly higher
concentrations result in initiation of the
oxidative burst. At the highest concentrations,
IL-8 induces degranulation of neutrophils (27).
In addition, many chemoattractant molecules
exert a priming effect. That is, alone they
stimulate the oxidative response only mildly,
but they dramatically enhance the subsequent
response to other stimuli. A notable example of
this phenomenon is the strong priming effect
of LPS on the fMLP response (28). In this case,
exposure of the neutrophil to LPS induces
assembly of the NADPH oxidase machinery on
the membrane; fMLP stimulation then induces
activation of this machinery (29). In contrast to
receptor priming, another critical feature of the
stimulation process is the desensitization to previously encountered ligands. Stimulation of the
neutrophil by a chemoattractant often results
in endocytosis of the corresponding receptor,
thus leading to a desensitization of the neutrophil to repeated stimulation with the same
molecule (30, 31). The rich and varied input
received by a neutrophil during this nal leg of
the activation process is complex, and the exact
effects of priming, desensitization, and signaling are incompletely understood. Regardless,
the end result of this signaling cacophony is
unambiguous: The neutrophil begins to implement its regime of microbial killing, executing
programs of phagocytosis, degranulation, and
NETosis (i.e., the process of setting neutrophil
463
ARI
17 February 2012
13:38
extracellular traps) (see the section on Neutrophils and the Elimination of Microbes,
below).
The initiation of these microbicidal actions
indicates the nal stage of the neutrophils
journey through the activation process. However, a prominent question remains largely
unanswered by the preceding exposition: What
exactly is meant by the (admittedly ambiguous)
phrase neutrophil activation? A quick scan
of the literature presents the inexperienced
reader with a sometimes rather conicting (and
overwhelming) view of neutrophil activation.
In fact, one could be (erroneously) led to
believe that neutrophil activation refers only to
direct stimulation of the oxidative burst, as this
has been the canonical in vitro activation assay
for decades. This is, however, an oversimplied view of a complex process. The myriad
interactions that occur during a neutrophils
journey toward an inammatory site must be

IY30CH19-Zychlinsky
a Capture
b Rolling
parsed by the complex neutrophilic signaling

mechanisms, a process that gradually leads
to complete activation and culminates in the
premiere killing functions of phagocytosis,
degranulation, and NETosis. It is, therefore,
more insightful to view neutrophil activation
as a continuum of processes, priming steps,
and signal cascades with varying effects and
outcomes, all focused on the realization of
one goal: the transition of naive, circulating
neutrophils to their microbe-eliminating,
tissue-resident counterparts (Figure 1).
NEUTROPHILS AND THE

ELIMINATION OF MICROBES
The basic instruction set of the activated
neutrophil is both effective and ruthless in
its simplicity: (1) kill microbes, (2) do no
harm to the host, and (3) when in doubt, see
rule 1. To fulll this antimicrobial agenda,
Firm adhesion
Neutrophil
PSGL-1,
L-selectin
ICAM
P-selectin and
E-selectin
Integrin
Phagocytosis
Endothelial cell
Degranulation
Cytokine secretion
NETs
Figure 1
Neutrophil recruitment to sites of inammation. The circulating neutrophil must recognize signs of
inammation and migrate to areas where its antimicrobial arsenal is needed for the elimination of infection.
(a) Close to the inammatory sites, stimulated endothelial cells expose a class of molecules, the selectins,
which serve to capture circulating neutrophils and tether them to the endothelium. (b) Selectin-mediated
rolling along chemoattractant gradients then ensues, followed by (c) integrin-mediated rm adhesion.
Subsequently, the neutrophil traverses through the endothelium and arrives at the site of inammation.
Here, the neutrophil releases cytokines that recruit other immune cells, and it begins to implement its
antimicrobial agenda. Among the processes employed are engulfment of microbes via receptor-mediated
phagocytosis, release of granular antimicrobial molecules through degranulation, and formation of
neutrophil extracellular traps (NETs).
464
Amulic et al.
IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
neutrophils possess an array of toxic weapons

that are carefully regulated through controlled
mechanisms. These antimicrobial weapons
vary considerably in their methods of action
and thus reect the neutrophils attempt to
exploit any and all weaknesses that microbes
might present during the course of infection.
An understanding of these weapons, their
action, and their method of release is critical
to understanding neutrophil function.

Granules and Degranulation

The neutrophil must safely transport a plethora
of dangerous substances through the bloodstream and then correctly deploy them at the
appropriate time. Therefore, it comes as no
surprise that a specialty storage organelle has
evolved in neutrophils: the granule. Expectedly, these structures are replete with specically tuned mechanics that address the unique
Granule type
Stage of
formation
Primary
(azurophilic)
Myeloblast
needs of neutrophils. Granules are, however,

far more than just latent repository organelles
for dangerous substances; they are active and indispensable participants in almost all neutrophil
activities during inammation.
As mentioned above, there are three
fundamental types of granules in neutrophils
(Figure 2). Azurophilic granules (also known
as peroxidase-positive or primary granules) are
the largest, measuring approximately 0.3 M
in diameter, and are the rst formed during
neutrophil maturation. They are named for
their ability to take up the basic dye azure A and
contain myeloperoxidase (MPO), an enzyme
critical in the oxidative burst (32, 33). Other
cargo of this granule class include the defensins,
lysozyme, bactericidal/permeability-increasing
protein (BPI), and a number of serine proteases:
neutrophil elastase (NE), proteinase 3 (PR3),
and cathepsin G (CG) (34). As such, these
granules are brimming with antimicrobial
Secondary
(specific)
Promyelocyte
Myelocyte
Inflammation:
recruitment and
activation of immune
cells upon infection or
injury; when
uncontrolled it leads to
tissue damage
Tertiary
(gelatinase)
Metamyelocyte
Secretory
vesicles
Band cell
PMN
Degranulation
propensity
Characteristic
proteins
Lysozyme
Complement receptor 1
Lactoferrin
Myeloperoxidase
Elastase
FcRIII
Gelatinase
Defensin
Other
proteins
Cathepsin G, PR3,
BPI, azurocidin,
sialidase,
-glucuronidase
Gp91phox/p22phox,
CD11b, collagenase,
hCAP18, NGAL, B12BP,
SLPI, haptoglobin,
pentraxin 3,
oroscomucoid,
2-microglobulin,
heparanase, CRISP3
Gp91phox/p22phox,
CD11b, MMP25,
arginase-1,
2-microglobulin,
CRISP3
Gp91phox/p22phox,
CD11b, MMP25, C1q-R,
FPR, alkaline
phosphatase, CD10,
CD13, CD14,
plasma proteins
Figure 2
Neutrophil granules. Neutrophil granules carry a rich variety of antimicrobials and signaling molecules. They are typically divided into
three types (primary or azurophilic, secondary or specic, and tertiary or gelatinase). Additionally, structures called secretory vesicles
are also considered to be a granule subset. Considerable overlap exists in the cargo of the different granules, and their contents seem
determined by the timepoint during hematopoiesis at which they are produced (5). Granules also differ in their ability to mobilize, with
secretory vesicles being the rst to fuse with the plasma membrane and the azurophilic granules demonstrating the least degranulation
propensity.
465
ARI
17 February 2012
13:38
compounds and function as a primary repository for the molecular weaponry of neutrophils.
The second class of granules, the specic (or
secondary) granules, are smaller (0.1 M
diameter), do not contain MPO, and are characterized by the presence of the glycoprotein
lactoferrin. These granules are formed after
azurophilic granules; they also contain a wide
range of antimicrobial compounds including
NGAL, hCAP-18, and lysozyme (33, 35). The
third class, the gelatinase (tertiary) granules, are
also MPO-negative, are smaller than specic
granules, and contain few antimicrobials,
but they serve as a storage location for a
number of metalloproteases, such as gelatinase
and leukolysin. These granules are also the
last population of granules formed during
neutrophil maturation (5). Finally, a fourth set
of structures, the secretory vesicles, are also
commonly considered part of the neutrophil
granule family. In contrast to the classical
granules, these do not bud from the Golgi,
but instead are formed through endocytosis
in the end stages of neutrophil maturation
(36). Consequently, their cargo consists predominantly of plasma-derived proteins such as
albumin. The membrane of secretory vesicles
serves as a reservoir for a number of important
membrane-bound molecules employed during
neutrophil migration.
As a neutrophil proceeds through activation,
granules are mobilized and fuse with either the
plasma membrane or the phagosome, releasing
their contents into the respective environment.
In both cases, the membrane of the granule
becomes a permanent part of the target membrane, thus altering its molecular composition
(6). The different classes of granules demonstrate varying propensities for mobilization in
response to inammatory signals: Azurophilic
granules are the most difcult to mobilize, followed by specic granules, gelatinase granules,
and nally, secretory vesicles (3741). The
underlying mechanisms for this differential
mobilization are not entirely understood, although regulation of intracellular calcium levels
appears to play a salient role (32, 39). Because
of this varying mobilization propensity, each

IY30CH19-Zychlinsky
466
Amulic et al.
granule subset has been traditionally associated

with a particular stage of neutrophil activation.
After neutrophils contact the endothelium,
stimulation through selectins and chemoattractants induces mobilization of secretory vesicles, whose membranes are rich in key factors
necessary for continued activation of the neutrophil, including, among others, the 2 integrins, complement and fMLP receptors, as well
as the FcRIII receptor CD16 (5, 38, 39, 42).
Fusion of the secretory vesicles with the plasma
membrane exposes these components to the external environment. This results in the transition to rm adhesion, mediated by 2 integrin
interaction with the endothelium. As they proceed through the endothelium, neutrophils are
exposed to further activation signals that initiate
mobilization of gelatinase granules, thereby releasing metalloproteases. The activity of these
proteases may help neutrophils traverse the
basement membrane, although this has not
been conclusively demonstrated (43, 44).
At the inammatory site, complete activation of the neutrophil ensues, prompting
initiation of the oxidative burst and mobilization of the azurophilic and specic granules.
These granules either fuse with the phagosome
(see section on Phagocytosis, below), contributing to the antimicrobial activities of this
compartment, or fuse with the plasma membrane, releasing their potent antimicrobials
into the tissue. The fusion of specic granules
with the plasma or phagosomal membrane is of
particular importance for the oxidative burst,
as avocytochrome b558, a component of the
NADPH oxidase machinery, resides in the
specic granule membrane (45). This fusion
permits assembly of the NADPH oxidase complex and allows reactive oxygen species (ROS)
production both inside the phagolysosome and
outside of the cell. Degranulation of primary
and secondary granules contributes to the
creation of an antimicrobial milieu at the inammatory site and produces an environment
inhospitable to invading pathogens.
The release of granular proteins during degranulation presents the astute observer with
a tempting proposition: Could these granular

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
components also serve as signaling molecules

for subsequent inammatory cell recruitment?
Recent studies have provided experimental evidence suggesting this does seem to be the case:
Granule proteins from neutrophils, including
PR3 and azurocidin, can induce monocyte recruitment. Furthermore, neutrophil granule
proteins may increase macrophage bacterial
clearance by enhancing phagocytosis (46). This
could be advantageous in situations in which the
extracellular concentration of released granule
proteins is insufcient to exert extensive microbicidal effects. In such cases, the granule proteins would instead operate as signaling and recruitment factors (see section on Neutrophils
in Immune Cell Cross Talk, below).
By necessity, most data on neutrophil
degranulation and its effects on neutrophil activity have been acquired through biochemical
approaches performed exclusively in vitro. A
pertinent question therefore presents itself: Is
this process truly relevant during the in vivo
inammatory response? The data here are
sparse, and understandably so: Historically, the
possibilities for such an in vivo observation have
been restrained by technical limitations. Most
evidence for in vivo degranulation relies on
observation of increased levels of extracellular
granular proteins at inammatory sites. Even
so, release of granular components could occur
primarily through other means, most notably
through formation of neutrophil extracellular
traps, cell damage, or cell lysis. With the
advent of intravital microscopy techniques,
direct observation of the degranulation process
in vivo may soon be realized.
Antimicrobial Proteins
Neutrophils produce a plethora of peptides and
proteins that directly or indirectly kill microbes
(Table 1). Many of these antimicrobials were
identied through biochemical fractionation of
neutrophil extracts, and their in vitro activity
is easily demonstrated in optimized conditions;
nonetheless, showing in vivo relevance is challenging. The diversity of antimicrobials suggests that some of them evolved to act together,
whereas others may be redundant. One of the

challenges in understanding the neutrophils
antimicrobial mechanisms is to study their
function during concerted action and in conditions that mimic an infection site. Therefore,
testing the relevance of antimicrobials in vivo
is essential. This is, however, particularly challenging; ablation of a single antimicrobial gene
may only subtly affect immune defense. In addition, much biochemical identication of neutrophil antimicrobials has been performed in
rabbits and humans, species with abundant neutrophils. Mice, which are genetically tractable,
have neutrophils that function differently from
those of other species. Indeed, as already mentioned, mice lack the genes for some antimicrobials identied in humans. Interestingly, there
are few clinically relevant innate immune deciencies that directly link antimicrobial activity with a particular mutation. Thus, with few
exceptions, evidence for clinical or biological
relevance of these molecules is still lacking.
There are three main types of antimicrobials: (a) cationic peptides and proteins that
bind to microbial membranes, (b) enzymes,
and (c) proteins that deprive microorganisms
of essential nutrients. Here we present an
overview of this rich eld of investigation.
There are more than 800 antimicrobial
peptides described in nature, some of them
highly conserved throughout evolution (47).
These peptides are often charged, a feature that
probably promotes their initial interaction with
microbial surfaces. Under articial conditions,
many of these peptides disrupt the membrane
integrity. Because in vitro tests are often executed at high antimicrobial concentrations to
obtain maximal microbial killing in the shortest
possible time, it is unclear whether this disruption reects their mechanism of action under
physiological conditions. Alternatively, some
antimicrobials are thought to disrupt essential
microbial functions, such as DNA replication,
transcription, or production of energy. Little
is known about antimicrobial concentrations
achieved at inammatory sites or in the phagosome. This information, as well as information
about the synergistic interactions of different
467
IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
Table 1 Mechanism of action of neutrophil antimicrobial proteins

