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Neutrophil Function:
From Mechanisms to Disease
Borko Amulic, Christel Cazalet,
Garret L. Hayes, Kathleen D. Metzler,
and Arturo Zychlinsky∗
Department of Cellular Microbiology, Max Planck Institute for Infection Biology,
Charit´eplatz 1, 10117 Berlin, Germany; email:,,,,

Annu. Rev. Immunol. 2012. 30:459–89


First published online as a Review in Advance on
January 3, 2012

inflammation, antimicrobial, granule, phagocytosis, NET

The Annual Review of Immunology is online at


This article’s doi:
c 2012 by Annual Reviews.
All rights reserved

All authors contributed equally to the work and
are listed alphabetically.

Neutrophils are the most abundant white blood cells in circulation,
and patients with congenital neutrophil deficiencies suffer from severe
infections that are often fatal, underscoring the importance of these
cells in immune defense. In spite of neutrophils’ relevance in immunity,
research on these cells has been hampered by their experimentally intractable nature. Here, we present a survey of basic neutrophil biology,
with an emphasis on examples that highlight the function of neutrophils
not only as professional killers, but also as instructors of the immune
system in the context of infection and inflammatory disease. We focus
on emerging issues in the field of neutrophil biology, address questions
in this area that remain unanswered, and critically examine the experimental basis for common assumptions found in neutrophil literature.




17 February 2012


Annu. Rev. Immunol. 2012.30:459-489. Downloaded from
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In the late nineteenth century, Paul Ehrlich,
dissatisfied with what he considered an inexcusable disinterest in the white blood cell,
began to utilize newly developed cell-staining
techniques to examine subpopulations of leukocytes. His experimentation led to a new appreciation for the heterogeneity of white blood cells
and to the discovery of several novel leukocyte
subpopulations. Ehrlich named one of these
newly discovered cell types, characterized by a
“polymorphous nucleus” and a tendency to retain neutral dyes, the “neutrophil” (1) (see also
the sidebar, A Natural History of Neutrophils).
The function of neutrophils was initially
shrouded in considerable mystery; their conspicuous presence during infections led several
researchers to arrive hastily at a rather ironic
conclusion: They surmised that neutrophils
promote infection, serving as cellular shuttles
for bacteria (2). Their actual function, that of
antimicrobial actors in the immune response,
was eventually demonstrated conclusively by a
contemporary of Ehrlich, Elie Metchnikoff, an

Phagocytes are ancient cells that evolved to allow multicellular
organisms to thrive in the face of constant competition with microbes for resources. Metchnikoff ’s seminal theory of cellular
immunity was based on comparative embryology and observations of phagocytes in various simple organisms, including the microscopic crustacean Daphnia. Remarkably, even the slime mold
Dictyostelium discoideum has phagocytic cells that protect it from
infection (200). The short-lived neutrophil with a lobulated nucleus and granule-packed cytoplasm is a more recent evolutionary
adaptation. In insects, phagocytes are long lived and have round
nuclei. They do, however, produce hydrogen peroxide and carry
distinct classes of granules (201). Bony fish and frogs have bona
fide neutrophils that are functionally similar to mammalian ones
(202, 203). In both zebrafish and rodents, neutrophils are less
abundant than in humans, comprising only 15–20% of immune
cells. In chimpanzees, neutrophils account for more than 50% of
the differential blood count (204).


Amulic et al.

early and enthusiastic evolutionary biologist interested in the phagocytic capacity of cells.
Metchnikoff demonstrated that injury of
starfish embryos resulted in recruitment of
phagocytic cells to the site of injury (3). He
theorized (correctly) that these cells migrate to
injured sites and participate in microbe digestion. Remarkably, this prescient view of neutrophil action still aptly summarizes, more than
a century later, the basic role of neutrophils
in immunity. The uniquely lobulated nucleus
of the neutrophil also inspired Metchnikoff to
rename these cells: He called them polymorphonuclear leukocytes (or PMNs), a title that
still enjoys frequent use and that is used interchangeably with neutrophil throughout this review. Together with two other developmentally
related cell types, the eosinophils and basophils
(also discovered by Ehrlich), PMNs form the
granulocyte family of white blood cells, a family whose hallmark is the presence of “granules,”
unique storage structures important in antimicrobial functions (see section on Granules and
Degranulation, below).
Neutrophils were discovered at the dawn
of the immunological sciences; consequently,
elucidation of their role in the immune response has been an ongoing process stretching
over more than a century. We now know that
they are key components of the innate immune
response and vital in immune function; unfortunately, their importance has often been overshadowed by breakthroughs in the study of the
adaptive immune response (4). Admittedly, this
situation is exacerbated by neutrophils’ notorious experimental intractability: They exhibit a
short life span and are terminally differentiated,
preventing growth in tissue culture. The standard tools of molecular biology, such as transfection and RNA interference, are of little use
when applied to these cells, and immortalized
“neutrophil-like” cell lines rarely reflect the
functional diversification of neutrophils. Furthermore, neutrophil-like cells studied in the
isolation of a culture dish most certainly do not
mimic the complex biological reality in tissues
or circulation. Conclusions from in vitro studies should, therefore, be carefully interpreted.

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17 February 2012


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

infection. Indeed, the number of neutrophils
drastically increases during infection and some
diseases. Interestingly, neutrophils circulate
for only approximately 6–8 h and are among
the shortest-lived cells in the human body.
Although the reason for this short life is unclear,
it may ensure neutrophil integrity; this hypothesis is bolstered by observations that apoptosis
prevents the release of noxious molecules.
Still, the question of why evolution opted for
eliminating neutrophils quickly as opposed
to reducing leakage of their dangerous cargo
remains an unanswered and intriguing mystery.
Mature neutrophils emerge from the bone
marrow intent on pursuing one simple, yet
essential, question: Has host integrity been
compromised by potentially harmful invaders?
Should the answer prove to be “yes,” the
neutrophil must swiftly enact a carefully
choreographed process to locate, attack, and
destroy the potential threat. At its disposal is
an impressive arsenal of antimicrobial weapons
that are deadly, indiscriminate, and brutish in
their application. Although effective in their
destructive capacity, these weapons can prove
to be just as dangerous to the host cells as to
their intended targets, the microbial invaders.
Therefore, their deployment must be executed
with exquisite precision and timing, at locations
where they are both contained and effective.
How then does the neutrophil locate and
identify infections? How does it transition
at the correct time and place from an inactive cellular bystander to a fully activated
microbial killing machine? This transition
process, during which the neutrophil integrates a complex barrage of environmental
cues and translates them into specific 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 • Neutrophil Functions


