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Chariteplatz 1, 10117 Berlin, Germany; email: amulic@mpiib-berlin.mpg.de,

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Annu. Rev. Immunol. 2012. 30:45989 Keywords

First published online as a Review in Advance on inammation, antimicrobial, granule, phagocytosis, NET
January 3, 2012

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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 in-
tractable 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 experi-
mental 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 in-
excusable disinterest in the white blood cell,
began to utilize newly developed cell-staining
techniques to examine subpopulations of leuko-
cytes. His experimentation led to a new appreci-
ation 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 re-
tain neutral dyes, the neutrophil (1) (see also
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
the sidebar, A Natural History of Neutrophils).
The function of neutrophils was initially
shrouded in considerable mystery; their con-
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spicuous 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 mi-
crobes for resources. Metchnikoff s seminal theory of cellular
immunity was based on comparative embryology and observa-
tions of phagocytes in various simple organisms, including the mi-
croscopic 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 nu-
cleus 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 in-
terested 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 diges-
tion. Remarkably, this prescient view of neu-
trophil 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 polymor-
phonuclear leukocytes (or PMNs), a title that
still enjoys frequent use and that is used inter-
changeably with neutrophil throughout this re-
view. 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 fam-
ily whose hallmark is the presence of granules,
unique storage structures important in antimi-
crobial 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 re-
sponse 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; unfor-
tunately, their importance has often been over-
shadowed by breakthroughs in the study of the
adaptive immune response (4). Admittedly, this
situation is exacerbated by neutrophils notori-
ous experimental intractability: They exhibit a
short life span and are terminally differentiated,
preventing growth in tissue culture. The stan-
dard tools of molecular biology, such as trans-
fection 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. Fur-
thermore, 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 stud-
ies should, therefore, be carefully interpreted.
 IY30CH19-Zychlinsky ARI 17 February 2012 13:38
Unfortunately, in vivo studies of neutrophil
function also raise concerns. Mouse neu-
trophils, 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 neu-
trophil and its functions. The extensive nature
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
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
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emphasizing recent advances in neutrophil re-
search 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 in-
struction 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 syn-
thesize 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 struc-
tures. The content of these structures is dic-
tated 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 in-
dividuals: 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 hypoth-
esis 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 in-
active cellular bystander to a fully activated
microbial killing machine? This transition
process, during which the neutrophil inte-
grates 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 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
Selectins:
transmembrane weapons. The initiation of these processes oc-
glycoproteins that curs in the bloodstream, where the neutrophil
mediate cell adhesion acts as a monitor for host distress, patrolling
via binding to sugar vessels and vigilantly seeking out indications of
moieties
an incipient inammatory response.
Integrins:
transmembrane
receptors that mediate NEUTROPHIL ACTIVATION
attachment to the
extracellular matrix, as At inammatory sites, bacterial-derived and
well as direct cell-cell host-produced inammatory signals are
interaction and
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
abundant; these compounds stimulate the
signaling
endothelial cells near the inammatory site.
Oxidative/respiratory These stimulants, such as the bacterial-derived
burst: a rapid increase
lipopolysaccharide (LPS) and fMLP, as well
by Harvard University on 09/10/13. For personal use only.
in oxygen
consumption upon as the classical chemoattractants and cytokines
neutrophil activation tumor necrosis factor (TNF)-, interleukin
due to production of (IL)-1, and IL-17, prompt endothelial cells to
ROS by the NADPH produce adhesion molecules on their luminal
oxidase
side: the P-selectins, E-selectins, and several
members of the integrin superfamily, the
ICAMs (5). As neutrophils traverse the circu-
latory 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 constitu-
tively expressed proteins are critical for recog-
nition 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 engage-
ment of PSGL-1 and L-selectin on neutrophils
activates a variety of kinases, including Src
462 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 adhe-
sion 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 lamel-
lipodium 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 trans-
migration 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, conclu-
sive 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 chemoat-
tractants and inammatory stimulants, both
host derived and of pathogenic origin. These
compounds will now be the primary dictators
of neutrophil behavior and assume respon-
sibility for initiating the concluding steps of
neutrophil activation. In the interstitial space,
the neutrophil follows chemotactic gradients
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
toward the invading microbes, pursuing host-
produced cytokines (e.g., IL-8) and, in parallel,
pathogen-derived chemoattractants (e.g.,
fMLP). During this process, these chemoat-
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tractants 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 sig-
naling cascade dominated by the MAPK/ERK
pathway (22, 23). Downstream molecules
prompt assembly of the oxidative burst ma-
chinery, 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 mi-
crobes (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 chemoattrac-
tants 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 ac-
tivators, 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 pre-
viously encountered ligands. Stimulation of the
neutrophil by a chemoattractant often results
in endocytosis of the corresponding receptor,
thus leading to a desensitization of the neu-
trophil 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 signal-
ing are incompletely understood. Regardless,
the end result of this signaling cacophony is
unambiguous: The neutrophil begins to imple-
ment its regime of microbial killing, executing
programs of phagocytosis, degranulation, and
NETosis (i.e., the process of setting neutrophil
www.annualreviews.org Neutrophil Functions 463
 IY30CH19-Zychlinsky ARI 17 February 2012 13:38