Antimicrobial mechanisma
Antimicrobial peptide
Cationic antimicrobial peptides
-defensins (HNP-1, HNP-2,
HNP-3, HNP-4)

Permeabilize membrane bilayers containing negatively charged

phospholipids
Inhibit DNA, RNA as well as protein biosynthesis
Inhibition of bacterial cell wall synthesis
LL-37
Transmembrane pore-forming
BPI
Increase bacterial permeability and hydrolysis of bacterial

phospholipids by binding to LPS
Histones
Unknown mechanism

Proteolytic enzymes
Lysozyme
Degrades bacterial cell wall
Proteinase 3 (PR3)
Mechanism independent of a proteolytic activity by binding to the

bacterial membrane
Neutrophil elastase (NE),

cathepsin G (CG)

Azurocidin
Cleaves bacterial virulence factors and outer membrane

proteins
Mechanism independent of a proteolytic activity by binding to
the bacterial membrane
Mechanism independent of a proteolytic activity by binding to the

bacterial membrane
Metal chelator proteins

Lactoferrin

Calprotectin
a
Alters bacterial growth by binding to iron, an essential bacterial

nutrient
Binds to the lipid A part of LPS, causing a release of LPS from
the cell wall and an increase in membrane permeability
Alters bacterial growth by sequestering manganese and zinc
Only direct actions of neutrophil antimicrobial proteins on microbes are listed in the table.
antimicrobials, is essential for designing appropriate in vitro conditions to probe mechanisms

of action.
The neutrophil cationic antimicrobial
peptides include defensins and cathelicidins.
Neutrophils mostly produce -defensins, a
protein family whose members possess multiple disulde bonds and whose structures may
change under physiological conditions and
increase their activity (48). A surprising number of functions are assigned to defensins, but
none have been validated in vivo. Interestingly,
inhibition of bacterial cell wall synthesis (49)
was recently shown at low concentrations that
may be more similar to those present at inammatory sites. Cathelicidins, including the wellstudied LL-37, are proteolytically processed
468
Amulic et al.
from larger proteins, and in addition to their

antimicrobial activity, they may potentiate
DNA activation of dendritic cells (DCs) (50).
Neutrophils also contain a number of
full-length cationic antimicrobial proteins,
including BPI and histones. BPI is cationic
and binds LPS avidly, much like its structural
cousin the LPS binding protein. BPI binding to
LPS results in increased bacterial permeability
and hydrolysis of bacterial phospholipids; cell
death then follows (51). Interestingly, histones
are extremely effective antimicrobials and
were one of the rst antimicrobials described
(52). The signicance of histones (and of the
peptides derived from them) as microbials
remains to be demonstrated in vivo (53).
Given their dual role as an architectural

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
scaffold for DNA and as antimicrobials, their

in vivo signicance is particularly difcult to
demonstrate.
The second class of neutrophil antimicrobials encompasses a broad assortment of
proteolytic enzymes that participate in microbe
destruction. Lysozyme destroys the bacterial
wall, making it an obvious antimicrobial, as
shown in mice decient in this enzyme (54).
Surprisingly, this occurred independently of its
enzymatic activity (55). Neutrophils also contain several serine proteases (including PR3,
CG, and NE, collectively known as the serprocidins) that exhibit differing specicities. They
are tightly regulated intra- and extracellularly
by serpins, indicating that their activity is
deployed under specic conditions. NE cleaves
enterobacterial virulence factors with high
specicity (56), indicating the possibility of the
coevolution of microbial virulence factors and
antimicrobial effectors. Of further interest, NE
mutations in humans, but not genetic ablation
of this enzyme in mice, result in neutropenia.
This can be rescued by the administration of
recombinant granulocyte macrophage colonystimulating factor (GM-CSF); however, these
patients still exhibit signicant susceptibility
to infections. Mice decient in NE or CG
are highly susceptible to bacterial and fungal
infections (57, 58). Another protein, azurocidin, is a member of the same family but lacks
protease activity. Unexpectedly, it still kills
microbes, suggesting that these proteins may
all have antimicrobial activity independent
of proteolysis, perhaps as a result of their
cationicity. These serine proteases also play a
salient role in autoimmunity (see discussion in
section on Autoimmunity, below) (59).
The nal class of neutrophil antimicrobials
consists of a number of proteins that chelate
essential metals from microbes and possibly
impact bacterial growth. Two of these chelators are lactoferrin, rst identied in milk,
which binds preferentially to iron, and calprotectin (also called S100A and many other
names), which sequesters zinc (60) and results in
nutritional immunity (61).
Reactive Oxygen Species

Upon activation, neutrophils produce ROS in
a process called the respiratory burst. It is misleading to think of ROS as a single entity because they differ in their stability, reactivity, and
permeability to membranes (62). However, all
ROS can modify and damage other molecules,
properties exploited by the host cell for signaling and antimicrobial action.
The NADPH oxidase complex assembles
on the phagosomal and plasma membranes
and begins the reactive oxygen cascade by
reducing molecular oxygen to superoxide.
Downstream of superoxide, many potential
reactions can occur (for details, see References
6264). Superoxide, though not a strong
oxidant, rapidly dismutates, forming hydrogen
peroxide. Superoxide can also react with nitric
oxide, which is produced at high levels at
inammatory sites, to form peroxynitrite, a
strong oxidant. Upon degranulation into the
phagosome, MPO can react with hydrogen
peroxide to produce various reactive species,
including hypohalous acids. Hypochlorous
acid, thought to be the major product of MPO
in the phagosome, is more reactive than superoxide and is antimicrobial in vitro. Thus, it
is assumed to have direct antimicrobial effects
in the phagosome. However, a theoretical
model of the phagosome suggests that most of
the hypochlorous acid produced would react
with host proteins before reaching the bacterium. This model predicts that chloramines,
produced when hypochlorous acid reacts
with amine groups, may be the most relevant
antimicrobial actors in the phagosome (65).
ROS are clearly important for neutrophil
antimicrobial activity: Neutrophils from
chronic granulomatous disease (CGD) patients
kill microbes poorly, making these patients
susceptible to many infections. Interestingly,
CGD patients can control catalase-negative
bacteria, which produce, but do not degrade,
their own hydrogen peroxide, thus providing
a substrate for reactions downstream in the
reactive oxygen cascade (66). NADPH oxidase is also implicated in the regulation of
Chronic
granulomatous
disease (CGD):
caused by mutations
rendering the
NADPH oxidase
nonfunctional,
characterized by
susceptibility to
infection and
autoinammation
469
ARI
17 February 2012
13:38
inammation, which explains why CGD

patients often suffer from autoinammatory
diseases (67).
Paradoxically, although MPO is required
for neutrophil microbicidal activity in vitro,
MPO-decient individuals do not have striking
clinical manifestations (68, 69). Some MPOdecient individuals suffer from frequent or severe infections, especially with Candida species,
and a few have been mistaken for CGD patients.
However, most MPO-decient individuals in
the developed world have apparently normal
immunity. The mild effects of MPO deciency
suggest that MPOs products are not essential
for antimicrobial action. Indeed, in the absence
of MPO, other reactive species (e.g., superoxide, hydrogen peroxide, hydroperoxyl radical,
peroxynitrite) can still be produced in the
neutrophil phagosome; hydroperoxyl radical is
predicted to be present at antimicrobial concentrations (65). However, there may be a broader
reason for this discrepancy. Modern technologies can distinguish between individuals who
are partially and completely MPO decient,
and partial MPO deciency does not correlate
with pathology (70). Residual activity of MPO
may be sufcient for antimicrobial activity: In
the case of CGD, even 1% of normal NADPH
oxidase activity leads to an improved prognosis
(71). Epidemiological studies distinguishing
the degrees of MPO deciency and their
correlation with clinical manifestations may be
necessary to understand the function of MPO.
In addition to direct antimicrobial action,
ROS can modify host molecules. Because
these species are highly reactive, they are often
thought to be too nonspecic to be involved in
signaling. However, specicity can be achieved
on the submolecular level, by cellular redox
buffering systems and by limited diffusion of
ROS owing to their short half-lives (72). A
well-studied example of ROS in signaling is
the reversible regulation of various targets
(including phosphatases, metalloproteinases,
and caspases) by direct oxidation of cysteine
residues. In addition, neutrophil granule
proteases can be regulated by oxidative inactivation of their inhibitors or by direct oxidation

IY30CH19-Zychlinsky
470
Amulic et al.
(73, 74). Furthermore, superoxide generation

leads to an ionic inux into the phagosome to
compensate for charge; this may activate granule proteases by releasing them from their putative matrix (75). There is controversy around
which ions and which channel are responsible
for charge compensation, but this theory of
protease activation is certainly intriguing (69).
Studies of ROS are hampered by various
technical issues. Ideally, a probe for ROS
should be specic, targetable to particular
intracellular compartments, and capable of
being used in vivo. Traditional probes for
ROS do not meet these specications; in
addition, the probes often become radical
species (76). One promising new approach
for ROS detection that meets these criteria is
the use of redox-sensitive uorescent proteinbased probes, such as roGFP and HyPer
(76). Other methods that can be used in vivo
include transcription proling of superoxide
or hydrogen peroxidesensitive genes as well
as the detection of relatively stable products of
reactive oxygen using mass spectrometry (76).
Phagocytosis
Phagocytosis is the major mechanism to remove pathogens and cell debris. It is an active,
receptor-mediated process during which a particle is internalized by the cell membrane into
a vacuole called the phagosome. As with other
phagocytes, the mechanistic details of internalization depend on the type of interaction between the neutrophil and the microorganism.
Interaction can be direct, through recognition
of PAMPs by pattern-recognition receptors, or
opsonin mediated. The latter mechanism is better characterized and includes two prototypical
examples: FcR-mediated phagocytosis, which
relies on the formation of pseudopod extensions
for engulfment of IgG-opsonized particles, and
complement receptor-mediated phagocytosis,
which does not require membrane extensions
or pseudopods (77).
After engulfment, the nascent phagosome
is relatively benign to microorganisms, acquiring its lethal properties only after a drastic

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
maturation process. Our understanding of

this process is largely based on studies in
macrophages, and although these are certainly
instructive, essential differences exist in neutrophils. Macrophage phagocytosis follows an
endocytic maturation pathway: In neutrophils,
phagosome maturation happens upon fusion of
granules to the phagosome, whereby delivery
of antimicrobial molecules into the phagosomal lumen occurs. Simultaneously, assembly
of the NADPH oxidase on the phagosomal
membrane allows ROS production, and jointly,
these two mechanisms create an environment
toxic to most pathogens. Neutrophil phagosomal pH regulation also differs signicantly
from that observed in macrophages. While the
macrophage phagosome gradually acidies,
neutrophil phagosomal pH is initially alkaline
(78) and remains neutral for prolonged periods
of time (79). The maintenance of this alkaline
pH is essential for the activation of the major
serine proteases NE and CG, and it is sustained
via NADPH oxidase activity, despite continuing fusion of acidic granules. Key events of
the maturation process are described in more
detail in Reference 80.
Not all pathogens succumb to the hostile
environment of the phagosome. In fact, some
have evolved strategies to survive inside neutrophils. These strategies include interfering
with engulfment, modulating phagosome
maturation, and creating a more hospitable
intraphagosomal environment. The polysaccharide capsule expressed by Staphylococcus
aureus confers antiphagocytic properties (81).
Helicobacter pylori can disrupt targeting of
NADPH oxidase to the phagosome so that
superoxide anions accumulate extracellularly
rather than in the phagosome (82). Francisella
tularensis prevents triggering of the oxidative
burst and also inhibits ROS production in
response to other stimuli (83). Finally, other
pathogens, such as Salmonella typhimurium and
Streptococcus pyogenes, can efciently block granule fusion with the phagosome (84, 85). The
variety of mechanisms evolved by intracellular
pathogens to resist killing and enable survival
within the phagosome further emphasizes the
importance of phagocytosis in the innate

immune defense.
Neutrophil Extracellular Traps

Upon stimulation, neutrophils can undergo
NETosis, an active form of cell death that
leads to release of decondensed chromatin into
the extracellular space (86, 87). The brous
structures termed NETs contain histones as
well as antimicrobial granular and cytoplasmic
proteins (88). NETs trap many types of microbes ex vivo and have been found in various
disease models in vivo; they are thought to
kill microbes by exposing them to high local
concentrations of antimicrobials (89).
The mechanism of NET formation is not
completely understood. The reactive oxygen
pathway is involved, as NADPH oxidase and
MPO are required for NET formation in response to chemical and biological stimuli (87,
90, 91). Nitric oxide donors can induce NETs
via a mechanism that also requires ROS (90), a
nding that awaits genetic conrmation. All activators of NET formation tested so far require
ROS production. S. aureus may be an exception,
although those experiments were done using
pharmacological inhibitors, not cells decient
in ROS production (92). Upstream of NADPH
oxidase, the Raf-MEK-ERK pathway is implicated in NET formation (93), but further along
in the process, NE translocates from the granules to the nucleus and degrades histones, leading to chromatin decondensation (94). Histone
citrullination may also play a role in NET formation, although this has not been conrmed
in primary human neutrophils (9597). Autophagy is also thought to be required for NET
formation, but this has so far been shown only
using a nonspecic inhibitor of autophagy (98).
The majority of research on NETs has been
conducted ex vivo. Ideally, to test the relevance
of NETs, a NETs knockout organism should
be generated to investigate its response to
pathogens. Unfortunately, it is not possible to
eliminate the main components of NETs
DNA and histonesfrom an infection model.
Moreover, the factors that are important for
Autophagy: a process
in which cellular
contents are degraded
in lysosomes,
especially in
conditions of nutrient
scarcity and infection
471
IY30CH19-Zychlinsky
ARI