two constitutively expressed proteins are critical for recognition of the endothelial inflammatory signals: the glycoprotein P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin (9. the neutrophil must negotiate a path through the endothelium into the underlying tissue. After selectin-mediated rolling. chemoattractants. a complex interaction between (a) the neutrophil integrins and their endothelial partners and (b) neutrophil surface proteins and various endothelial junction molecules results in transmigration through the endothelial junction (13). as well as direct cell-cell interaction and signaling Oxidative/respiratory burst: a rapid increase in oxygen consumption upon neutrophil activation due to production of ROS by the NADPH oxidase 462 17 February 2012 13:38 cells. What changes occur in the neutrophil at this early time point? The engagement of PSGL-1 and L-selectin on neutrophils activates a variety of kinases. Selectins: transmembrane glycoproteins that mediate cell adhesion via binding to sugar moieties Integrins: transmembrane receptors that mediate attachment to the extracellular matrix. This is followed by a characteristic “rolling” of neutrophils along the endothelium. including Src Amulic et al. patrolling vessels and vigilantly seeking out indications of an incipient inflammatory response. Upon arrival at an endothelial cell junction. 8). Rev.30:459-489. It is here that the complex activation cascade begins and the neutrophil commitment to microbial killing commences. The initiation of these processes occurs in the bloodstream. 14). 18).annualreviews. This cascade initiates a number of changes in neutrophil biology and sets the stage for integrin activation and firm adhesion. Upon random contact with the endothelium. the postcapillary venules. This integrin engagement. bacterial-derived and host-produced inflammatory signals are abundant. the cytoskeleton is rebuilt and targeted toward movement along chemotactic gradients. For personal use only. resulting in arrest of neutrophil rolling and firm adhesion. As the neutrophil rolls along the by Harvard University on 09/10/13. Now firmly adhered. interaction with selectins. prepares the neutrophil for its final chemotactic pursuit: The cell spreads. In a process dependent on β2 integrins and ICAMs. upon arriving at the infection site. as well as the classical chemoattractants and cytokines tumor necrosis factor (TNF)-α. As neutrophils traverse the circulatory system. The β2 integrins then engage their endothelial ligands. prompt endothelial cells to produce adhesion molecules on their luminal side: the P-selectins. These stimulants. and bacterial products results in activation and clustering of the β2 integrins on the surface of the neutrophil (15. the ICAMs (5). interleukin (IL)-1β. the neutrophil must navigate the basement membrane. these compounds stimulate the endothelial cells near the inflammatory site. Speculation abounds that granule proteases assist in this migration by digesting the protein mesh . neutrophils crawl along the vessel wall until a preferred site of transmigration is reached (19–21). a protein mesh consisting largely of laminins and collagen type IV. it will seek the insulting pathogens and unleash its extensive arsenal of antimicrobial weapons. resulting in selectin-mediated tethering of neutrophils to the vessel wall. 2012. firm adhesion is characterized by the arrest of neutrophil rolling in preparation for transendothelial migration (13. Ultimately. family kinases. they continuously and randomly probe the vessel wall. On the surface of neutrophils. 16). such as the bacterial-derived lipopolysaccharide (LPS) and fMLP. and several members of the integrin superfamily. where the neutrophil acts as a monitor for host distress. NEUTROPHIL ACTIVATION At inflammatory sites. as well as continuing input from inflammatory chemoattractants and cytokines. producing a leading-edge lamellipodium where chemokine and phagocytic receptors are concentrated. 10). are often the best-suited location for neutrophils to encounter the stimulated endothelial cells (7. Syk. and p38 mitogen-activated protein kinase (11–13). these molecules engage the P.and E-selectins of endothelial cells. and IL-17. cytokines. where flow dynamics and the constricted space are particularly amenable to increased random probing. Once through the endothelial lining. and initiation of the neutrophil oxidative burst begins (17. Immunol. phosphoinositide 3-kinase (PI3K). members of the ICAM-1 immunoglobulin superfamily. neutrophils enter a “firm adhesion” state mediated by the β2 integrin family of proteins (LFA-1 and Mac-1 proteins on the neutrophil). E-selectins. Downloaded from www.IY30CH19-Zychlinsky ARI Annu.

That is. alone they stimulate the oxidative response only mildly. Downstream molecules prompt assembly of the oxidative burst machinery. pathogen-derived chemoattractants (e..g.g. As the neutrophil nears its target. fMLP). The complex signaling cascade leading to final neutrophil activation has several facets worthy of note. Concomitantly. the process of setting neutrophil www. A notable example of this phenomenon is the strong priming effect of LPS on the fMLP response (28).org by Harvard University on 09/10/13.. the neutrophil follows chemotactic gradients toward the invading microbes. • Neutrophil Functions 463 . Furthermore. 26). as individual chemoattractants may have very different effects on neutrophil physiology at different concentrations.g. In neutrophils. the stimulation of FPR1 triggers the release of ATP. collectively called pathogen-associated molecular patterns (PAMPs). 2012. and DNA (TLR9).annualreviews. and the exact effects of priming. and signaling are incompletely understood. IL-8 stimulates L-selectin shedding and increased expression of β2 integrins. whose autocrine action through activation of purinergic receptors is critical for the initiation of effective functional responses in neutrophils (24). a phenomenon exemplified by one of the key neutrophil-recruiting chemokines and activators.e. the environment is awash in a soup of chemoattractants and inflammatory stimulants. desensitization. bacterial lipopeptides (TLR2).annualreviews. flagellin (TLR5). and their stimulation contributes to further activation. Regardless. At low concentrations. slightly higher concentrations result in initiation of the oxidative burst. In addition. the neutrophil is now fully in an antimicrobial attack state. both host derived and of pathogenic origin. a hallmark of neutrophil activation. all but one of these receptors (TLR3) are constitutively expressed. 31). The rich and varied input received by a neutrophil during this final leg of the activation process is complex.Annu.. which initiate a signaling cascade dominated by the MAPK/ERK pathway (22. Perhaps the best-known example of this family is the Toll-like receptors (TLRs). exposure of the neutrophil to LPS induces assembly of the NADPH oxidase machinery on the membrane. the neutrophil finds itself in a much different inflammatory milieu: Here. IL-8 induces degranulation of neutrophils (27). in parallel. the neutrophil halts and begins the final release of its antimicrobial arsenal. pursuing hostproduced cytokines (e. fMLP stimulation then induces activation of this machinery (29). In this case. however. the end result of this signaling cacophony is unambiguous: The neutrophil begins to implement its regime of microbial killing. as is the case with the fMLP receptor FPR1 or the chemokine receptors). Downloaded from www. During this process. including LPS (TLR4). Stimulation of the neutrophil by a chemoattractant often results in endocytosis of the corresponding receptor. continued activation by chemoattractants further stimulates the oxidative response and degranulation. they are responsible for recognizing a number of pathogen-derived compounds. another critical feature of the stimulation process is the desensitization to previously encountered ligands. and NETosis (i. conclusive experimental evidence for this is lacking. These compounds will now be the primary dictators of neutrophil behavior and assume responsibility for initiating the concluding steps of neutrophil activation. Upon finally reaching a point of high chemoattractant concentration. induction of the oxidative burst (25. The movement to ever-higher concentrations of chemoattractant is key in this process. these chemoattractants bind to their respective neutrophil receptors (often G protein–coupled receptors. IY30CH19-Zychlinsky ARI 17 February 2012 13:38 subsequent to degranulation. Once the endothelial barrier has been traversed. is activated through recognition of specific nonself patterns present on many microbes (25). 23). executing programs of phagocytosis. e. the pattern-recognition receptors.. but they dramatically enhance the subsequent response to other stimuli. For personal use only. In contrast to receptor priming. IL-8. Rev. At the highest concentrations.30:459-489. IL-8) and. many chemoattractant molecules exert a “priming” effect. degranulation. where no discernible gradient exists. a family of molecules. thus leading to a desensitization of the neutrophil to repeated stimulation with the same molecule (30. In the interstitial space.