extracellular traps) (see the section on Neu-
trophils and the Elimination of Microbes,
below).
The initiation of these microbicidal actions
indicates the nal stage of the neutrophils
journey through the activation process. How-
ever, 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.
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
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).

In fact, one could be (erroneously) led to

believe that neutrophil activation refers only to
NEUTROPHILS AND THE
direct stimulation of the oxidative burst, as this
ELIMINATION OF MICROBES
has been the canonical in vitro activation assay The basic instruction set of the activated
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for decades. This is, however, an oversimpli- neutrophil is both effective and ruthless in
ed view of a complex process. The myriad its simplicity: (1) kill microbes, (2) do no
interactions that occur during a neutrophils harm to the host, and (3) when in doubt, see
journey toward an inammatory site must be rule 1. To fulll this antimicrobial agenda,

a Capture b Rolling c Firm adhesion

Neutrophil

Integrin
P-selectin and ICAM
PSGL-1, E-selectin
L-selectin

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
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
needs of neutrophils. Granules are, however,
far more than just latent repository organelles
for dangerous substances; they are active and in-
Inflammation:
dispensable participants in almost all neutrophil recruitment and
activities during inammation. activation of immune
As mentioned above, there are three cells upon infection or
fundamental types of granules in neutrophils injury; when
uncontrolled it leads to
(Figure 2). Azurophilic granules (also known
tissue damage
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

The neutrophil must safely transport a plethora
of dangerous substances through the blood-
stream and then correctly deploy them at the
appropriate time. Therefore, it comes as no
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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

surprise that a specialty storage organelle has protein (BPI), and a number of serine proteases:
evolved in neutrophils: the granule. Expect- neutrophil elastase (NE), proteinase 3 (PR3),
edly, these structures are replete with speci- and cathepsin G (CG) (34). As such, these
cally tuned mechanics that address the unique granules are brimming with antimicrobial

Primary Secondary Tertiary Secretory

Granule type (azurophilic) (specific) (gelatinase) vesicles

Stage of Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cell

formation PMN

Degranulation
propensity

Characteristic Lysozyme Complement receptor 1

proteins
Myeloperoxidase Lactoferrin FcRIII

Elastase Gelatinase

Defensin

Other Cathepsin G, PR3, Gp91phox/p22phox, Gp91phox/p22phox, Gp91phox/p22phox,

proteins BPI, azurocidin, CD11b, collagenase, CD11b, MMP25, CD11b, MMP25, C1q-R,
sialidase, hCAP18, NGAL, B12BP, arginase-1, FPR, alkaline
-glucuronidase SLPI, haptoglobin, 2-microglobulin, phosphatase, CD10,
pentraxin 3, CRISP3 CD13, CD14,
oroscomucoid, plasma proteins
2-microglobulin,
heparanase, CRISP3

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.