Cystic fibrosis:
caused by defects in
the CFTR ion
transporter,
characterized by thick,
sticky mucus and
decreases in lung and
digestive function
17 February 2012
13:38
NET formation, such as NADPH oxidase,

MPO, and NE, are also critical for other antimicrobial neutrophil functions. For now, the
evidence for the relevance of NETs is indirect.
On the one hand, bacteria that express DNases
as virulence factors disseminate more efciently
in the host, which may point to evolutionary
pressure to avoid entrapment by NETs (99,
100). In addition, a persistent Aspergillus
infection in a CGD patient was cleared after
gene therapy, which restored NADPH oxidase
activity, NET formation, and NET-mediated
but not phagocytosis-mediated killing by the
patients neutrophils ex vivo (101). On the other
hand, the immune system has redundant mechanisms to ght infection, and it may be that
NETs are especially important under certain
conditions, such as during infections with large
pathogens that are not readily phagocytosed.
NETs can also have detrimental effects on
the host. Because NETs expose self molecules
extracellularly, they lead to autoimmunity:
NETs have been implicated in systemic
lupus erythematosus (SLE), an autoimmune
disease characterized by the formation of
autoantibodies, often against chromatin and
neutrophil components (102106) (see section
on Autoimmunity, below). Platelet-induced
NETs, formed during sepsis, are associated
with hepatotoxicity due to tissue damage
(107). Platelets also bind to NETs, raising the
possibility that NETs nucleate blood clots in
the context of deep vein thrombosis (108).
NETs have also been observed in the airway
uids of cystic brosis patients, where they
may increase the viscosity of the sputum and
decrease lung function (109).
NEUTROPHILS IN IMMUNE
CELL CROSS TALK
Neutrophils participate in the communication networks that form the foundations of
immunity, issuing instructions to practically
all other immune cells. As one of the rst cell
types to arrive at sites of infection, neutrophils
secrete cytokines and chemokines critical in the
unfolding of the inammatory response and in
472
Amulic et al.
establishing the correct environmental conditions to launch the adaptive immune response.
The cytokines released by PMNs are often
synthesized de novo. Although neutrophils
transcribe little after leaving the bone marrow,
once activated, these cells undergo a transcriptional burst that results in the synthesis
of signaling molecules (110, 111). Compared
with other immune cells (e.g., macrophages),
neutrophils typically produce lower amounts
of cytokines per cell, but they are so abundant
at inammatory sites that their contribution
to total cytokine levels is signicant (4). Furthermore, neutrophil-secreted proteases can
modulate signaling networks in vivo through
cytokine processing (112).
The initial neutrophil cytokine response is
an appeal for immunological reinforcement.
The most abundantly produced cytokine, IL-8,
primarily serves to recruit other neutrophils
(113). Similarly, neutrophil-derived proinammatory IL-1 and TNF- induce other cells
to produce neutrophil chemoattractants (114,
115) (for a comprehensive list of cytokines
produced by neutrophils, please see References
115, 116). In addition to cytokines, neutrophils
release other signaling mediators, including
granule contents (117), lipids (118), and ROS
such as hydrogen peroxide (119). They also
communicate via cell-cell contact (120). Here
we provide examples of how neutrophils
interact with other cells to shape the immune
response (see Figure 3).
Monocytes and Macrophages

As they respond to infection or injury,
neutrophils and their relatives in the monocyte/macrophage lineage coordinate their
activities, leading to alternating waves of recruitment of these two cell types. Macrophages
and patrolling monocytes are among the initial
detectors of PAMPs and endogenous activators,
the danger-associated molecular patterns (121),
and these cells work to summon large numbers
of neutrophils to the inammatory locus. The
inux of neutrophils is followed closely by the
arrival of monocytes, suggesting a causal link
IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
Tissue
T cell
IFN-
T cell
Activation and
differentiation
ROS?
Arginase?
DC
Activation
Lymph node
DC
DC
NK cell
IL-12

CD8+
T cell
Neutrophil
IFN-
Macrophage
Crosspriming
DC
Antigen
presentation
CD4+
T cell Th1
Neutrophil
Activation
Neutrophil
Bacteria
Blood
Neutrophil
Monocyte
DC
Figure 3
Neutrophil communication with other immune cells. Neutrophils interact with a variety of cell types. They are important both for
recruitment of monocytes and dendritic cells (DCs) to infected tissues and for enhancement of macrophage and DC activity. In
contrast, in the lymph nodes, neutrophils impede DC function by inhibiting antigen presentation to CD4+ cells. Neutrophils also
interact with the adaptive arm of the immune system: They can act as antigen-presenting cells by cross-presenting antigen to CD8+ T
cells; they also secrete IL-12, which activates T cells. T cells, in turn, activate neutrophils by secreting IFN-. Finally, neutrophils,
DCs and natural killer (NK) cells colocalize and enhance each others activity via receptor-receptor interactions and soluble mediators.
behind these temporal dynamics. Indeed, neutrophils recruit monocytes via several different
mechanisms. They express classical monocyte
chemoattractants such as CCL2 (MCP-1)
(122), CCL3 (MIP-1) (123), CCL20 (MIP3), and CCL19 (MIP-3) (124). Additionally,
and perhaps more unexpectedly, neutrophils
use granule proteins to induce extravasation
of monocytes in vivo, as shown for LL-37,
azurocidin (HBP/CAP37), and CG (125127).
Monocyte recruitment is also affected indirectly
by neutrophils: via upregulation of endothelial
adhesion factors, increase of transendothelial
permeability, enhancement of production of
chemoattractants by other cell types, and modulation of the activities of these chemokines
via proteolytic processing (reviewed in 128).
In addition to recruitment, neutrophils modulate monocyte and macrophage cytokine
production (128), directly enhancing their
microbicidal activity (129). The circuitous

nature of the cross talk of these two cell types
becomes obvious during inammation abatement: Monocytes, recruited by neutrophils
and differentiated into macrophages, repress
further neutrophil chemotaxis and ensure
the appropriate removal of their postmortem
remains (see section on Neutrophils and
Resolution of Inammation, below).
Dendritic Cells
Neutrophils can also recruit and activate
DCs in vivo. This was recently illustrated
in a mouse model of Leishmaniasis, where
subcutaneous inoculation of Leishmania major
triggered a massive and rapid inltration of
neutrophils (130). These cells secrete the
chemokine CCL3, recruiting DCs to the
site of inoculation and initiating a protective
473
IY30CH19-Zychlinsky
ARI

DC-SIGN:
dendritic cellspecic
intercellular adhesion
molecule-3-grabbing
nonintegrin
Granulocyte
receptor 1 (Gr1):
the anti-Gr1 antibody
RB6-8C5 reacts with
both Ly6G (specic
for neutrophils) and
Ly6C (present on
many immune cell
types)
Th17 cells: subset of
T helper cells that
produce IL-17,
important in
inammation and
implicated in
autoimmunity
17 February 2012
13:38
Th1 response (131). Interestingly, activated

neutrophils can induce the maturation of DCs
in vitro through specic receptor-receptor
interactions between Mac-1 and DC-SIGN,
leading to local secretion of TNF- (120).
In this case, the reduced levels of cytokine
production foster specicity, as only proximal
DCs receive the maturation signal. A similar
activation model was earlier proposed for Toxoplasma gondii (132). Neutrophil-activated DCs
produce the proinammatory cytokine IL-12
and induce proliferation of T cells (120, 132).
However, some of these experiments should
be interpreted cautiously because they are
based on the injection of the anti-Gr1 antibody
(RB6), which depletes neutrophils but may also
result in depletion of many other cell types in
mice. The anti-Ly6G monoclonal antibody is
more specic and hence a better reagent for this
type of experiment (133). The crucial role of
neutrophils in DC activation was recently conrmed using anti-Ly6G antibody depletion: In
Mycobacterium tuberculosis infection, timely trafcking of DCs to lymph nodes and activation of
CD4+ T cells were both dependent on PMNs.
Furthermore, this study demonstrated that
DCs presented bacterial antigens when they
ingested infected neutrophils just as efciently
as they did via direct uptake of Mycobacterium
(134). In sharp contrast to the above ndings,
a separate study using an immunization model
showed that neutrophils recruited to lymph
nodes compete for antigen with DCs and
macrophages and that these neutrophils inhibit
their interactions with T cells (135). It is possible that neutrophils have site-specic effects on
DCs and can be stimulatory at peripheral sites
and inhibitory in the lymph nodes. Neutrophils
exhibit fascinating and somewhat enigmatic behavior in the lymph nodes, where they engage
in swarming activity in response to parasitic
infection (136). The functions and mechanistic
details of these swarms are unknown and
represent questions of immense interest.
Natural Killer Cells

Studies of interactions between neutrophil and
natural killer (NK) cells have historically been
474
Amulic et al.
performed in vitro, and their interpretation is

frustratingly difcult owing to the questionable purity of cell preparations. Recently, it
was shown that neutrophils, NK cells, and DCs
interact in a menage a` trois involving both
cytokine signaling and direct cell-cell contact
(137, 138). In one report, infection of mice
with Legionella pneumophila triggered production of IFN- by NK cells; this was dependent
on both PMN-derived IL-18 and DC-derived
IL-12 (137). Similarly, human neutrophils, NK
cells, and DCs colocalize at inammatory sites,
and a positive feedback loop has been proposed
on the basis of in vitro data. In this scheme, neutrophils interact with a specic subset of DCs,
(via CD18-ICAM-1 interactions), prompting
the DCs to produce IL-12p70, which in turn
stimulates IFN- production by NK cells and
further activates neutrophils. Simultaneously,
neutrophils also activate NK cells by direct contact (139). Additional in vitro interactions between neutrophils and NK cells are extensively
reviewed in Reference 138.
Lymphocytes
A surprising nding in recent years is the extensive cross talk between cells located at opposite
ends of the immune spectrum. Previously
thought to belong to isolated compartments,
neutrophils and T cells shape and impact
each others functions, both qualitatively and
quantitatively (140). Neutrophils affect T cell
function indirectly via DCs, as outlined above,
but can also inuence T cell function directly.
PMNs secrete IL-12, which may be crucial for
Th1 cell differentiation (141, 142). They also
express several T cell chemoattractants (116)
as well as B cell development and maturation
factors (143, 144). Cytokine communication
occurs in both directions: For instance, IFN-,
which is secreted by T cells, prolongs neutrophil life span, induces gene expression, and
increases phagocytic capacity (145). The T
helper 17 (Th17) cell subset secretes IL-17,
a key cytokine in the control of neutrophil
dynamics, which acts by upregulating expression of CXCL8 (IL-8), G-CSF, and TNF-

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
by epithelial, endothelial, and stromal cells

(146). Collectively, these Th17-associated
cytokines increase granulopoeisis as well as the
recruitment and life span of neutrophils.
Neutrophils potentially have suppressive effects on T cells via two proposed mechanisms:
(a) L-arginine depletion by release of arginase,
which inhibits T cell responses in vitro (147),
and (b) hydrogen peroxidemediated suppression, as proposed in a cancer model (119) (see
section on Cancer, below). Direct evidence of
such interactions in vivo is still missing.
Interestingly, neutrophils inuence CD8+
T cell responses by cross-presenting exogenous
antigens in vivo. Using mice in which professional antigen-presenting cells do not express
functional MHC class I, Beauvillain et al. (148)
showed that antigen-pulsed neutrophils can
induce differentiation of cytotoxic T cells.
These striking ndings imply that neutrophils
have characteristics of antigen-presenting cells.
Neutrophils also appear capable of expressing
MHC class II and costimulatory molecules
under inammatory conditions (149151),
and they can present antigen to CD4+ T cells
in vitro (152154). However, the functional
signicance for protective immunity remains
unclear, especially in light of the nding that
mouse neutrophils that migrate to the lymph
node have a negative effect on CD4 responses
in an immunization system (135). In humans,
there are large variations in the ability of
donors to express MHC class II (149, 151),
suggesting concomitant variations in the ability
to activate T cells, a nding that could have
implications for susceptibility to autoimmune
diseases. Therefore, neutrophil modulation of
adaptive immunity seems to be highly complex
and is only now starting to be unraveled.
NEUTROPHILS AND
RESOLUTION OF
INFLAMMATION
The lethal cargo of neutrophils is not only
destructive toward invading microbes, but
also harmful to host cells. Thus, neutrophil
deployment must be tightly controlled.
Although some collateral damage to host

tissues is inevitable during infection, neutrophils must be removed before they have
serious, detrimental effects on inamed tissues.
Resolution of inammation is an active process
that limits further leukocyte inltration and
removes apoptotic cells from inamed sites.
This process is essential for maintenance of
tissue homeostasis and, if impeded, leads to
nonresolving inammation, a problematic
condition that contributes to many diseases.
Ulcerative colitis: a
type of inammatory
bowel disease
characterized by ulcers
and tissue erosion in
the colon and rectum
Apoptosis and Clearance