(2) do no harm to the host. .30:459-489. and NETosis. Here. release of granular antimicrobial molecules through degranulation. (b) Selectin-mediated rolling along chemoattractant gradients then ensues. However. therefore. Among the processes employed are engulfment of microbes via receptor-mediated phagocytosis. degranulation. however. the neutrophil releases cytokines that recruit other immune cells. below).org by Harvard University on 09/10/13. an oversimplified view of a complex process. a process that gradually leads to complete activation and culminates in the premiere killing functions of phagocytosis. more insightful to view neutrophil activation as a continuum of processes. followed by (c) integrin-mediated firm adhesion. see rule 1. 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 conflicting (and overwhelming) view of neutrophil activation. The circulating neutrophil must recognize signs of inflammation and migrate to areas where its antimicrobial arsenal is needed for the elimination of infection. tissue-resident counterparts (Figure 1). and signal cascades with varying effects and outcomes. circulating neutrophils to their microbe-eliminating. It is. 2012. one could be (erroneously) led to believe that neutrophil activation refers only to direct stimulation of the oxidative burst. the neutrophil traverses through the endothelium and arrives at the site of inflammation. IY30CH19-Zychlinsky a Capture b Rolling parsed by the complex neutrophilic signaling mechanisms. For personal use only. and (3) when in doubt. c Firm adhesion Neutrophil PSGL-1. The initiation of these microbicidal actions indicates the final stage of the neutrophil’s journey through the activation process. L-selectin ICAM P-selectin and E-selectin Integrin Phagocytosis Endothelial cell Degranulation Cytokine secretion NETs Figure 1 Neutrophil recruitment to sites of inflammation. 464 Amulic et al. as this has been the canonical in vitro activation assay for decades. all focused on the realization of one goal: the transition of naive. (a) Close to the inflammatory sites. stimulated endothelial cells expose a class of molecules. and it begins to implement its antimicrobial agenda. the selectins. NEUTROPHILS AND THE ELIMINATION OF MICROBES The basic instruction set of the activated neutrophil is both effective and ruthless in its simplicity: (1) kill microbes. This is. To fulfill this antimicrobial agenda. In fact.annualreviews. Rev.ARI 17 February 2012 13:38 extracellular traps) (see the section on Neutrophils and the Elimination of Microbes. priming steps. Subsequently. which serve to capture circulating neutrophils and tether them to the endothelium. Downloaded from www. and formation of neutrophil extracellular traps (NETs). The myriad interactions that occur during a neutrophil’s journey toward an inflammatory site must be Annu. Immunol.

β-glucuronidase Gp91phox/p22phox.30:459-489. collagenase. FPR. oroscomucoid. As such. Additionally. These antimicrobial weapons vary considerably in their methods of action and thus reflect the neutrophil’s attempt to exploit any and all weaknesses that microbes might present during the course of infection. arginase-1. NGAL. haptoglobin. and cathepsin G (CG) (34). Granules also differ in their ability to mobilize. Annu. MMP25. Rev. Expectedly. hCAP18. • Neutrophil Functions 465 . and are the first formed during neutrophil maturation. As mentioned above.3 μM in diameter. Azurophilic granules (also known as peroxidase-positive or primary granules) are the largest. MMP25. They are named for their ability to take up the basic dye azure A and contain myeloperoxidase (MPO).org by Harvard University on 09/10/13. 33). with secretory vesicles being the first to fuse with the plasma membrane and the azurophilic granules demonstrating the least degranulation propensity.annualreviews. far more than just latent repository organelles for dangerous substances. alkaline phosphatase. and tertiary or gelatinase). pentraxin 3. Other cargo of this granule class include the defensins.annualreviews. BPI. Considerable overlap exists in the cargo of the different granules. measuring approximately 0. Therefore. Granules are. For personal use only. sialidase. there are three fundamental types of granules in neutrophils (Figure 2). an enzyme critical in the oxidative burst (32. Immunol. They are typically divided into three types (primary or azurophilic. An understanding of these weapons. CRISP3 Gp91phox/p22phox. they are active and indispensable participants in almost all neutrophil activities during inflammation. C1q-R. heparanase. Downloaded from www. these structures are replete with specifically tuned mechanics that address the unique Granule type Stage of formation Primary (azurophilic) Myeloblast needs of neutrophils. Granules and Degranulation The neutrophil must safely transport a plethora of dangerous substances through the bloodstream and then correctly deploy them at the appropriate time. these granules are brimming with antimicrobial Secondary (specific) Promyelocyte Myelocyte Inflammation: recruitment and activation of immune cells upon infection or injury. CD13. CD11b. proteinase 3 (PR3). structures called secretory vesicles are also considered to be a granule subset. 2012. CD11b. CD11b. plasma proteins Figure 2 Neutrophil granules. CD10. lysozyme. β2-microglobulin. their action. B12BP. CD14. bactericidal/permeability-increasing protein (BPI). azurocidin. www. CRISP3 Gp91phox/p22phox.IY30CH19-Zychlinsky ARI 17 February 2012 13:38 neutrophils possess an array of toxic weapons that are carefully regulated through controlled mechanisms. PR3. and their contents seem determined by the timepoint during hematopoiesis at which they are produced (5). and a number of serine proteases: neutrophil elastase (NE). and their method of release is critical to understanding neutrophil function. when uncontrolled it leads to tissue damage Tertiary (gelatinase) Metamyelocyte Secretory vesicles Band cell PMN Degranulation propensity Characteristic proteins Lysozyme Complement receptor 1 Lactoferrin Myeloperoxidase Elastase FcγRIII Gelatinase Defensin Other proteins Cathepsin G. Neutrophil granules carry a rich variety of antimicrobials and signaling molecules. however. secondary or specific. it comes as no surprise that a specialty storage organelle has evolved in neutrophils: the granule. β2-microglobulin.