www.annualreviews.org Neutrophil Functions 465

 IY30CH19-Zychlinsky
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
by Harvard University on 09/10/13. For personal use only.
ARI 17 February 2012 13:38
compounds and function as a primary reposi-
tory 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 char-
acterized 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 pre-
dominantly 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 mem-
brane, thus altering its molecular composition
(6). The different classes of granules demon-
strate varying propensities for mobilization in
response to inammatory signals: Azurophilic
granules are the most difcult to mobilize, fol-
lowed by specic granules, gelatinase granules,
and nally, secretory vesicles (3741). The
underlying mechanisms for this differential
mobilization are not entirely understood, al-
though regulation of intracellular calcium levels
appears to play a salient role (32, 39). Because
of this varying mobilization propensity, each
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 chemoattrac-
tants induces mobilization of secretory vesi-
cles, whose membranes are rich in key factors
necessary for continued activation of the neu-
trophil, including, among others, the 2 inte-
grins, 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 ex-
ternal environment. This results in the transi-
tion to rm adhesion, mediated by 2 integrin
interaction with the endothelium. As they pro-
ceed through the endothelium, neutrophils are
exposed to further activation signals that initiate
mobilization of gelatinase granules, thereby re-
leasing 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 acti-
vation of the neutrophil ensues, prompting
initiation of the oxidative burst and mobiliza-
tion of the azurophilic and specic granules.
These granules either fuse with the phagosome
(see section on Phagocytosis, below), con-
tributing to the antimicrobial activities of this
compartment, or fuse with the plasma mem-
brane, 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 com-
plex 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 in-
ammatory site and produces an environment
inhospitable to invading pathogens.
The release of granular proteins during de-
granulation 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 evi-
dence suggesting this does seem to be the case:
Granule proteins from neutrophils, including
PR3 and azurocidin, can induce monocyte re-
cruitment. 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 micro-
bicidal effects. In such cases, the granule pro-
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
teins would instead operate as signaling and re-
cruitment factors (see section on Neutrophils
in Immune Cell Cross Talk, below).
By necessity, most data on neutrophil
by Harvard University on 09/10/13. For personal use only.
degranulation and its effects on neutrophil ac-
tivity 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 chal-
lenging. The diversity of antimicrobials sug-
gests 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 con-
ditions that mimic an infection site. Therefore,
testing the relevance of antimicrobials in vivo
is essential. This is, however, particularly chal-
lenging; ablation of a single antimicrobial gene
may only subtly affect immune defense. In ad-
dition, much biochemical identication of neu-
trophil antimicrobials has been performed in
rabbits and humans, species with abundant neu-
trophils. Mice, which are genetically tractable,
have neutrophils that function differently from
those of other species. Indeed, as already men-
tioned, mice lack the genes for some antimicro-
bials identied in humans. Interestingly, there
are few clinically relevant innate immune de-
ciencies that directly link antimicrobial activ-
ity 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 antimicro-
bials: (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 exe-
cuted at high antimicrobial concentrations to
obtain maximal microbial killing in the shortest
possible time, it is unclear whether this disrup-
tion 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 phago-
some. This information, as well as information
about the synergistic interactions of different
www.annualreviews.org Neutrophil Functions 467
 IY30CH19-Zychlinsky ARI 17 February 2012 13:38

Table 1 Mechanism of action of neutrophil antimicrobial proteins

Antimicrobial peptide Antimicrobial mechanisma
Cationic antimicrobial peptides
-defensins (HNP-1, HNP-2,  Permeabilize membrane bilayers containing negatively charged
HNP-3, HNP-4) 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
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

Proteinase 3 (PR3) Mechanism independent of a proteolytic activity by binding to the

bacterial membrane
Neutrophil elastase (NE),  Cleaves bacterial virulence factors and outer membrane
cathepsin G (CG) proteins
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 Mechanism independent of a proteolytic activity by binding to

the bacterial membrane
Azurocidin Mechanism independent of a proteolytic activity by binding to the
bacterial membrane
Metal chelator proteins
Lactoferrin  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
Calprotectin Alters bacterial growth by sequestering manganese and zinc

a
Only direct actions of neutrophil antimicrobial proteins on microbes are listed in the table.

antimicrobials, is essential for designing appro-
priate 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 multi-
ple disulde bonds and whose structures may
change under physiological conditions and
increase their activity (48). A surprising num-
ber 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 inam-
matory sites. Cathelicidins, including the well-
studied LL-37, are proteolytically processed
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