Apoptosis is a central aspect of inammation
resolution. Once neutrophils have executed
their antimicrobial agenda, they die via a builtin cell-death program. However, not only does
apoptosis reduce the number of neutrophils
present, it also produces signals that abrogate further neutrophil recruitment. Phagocytosis of apoptotic neutrophils also reprograms
macrophages to adopt an anti-inammatory
phenotype.
Neutrophil death is inuenced by inammatory mediators such as GM-CSF and LPS and
by environmental conditions such as hypoxia,
all of which prolong neutrophil survival. The
signaling networks that regulate survival have
also been well characterized. These networks
also control the expression of known antiapoptotic (Mcl-1 and A1) or proapoptotic proteins
(Bad, Bax, Bak, and Bid), and they also activate
caspases (for an extensive review, see Reference
155). Given that neutrophils are terminally
differentiated, it is unexpected that molecules
controlling cell proliferation regulate survival.
Proposed to have prosurvival effects, one such
protein is survivin. It is expressed more highly
in immature neutrophils than in mature ones,
but its expression can be restored in mature
cells by inammatory signals such as G-CSF or
GM-CSF. In line with these ndings, survivin
is also highly expressed in neutrophils at sites
of inammation, such as cystic brosis sputum,
appendix inltrates, and intestines of patients
with ulcerative colitis (156).
475
IY30CH19-Zychlinsky
ARI

Wegeners
granulomatosis:
vasculitis affecting the
lungs, nose, and
kidneys; inammation
leads to reduced blood
ow, tissue
destruction, and
damage of vital organs
Prostaglandins and
leukotrienes: lipids
synthesized by
cyclooxygenases and
5-lipoxygenase,
respectively, in the
arachidonic acid
pathway; have
proinammatory
functions including
leukocyte recruitment
17 February 2012
13:38
Similarly, cyclin-dependent kinases function as prosurvival factors in neutrophils.

Pharmacological inhibition of these cell cycle
regulators induce caspase-dependent apoptosis
and block life-span extension by survival factors
(157). More recently, prosurvival effects were
also attributed to proliferating cell nuclear
antigen (PCNA). This factor usually resides
in the nucleus, where it is involved in DNA
replication, but in neutrophils, it associates
with procaspases in the cytosol and is thought
to prevent their activation. During apoptosis,
PCNA is targeted for proteosomal degradation,
which correlates with an increase in caspase-3
and caspase-8 activities. This mechanism is relevant in Wegeners granulomatosis and sepsis,
where stabilization of PCNA is associated with
resistance of neutrophils to apoptosis (158).
Equally important for the resolution of inammation is the proper removal of apoptotic
cells. This relies on the release of nd-me
signals at early stages of cell death, which attract phagocytes. Likewise, distinct eat me
signals are required for specic recognition of
apoptotic cells. Ingestion of apoptotic cells by
macrophages drives the production of the antiinammatory cytokines tumor growth factor
(TGF)- and IL-10 (155). Failure to clear these
apoptotic cells, by contrast, results in secondary
necrosis and release of products that generate
proinammatory signals (Figure 4).
Lipid Mediator Class Switch

Soluble mediators play a crucial role in the
resolution of inammation. In neutrophils,
a particularly prominent role is assumed by
lipid mediators. The successful progression
of inammation appears to hinge on a shift
in the composition of secreted lipids. At early
stages of inammation, neutrophils synthesize
proinammatory lipid mediators, such as
prostaglandins and leukotrienes. These are
derived from arachidonate precursor molecules
and are synthesized through the cyclooxygenase and lipoxygenase pathways. During
the later stages of the inammatory response,
neutrophils interact with various cell types in
476
Amulic et al.
their vicinity (epithelial cells, endothelial cells,

broblasts, platelets, and leukocytes) and participate in the transcellular biosynthesis of lipid
mediators with anti-inammatory and proresolving activities, such as lipoxins, resolvins, and
protectins. A major lipid mediator class switch
thus exists, governed by temporally regulated
expression of different lipoxygenases and the
mobilization of different fatty acid substrates.
The different biosynthesis pathways of proresolving lipid mediators have been reviewed in
detail elsewhere (118). Interestingly, microorganisms are also a source of lipid precursors
that can be used by neutrophils for resolvin
synthesis. Thus, microbes also likely participate
in synthesis of mediators with proresolving
functions at the site of infection (159, 160).
How do lipid mediators contribute to
the termination of inammation? Lipoxins,
resolvins, and protectins exert cell-type specic
effects, promoting monocyte/macrophage
recruitment and activation while inhibiting
neutrophil functions. The inhibitory effect
extends to all essential steps of neutrophil
responses: migration, adhesion, and activation.
All three lipid mediators reduce neutrophil
recruitment, a process that involves the lipoxinA4 receptor and the leukotriene B4 receptor
(BLT1) (161167). Ariel et al. (168) also proposed an interesting mechanism of action for
lipoxins, resolvins, and protectins in clearing inammatory sites. They showed that neutrophil
exposure to these lipids increases expression
of CCR5 on the surface of late apoptotic neutrophils, leading to efcient sequestration of the
chemoattractants CCL3 and CCL5. The sequestration of these chemokines means they are
unavailable to recruit neutrophils to inamed
sites (168) (Figure 4). This mechanism complements other anti-inammatory processes
in which chemokines are inactivated by neutrophil proteases. Of these lipids, lipoxins are
the most completely understood. In addition to
neutrophil recruitment, lipoxins can inhibit the
shedding of L-selectin and the upregulation of
2 integrins in response to proinammatory
stimuli, thereby reducing adhesion of neutrophils to endothelial cells (169, 170). Finally,
IY30CH19-Zychlinsky
ARI
Initiation
of inflammation
17 February 2012
Leukotrienes
13:38
TNF-
Prostaglandins
Lipoxins
Resolvins
Protectins
IL-10
TGF-
Resolution
of inflammation
Platelets
Monocyte
Lipoxins

Neutrophil
Lipoxin
Resolvins
Protectins
Macrophage
Apoptotic
neutrophil
Chemokines
Chemokines CCR5
IL-10
TGF-
PGE-2
Chemokine clearance
Leukotrienes
Prostaglandins
Microorganisms
TNF-
IL-6
NETotic
neutrophil
?
Macrophage
Figure 4
From inammation to homeostasis: neutrophil apoptosis and lipid mediator class switching in the resolution of inammation. At the
site of infection, resident macrophages initiate an inammatory response, secreting proinammatory cytokines and chemokines that
alert the immune system and promote neutrophil recruitment. In the early stages of inammation, microbes trigger the production of
proinammatory lipid mediators, such as leukotrienes and prostaglandins, which also recruit neutrophils. As inammation progresses, a
switch occurs, and anti-inammatory lipid mediators such as lipoxins, resolvins, and protectins are produced. Notably, interaction of
neutrophils with platelets induces the production of lipoxins. Anti-inammatory lipid mediators initiate the resolution of inammation
by blocking neutrophil and promoting monocyte recruitment. Monocytes differentiated into macrophages ingest apoptotic neutrophils,
driving the production of the anti-inammatory cytokines tumor growth factor (TGF)- and IL-10 and prostaglandin-E2 (PGE-2),
which drive the lipid mediator class switch. Proresolving lipid mediators also promote the expression of CCR5 on the surface of
apoptotic neutrophils, providing a means of scavenging chemokines. Chemokine clearance upon phagocytosis of apoptotic neutrophils
by macrophages further contributes to the reduction of neutrophil inltration and the return to tissue homeostasis. The contribution of
macrophages to the clearance of NETotic neutrophils, and how this could impact inammation resolution, is currently unknown. A
timeline of the inammation process from initiation to resolution is summarized in the upper part of the gure.
lipoxins also impact neutrophil activation by

inhibiting ROS and peroxynitrite production,
NF-B activation, and IL-8 expression (170).
In addition to directly impacting neutrophil functions, lipid mediators promote
nonphlogistic (noninammatory) phagocytosis of apoptotic neutrophils by monocytes
and macrophages. In the presence of antiinammatory lipids, engulfment of apoptotic
neutrophils is not accompanied by the release of
proinammatory mediators, as typically occurs
during macrophage activation. Instead, production of the anti-inammatory cytokines TGF-
and IL-10 is increased (163, 171).
Disorders Associated with

Nonresolved Inflammation
The failure of neutrophils to apoptose or malfunctions in the removal of their apoptotic remains result in chronic inammation. These
conditions lead to the accumulation of cytotoxic substances and are associated with severe
pathologies, including cystic brosis, chronic
obstructive pulmonary disease (COPD), and
rheumatoid arthritis (RA). The severity of inammation often directly correlates with poor
clinical outcome.
COPD is a major cause of death in industrialized nations, where smoking is a prime
Chronic obstructive
pulmonary disease
(COPD): lung disease
caused by noxious
particles or gas, e.g.,
tobacco smoking;
inammation leads to
lung obstruction
477
IY30CH19-Zychlinsky
ARI

Rheumatoid arthritis
(RA): chronic
inammatory disease
that affects many
tissues and organs but
primarily synovial
joints; severe
inammation causes
deformity
478
17 February 2012
13:38
instigator of this disease. A chronic neutrophil

inltration in the lungs of COPD patients
promotes tissue damage and organ dysfunction. One of the key molecules controlling
the inammatory response in the lung is
leukotriene A4 hydrolase (LTA4H). This
enzyme has two opposing activities. First, its
hydrolase activity converts leukotriene A4 into
leukotriene B4, a potent neutrophil chemoattractant and proinammatory agent. Second,
LTA4H is an aminopeptidase that inactivates
a specic neutrophil chemoattractant, the
proline-glycine-proline tripeptide (PGP), thus
conferring the enzyme with anti-inammatory
properties. Interestingly, tobacco smoke selectively inhibits only the aminopeptidase activity
of LTA4H, promoting the accumulation of
both leukotriene B4 and PGP. This in turn
promotes neutrophil recruitment and fuels
chronic lung inammation (172).
Another prime example of a disease linked to
nonresolving inammation is RA. Neutrophils
are the most abundant leukocytes present in the
synovial uid of RA patients, and their role in
pathogenesis has been demonstrated in several
animal models. These models primarily used
neutrophil depletion or adoptive transfer of
wild-type neutrophils in leukotriene-decient
mice (173175). In one model, synthesis
of leukotriene B4 by neutrophils in joints
is essential for disease development (174).
Leukotriene B4 can act in an autocrine manner
via the neutrophil receptor BLT1 to promote
the recruitment of a rst wave of neutrophils
into the joint. Later, the recruitment of a
second wave of neutrophils is independent of
this leukotriene B4BLT1 pathway. At this
stage, immune complexes are essential for
stimulating inltrating neutrophils to deliver
IL-1 into the joint. This in turn induces the
production of chemokines by synovial tissue
cells and sustains neutrophil recruitment (175,
176). These studies exemplify the complex
regulation cascades involving lipids, cytokines,
and chemokines that orchestrate neutrophil
recruitment in chronic inammation. They
also demonstrate the cross talk between neutrophils and other immune cells discussed in
Amulic et al.
the previous section. It is, however, unknown

whether all neutrophils are capable of adapting
to the changing chemoattractant environment
or if different subsets of neutrophils are successively involved. The relevance of this model
in human disease remains to be established,
although the clinical similarities between this
mouse model and human RA are encouraging.
NEUTROPHILS IN DISEASE
Neutrophils are prominent players in the innate
immune response and the clearance of infection, a subject addressed in several prominent
reviews. However, neutrophil action can also
support disease progression in other illnesses.
A host of autoimmune disorders belong to this
category. In addition, certain malignant cancers
are also prime examples of illnesses in which
neutrophils play a salient role.
Cancer
The link between cancer and inammation
was noted as early as 1863 by Rudolf Virchow
(177). Since then, it has been proposed that
neutrophil-derived ROS have the potential to
initiate tumor formation by genotoxic stress
and induction of genomic instability. Although
this has been demonstrated in vitro (178, 179),
convincing evidence for PMN-mediated DNA
mutagenesis in vivo is still lacking. Neutrophils
do, however, impact cancer progression.
They are abundant in tumors and inuence
tumor development through several secreted
mediators, including cytokines, ROS, and
matrix-degrading proteases (reviewed in Reference 180). The majority of ndings support
a protumor and antihost effect of these
cells; clinical studies indicate that neutrophil
inltration of tumors is associated with poorer
prognosis (181, 182). Indeed, some cancers
seem to actively recruit neutrophils through
production of IL-8 (183). In agreement with
this, antibody depletion of neutrophils reduces
tumor growth (184). The protumor function
of neutrophils operates at multiple levels,
including production of angiogenic factors
(185), enhancement of metastasis (186), and

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
suppression of the antitumor immune response