and contain few antimicrobials. secretory vesicles (37–41). The second class of granules. At the inflammatory site. The third class. Consequently. Fusion of the secretory vesicles with the plasma membrane exposes these components to the external environment. such as gelatinase and leukolysin. In contrast to the classical granules. as well as the FcγRIII receptor CD16 (5. Finally. Rev. 39). 42). After neutrophils contact the by Harvard University on 09/10/13. granules are mobilized and fuse with either the plasma membrane or the phagosome. The release of granular proteins during degranulation presents the astute observer with a tempting proposition: Could these granular . IY30CH19-Zychlinsky 466 Amulic et al. are also commonly considered part of the neutrophil granule family. The membrane of secretory vesicles serves as a reservoir for a number of important membrane-bound molecules employed during neutrophil migration. The different classes of granules demonstrate varying propensities for mobilization in response to inflammatory signals: Azurophilic granules are the most difficult to mobilize. Immunol. gelatinase granules. 39. below). complement and fMLP receptors. resides in the specific granule membrane (45). are smaller (0. but instead are formed through endocytosis in the end stages of neutrophil maturation (36). the gelatinase (tertiary) granules. This fusion permits assembly of the NADPH oxidase complex and allows reactive oxygen species (ROS) production both inside the phagolysosome and outside of the cell. Degranulation of primary and secondary granules contributes to the creation of an antimicrobial milieu at the inflammatory site and produces an environment inhospitable to invading pathogens. the specific (or secondary) granules. and are characterized by the presence of the glycoprotein lactoferrin. Downloaded from www. prompting initiation of the oxidative burst and mobilization of the azurophilic and specific granules. As a neutrophil proceeds through activation. whose membranes are rich in key factors necessary for continued activation of the neutrophil. complete activation of the neutrophil ensues. releasing their contents into the respective environment. hCAP-18. do not contain MPO. a fourth set of structures. mediated by β2 integrin interaction with the endothelium. Because of this varying mobilization propensity. each Annu.ARI 17 February 2012 13:38 compounds and function as a primary repository for the molecular weaponry of neutrophils.30:459-489. their cargo consists predominantly of plasma-derived proteins such as albumin.1 μM diameter). thereby releasing metalloproteases. including. they also contain a wide range of antimicrobial compounds including NGAL. The underlying mechanisms for this differential mobilization are not entirely understood. or fuse with the plasma membrane. followed by specific granules. These granules are formed after azurophilic granules. but they serve as a storage location for a number of metalloproteases. and lysozyme (33. contributing to the antimicrobial activities of this compartment. This results in the transition to firm adhesion. the β2 integrins. these do not bud from the Golgi. although this has not been conclusively demonstrated (43. stimulation through selectins and chemoattractants induces mobilization of secretory vesicles. a component of the NADPH oxidase machinery. These granules either fuse with the phagosome (see section on Phagocytosis. among others. The fusion of specific granules with the plasma or phagosomal membrane is of particular importance for the oxidative burst. are smaller than specific granules. the membrane of the granule becomes a permanent part of the target membrane. 35). granule subset has been traditionally associated with a particular stage of neutrophil activation. As they proceed through the endothelium. In both cases. neutrophils are exposed to further activation signals that initiate mobilization of gelatinase granules.annualreviews. 2012. releasing their potent antimicrobials into the tissue. although regulation of intracellular calcium levels appears to play a salient role (32. are also MPO-negative. thus altering its molecular composition (6). These granules are also the last population of granules formed during neutrophil maturation (5). For personal use only. and finally. The activity of these proteases may help neutrophils traverse the basement membrane. 44). 38. the secretory vesicles. as flavocytochrome b558.

Because in vitro tests are often executed at high antimicrobial concentrations to obtain maximal microbial killing in the shortest possible time. direct observation of the degranulation process in vivo may soon be realized. Interestingly. many of these peptides disrupt the membrane integrity. most data on neutrophil degranulation and its effects on neutrophil activity have been acquired through biochemical approaches performed exclusively in vitro. Even so. it is unclear whether this disruption reflects their mechanism of action under physiological conditions. most notably through formation of neutrophil extracellular traps. In addition. Here we present an overview of this rich field of investigation. Immunol. By necessity. which are genetically tractable. (b) enzymes. and their in vitro activity is easily demonstrated in optimized conditions.annualreviews. some of them highly conserved throughout evolution (47). Under artificial conditions. there are few clinically relevant innate immune deficiencies that directly link antimicrobial activity with a particular mutation. have neutrophils that function differently from those of other species. Therefore. a feature that probably promotes their initial interaction with microbial surfaces. This could be advantageous in situations in which the extracellular concentration of released granule proteins is insufficient to exert extensive microbicidal effects. Downloaded from www. Alternatively. There are more than 800 antimicrobial peptides described in nature. however.annualreviews. below).org by Harvard University on 09/10/13. whereas others may be redundant. as already • Neutrophil Functions 467 . particularly challenging. or production of energy. Mice. with few exceptions. There are three main types of antimicrobials: (a) cationic peptides and proteins that bind to microbial membranes. This information. transcription. evidence for clinical or biological relevance of these molecules is still lacking. mice lack the genes for some antimicrobials identified in humans. In such cases. Indeed. including PR3 and azurocidin. A pertinent question therefore presents itself: Is this process truly relevant during the in vivo inflammatory response? The data here are sparse. Many of these antimicrobials were identified through biochemical fractionation of neutrophil extracts. species with abundant neutrophils. showing in vivo relevance is challenging.30:459-489. and (c) proteins that deprive microorganisms of essential nutrients. testing the relevance of antimicrobials in vivo is essential. neutrophil granule proteins may increase macrophage bacterial clearance by enhancing phagocytosis (46). ablation of a single antimicrobial gene may only subtly affect immune defense. Thus. These peptides are often charged. Furthermore. For personal use only. release of granular components could occur primarily through other means. Rev. This is. Most evidence for in vivo degranulation relies on observation of increased levels of extracellular granular proteins at inflammatory sites. can induce monocyte recruitment. or cell lysis. Little is known about antimicrobial concentrations achieved at inflammatory sites or in the phagosome. the granule proteins would instead operate as signaling and recruitment factors (see section on Neutrophils in Immune Cell Cross Talk. the possibilities for such an in vivo observation have been restrained by technical limitations. as well as information about the synergistic interactions of different www. such as DNA replication. 2012. The diversity of antimicrobials suggests that some of them evolved to act together. IY30CH19-Zychlinsky ARI 17 February 2012 13:38 components also serve as signaling molecules for subsequent inflammatory cell recruitment? Recent studies have provided experimental evidence suggesting this does seem to be the case: Granule proteins from neutrophils. some antimicrobials are thought to disrupt essential microbial functions. and understandably so: Historically. Antimicrobial Proteins Neutrophils produce a plethora of peptides and proteins that directly or indirectly kill microbes (Table 1). cell damage. nonetheless. With the advent of intravital microscopy techniques. much biochemical identification of neutrophil antimicrobials has been performed in rabbits and humans. One of the challenges in understanding the neutrophil’s antimicrobial mechanisms is to study their function during concerted action and in conditions that mimic an infection site.