468 Amulic et al.

 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 antimi-
crobials 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 con-
tain several serine proteases (including PR3,
CG, and NE, collectively known as the serpro-
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
cidins) that exhibit differing specicities. They
are tightly regulated intra- and extracellularly
by serpins, indicating that their activity is
deployed under specic conditions. NE cleaves
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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 colony-
stimulating 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, azuro-
cidin, 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 chela-
tors are lactoferrin, rst identied in milk,
which binds preferentially to iron, and cal-
protectin (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 mis- Chronic
leading to think of ROS as a single entity be- granulomatous
cause they differ in their stability, reactivity, and disease (CGD):
permeability to membranes (62). However, all
caused by mutations
rendering the
ROS can modify and damage other molecules, NADPH oxidase
properties exploited by the host cell for signal- nonfunctional,
ing and antimicrobial action. characterized by
The NADPH oxidase complex assembles susceptibility to
on the phagosomal and plasma membranes
infection and
autoinammation
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 su-
peroxide 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 bac-
terium. 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 ox-
idase is also implicated in the regulation of
www.annualreviews.org Neutrophil Functions 469
 IY30CH19-Zychlinsky
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
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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 MPO-
decient individuals suffer from frequent or se-
vere 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., superox-
ide, hydrogen peroxide, hydroperoxyl radical,
peroxynitrite) can still be produced in the
neutrophil phagosome; hydroperoxyl radical is
predicted to be present at antimicrobial concen-
trations (65). However, there may be a broader
reason for this discrepancy. Modern technolo-
gies 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 inacti-
vation of their inhibitors or by direct oxidation
470 Amulic et al.
(73, 74). Furthermore, superoxide generation
leads to an ionic inux into the phagosome to
compensate for charge; this may activate gran-
ule proteases by releasing them from their pu-
tative 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 protein-
based 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 re-
move pathogens and cell debris. It is an active,
receptor-mediated process during which a par-
ticle is internalized by the cell membrane into
a vacuole called the phagosome. As with other
phagocytes, the mechanistic details of internal-
ization depend on the type of interaction be-
tween the neutrophil and the microorganism.
Interaction can be direct, through recognition
of PAMPs by pattern-recognition receptors, or
opsonin mediated. The latter mechanism is bet-
ter 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, acquir-
ing 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 neu-
trophils. 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 phagoso-
mal lumen occurs. Simultaneously, assembly
of the NADPH oxidase on the phagosomal
membrane allows ROS production, and jointly,
these two mechanisms create an environment
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
toxic to most pathogens. Neutrophil phago-
somal pH regulation also differs signicantly
from that observed in macrophages. While the
macrophage phagosome gradually acidies,
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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 contin-
uing 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 neu-
trophils. These strategies include interfering
with engulfment, modulating phagosome
maturation, and creating a more hospitable
intraphagosomal environment. The polysac-
charide 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 gran-
ule 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.
Autophagy: a process
in which cellular
Neutrophil Extracellular Traps
contents are degraded
Upon stimulation, neutrophils can undergo in lysosomes,
NETosis, an active form of cell death that especially in
conditions of nutrient
leads to release of decondensed chromatin into
scarcity and infection
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 mi-
crobes 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 re-
sponse 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 ac-
tivators 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 impli-
cated in NET formation (93), but further along
in the process, NE translocates from the gran-
ules to the nucleus and degrades histones, lead-
ing to chromatin decondensation (94). Histone
citrullination may also play a role in NET for-
mation, although this has not been conrmed
in primary human neutrophils (9597). Au-
tophagy 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
www.annualreviews.org Neutrophil Functions 471
 IY30CH19-Zychlinsky ARI 17 February 2012 13:38
NET formation, such as NADPH oxidase,
MPO, and NE, are also critical for other an-
timicrobial neutrophil functions. For now, the
Cystic fibrosis:
caused by defects in evidence for the relevance of NETs is indirect.
the CFTR ion On the one hand, bacteria that express DNases
transporter, as virulence factors disseminate more efciently
characterized by thick, in the host, which may point to evolutionary
sticky mucus and
pressure to avoid entrapment by NETs (99,
decreases in lung and
digestive function 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
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
patients neutrophils ex vivo (101). On the other
hand, the immune system has redundant mech-
anisms to ght infection, and it may be that
NETs are especially important under certain
by Harvard University on 09/10/13. For personal use only.
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 communica-
tion 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 condi-
tions 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 tran-
scriptional 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). Fur-
thermore, 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 proinam-
matory 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 mono-
cyte/macrophage lineage coordinate their
activities, leading to alternating waves of re-
cruitment 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
Activation and CD8+ Lymph node
IFN-
differentiation DC T cell
Crosspriming
ROS?
T cell Arginase? Neutrophil
IFN- Activation DC