(119, 187). Using the anti-Ly6G antibody,
Fridlender and colleagues (187) depleted neutrophils and conrmed their tumorigenic role.
Moreover, the study showed that neutrophils in
the tumor microenvironment could, under certain circumstances, be induced to target their
cytotoxic arsenal at tumor cells, whose growth
they usually help to fuel. Pharmacological
inhibition of TGF- signaling led tumorassociated neutrophils to assume a heightened
proinammatory state, causing a reduction in
tumor growth. These alternatively activated
neutrophils underwent a complete reversal in
their effect on CD8+ T cells, serving to activate
rather than suppress these cells. Differential
neutrophil responses were also demonstrated in
a melanoma study. In this instance, increased
systemic levels of the acute-phase protein
serum amyloid A (SAA-1) induced neutrophils
to secrete the anti-inammatory cytokine IL10, which also inhibited T cell responses. Cross
talk with invariant NKT cells could counter
this response, restoring a proinammatory
activation status (188). Thus, investigation of
neutrophils in cancer has revealed considerable
plasticity in their responses. Although little
evidence currently supports the existence of
different populations, it is likely that neutrophil
responses are more exible and less stereotyped
than previously thought.
Another major mechanism of tumor escape
from immune control has recently been
attributed to a heterogeneous category of immature myeloid cells, called myeloid-derived
suppressor cells (MDSCs) (189). In healthy
individuals, MDSCs are found in the bone
marrow, where they differentiate into mature
neutrophils and monocytes. In cancer and
some autoimmune and infectious diseases,
differentiation is partially blocked, leading to
accumulation of these precursors, which act as
powerful suppressors of T cell functions. MDSCs have characteristics of neutrophils, and in
mice, they are typically detected using the neutrophil surface markers CD11b+ and Gr-1+ ,
although they consist of variable proportions
of monocytic and granulocytic cells (189). In
human renal cell carcinoma, MDSCs have

identical morphology and express the same surface markers as do activated neutrophils (190,
191). MDSCs inhibit T cell proliferation by
limiting L-arginine availability via arginase and
NOS activities, both of which use this amino
acid as a substrate (189, 191, 192). Furthermore,
MDSCs are strong producers of ROS, which
suppresses T cell responses (119, 192). Interfering with the release of MDSCs or using drug
interventions to polarize neutrophil responses
in the tumor microenvironment could represent novel therapeutic strategies against cancer.
Acute-phase
proteins: secreted by
liver, concentration in
plasma changes by
25% or more during
inammation
Autoimmunity
Deregulated neutrophil cell death and/or
clearance often accompanies autoimmune syndromes (193195) and may play a major role
in disease pathogenesis, given that release of
proteolytic and cytotoxic molecules from neutrophils can trigger organ damage. Neutrophil
products act as both targets and mediators of
autoimmunity. MPO and PR3 are the main targets of antineutrophil cytoplasmic antibodies
(ANCA), autoantibodies directed against antigens present in the cytoplasm of neutrophils.
Wegeners granulomatosis is consistently associated with the presence of ANCA. Furthermore, the extent of organ damage in patients
with Wegeners granulomatosis correlates with
the PMN inltrate rather than with traditional
autoimmunity parameters such as T cell activation or autoantibody titers (196). Likewise,
ANCA bind MPO and PR3 expressed on the
surface of activated neutrophils, promoting
degranulation and release of chemoattractants
and ROS, which together lead to a vicious
cycle of tissue damage and inammation. Early
reports also suggest that, in an inammatory environment, ANCA accelerate ROS-dependent
neutrophil apoptosis, suggesting a feed-forward
cycle culminating in organ damage (194, 195).
SLE is another chronic autoimmune disease
affecting multiple tissues and organs. Autoantibodies produced in SLE are predominantly
either ANCA or directed against chromatin.
Although neutrophils had long been suspected
479
IY30CH19-Zychlinsky
ARI

Vasculitis:
inammation of blood
vessels
480
17 February 2012
13:38
to be causative agents, their role in SLE pathogenesis remained elusive. The recent discovery
of a link between SLE and NET formation
has helped to shed light on this quandary.
It was proposed that TNF- and IFN-
prime cells for NET formation in response to
anti-PR3, antiribonucleoprotein, anti-HNP,
or anti-LL-37 autoantibodies (103, 104, 106).
Thus, high levels of inammatory cytokines in
autoimmune patients are believed to sensitize
neutrophils to NETosis, whereas autoantibodies may trigger a switch from apoptosis to
NETosis. Additional evidence suggesting a
role for NETs in autoimmune pathology was
obtained when NETs were identied in renal
and/or skin biopsies from patients with SLE
and small vessel vasculitis (103106). Several
studies have reported the presence of a particular subset of neutrophils in PBMC preparations
from pediatric and adult SLE patients. These
low-density granulocytes display phenotypic
characteristics of immature neutrophils with
nonsegmented nuclei and higher expression
of MPO, NE, and defensin-3, and they may
be related to the MDSCs discussed previously
(see section on Cancer, above) (197, 198).
An increased capacity to form NETs and a
heightened cytotoxicity toward endothelial
cells could bestow them with pathogenic
properties in lupus (105).
Because NETs appear to be formed during
autoimmune disease, their timely removal may
be an essential mechanism for maintaining
tissue homeostasis. Human serum contains the
nuclease DNase I, which degrades NETs in
vitro. Notably, a familial form of SLE is linked
to a mutation in DNase I (199). Furthermore,
in a cohort of SLE patients, 36% exhibited
either elevated titers of autoantibodies directed
against NET components or inhibitors of
DNase I, both of which may protect NETs
from degradation. Most notably, impaired
NET degradation correlates with development
of lupus nephritis, one of the most severe
manifestations of SLE (102).
Can it be that NETs play a general role
in modulation of autoimmune responses? We
know that NETs induce plasmacytoid DCs
Amulic et al.
to produce IFN-, a central cytokine in SLE

pathogenesis (103, 104). However, it remains to
be determined if DCs can present NET components or if they contribute to autoreactive B
cell activation. It is also possible that NETs are
involved in other autoimmune diseases. Should
this prove to be the case, understanding the
role of NETs may provide critical insights into
the role of microbial infections as a trigger of
autoimmunity.
CONCLUDING REMARKS
Neutrophils are specialized phagocytes that
arose as an evolutionary adaptation in vertebrates to coordinate and execute one of the most
fundamental physiological responses: inammation. They are endowed with antimicrobial
mechanisms that make them the preeminent
microbe exterminators of the immune system.
In addition to this important role, PMNs also
network with many other immune cells and
help regulate the initiation of specic T and
B cell immunity. However, neutrophils do not
always act in ways benecial to the host: Uncontrolled neutrophil responses can exacerbate and
even cause autoimmune and inammatory diseases. Many challenges remain in understanding neutrophil function: Is there specialization
among PMNs? Are they more plastic than we
suspect? How do they make decisions before
deploying their armamentaria? How do they
kill microbes? How specic are their instructions to other cells? Answering these questions
will better dene neutrophils role in defense
and disease and will provide a rational path for
pursuing new therapies. Moreover, neutrophils
can potentially provide insights into several
unique aspects of basic cell biology. Their strikingly short life spans make them excellent models for investigating cell death, whereas their
reliance on ROS as biochemical effectors may
reveal novel ways for relaying intracellular
signals. The uniquely lobulated neutrophil
nucleus is a feat of higher-order nuclear
architecture that is just beginning to yield
its secrets. In short, exciting times await the
humble neutrophil.
IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank Diane Schad for assistance with graphic design and Cornelia Heinz for administrative
help. G.H. is an Alexander von Humboldt Foundation Scholar, and B.A. is supported by an EMBO
Long-Term Fellowship.

LITERATURE CITED
1. Ehrlich P. 1880. Methodologische Beitrage zur Physiologie und Pathologie der verschiedenen Formen
der Leukocyten. Z. Klin. Med. 1:55360
2. Waller A. 1846. Microscopic observation on the perforation of capillaries by the corpuscles of blood and
the origin of mucus and pus globules. Lond. Edinburgh Philos. Mag. J. Sci. 2:27180
3. Metchnikoff E. 1893. Lecon sur la pathologie comparee de inammation. Ann. Inst. Pasteur 7:34857
4. Nathan C. 2006. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6:17382
5. Borregaard N. 2010. Neutrophils, from marrow to microbes. Immunity 33:65770
6. Borregaard N, Cowland JB. 1997. Granules of the human neutrophilic polymorphonuclear leukocyte.
Blood 89:350321
7. Finger EB, Puri KD, Alon R, Lawrence MB, von Andrian UH, Springer TA. 1996. Adhesion through
L-selectin requires a threshold hydrodynamic shear. Nature 379:26669
8. Lawrence MB, Kansas GS, Kunkel EJ, Ley K. 1997. Threshold levels of uid shear promote leukocyte
adhesion through selectins (CD62L,P,E). J. Cell Biol. 136:71727
9. Kansas GS. 1996. Selectins and their ligands: current concepts and controversies. Blood 88:325987
10. McEver RP, Cummings RD. 1997. Role of PSGL-1 binding to selectins in leukocyte recruitment.
J. Clin. Investig. 100:S97103
11. Yago T, Shao B, Miner JJ, Yao L, Klopocki AG, et al. 2010. E-selectin engages PSGL-1 and CD44
through a common signaling pathway to induce integrin L2-mediated slow leukocyte rolling. Blood
116:48594
12. Mueller H, Stadtmann A, Van Aken H, Hirsch E, Wang D, et al. 2010. Tyrosine kinase Btk regulates
E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C
(PLC) 2 and PI3K pathways. Blood 115:311827
13. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. 2007. Getting to the site of inammation: the leukocyte
adhesion cascade updated. Nat. Rev. Immunol. 7:67889
14. Campbell JJ, Hedrick J, Zlotnik A, Siani MA, Thompson DA, Butcher EC. 1998. Chemokines and the
arrest of lymphocytes rolling under ow conditions. Science 279:38184
15. Constantin G, Majeed M, Giagulli C, Piccio L, Kim JY, et al. 2000. Chemokines trigger immediate 2
integrin afnity and mobility changes: differential regulation and roles in lymphocyte arrest under ow.
Immunity 13:75969
16. Liu S, Kiosses WB, Rose DM, Slepak M, Salgia R, et al. 2002. A fragment of paxillin binds the 4
integrin cytoplasmic domain (tail) and selectively inhibits 4-mediated cell migration. J. Biol. Chem.
277:2088794
17. Goldnger LE, Han J, Kiosses WB, Howe AK, Ginsberg MH. 2003. Spatial restriction of 4 integrin phosphorylation regulates lamellipodial stability and 41-dependent cell migration. J. Cell Biol.
162:73141
18. Xu J, Wang F, Van Keymeulen A, Herzmark P, Straight A, et al. 2003. Divergent signals and cytoskeletal
assemblies regulate self-organizing polarity in neutrophils. Cell 114:20114
19. Schenkel AR, Chew TW, Muller WA. 2004. Platelet endothelial cell adhesion molecule deciency or
blockade signicantly reduces leukocyte emigration in a majority of mouse strains. J. Immunol. 173:6403
8
481
ARI
17 February 2012
13:38
20. Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, Kubes P. 2006. Intraluminal crawling of
neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade.
J. Exp. Med. 203:256975
21. Wong CH, Heit B, Kubes P. 2010. Molecular regulators of leucocyte chemotaxis during inammation.
Cardiovasc. Res. 86:18391
22. Zarbock A, Ley K. 2008. Mechanisms and consequences of neutrophil interaction with the endothelium.
Am. J. Pathol. 172:17
23. Selvatici R, Falzarano S, Mollica A, Spisani S. 2006. Signal transduction pathways triggered by selective
formylpeptide analogues in human neutrophils. Eur. J. Pharmacol. 534:111
24. Chen Y, Yao Y, Sumi Y, Li A, To UK, et al. 2010. Purinergic signaling: a fundamental mechanism in
neutrophil activation. Sci. Signal. 3:ra45
25. Sabroe I, Dower SK, Whyte MK. 2005. The role of Toll-like receptors in the regulation of neutrophil
migration, activation, and apoptosis. Clin. Infect. Dis. 41(Suppl. 7):S42126
26. Parker LC, Whyte MK, Dower SK, Sabroe I. 2005. The expression and roles of Toll-like receptors in
the biology of the human neutrophil. J. Leukoc. Biol. 77:88692
27. Ley K. 2002. Integration of inammatory signals by rolling neutrophils. Immunol. Rev. 186:818
28. Guthrie LA, McPhail LC, Henson PM, Johnston RB Jr. 1984. Priming of neutrophils for enhanced
release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the
superoxide-producing enzyme. J. Exp. Med. 160:165671
29. El-Benna J, Dang PM, Gougerot-Pocidalo MA. 2008. Priming of the neutrophil NADPH oxidase
activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane. Semin.
Immunopathol. 30:27989
30. Didsbury JR, Uhing RJ, Tomhave E, Gerard C, Gerard N, Snyderman R. 1991. Receptor class desensitization of leukocyte chemoattractant receptors. Proc. Natl. Acad. Sci. USA 88:1156468
31. Claing A, Laporte SA, Caron MG, Lefkowitz RJ. 2002. Endocytosis of G proteincoupled receptors:
roles of G proteincoupled receptor kinases and -arrestin proteins. Prog. Neurobiol. 66:6179
32. Nusse O, Lindau M. 1988. The dynamics of exocytosis in human neutrophils. J. Cell Biol. 107:211723
33. Lacy P. 2005. The role of Rho GTPases and SNAREs in mediator release from granulocytes. Pharmacol.
Ther. 107:35876
34. Faurschou M, Borregaard N. 2003. Neutrophil granules and secretory vesicles in inammation. Microbes
Infect. 5:131727
35. Faurschou M, Borregaard N. 2003. Neutrophil granules and secretory vesicles in inammation. Microbes
Infect. 5:131727
36. Borregaard N, Sorensen OE, Theilgaard-Monch K. 2007. Neutrophil granules: a library of innate
immunity proteins. Trends Immunol. 28:34045
37. Borregaard N, Kjeldsen L, Lollike K, Sengelov H. 1992. Ca2+ -dependent translocation of cytosolic
proteins to isolated granule subpopulations and plasma membrane from human neutrophils. FEBS Lett.
304:19597
38. Borregaard N, Kjeldsen L, Sengelov H, Diamond MS, Springer TA, et al. 1994. Changes in subcellular
localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils
during stimulation with inammatory mediators. J. Leukoc. Biol. 56:8087
39. Sengelov H, Kjeldsen L, Borregaard N. 1993. Control of exocytosis in early neutrophil activation.
J. Immunol. 150:153543
40. Kjeldsen L, Bjerrum OW, Askaa J, Borregaard N. 1992. Subcellular localization and release of human
neutrophil gelatinase, conrming the existence of separate gelatinase-containing granules. Biochem. J.
287(Pt. 2):60310
41. Kjeldsen L, Bainton DF, Sengelov H, Borregaard N. 1993. Structural and functional heterogeneity among peroxidase-negative granules in human neutrophils: identication of a distinct gelatinasecontaining granule subset by combined immunocytochemistry and subcellular fractionation. Blood
82:318391
42. Faurschou M, Sorensen OE, Johnsen AH, Askaa J, Borregaard N. 2002. Defensin-rich granules of human
neutrophils: characterization of secretory properties. Biochim. Biophys. Acta Mol. Cell Res. 1591:2935

IY30CH19-Zychlinsky
482
Amulic et al.