but none have been validated in vivo. antimicrobials. Proteolytic enzymes Lysozyme Degrades bacterial cell wall Proteinase 3 (PR3) Mechanism independent of a proteolytic activity by binding to the bacterial membrane Neutrophil elastase (NE). 2012. Interestingly. Immunol. cathepsin G (CG)   Azurocidin Cleaves bacterial virulence factors and outer membrane proteins Mechanism independent of a proteolytic activity by binding to the bacterial membrane Mechanism independent of a proteolytic activity by binding to the bacterial membrane Metal chelator proteins Lactoferrin   Calprotectin a Alters bacterial growth by binding to iron. including BPI and histones. are proteolytically processed 468 Amulic et al. from larger proteins. A surprising number of functions are assigned to defensins. histones are extremely effective antimicrobials and were one of the first antimicrobials described (52). a protein family whose members possess multiple disulfide bonds and whose structures may change under physiological conditions and increase their activity (48). BPI binding to LPS results in increased bacterial permeability and hydrolysis of bacterial by Harvard University on 09/10/13. HNP-2. Neutrophils also contain a number of full-length cationic antimicrobial proteins.30:459-489. much like its structural cousin the LPS binding protein. inhibition of bacterial cell wall synthesis (49) was recently shown at low concentrations that may be more similar to those present at inflammatory sites. The neutrophil cationic antimicrobial peptides include defensins and cathelicidins. Given their dual role as an architectural . Downloaded from www. Neutrophils mostly produce α-defensins. HNP-4)    Permeabilize membrane bilayers containing negatively charged phospholipids Inhibit DNA. HNP-3. causing a release of LPS from the cell wall and an increase in membrane permeability Alters bacterial growth by sequestering manganese and zinc Only direct actions of neutrophil antimicrobial proteins on microbes are listed in the table. For personal use only. Cathelicidins. BPI is cationic and binds LPS avidly. cell death then follows (51). Interestingly. including the wellstudied LL-37.IY30CH19-Zychlinsky ARI 17 February 2012 13:38 Table 1 Mechanism of action of neutrophil antimicrobial proteins Antimicrobial mechanisma Antimicrobial peptide Cationic antimicrobial peptides α-defensins (HNP-1. and in addition to their antimicrobial activity. Rev.annualreviews. is essential for designing appropriate in vitro conditions to probe mechanisms of action. they may potentiate DNA activation of dendritic cells (DCs) (50). 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 Annu. The significance of histones (and of the peptides derived from them) as microbials remains to be demonstrated in vivo (53). an essential bacterial nutrient Binds to the lipid A part of LPS.

all ROS can modify and damage other molecules. azurocidin. a theoretical model of the phagosome suggests that most of the hypochlorous acid produced would react with host proteins before reaching the bacterium. CGD patients can control catalase-negative bacteria. making it an obvious antimicrobial. The NADPH oxidase complex assembles on the phagosomal and plasma membranes and begins the reactive oxygen cascade by reducing molecular oxygen to superoxide. thought to be the major product of MPO in the phagosome. this occurred independently of its enzymatic activity (55). Downstream of superoxide. collectively known as the serprocidins) that exhibit differing specificities. Thus. Surprisingly. below) (59). their own hydrogen peroxide. a strong oxidant. Neutrophils also contain several serine proteases (including PR3. including hypohalous acids. CG. which binds preferentially to iron. characterized by susceptibility to infection and autoinflammation 469 . However. Reactive Oxygen Species Upon activation. Lysozyme destroys the bacterial wall. However. The second class of neutrophil antimicrobials encompasses a broad assortment of proteolytic enzymes that participate in microbe destruction. This model predicts that chloramines. Interestingly. forming hydrogen peroxide. first identified in milk. NE cleaves enterobacterial virulence factors with high specificity (56).30:459-489. and permeability to membranes (62). Mice deficient in NE or CG are highly susceptible to bacterial and fungal infections (57. This can be rescued by the administration of recombinant granulocyte macrophage colonystimulating factor (GM-CSF). thus providing a substrate for reactions downstream in the reactive oxygen cascade (66).annualreviews. indicating the possibility of the coevolution of microbial virulence factors and antimicrobial effectors. it still kills microbes. and calprotectin (also called S100A and many other names). however. Immunol. Two of these chelators are lactoferrin. Upon degranulation into the phagosome. many potential reactions can occur (for details. indicating that their activity is deployed under specific conditions. NADPH oxidase is also implicated in the regulation of • Neutrophil Functions Chronic granulomatous disease (CGD): caused by mutations rendering the NADPH oxidase nonfunctional. The final class of neutrophil antimicrobials consists of a number of proteins that chelate essential metals from microbes and possibly impact bacterial growth. as shown in mice deficient in this enzyme (54). perhaps as a result of their cationicity. Hypochlorous acid. ROS are clearly important for neutrophil antimicrobial activity: Neutrophils from chronic granulomatous disease (CGD) patients kill microbes poorly. produced when hypochlorous acid reacts with amine groups. these patients still exhibit significant susceptibility to infections. suggesting that these proteins may all have antimicrobial activity independent of proteolysis. may be the most relevant antimicrobial actors in the phagosome (65). but do not degrade. is a member of the same family but lacks protease activity. They are tightly regulated intra. making these patients susceptible to many infections. Downloaded from www. their in vivo significance is particularly difficult to demonstrate. though not a strong oxidant. properties exploited by the host cell for signaling and antimicrobial action. Superoxide can also react with nitric oxide. For personal use only. which is produced at high levels at inflammatory sites. rapidly dismutates.Annu. IY30CH19-Zychlinsky ARI 17 February 2012 13:38 scaffold for DNA and as antimicrobials. and NE. Rev. it is assumed to have direct antimicrobial effects in the phagosome. MPO can react with hydrogen peroxide to produce various reactive species. Superoxide. 2012.annualreviews. These serine proteases also play a salient role in autoimmunity (see discussion in section on Autoimmunity. reactivity. Unexpectedly. result in neutropenia. is more reactive than superoxide and is antimicrobial in by Harvard University on 09/10/13. but not genetic ablation of this enzyme in mice. 58). to form peroxynitrite. Another protein.and extracellularly by serpins. Of further interest. see References 62–64). NE mutations in humans. neutrophils produce ROS in a process called the respiratory burst. It is misleading to think of ROS as a single entity because they differ in their stability. which produce. which sequesters zinc (60) and results in “nutritional immunity” (61).