DC
NK cell

IL-12 DC Antigen
presentation
CD4+
Macrophage Neutrophil T cell Th1
Activation Neutrophil
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org

Bacteria
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Neutrophil Monocyte DC
Blood

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, neu-
trophils recruit monocytes via several different
mechanisms. They express classical monocyte
chemoattractants such as CCL2 (MCP-1)
(122), CCL3 (MIP-1) (123), CCL20 (MIP-
3), 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 mod-
ulation of the activities of these chemokines
via proteolytic processing (reviewed in 128).
In addition to recruitment, neutrophils mod-
ulate 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 abate-
ment: 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

www.annualreviews.org Neutrophil Functions 473

 IY30CH19-Zychlinsky ARI 17 February 2012 13:38
Th1 response (131). Interestingly, activated
neutrophils can induce the maturation of DCs
in vitro through specic receptor-receptor
DC-SIGN:
dendritic cellspecic interactions between Mac-1 and DC-SIGN,
intercellular adhesion leading to local secretion of TNF- (120).
molecule-3-grabbing In this case, the reduced levels of cytokine
nonintegrin production foster specicity, as only proximal
Granulocyte DCs receive the maturation signal. A similar
receptor 1 (Gr1): activation model was earlier proposed for Tox-
the anti-Gr1 antibody
oplasma gondii (132). Neutrophil-activated DCs
RB6-8C5 reacts with
both Ly6G (specic produce the proinammatory cytokine IL-12
for neutrophils) and and induce proliferation of T cells (120, 132).
Ly6C (present on However, some of these experiments should
many immune cell
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
be interpreted cautiously because they are
types)
based on the injection of the anti-Gr1 antibody
Th17 cells: subset of (RB6), which depletes neutrophils but may also
T helper cells that
result in depletion of many other cell types in
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produce IL-17,
important in mice. The anti-Ly6G monoclonal antibody is
inammation and more specic and hence a better reagent for this
implicated in type of experiment (133). The crucial role of
autoimmunity neutrophils in DC activation was recently con-
rmed using anti-Ly6G antibody depletion: In
Mycobacterium tuberculosis infection, timely traf-
cking 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 possi-
ble 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 be-
havior 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 question-
able 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 produc-
tion 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, neu-
trophils 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 con-
tact (139). Additional in vitro interactions be-
tween neutrophils and NK cells are extensively
reviewed in Reference 138.
Lymphocytes
A surprising nding in recent years is the exten-
sive 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 neu-
trophil 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 expres-
sion 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 ef-
fects 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 suppres-
sion, 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+
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
T cell responses by cross-presenting exogenous
antigens in vivo. Using mice in which profes-
sional antigen-presenting cells do not express
functional MHC class I, Beauvillain et al. (148)
by Harvard University on 09/10/13. For personal use only.
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, neu-
trophils must be removed before they have
Ulcerative colitis: a
serious, detrimental effects on inamed tissues. type of inammatory
Resolution of inammation is an active process bowel disease
that limits further leukocyte inltration and characterized by ulcers
removes apoptotic cells from inamed sites. and tissue erosion in
the colon and rectum
This process is essential for maintenance of
tissue homeostasis and, if impeded, leads to
nonresolving inammation, a problematic
condition that contributes to many diseases.
Apoptosis and Clearance
Apoptosis is a central aspect of inammation
resolution. Once neutrophils have executed
their antimicrobial agenda, they die via a built-
in cell-death program. However, not only does
apoptosis reduce the number of neutrophils
present, it also produces signals that abro-
gate further neutrophil recruitment. Phagocy-
tosis of apoptotic neutrophils also reprograms
macrophages to adopt an anti-inammatory
phenotype.
Neutrophil death is inuenced by inamma-
tory 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 antiapo-
ptotic (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).
www.annualreviews.org Neutrophil Functions 475
 IY30CH19-Zychlinsky ARI 17 February 2012 13:38
Similarly, cyclin-dependent kinases func-
tion as prosurvival factors in neutrophils.
Pharmacological inhibition of these cell cycle
Wegeners
granulomatosis: regulators induce caspase-dependent apoptosis
vasculitis affecting the and block life-span extension by survival factors
lungs, nose, and (157). More recently, prosurvival effects were
kidneys; inammation also attributed to proliferating cell nuclear
leads to reduced blood
antigen (PCNA). This factor usually resides
ow, tissue
destruction, and in the nucleus, where it is involved in DNA
damage of vital organs replication, but in neutrophils, it associates
Prostaglandins and with procaspases in the cytosol and is thought
leukotrienes: lipids to prevent their activation. During apoptosis,
synthesized by PCNA is targeted for proteosomal degradation,
cyclooxygenases and
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
which correlates with an increase in caspase-3
5-lipoxygenase,
and caspase-8 activities. This mechanism is rel-
respectively, in the
arachidonic acid evant in Wegeners granulomatosis and sepsis,
pathway; have where stabilization of PCNA is associated with
by Harvard University on 09/10/13. For personal use only.
proinammatory resistance of neutrophils to apoptosis (158).
functions including Equally important for the resolution of in-
leukocyte recruitment
ammation is the proper removal of apoptotic
cells. This relies on the release of nd-me
signals at early stages of cell death, which at-
tract 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 anti-
inammatory 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 cyclooxy-
genase 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 par-
ticipate in the transcellular biosynthesis of lipid
mediators with anti-inammatory and prore-
solving 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 prore-
solving lipid mediators have been reviewed in
detail elsewhere (118). Interestingly, microor-
ganisms 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 lipoxin-
A4 receptor and the leukotriene B4 receptor
(BLT1) (161167). Ariel et al. (168) also pro-
posed an interesting mechanism of action for
lipoxins, resolvins, and protectins in clearing in-
ammatory sites. They showed that neutrophil
exposure to these lipids increases expression
of CCR5 on the surface of late apoptotic neu-
trophils, leading to efcient sequestration of the
chemoattractants CCL3 and CCL5. The se-
questration of these chemokines means they are
unavailable to recruit neutrophils to inamed
sites (168) (Figure 4). This mechanism com-
plements other anti-inammatory processes
in which chemokines are inactivated by neu-
trophil 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 neu-
trophils to endothelial cells (169, 170). Finally,
 IY30CH19-Zychlinsky ARI 17 February 2012 13:38