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
43. Delclaux C, Delacourt C, DOrtho MP, Boyer V, Lafuma C, Harf A. 1996. Role of gelatinase B and
elastase in human polymorphonuclear neutrophil migration across basement membrane. Am. J. Respir.
Cell Mol. Biol. 14:28895
44. Singer II, Scott S, Kawka DW, Kazazis DM. 1989. Adhesomes: specic granules containing receptors
for laminin, C3bi/brinogen, bronectin, and vitronectin in human polymorphonuclear leukocytes and
monocytes. J. Cell Biol. 109:316982
45. Jesaitis AJ, Buescher ES, Harrison D, Quinn MT, Parkos CA, et al. 1990. Ultrastructural localization
of cytochrome b in the membranes of resting and phagocytosing human granulocytes. J. Clin. Investig.
85:82135
46. Soehnlein O, Zernecke A, Weber C. 2009. Neutrophils launch monocyte extravasation by release of
granule proteins. Thromb. Haemost. 102:198205
47. Brogden KA. 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev.
Microbiol. 3:23850
48. Schroeder BO, Wu Z, Nuding S, Groscurth S, Marcinowski M, et al. 2011. Reduction of disulphide
bonds unmasks potent antimicrobial activity of human -defensin 1. Nature 469:41923
49. Schneider T, Kruse T, Wimmer R, Wiedemann I, Sass V, et al. 2010. Plectasin, a fungal defensin, targets
the bacterial cell wall precursor Lipid II. Science 328:116872
50. Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, et al. 2007. Plasmacytoid dendritic cells
sense self-DNA coupled with antimicrobial peptide. Nature 449:56469
51. Canny G, Levy O. 2008. Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites. Trends. Immunol. 29:54147
52. Hirsch JG. 1958. Bactericidal action of histone. J. Exp. Med. 108:92544
53. Park CB, Yi KS, Matsuzaki K, Kim MS, Kim SC. 2000. Structure-activity analysis of buforin II, a histone
H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of
buforin II. Proc. Natl. Acad. Sci. USA 97:824550
54. Markart P, Korfhagen TR, Weaver TE, Akinbi HT. 2004. Mouse lysozyme M is important in pulmonary
host defense against Klebsiella pneumoniae infection. Am. J. Respir. Crit. Care Med. 169:45458
55. Nash JA, Ballard TN, Weaver TE, Akinbi HT. 2006. The peptidoglycan-degrading property of lysozyme
is not required for bactericidal activity in vivo. J. Immunol. 177:51926
56. Weinrauch Y, Drujan D, Shapiro SD, Weiss J, Zychlinsky A. 2002. Neutrophil elastase targets virulence
factors of enterobacteria. Nature 417:9194
57. Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, et al. 1998. Mice lacking neutrophil elastase
reveal impaired host defense against gram negative bacterial sepsis. Nat. Med. 4:61518
58. Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, Roes J. 2000. Impaired immunity and
enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12:201
10
59. Campanelli D, Detmers PA, Nathan CF, Gabay JE. 1990. Azurocidin and a homologous serine protease
from neutrophils. Differential antimicrobial and proteolytic properties. J. Clin. Investig. 85:90415
60. Corbin BD, Seeley EH, Raab A, Feldmann J, Miller MR, et al. 2008. Metal chelation and inhibition of
bacterial growth in tissue abscesses. Science 319:96265
61. Weinberg ED. 1975. Nutritional immunity. Hosts attempt to withhold iron from microbial invaders.
J. Am. Med. Assoc. 231:3941
62. Hampton MB, Kettle AJ, Winterbourn CC. 1998. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92:300717
63. Bogdan C, Rollinghoff M, Diefenbach A. 2000. Reactive oxygen and reactive nitrogen intermediates in
innate and specic immunity. Curr. Opin. Immunol. 12:6476
64. Winterbourn CC. 2008. Reconciling the chemistry and biology of reactive oxygen species. Nat. Chem.
Biol. 4:27886
65. Winterbourn CC, Hampton MB, Livesey JH, Kettle AJ. 2006. Modeling the reactions of superoxide
and myeloperoxidase in the neutrophil phagosome: implications for microbial killing. J. Biol. Chem.
281:3986069
66. Segal BH, Leto TL, Gallin JI, Malech HL, Holland SM. 2000. Genetic, biochemical, and clinical features
of chronic granulomatous disease. Medicine 79:170200
483
ARI
17 February 2012
13:38
67. Seger RA. 2010. Chronic granulomatous disease: recent advances in pathophysiology and treatment.
Neth. J. Med. 68:33440
68. Klebanoff SJ. 2005. Myeloperoxidase: friend and foe. J. Leukoc. Biol. 77:598625
69. Nauseef WM. 2007. How human neutrophils kill and degrade microbes: an integrated view. Immunol.
Rev. 219:88102
70. Kutter D. 1998. Prevalence of myeloperoxidase deciency: population studies using Bayer-Technicon
automated hematology. J. Mol. Med. 76:66975
71. Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, et al. 2010. Residual NADPH oxidase and survival
in chronic granulomatous disease. N. Engl. J. Med. 363:260010
72. Nathan C. 2003. Specicity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling.
J. Clin. Investig. 111:76978
73. Johnson D, Travis J. 1979. Oxidative inactivation of human -1-proteinase inhibitor - further evidence
for methionine at the reactive center. J. Biol. Chem. 254:402226
74. Shao BH, Belaaouaj A, Verlinde C, Fu XY, Heinecke JW. 2005. Methionine sulfoxide and proteolytic
cleavage contribute to the inactivation of cathepsin G by hypochlorous acidan oxidative mechanism
for regulation of serine proteinases by myeloperoxidase. J. Biol. Chem. 280:2931121
75. Reeves EP, Lu H, Lortat-Jacob H, Messina CGM, Bolsover S, et al. 2002. Killing activity of neutrophils
is mediated through activation of proteases by K+ ux. Nature 416:29197
76. Murphy MP, Holmgren A, Larsson NG, Halliwell B, Chang CJ, et al. 2011. Unraveling the biological
roles of reactive oxygen species. Cell Metab. 13:36166
77. Underhill DM, Ozinsky A. 2002. Phagocytosis of microbes: complexity in action. Annu. Rev. Immunol.
20:82552
78. Segal AW, Geisow M, Garcia R, Harper A, Miller R. 1981. The respiratory burst of phagocytic cells is
associated with a rise in vacuolar pH. Nature 290:4069
79. Jankowski A, Scott CC, Grinstein S. 2002. Determinants of the phagosomal pH in neutrophils. J. Biol.
Chem. 277:605966
80. Lee WL, Harrison RE, Grinstein S. 2003. Phagocytosis by neutrophils. Microbes Infect. 5:1299306
81. Luong TT, Lee CY. 2002. Overproduction of type 8 capsular polysaccharide augments Staphylococcus
aureus virulence. Infect. Immun. 70:338995
82. Allen LA, Beecher BR, Lynch JT, Rohner OV, Wittine LM. 2005. Helicobacter pylori disrupts NADPH
oxidase targeting in human neutrophils to induce extracellular superoxide release. J. Immunol. 174:3658
67
83. McCaffrey RL, Schwartz JT, Lindemann SR, Moreland JG, Buchan BW, et al. 2010. Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis. J. Leukoc. Biol. 88:791805
84. Joiner KA, Ganz T, Albert J, Rotrosen D. 1989. The opsonizing ligand on Salmonella typhimurium
inuences incorporation of specic, but not azurophil, granule constituents into neutrophil phagosomes.
J. Cell Biol. 109:277182
85. Staali L, Bauer S, Morgelin M, Bjorck L, Tapper H. 2006. Streptococcus pyogenes bacteria modulate membrane trafc in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes.
Cell. Microbiol. 8:690703
86. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, et al. 2004. Neutrophil extracellular
traps kill bacteria. Science 303:153235
87. Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, et al. 2007. Novel cell death program leads to
neutrophil extracellular traps. J. Cell Biol. 176:23141
88. Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, et al. 2009. Neutrophil extracellular traps
contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS
Pathog. 5:e1000639
89. Papayannopoulos V, Zychlinsky A. 2009. NETs: a new strategy for using old weapons. Trends Immunol.
30:51321
90. Patel S, Kumar S, Jyoti A, Srinag BS, Keshari RS, et al. 2010. Nitric oxide donors release extracellular
traps from human neutrophils by augmenting free radical generation. Nitric Oxide 22:22634
91. Metzler KD, Fuchs TA, Nauseef WM, Reumaux D, Roesler J, et al. 2011. Myeloperoxidase is required
for neutrophil extracellular trap formation: implications for innate immunity. Blood 117:95359

IY30CH19-Zychlinsky
484
Amulic et al.