metalloproteinases. and complement receptor-mediated phagocytosis. However. by cellular redox buffering systems and by limited diffusion of ROS owing to their short half-lives (72). this may activate granule proteases by releasing them from their putative matrix (75). in addition. Because these species are highly reactive. Paradoxically. Furthermore. in the absence of MPO. Immunol. receptor-mediated process during which a particle is internalized by the cell membrane into a vacuole called the phagosome. the probes often become radical species (76). However. a probe for ROS should be specific. other reactive species (e. 69).org by Harvard University on 09/10/13.annualreviews. most MPO-deficient individuals in the developed world have apparently normal immunity. Indeed. 74). they are often thought to be too nonspecific to be involved in signaling. specificity can be achieved on the submolecular level.g. and partial MPO deficiency does not correlate with pathology (70). peroxynitrite) can still be produced in the neutrophil phagosome. In addition. through recognition of PAMPs by pattern-recognition receptors. 2012. Rev. neutrophil granule proteases can be regulated by oxidative inactivation of their inhibitors or by direct oxidation Annu. In addition to direct antimicrobial action. and a few have been mistaken for CGD patients. such as roGFP and HyPer (76). there may be a broader reason for this discrepancy. The latter mechanism is better characterized and includes two prototypical examples: FcγR-mediated phagocytosis. As with other phagocytes. targetable to particular intracellular compartments. Phagocytosis Phagocytosis is the major mechanism to remove pathogens and cell debris. especially with Candida species. There is controversy around which ions and which channel are responsible for charge compensation. Modern technologies can distinguish between individuals who are partially and completely MPO deficient. acquiring its lethal properties only after a drastic . which does not require membrane extensions or pseudopods (77). A well-studied example of ROS in signaling is the reversible regulation of various targets (including phosphatases. ROS can modify host molecules. Ideally. However. which explains why CGD patients often suffer from autoinflammatory diseases (67). even 1% of normal NADPH oxidase activity leads to an improved prognosis (71). Other methods that can be used in vivo include transcription profiling of superoxide or hydrogen peroxide–sensitive genes as well as the detection of relatively stable products of reactive oxygen using mass spectrometry (76). Residual activity of MPO may be sufficient for antimicrobial activity: In the case of CGD. and caspases) by direct oxidation of cysteine residues. superoxide generation leads to an ionic influx into the phagosome to compensate for charge. Studies of ROS are hampered by various technical issues. Some MPOdeficient individuals suffer from frequent or severe infections. hydroperoxyl radical. superoxide. the mechanistic details of internalization depend on the type of interaction between the neutrophil and the microorganism. hydrogen peroxide. One promising new approach for ROS detection that meets these criteria is the use of redox-sensitive fluorescent proteinbased probes. It is an active. For personal use only. The mild effects of MPO deficiency suggest that MPO’s products are not essential for antimicrobial action. but this theory of protease activation is certainly intriguing (69). Interaction can be direct. and capable of being used in vivo.. Epidemiological studies distinguishing the degrees of MPO deficiency and their correlation with clinical manifestations may be necessary to understand the function of MPO. which relies on the formation of pseudopod extensions for engulfment of IgG-opsonized particles. After engulfment.ARI 17 February 2012 13:38 inflammation. (73. MPO-deficient individuals do not have striking clinical manifestations (68. Downloaded from www. Traditional probes for ROS do not meet these specifications. although MPO is required for neutrophil microbicidal activity in vitro. IY30CH19-Zychlinsky 470 Amulic et al. the nascent phagosome is relatively benign to microorganisms. hydroperoxyl radical is predicted to be present at antimicrobial concentrations (65).30:459-489. or opsonin mediated.

despite continuing fusion of acidic granules. The fibrous structures termed NETs contain histones as well as antimicrobial granular and cytoplasmic proteins (88). Nitric oxide donors can induce NETs via a mechanism that also requires ROS (90). and it is sustained via NADPH oxidase activity. In fact. 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. NETs trap many types of microbes ex vivo and have been found in various disease models in vivo. although those experiments were done using pharmacological inhibitors. and jointly. aureus may be an exception. neutrophil phagosomal pH is initially alkaline (78) and remains neutral for prolonged periods of time (79). modulating phagosome maturation. Rev. For personal use only. S. leading to chromatin decondensation (94). whereby delivery of antimicrobial molecules into the phagosomal lumen occurs. not cells deficient in ROS production (92).org • Neutrophil Functions Autophagy: a process in which cellular contents are degraded in lysosomes. assembly of the NADPH oxidase on the phagosomal membrane allows ROS production. The mechanism of NET formation is not completely understood. but further along in the process. and creating a more hospitable intraphagosomal environment. 91).30:459-489. especially in conditions of nutrient scarcity and infection 471 . phagosome maturation happens upon fusion of granules to the phagosome. These strategies include interfering with engulfment. the factors that are important for www. other pathogens. but this has so far been shown only using a nonspecific inhibitor of autophagy (98). The reactive oxygen pathway is involved. Unfortunately. All activators of NET formation tested so far require ROS production. neutrophils can undergo NETosis. Histone citrullination may also play a role in NET formation. Key events of the maturation process are described in more detail in Reference 80. The majority of research on NETs has been conducted ex vivo. the Raf-MEK-ERK pathway is implicated in NET formation (93). Immunol. these two mechanisms create an environment toxic to most pathogens. Francisella tularensis prevents triggering of the oxidative burst and also inhibits ROS production in response to other stimuli (83). some have evolved strategies to survive inside neutrophils. essential differences exist in neutrophils. Upstream of NADPH oxidase. as NADPH oxidase and MPO are required for NET formation in response to chemical and biological stimuli (87. to test the relevance of NETs.annualreviews. Macrophage phagocytosis follows an endocytic maturation pathway: In neutrophils.Annu. NE translocates from the granules to the nucleus and degrades histones. an active form of cell death that leads to release of decondensed chromatin into the extracellular space (86. they are thought to kill microbes by exposing them to high local concentrations of antimicrobials (89). Finally. Moreover. Helicobacter pylori can disrupt targeting of NADPH oxidase to the phagosome so that superoxide anions accumulate extracellularly rather than in the phagosome (82). Neutrophil phagosomal pH regulation also differs significantly from that observed in macrophages. although this has not been confirmed in primary human neutrophils (95–97). 2012. The maintenance of this alkaline pH is essential for the activation of the major serine proteases NE and CG. Simultaneously. 85). 90. The polysaccharide capsule expressed by Staphylococcus aureus confers antiphagocytic properties (81). such as Salmonella typhimurium and Streptococcus pyogenes. a “NETs knockout” organism should be generated to investigate its response to pathogens. Neutrophil Extracellular Traps Upon stimulation. While the macrophage phagosome gradually acidifies. IY30CH19-Zychlinsky ARI 17 February 2012 13:38 maturation process. Autophagy is also thought to be required for NET formation. Downloaded from www. a finding that awaits genetic by Harvard University on 09/10/13.annualreviews. and although these are certainly instructive. Our understanding of this process is largely based on studies in macrophages. can efficiently block granule fusion with the phagosome (84. it is not possible to eliminate the main components of NETs— DNA and histones—from an infection model. Not all pathogens succumb to the hostile environment of the phagosome. Ideally. 87).