Initiation Resolution
of inflammation Leukotrienes Prostaglandins TNF- Lipoxins Resolvins Protectins IL-10 TGF- of inflammation

Platelets

Monocyte

Lipoxins
Neutrophil

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

Macrophage
Apoptotic
neutrophil Chemokines CCR5
IL-10
Leukotrienes TGF-
Prostaglandins PGE-2
by Harvard University on 09/10/13. For personal use only.

Chemokines Chemokine clearance

Microorganisms ?
TNF- NETotic
IL-6 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 Disorders Associated with

inhibiting ROS and peroxynitrite production,
NF-B activation, and IL-8 expression (170).
In addition to directly impacting neu-
trophil functions, lipid mediators promote
nonphlogistic (noninammatory) phagocyto-
sis of apoptotic neutrophils by monocytes
and macrophages. In the presence of anti-
inammatory lipids, engulfment of apoptotic
neutrophils is not accompanied by the release of
proinammatory mediators, as typically occurs
during macrophage activation. Instead, produc-
tion of the anti-inammatory cytokines TGF-
and IL-10 is increased (163, 171).
Nonresolved Inflammation
The failure of neutrophils to apoptose or mal-
functions in the removal of their apoptotic re-
mains result in chronic inammation. These
conditions lead to the accumulation of cyto-
toxic substances and are associated with severe Chronic obstructive
pathologies, including cystic brosis, chronic pulmonary disease
(COPD): lung disease
obstructive pulmonary disease (COPD), and
caused by noxious
rheumatoid arthritis (RA). The severity of in-
particles or gas, e.g.,
ammation often directly correlates with poor
tobacco smoking;
clinical outcome. inammation leads to
COPD is a major cause of death in indus- lung obstruction
trialized nations, where smoking is a prime