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
92. Pilsczek FH, Salina D, Poon KKH, Fahey C, Yipp BG, et al. 2010. A novel mechanism of rapid nuclear
neutrophil extracellular trap formation in response to Staphylococcus aureus. J. Immunol. 185:741325
93. Hakkim A, Fuchs TA, Martinez NE, Hess S, Prinz H, et al. 2011. Activation of the Raf-MEK-ERK
pathway is required for neutrophil extracellular trap formation. Nat. Chem. Biol. 7:7577
94. Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A. 2010. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J. Cell Biol. 191:67791
95. Neeli I, Khan SN, Radic M. 2008. Histone deimination as a response to inammatory stimuli in neutrophils. J. Immunol. 180:1895902
96. Wang Y, Li M, Stadler S, Correll S, Li P, et al. 2009. Histone hypercitrullination mediates chromatin
decondensation and neutrophil extracellular trap formation. J. Cell Biol. 184:20513
97. Li P, Li M, Lindberg MR, Kennett MJ, Xiong N, Wang Y. 2010. PAD4 is essential for antibacterial
innate immunity mediated by neutrophil extracellular traps. J. Exp. Med. 207:185362
98. Remijsen Q, Vanden Berghe T, Wirawan E, Asselbergh B, Parthoens E, et al. 2011. Neutrophil extracellular trap cell death requires both autophagy and superoxide generation. Cell Res. 21:290304
99. Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A, Henriques-Normark B. 2006. An endonuclease
allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr. Biol. 16:4017
100. Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, et al. 2006. DNase expression allows the
pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr. Biol. 16:396400
101. Bianchi M, Hakkim A, Brinkmann V, Siler U, Seger RA, et al. 2009. Restoration of NET formation by
gene therapy in CGD controls aspergillosis. Blood 114:261922
102. Hakkim A, Furnrohr BG, Amann K, Laube B, Abed UA, et al. 2010. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc. Natl. Acad. Sci. USA 107:981318
103. Garcia-Romo GS, Caielli S, Vega B, Connolly J, Allantaz F, et al. 2011. Netting neutrophils are major
inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci. Transl. Med. 3:73ra20
104. Lande R, Ganguly D, Facchinetti V, Frasca L, Conrad C, et al. 2011. Neutrophils activate plasmacytoid
dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci. Transl.
Med. 3:73ra19
105. Villanueva E, Yalavarthi S, Berthier CC, Hodgin JB, Khandpur R, et al. 2011. Netting neutrophils
induce endothelial damage, inltrate tissues, and expose immunostimulatory molecules in systemic lupus
erythematosus. J. Immunol. 187:53852
106. Kessenbrock K, Krumbholz M, Schonermarck U, Back W, Gross WL, et al. 2009. Netting neutrophils
in autoimmune small-vessel vasculitis. Nat. Med. 15:62325
107. Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, et al. 2007. Platelet TLR4 activates neutrophil
extracellular traps to ensnare bacteria in septic blood. Nat. Med. 13:46369
108. Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, et al. 2010. Extracellular DNA traps
promote thrombosis. Proc. Natl. Acad. Sci. USA 107:1588085
109. Marcos V, Zhou Z, Yildirim AO, Bohla A, Hector A, et al. 2010. CXCR2 mediates NADPH oxidaseindependent neutrophil extracellular trap formation in cystic brosis airway inammation. Nat. Med.
16:101823
110. Subrahmanyam YV, Yamaga S, Prashar Y, Lee HH, Hoe NP, et al. 2001. RNA expression patterns
change dramatically in human neutrophils exposed to bacteria. Blood 97:245768
111. Kobayashi SD, Voyich JM, Buhl CL, Stahl RM, DeLeo FR. 2002. Global changes in gene expression
by human polymorphonuclear leukocytes during receptor-mediated phagocytosis: Cell fate is regulated
at the level of gene expression. Proc. Natl. Acad. Sci. USA 99:69016
112. Meyer-Hoffert U, Wiedow O. 2010. Neutrophil serine proteases: mediators of innate immune responses.
Curr. Opin. Hematol. 18:1924
113. Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA. 2000. The neutrophil as
a cellular source of chemokines. Immunol. Rev. 177:195203
114. Sica A, Matsushima K, Van Damme J, Wang JM, Polentarutti N, et al. 1990. IL-1 transcriptionally
activates the neutrophil chemotactic factor/IL-8 gene in endothelial cells. Immunology 69:54853
115. Kasama T, Miwa Y, Isozaki T, Odai T, Adachi M, Kunkel SL. 2005. Neutrophil-derived cytokines:
potential therapeutic targets in inammation. Curr. Drug Targets Inamm. Allergy 4:2739
485
ARI
17 February 2012
13:38
116. Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA. 2000. The neutrophil as
a cellular source of chemokines. Immunol. Rev. 177:195203
117. Yang D, de la Rosa G, Tewary P, Oppenheim JJ. 2009. Alarmins link neutrophils and dendritic cells.
Trends Immunol. 30:53137
118. Serhan CN. 2007. Resolution phase of inammation: novel endogenous anti-inammatory and proresolving lipid mediators and pathways. Annu. Rev. Immunol. 25:10137
119. Schmielau J, Finn OJ. 2001. Activated granulocytes and granulocyte-derived hydrogen peroxide are
the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res.
61:475660
120. van Gisbergen KP, Sanchez-Hernandez M, Geijtenbeek TB, van Kooyk Y. 2005. Neutrophils mediate
immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and
DC-SIGN. J. Exp. Med. 201:128192
121. Cailhier JF, Partolina M, Vuthoori S, Wu S, Ko K, et al. 2005. Conditional macrophage ablation
demonstrates that resident macrophages initiate acute peritoneal inammation. J. Immunol. 174:233642
122. Yoshimura T, Takahashi M. 2007. IFN--mediated survival enables human neutrophils to produce
MCP-1/CCL2 in response to activation by TLR ligands. J. Immunol. 179:194249
123. Katsura T, Kobayashi K, Hosaka M, Sugihara S, Kasama T, et al. 1993. Desensitization of delayed-type
hypersensitivity in mice: suppressive environment. Med. Inamm. 2:20510
124. Scapini P, Laudanna C, Pinardi C, Allavena P, Mantovani A, et al. 2001. Neutrophils produce biologically
active macrophage inammatory protein-3 (MIP-3)/CCL20 and MIP-3/CCL19. Eur. J. Immunol.
31:198188
125. Soehnlein O, Zernecke A, Eriksson EE, Rothfuchs AG, Pham CT, et al. 2008. Neutrophil secretion
products pave the way for inammatory monocytes. Blood 112:146171
126. De Y, Chen Q, Schmidt AP, Anderson GM, Wang JM, et al. 2000. LL-37, the neutrophil granuleand epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to
chemoattract human peripheral blood neutrophils, monocytes, and T cells. J. Exp. Med. 192:106974
127. Chertov O, Ueda H, Xu LL, Tani K, Murphy WJ, et al. 1997. Identication of human neutrophilderived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils.
J. Exp. Med. 186:73947
128. Soehnlein O, Weber C, Lindbom L. 2009. Neutrophil granule proteins tune monocytic cell function.
Trends Immunol. 30:53846
129. Soehnlein O, Kai-Larsen Y, Frithiof R, Sorensen OE, Kenne E, et al. 2008. Neutrophil primary granule
proteins HBP and HNP1-3 boost bacterial phagocytosis by human and murine macrophages. J. Clin.
Investig. 118:3491502
130. Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, et al. 2008. In vivo imaging reveals an
essential role for neutrophils in Leishmaniasis transmitted by sand ies. Science 321:97074
131. Charmoy M, Brunner-Agten S, Aebischer D, Auderset F, Launois P, et al. 2010. Neutrophil-derived
CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation
in resistant mice. PLoS Pathog. 6:e1000755
132. Bennouna S, Bliss SK, Curiel TJ, Denkers EY. 2003. Cross-talk in the innate immune system: neutrophils
instruct recruitment and activation of dendritic cells during microbial infection. J. Immunol. 171:605258
133. Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JE. 2008. Use of Ly6G-specic monoclonal
antibody to deplete neutrophils in mice. J. Leukoc. Biol. 83:6470
134. Blomgran R, Ernst JD. 2011. Lung neutrophils facilitate activation of naive antigen-specic CD4+
T cells during Mycobacterium tuberculosis infection. J. Immunol. 186:711019
135. Yang CW, Strong BS, Miller MJ, Unanue ER. 2010. Neutrophils inuence the level of antigen presentation during the immune response to protein antigens in adjuvants. J. Immunol. 185:292734
136. Chtanova T, Schaeffer M, Han SJ, van Dooren GG, Nollmann M, et al. 2008. Dynamics of neutrophil
migration in lymph nodes during infection. Immunity 29:48796
137. Sporri R, Joller N, Hilbi H, Oxenius A. 2008. A novel role for neutrophils as critical activators of NK
cells. J. Immunol. 181:712130
138. Costantini C, Cassatella MA. 2011. The defensive alliance between neutrophils and NK cells as a novel
arm of innate immunity. J. Leukoc. Biol. 89:22133

IY30CH19-Zychlinsky
486
Amulic et al.

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
139. Costantini C, Calzetti F, Perbellini O, Micheletti A, Scarponi C, et al. 2011. Human neutrophils interact
with both 6-sulfo LacNAc+ DC and NK cells to amplify NK-derived IFN: role of CD18, ICAM-1,
and ICAM-3. Blood 117:167786
140. Muller I, Munder M, Kropf P, Hansch GM. 2009. Polymorphonuclear neutrophils and T lymphocytes:
strange bedfellows or brothers in arms? Trends Immunol. 30:52230
141. Romani L, Mencacci A, Cenci E, Spaccapelo R, Del Sero G, et al. 1997. Neutrophil production of IL-12
and IL-10 in candidiasis and efcacy of IL-12 therapy in neutropenic mice. J. Immunol. 158:534956
142. Tateda K, Moore TA, Deng JC, Newstead MW, Zeng X, et al. 2001. Early recruitment of neutrophils
determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia.
J. Immunol. 166:335561
143. Scapini P, Bazzoni F, Cassatella MA. 2008. Regulation of B-cell-activating factor (BAFF)/B lymphocyte
stimulator (BLyS) expression in human neutrophils. Immunol. Lett. 116:16
144. Huard B, McKee T, Bosshard C, Durual S, Matthes T, et al. 2008. APRIL secreted by neutrophils
binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa. J. Clin. Investig.
118:288795
145. Ellis TN, Beaman BL. 2004. Interferon- activation of polymorphonuclear neutrophil function.
Immunology 112:212
146. Ouyang W, Kolls JK, Zheng Y. 2008. The biological functions of T helper 17 cell effector cytokines in
inammation. Immunity 28:45467
147. Munder M, Schneider H, Luckner C, Giese T, Langhans CD, et al. 2006. Suppression of T-cell functions
by human granulocyte arginase. Blood 108:162734
148. Beauvillain C, Delneste Y, Scotet M, Peres A, Gascan H, et al. 2007. Neutrophils efciently cross-prime
naive T cells in vivo. Blood 110:296573
149. Gosselin EJ, Wardwell K, Rigby WF, Guyre PM. 1993. Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-, and IL-3.
J. Immunol. 151:148290
150. Mudzinski SP, Christian TP, Guo TL, Cirenza E, Hazlett KR, Gosselin EJ. 1995. Expression of HLADR (major histocompatibility complex class II) on neutrophils from patients treated with granulocytemacrophage colony-stimulating factor for mobilization of stem cells. Blood 86:245253
151. Reinisch W, Lichtenberger C, Steger G, Tillinger W, Scheiner O, et al. 2003. Donor dependent,
interferon- induced HLA-DR expression on human neutrophils in vivo. Clin. Exp. Immunol. 133:476
84
152. Fanger NA, Liu C, Guyre PM, Wardwell K, ONeil J, et al. 1997. Activation of human T cells by major
histocompatability complex class II expressing neutrophils: proliferation in the presence of superantigen,
but not tetanus toxoid. Blood 89:412835
153. Radsak M, Iking-Konert C, Stegmaier S, Andrassy K, Hansch GM. 2000. Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted
antigen-dependent induction of T-cell proliferation. Immunology 101:52130
154. Culshaw S, Millington OR, Brewer JM, McInnes IB. 2008. Murine neutrophils present Class II restricted
antigen. Immunol. Lett. 118:4954
155. Kennedy AD, DeLeo FR. 2009. Neutrophil apoptosis and the resolution of infection. Immunol. Res.
43:2561
156. Altznauer F, Martinelli S, Youse S, Thurig C, Schmid I, et al. 2004. Inammation-associated cell
cycleindependent block of apoptosis by survivin in terminally differentiated neutrophils. J. Exp. Med.
199:134354
157. Rossi AG, Sawatzky DA, Walker A, Ward C, Sheldrake TA, et al. 2006. Cyclin-dependent kinase
inhibitors enhance the resolution of inammation by promoting inammatory cell apoptosis. Nat. Med.
12:105664
158. Witko-Sarsat V, Mocek J, Bouayad D, Tamassia N, Ribeil JA, et al. 2010. Proliferating cell nuclear
antigen acts as a cytoplasmic platform controlling human neutrophil survival. J. Exp. Med. 207:263145
159. Arita M, Clish CB, Serhan CN. 2005. The contributions of aspirin and microbial oxygenase to the
biosynthesis of anti-inammatory resolvins: novel oxygenase products from omega-3 polyunsaturated
fatty acids. Biochem. Biophys. Res. Commun. 338:14957
487
ARI
17 February 2012
13:38
160. Vance RE, Hong S, Gronert K, Serhan CN, Mekalanos JJ. 2004. The opportunistic pathogen Pseudomonas
aeruginosa carries a secretable arachidonate 15-lipoxygenase. Proc. Natl. Acad. Sci. USA 101:213539
161. Devchand PR, Arita M, Hong S, Bannenberg G, Moussignac RL, et al. 2003. Human ALX receptor
regulates neutrophil recruitment in transgenic mice: roles in inammation and host defense. FASEB J.
17:65259
162. Arita M, Ohira T, Sun YP, Elangovan S, Chiang N, Serhan CN. 2007. Resolvin E1 selectively interacts
with leukotriene B4 receptor BLT1 and ChemR23 to regulate inammation. J. Immunol. 178:391217
163. Schwab JM, Chiang N, Arita M, Serhan CN. 2007. Resolvin E1 and protectin D1 activate inammationresolution programmes. Nature 447:86974
164. Spite M, Norling LV, Summers L, Yang R, Cooper D, et al. 2009. Resolvin D2 is a potent regulator of
leukocytes and controls microbial sepsis. Nature 461:128791
165. Xu ZZ, Zhang L, Liu T, Park JY, Berta T, et al. 2010. Resolvins RvE1 and RvD1 attenuate inammatory
pain via central and peripheral actions. Nat. Med. 16:59297
166. Krishnamoorthy S, Recchiuti A, Chiang N, Yacoubian S, Lee CH, et al. 2010. Resolvin D1 binds human
phagocytes with evidence for proresolving receptors. Proc. Natl. Acad. Sci. USA 107:166065
167. Oh SF, Pillai PS, Recchiuti A, Yang R, Serhan CN. 2011. Pro-resolving actions and stereoselective
biosynthesis of 18S E-series resolvins in human leukocytes and murine inammation. J. Clin. Investig.
121:56981
168. Ariel A, Fredman G, Sun YP, Kantarci A, Van Dyke TE, et al. 2006. Apoptotic neutrophils and T cells
sequester chemokines during immune response resolution through modulation of CCR5 expression.
Nat. Immunol. 7:120916
169. Filep JG, Zouki C, Petasis NA, Hachicha M, Serhan CN. 1999. Anti-inammatory actions of lipoxin
A(4) stable analogs are demonstrable in human whole blood: modulation of leukocyte adhesion molecules
and inhibition of neutrophil-endothelial interactions. Blood 94:413242
170. Jozsef L, Zouki C, Petasis NA, Serhan CN, Filep JG. 2002. Lipoxin A4 and aspirin-triggered 15-epilipoxin A4 inhibit peroxynitrite formation, NF-B and AP-1 activation, and IL-8 gene expression in
human leukocytes. Proc. Natl. Acad. Sci. USA 99:1326671
171. Godson C, Mitchell S, Harvey K, Petasis NA, Hogg N, Brady HR. 2000. Cutting edge: lipoxins rapidly
stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages.
J. Immunol. 164:166367
172. Snelgrove RJ, Jackson PL, Hardison MT, Noerager BD, Kinloch A, et al. 2010. A critical role for LTA4H
in limiting chronic pulmonary neutrophilic inammation. Science 330:9094
173. Tanaka D, Kagari T, Doi H, Shimozato T. 2006. Essential role of neutrophils in anti-type II collagen
antibody and lipopolysaccharide-induced arthritis. Immunology 119:195202
174. Chen M, Lam BK, Kanaoka Y, Nigrovic PA, Audoly LP, et al. 2006. Neutrophil-derived leukotriene B4
is required for inammatory arthritis. J. Exp. Med. 203:83742
175. Kim ND, Chou RC, Seung E, Tager AM, Luster AD. 2006. A unique requirement for the leukotriene
B4 receptor BLT1 for neutrophil recruitment in inammatory arthritis. J. Exp. Med. 203:82935
176. Chou RC, Kim ND, Sadik CD, Seung E, Lan Y, et al. 2010. Lipid-cytokine-chemokine cascade drives
neutrophil recruitment in a murine model of inammatory arthritis. Immunity 33:26678
177. Virchow R. 1862 /1863. Die krankhaften Geschwulste:
30 Vorlesungen gehalten wahrend des Wintersemesters