org by Harvard University on 09/10/13. Here we provide examples of how neutrophils interact with other cells to shape the immune response (see Figure 3). On the one hand. please see References 115. 116). below). IL-8. primarily serves to recruit other neutrophils (113). an autoimmune disease characterized by the formation of autoantibodies. issuing instructions to practically all other immune cells. For now. a persistent Aspergillus infection in a CGD patient was cleared after gene therapy. NETs have also been observed in the airway fluids of cystic fibrosis patients. Platelet-induced NETs. sticky mucus and decreases in lung and digestive function 17 February 2012 13:38 NET formation. NEUTROPHILS IN IMMUNE CELL CROSS TALK Neutrophils participate in the communication networks that form the foundations of immunity. including granule contents (117).IY30CH19-Zychlinsky ARI Annu. such as during infections with large pathogens that are not readily phagocytosed. and it may be that NETs are especially important under certain conditions. such as NADPH oxidase. are also critical for other antimicrobial neutrophil functions. MPO. characterized by thick. the danger-associated molecular patterns (121). and ROS such as hydrogen peroxide (119). neutrophils release other signaling mediators. Monocytes and Macrophages As they respond to infection or injury. The most abundantly produced cytokine. The cytokines released by PMNs are often synthesized de novo. raising the possibility that NETs nucleate blood clots in the context of deep vein thrombosis (108). neutrophils secrete cytokines and chemokines critical in the unfolding of the inflammatory response and in 472 Amulic et al. neutrophil-derived proinflammatory IL-1β and TNF-α induce other cells to produce neutrophil chemoattractants (114. and NE. once activated. For personal use only. Cystic fibrosis: caused by defects in the CFTR ion transporter. but they are so abundant at inflammatory sites that their contribution to total cytokine levels is significant (4). 115) (for a comprehensive list of cytokines produced by neutrophils. lipids (118).. are associated with hepatotoxicity due to tissue damage (107). which may point to evolutionary pressure to avoid entrapment by NETs (99. Compared with other immune cells (e. the immune system has redundant mechanisms to fight infection. In addition to cytokines. bacteria that express DNases as virulence factors disseminate more efficiently in the host. The initial neutrophil cytokine response is an appeal for immunological reinforcement. suggesting a causal link .annualreviews. 111). establishing the correct environmental conditions to launch the adaptive immune response. Because NETs expose self molecules extracellularly. they lead to autoimmunity: NETs have been implicated in systemic lupus erythematosus (SLE). which restored NADPH oxidase activity. formed during sepsis. and these cells work to summon large numbers of neutrophils to the inflammatory locus. They also communicate via cell-cell contact (120). neutrophils typically produce lower amounts of cytokines per cell. Although neutrophils transcribe little after leaving the bone marrow. 2012. where they may increase the viscosity of the sputum and decrease lung function (109). often against chromatin and neutrophil components (102–106) (see section on Autoimmunity. As one of the first cell types to arrive at sites of infection. and NET-mediated but not phagocytosis-mediated killing by the patient’s neutrophils ex vivo (101).g. Macrophages and patrolling monocytes are among the initial detectors of PAMPs and endogenous activators. leading to alternating waves of recruitment of these two cell types. Rev. neutrophils and their relatives in the monocyte/macrophage lineage coordinate their activities. The influx of neutrophils is followed closely by the arrival of monocytes. macrophages). the evidence for the relevance of NETs is indirect. these cells undergo a transcriptional burst that results in the synthesis of signaling molecules (110. Furthermore. 100). In addition. NET formation. Similarly. Downloaded from www. neutrophil-secreted proteases can modulate signaling networks in vivo through cytokine processing (112). Platelets also bind to NETs. NETs can also have detrimental effects on the host. On the other hand. Immunol.30:459-489.

and perhaps more unexpectedly. recruited by neutrophils and differentiated into macrophages. CCL3 (MIP-1α) (123). These cells secrete the chemokine CCL3. neutrophils. Indeed. in the lymph nodes. 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. 2012. CD8+ T cell Neutrophil IFN-γ Macrophage Crosspriming DC Antigen presentation CD4+ T cell Th1 Neutrophil Activation Neutrophil Bacteria Blood Neutrophil Monocyte DC Figure 3 Neutrophil communication with other immune cells. repress further neutrophil chemotaxis and ensure the appropriate removal of their postmortem remains (see section on Neutrophils and Resolution of Inflammation. as shown for LL-37. neutrophils impede DC function by inhibiting antigen presentation to CD4+ cells. Dendritic Cells Neutrophils can also recruit and activate DCs in vivo. enhancement of production of chemoattractants by other cell types. They express classical monocyte chemoattractants such as CCL2 (MCP-1) (122). where subcutaneous inoculation of Leishmania major triggered a massive and rapid infiltration of neutrophils (130). and CCL19 (MIP-3β) (124). In addition to recruitment. below). behind these temporal dynamics. CCL20 (MIP3α). recruiting DCs to the site of inoculation and initiating a protective • Neutrophil Functions 473 . Neutrophils interact with a variety of cell types. neutrophils modulate monocyte and macrophage cytokine production (128). For personal use only.annualreviews. they also secrete IL-12. DCs and natural killer (NK) cells colocalize and enhance each other’s activity via receptor-receptor interactions and soluble mediators. and CG (125–127). This was recently illustrated in a mouse model of Leishmaniasis. Immunol. Finally. neutrophils use granule proteins to induce extravasation of monocytes in vivo. In contrast. Additionally. The circuitous nature of the cross talk of these two cell types becomes obvious during inflammation abatement: Monocytes. in turn. Monocyte recruitment is also affected indirectly by neutrophils: via upregulation of endothelial adhesion factors. and modulation of the activities of these chemokines via proteolytic processing (reviewed in 128).org by Harvard University on 09/10/13. azurocidin (HBP/CAP37). activate neutrophils by secreting IFN-γ.annualreviews. increase of transendothelial permeability. They are important both for recruitment of monocytes and dendritic cells (DCs) to infected tissues and for enhancement of macrophage and DC activity. Downloaded from www. neutrophils recruit monocytes via several different mechanisms. T cells. directly enhancing their microbicidal activity (129). Rev. which activates T cells.IY30CH19-Zychlinsky ARI 17 February 2012 13:38 Tissue T cell IFN-γ T cell Activation and differentiation ROS? Arginase? DC Activation Lymph node DC DC NK cell IL-12 Annu.30:459-489.