www.annualreviews.org Neutrophil Functions 477

 IY30CH19-Zychlinsky ARI 17 February 2012 13:38
instigator of this disease. A chronic neutrophil
inltration in the lungs of COPD patients
promotes tissue damage and organ dysfunc-
Rheumatoid arthritis
(RA): chronic tion. One of the key molecules controlling
inammatory disease the inammatory response in the lung is
that affects many leukotriene A4 hydrolase (LTA4H). This
tissues and organs but enzyme has two opposing activities. First, its
primarily synovial
hydrolase activity converts leukotriene A4 into
joints; severe
inammation causes leukotriene B4, a potent neutrophil chemoat-
deformity tractant and proinammatory agent. Second,
LTA4H is an aminopeptidase that inactivates
a specic neutrophil chemoattractant, the
proline-glycine-proline tripeptide (PGP), thus
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
conferring the enzyme with anti-inammatory
properties. Interestingly, tobacco smoke selec-
tively inhibits only the aminopeptidase activity
of LTA4H, promoting the accumulation of
by Harvard University on 09/10/13. For personal use only.
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 neu-
trophils and other immune cells discussed in
478 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 suc-
cessively 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 infec-
tion, 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 Ref-
erence 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 neu-
trophils and conrmed their tumorigenic role.
Moreover, the study showed that neutrophils in
the tumor microenvironment could, under cer-
tain circumstances, be induced to target their
cytotoxic arsenal at tumor cells, whose growth
they usually help to fuel. Pharmacological
inhibition of TGF- signaling led tumor-
associated neutrophils to assume a heightened
proinammatory state, causing a reduction in
tumor growth. These alternatively activated
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
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
by Harvard University on 09/10/13. For personal use only.
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 IL-
10, 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 im-
mature 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. MD-
SCs have characteristics of neutrophils, and in
mice, they are typically detected using the neu-
trophil 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 sur-
face markers as do activated neutrophils (190,
Acute-phase
191). MDSCs inhibit T cell proliferation by proteins: secreted by
limiting L-arginine availability via arginase and liver, concentration in
NOS activities, both of which use this amino plasma changes by
acid as a substrate (189, 191, 192). Furthermore, 25% or more during
inammation
MDSCs are strong producers of ROS, which
suppresses T cell responses (119, 192). Inter-
fering with the release of MDSCs or using drug
interventions to polarize neutrophil responses
in the tumor microenvironment could repre-
sent novel therapeutic strategies against cancer.
Autoimmunity
Deregulated neutrophil cell death and/or
clearance often accompanies autoimmune syn-
dromes (193195) and may play a major role
in disease pathogenesis, given that release of
proteolytic and cytotoxic molecules from neu-
trophils can trigger organ damage. Neutrophil
products act as both targets and mediators of
autoimmunity. MPO and PR3 are the main tar-
gets of antineutrophil cytoplasmic antibodies
(ANCA), autoantibodies directed against anti-
gens present in the cytoplasm of neutrophils.
Wegeners granulomatosis is consistently as-
sociated with the presence of ANCA. Further-
more, 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 acti-
vation 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 en-
vironment, 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. Autoan-
tibodies produced in SLE are predominantly
either ANCA or directed against chromatin.
Although neutrophils had long been suspected
www.annualreviews.org Neutrophil Functions 479
 IY30CH19-Zychlinsky ARI 17 February 2012 13:38
to be causative agents, their role in SLE patho-
genesis remained elusive. The recent discovery
of a link between SLE and NET formation
Vasculitis:
inammation of blood has helped to shed light on this quandary.
vessels 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 autoanti-
bodies may trigger a switch from apoptosis to
NETosis. Additional evidence suggesting a
Annu. Rev. Immunol. 2012.30:459-489. Downloaded from www.annualreviews.org
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
by Harvard University on 09/10/13. For personal use only.
studies have reported the presence of a particu-
lar 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
480 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 com-
ponents 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 verte-
brates to coordinate and execute one of the most
fundamental physiological responses: inam-
mation. 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: Uncon-
trolled neutrophil responses can exacerbate and
even cause autoimmune and inammatory dis-
eases. Many challenges remain in understand-
ing 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 instruc-
tions 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 strik-
ingly short life spans make them excellent mod-
els 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.

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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
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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,
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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
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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
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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

x Contents