1862/1863. Berlin: Hirschwald
178. Shacter E, Beecham EJ, Covey JM, Kohn KW, Potter M. 1988. Activated neutrophils induce prolonged
DNA damage in neighboring cells. Carcinogenesis 9:2297304
179. Dizdaroglu M, Olinski R, Doroshow JH, Akman SA. 1993. Modication of DNA bases in chromatin of
intact target human cells by activated human polymorphonuclear leukocytes. Cancer Res. 53:126972
180. Gregory AD, McGarry Houghton A. 2011. Tumor-associated neutrophils: new targets for cancer therapy. Cancer Res. 71:241116
181. Bellocq A, Antoine M, Flahault A, Philippe C, Crestani B, et al. 1998. Neutrophil alveolitis in bronchioloalveolar carcinoma: induction by tumor-derived interleukin-8 and relation to clinical outcome.
Am. J. Pathol. 152:8392

IY30CH19-Zychlinsky
488
Amulic et al.

IY30CH19-Zychlinsky
ARI
17 February 2012
13:38
182. Jensen HK, Donskov F, Marcussen N, Nordsmark M, Lundbeck F, von der Maase H. 2009. Presence of
intratumoral neutrophils is an independent prognostic factor in localized renal cell carcinoma. J. Clin.
Oncol. 27:470917
183. Ji H, Houghton AM, Mariani TJ, Perera S, Kim CB, et al. 2006. K-ras activation generates an inammatory response in lung tumors. Oncogene 25:210512
184. Pekarek LA, Starr BA, Toledano AY, Schreiber H. 1995. Inhibition of tumor growth by elimination of
granulocytes. J. Exp. Med. 181:43540
185. Shojaei F, Singh M, Thompson JD, Ferrara N. 2008. Role of Bv8 in neutrophil-dependent angiogenesis
in a transgenic model of cancer progression. Proc. Natl. Acad. Sci. USA 105:264045
186. Huh SJ, Liang S, Sharma A, Dong C, Robertson GP. 2010. Transiently entrapped circulating tumor
cells interact with neutrophils to facilitate lung metastasis development. Cancer Res. 70:607182
187. Fridlender ZG, Sun J, Kim S, Kapoor V, Cheng G, et al. 2009. Polarization of tumor-associated neutrophil phenotype by TGF-: N1 versus N2 TAN. Cancer Cell 16:18394
188. De Santo C, Arscott R, Booth S, Karydis I, Jones M, et al. 2010. Invariant NKT cells modulate the
suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A. Nat. Immunol.
11:103946
189. Gabrilovich DI, Nagaraj S. 2009. Myeloid-derived suppressor cells as regulators of the immune system.
Nat. Rev. Immunol. 9:16274
190. Zea AH, Rodriguez PC, Atkins MB, Hernandez C, Signoretti S, et al. 2005. Arginase-producing myeloid
suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion. Cancer Res. 65:304448
191. Rodriguez PC, Ernstoff MS, Hernandez C, Atkins M, Zabaleta J, et al. 2009. Arginase I-producing
myeloid-derived suppressor cells in renal cell carcinoma are a subpopulation of activated granulocytes.
Cancer Res. 69:155360
192. Kusmartsev S, Nefedova Y, Yoder D, Gabrilovich DI. 2004. Antigen-specic inhibition of CD8+
T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species. J. Immunol.
172:98999
193. Raza K, Scheel-Toellner D, Lee CY, Pilling D, Curnow SJ, et al. 2006. Synovial uid leukocyte apoptosis
is inhibited in patients with very early rheumatoid arthritis. Arthritis Res. Ther. 8:R120
194. Harper L, Cockwell P, Adu D, Savage CO. 2001. Neutrophil priming and apoptosis in anti-neutrophil
cytoplasmic autoantibody-associated vasculitis. Kidney Int. 59:172938
195. Ren Y, Tang J, Mok MY, Chan AW, Wu A, Lau CS. 2003. Increased apoptotic neutrophils and
macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus
erythematosus. Arthritis Rheum. 48:288897
196. Brouwer E, Huitema MG, Mulder AH, Heeringa P, van Goor H, et al. 1994. Neutrophil activation in
vitro and in vivo in Wegeners granulomatosis. Kidney Int. 45:112031
197. Hacbarth E, Kajdacsy-Balla A. 1986. Low density neutrophils in patients with systemic lupus erythematosus, rheumatoid arthritis, and acute rheumatic fever. Arthritis Rheum. 29:133442
198. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, et al. 2003. Interferon and granulopoiesis signatures
in systemic lupus erythematosus blood. J. Exp. Med. 197:71123
199. Yasutomo K, Horiuchi T, Kagami S, Tsukamoto H, Hashimura C, et al. 2001. Mutation of DNASE1
in people with systemic lupus erythematosus. Nat. Gen. 28:31314
200. Chen G, Zhuchenko O, Kuspa A. 2007. Immune-like phagocyte activity in the social amoeba. Science
317:67881
201. Ribeiro C, Brehelin M. 2006. Insect haemocytes: What type of cell is that? J. Insect Physiol. 52:41729
202. Martin JS, Renshaw SA. 2009. Using in vivo zebrash models to understand the biochemical basis of
neutrophilic respiratory disease. Biochem. Soc. Trans. 37:83037
203. Robert J, Ohta Y. 2009. Comparative and developmental study of the immune system in Xenopus. Dev.
Dyn. 238:124970
204. Hawkey CM. 1985. Analysis of hematologic ndings in healthy and sick adult chimpanzees
(Pan troglodytes). J. Med. Primatol. 14:32743
489
IY30-Frontmatter
ARI
17 February 2012
11:21
Annual Review of
Immunology
Contents
Volume 30, 2012

Decisions About Dendritic Cells: Past, Present, and Future

Ralph M. Steinman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
The Basel Institute for Immunology
Fritz Melchers p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p23
Regulation of Immune Responses by mTOR
Jonathan D. Powell, Kristen N. Pollizzi, Emily B. Heikamp,
and Maureen R. Horton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p39
Sphingosine-1-Phosphate and Lymphocyte Egress from Lymphoid Organs
Jason G. Cyster and Susan R. Schwab p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p69
Selection of Self-Reactive T Cells in the Thymus
Gretta L. Stritesky, Stephen C. Jameson, and Kristin A. Hogquist p p p p p p p p p p p p p p p p p p p p p p p95
Adaptive Immunity to Fungi
Marcel Wuthrich,
George S. Deepe, Jr., and Bruce Klein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 115

Microbial Translocation Across the GI Tract
Jason M. Brenchley and Daniel C. Douek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 149
The Response to and Repair of RAG-Mediated DNA Double-Strand Breaks
Beth A. Helmink and Barry P. Sleckman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 175
VLR-Based Adaptive Immunity
Thomas Boehm, Nathanael McCurley, Yoichi Sutoh, Michael Schorpp,
Masanori Kasahara, and Max D. Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 203
Immune Regulatory Function of B Cells
Claudia Mauri and Anneleen Bosma p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221
Lung Dendritic Cells in Respiratory Viral Infection and Asthma: From
Protection to Immunopathology
Bart N. Lambrecht and Hamida Hammad p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 243
Tolerance of Infections
Janelle S. Ayres and David S. Schneider p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 271
microRNA Regulation of Inammatory Responses
Ryan M. OConnell, Dinesh S. Rao, and David Baltimore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 295
ix
IY30-Frontmatter
ARI
17 February 2012
11:21
Reex Principles of Immunological Homeostasis

Ulf Andersson and Kevin J. Tracey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 313
Chromatin Topology and the Regulation of Antigen Receptor Assembly
Claudia Bossen, Robert Mansson, and Cornelis Murre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 337
Siglecs and Immune Regulation
Shiv Pillai, Ilka Arun Netravali, Annaiah Cariappa, and Hamid Mattoo p p p p p p p p p p p p p 357
Monogenic Autoimmunity
Mickie H. Cheng and Mark S. Anderson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 393

Germinal Centers
Gabriel D. Victora and Michel C. Nussenzweig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429
Neutrophil Function: From Mechanisms to Disease
Borko Amulic, Christel Cazalet, Garret L. Hayes, Kathleen D. Metzler,
and Arturo Zychlinsky p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 459
Signaling by Myeloid C-Type Lectin Receptors in Immunity and
Homeostasis
David Sancho and Caetano Reis e Sousa p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 491
Regulatory T Cells: Mechanisms of Differentiation and Function
Steven Z. Josefowicz, Li-Fan Lu, and Alexander Y. Rudensky p p p p p p p p p p p p p p p p p p p p p p p p p p 531
Pathogenesis of Human B Cell Lymphomas
Arthur L. Shaffer III, Ryan M. Young, and Louis M. Staudt p p p p p p p p p p p p p p p p p p p p p p p p p p p 565
Autophagy and the Immune System
Petric Kuballa, Whitney M. Nolte, Adam B. Castoreno, and Ramnik J. Xavier p p p p p p p 611
Innate Lymphoid Cells: Emerging Insights in Development, Lineage
Relationships, and Function
Hergen Spits and Tom Cupedo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 647
Cancer and Inammation: An Old Intuition with Rapidly Evolving New
Concepts
Giorgio Trinchieri p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 677
Transcriptional and Epigenetic Control of T Helper Cell Specication:
Molecular Mechanisms Underlying Commitment and Plasticity
Yuka Kanno, Golnaz Vahedi, Kiyoshi Hirahara, Kentner Singleton,
and John J. OShea p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 707
Induced CD4+ Foxp3+ Regulatory T Cells in Immune Tolerance
Angelina M. Bilate and Juan J. Lafaille p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 733
The Microbiome in Infectious Disease and Inammation
Kenya Honda and Dan R. Littman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 759
Contents

Neutrophil Function From Mechanism To Disease

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Neutrophil Function From Mechanism To Disease

Uploaded by

Copyright:

Available Formats

IY30CH19-Zychlinsky

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

Click here for quick links to

Annu. Rev. Immunol. 2012. 30:45989

First published online as a Review in Advance on

inammation, antimicrobial, granule, phagocytosis, NET

The Annual Review of Immunology is online at

This articles doi:

All authors contributed equally to the work and

Neutrophils are the most abundant white blood cells in circulation,

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

A NATURAL HISTORY OF NEUTROPHILS

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

Unfortunately, in vivo studies of neutrophil

infection. Indeed, the number of neutrophils

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

cells. Ultimately, upon arriving at the infection

family kinases, Syk, phosphoinositide 3-kinase

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

subsequent to degranulation; however, conclusive experimental evidence for this is lacking.

point of high chemoattractant concentration,

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

parsed by the complex neutrophilic signaling

NEUTROPHILS AND THE

neutrophils possess an array of toxic weapons

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

Granules and Degranulation

needs of neutrophils. Granules are, however,

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

granule subset has been traditionally associated

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

components also serve as signaling molecules

whereas others may be redundant. One of the

Table 1 Mechanism of action of neutrophil antimicrobial proteins

Permeabilize membrane bilayers containing negatively charged

Increase bacterial permeability and hydrolysis of bacterial

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

Degrades bacterial cell wall

Mechanism independent of a proteolytic activity by binding to the

Neutrophil elastase (NE),

Cleaves bacterial virulence factors and outer membrane

Mechanism independent of a proteolytic activity by binding to the

Metal chelator proteins

Alters bacterial growth by binding to iron, an essential bacterial

Alters bacterial growth by sequestering manganese and zinc

antimicrobials, is essential for designing appropriate in vitro conditions to probe mechanisms

from larger proteins, and in addition to their

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

scaffold for DNA and as antimicrobials, their

Reactive Oxygen Species

www.annualreviews.org Neutrophil Functions

inammation, which explains why CGD

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

(73, 74). Furthermore, superoxide generation

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

maturation process. Our understanding of

importance of phagocytosis in the innate

Neutrophil Extracellular Traps

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

NET formation, such as NADPH oxidase,

Monocytes and Macrophages

Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

microbicidal activity (129). The circuitous