prompting the DCs to produce IL-12p70. G-CSF. neutrophils also activate NK cells by direct contact (139). leading to local secretion of TNF-α (120). neutrophils interact with a specific subset of DCs. NK cells. a key cytokine in the control of neutrophil dynamics. In this scheme. NK cells. which depletes neutrophils but may also result in depletion of many other cell types in mice. as outlined above. which in turn stimulates IFN-γ production by NK cells and further activates neutrophils. where they engage in swarming activity in response to parasitic infection (136). Recently. The anti-Ly6G monoclonal antibody is more specific and hence a better reagent for this type of experiment (133). and their interpretation is frustratingly difficult owing to the questionable purity of cell preparations. Additional in vitro interactions between neutrophils and NK cells are extensively reviewed in Reference 138. important in inflammation and implicated in autoimmunity 17 February 2012 13:38 Th1 response (131). DC-SIGN: dendritic cell–specific intercellular adhesion molecule-3-grabbing nonintegrin Granulocyte receptor 1 (Gr1): the anti-Gr1 antibody RB6-8C5 reacts with both Ly6G (specific for neutrophils) and Ly6C (present on many immune cell types) Th17 cells: subset of T helper cells that produce IL-17. the reduced levels of cytokine production foster specificity. Downloaded from www. It is possible that neutrophils have site-specific effects on DCs and can be stimulatory at peripheral sites and inhibitory in the lymph nodes. prolongs neutrophil life span. timely trafficking of DCs to lymph nodes and activation of CD4+ T cells were both dependent on PMNs. Neutrophil-activated DCs produce the proinflammatory cytokine IL-12 and induce proliferation of T cells (120. which may be crucial for Th1 cell differentiation (141. and DCs colocalize at inflammatory sites. but can also influence T cell function directly. IFN-γ. some of these experiments should be interpreted cautiously because they are based on the injection of the anti-Gr1 antibody (RB6). In one report. Interestingly. (via CD18-ICAM-1 interactions). it was shown that neutrophils. and a positive feedback loop has been proposed on the basis of in vitro data. Immunol. which acts by upregulating expression of CXCL8 (IL-8). However. and increases phagocytic capacity (145).org by Harvard University on 09/10/13. The crucial role of neutrophils in DC activation was recently confirmed using anti-Ly6G antibody depletion: In Mycobacterium tuberculosis infection. Similarly. A similar activation model was earlier proposed for Toxoplasma gondii (132). The functions and mechanistic details of these swarms are unknown and represent questions of immense interest. Neutrophils exhibit fascinating and somewhat enigmatic behavior in the lymph nodes.annualreviews. as only proximal DCs receive the maturation signal. and DCs interact in a m´enage a` trois involving both cytokine signaling and direct cell-cell contact (137. Furthermore. which is secreted by T cells. this study demonstrated that DCs presented bacterial antigens when they ingested infected neutrophils just as efficiently as they did via direct uptake of Mycobacterium (134). 132). human neutrophils. Simultaneously. 138). and TNF-α .IY30CH19-Zychlinsky ARI Annu. For personal use only. In this case. 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). 2012. Natural Killer Cells Studies of interactions between neutrophil and natural killer (NK) cells have historically been 474 Amulic et al.30:459-489. activated neutrophils can induce the maturation of DCs in vitro through specific receptor-receptor interactions between Mac-1 and DC-SIGN. Neutrophils affect T cell function indirectly via DCs. The T helper 17 (Th17) cell subset secretes IL-17. 144). In sharp contrast to the above findings. Previously thought to belong to isolated compartments. Cytokine communication occurs in both directions: For instance. Rev. performed in vitro. infection of mice with Legionella pneumophila triggered production of IFN-γ by NK cells. induces gene expression. both qualitatively and quantitatively (140). Lymphocytes A surprising finding in recent years is the extensive cross talk between cells located at opposite ends of the immune spectrum. PMNs secrete IL-12. 142). neutrophils and T cells shape and impact each other’s functions. this was dependent on both PMN-derived IL-18 and DC-derived IL-12 (137). They also express several T cell chemoattractants (116) as well as B cell development and maturation factors (143.

org by Harvard University on 09/10/13. Bak. However.annualreviews. IY30CH19-Zychlinsky ARI 17 February 2012 13:38 by epithelial. Rev. Thus. NEUTROPHILS AND RESOLUTION OF INFLAMMATION The lethal cargo of neutrophils is not only destructive toward invading microbes. The signaling networks that regulate survival have also been well characterized. Neutrophils also appear capable of expressing MHC class II and costimulatory molecules under inflammatory conditions (149–151). especially in light of the finding that mouse neutrophils that migrate to the lymph node have a negative effect on CD4 responses in an immunization system (135). such as cystic fibrosis sputum. and they also activate caspases (for an extensive review. which inhibits T cell responses in vitro (147). Downloaded from www. these Th17-associated cytokines increase granulopoeisis as well as the recruitment and life span of neutrophils. and (b) hydrogen peroxide–mediated suppression. not only does apoptosis reduce the number of neutrophils • Neutrophil Functions 475 . as proposed in a cancer model (119) (see section on Cancer. and intestines of patients with ulcerative colitis (156). Proposed to have prosurvival effects. but its expression can be restored in mature cells by inflammatory signals such as G-CSF or GM-CSF. they die via a builtin cell-death program. Neutrophil death is influenced by inflammatory mediators such as GM-CSF and LPS and by environmental conditions such as hypoxia. Bax. neutrophil modulation of adaptive immunity seems to be highly complex and is only now starting to be unraveled. leads to “nonresolving inflammation. see Reference 155). Neutrophils potentially have suppressive effects on T cells via two proposed mechanisms: (a) L-arginine depletion by release of arginase. Using mice in which professional antigen-presenting cells do not express functional MHC class I.” a problematic condition that contributes to many diseases. In humans. and Bid). suggesting concomitant variations in the ability to activate T cells. These networks also control the expression of known antiapoptotic (Mcl-1 and A1) or proapoptotic proteins (Bad. endothelial. Therefore. Beauvillain et al. it also produces signals that abrogate further neutrophil recruitment. Although some collateral damage to host tissues is inevitable during infection. there are large variations in the ability of donors to express MHC class II (149. Interestingly.annualreviews. Ulcerative colitis: a type of inflammatory bowel disease characterized by ulcers and tissue erosion in the colon and rectum Apoptosis and Clearance Apoptosis is a central aspect of inflammation resolution. Given that neutrophils are terminally differentiated. Immunol. 2012. 151).30:459-489. neutrophils influence CD8+ T cell responses by cross-presenting exogenous antigens in vivo. a finding that could have implications for susceptibility to autoimmune diseases. These striking findings imply that neutrophils have characteristics of antigen-presenting cells. This process is essential for maintenance of tissue homeostasis and. if impeded. survivin is also highly expressed in neutrophils at sites of inflammation. Phagocytosis of apoptotic neutrophils also reprograms macrophages to adopt an anti-inflammatory phenotype. www. all of which prolong neutrophil survival. and stromal cells (146). but also harmful to host cells. below). Resolution of inflammation is an active process that limits further leukocyte infiltration and removes apoptotic cells from inflamed sites. For personal use only. Direct evidence of such interactions in vivo is still missing. Collectively. and they can present antigen to CD4+ T cells in vitro (152–154). In line with these findings. one such protein is survivin. it is unexpected that molecules controlling cell proliferation regulate survival. neutrophils must be removed before they have serious.Annu. detrimental effects on inflamed tissues. (148) showed that antigen-pulsed neutrophils can induce differentiation of cytotoxic T cells. the functional significance for protective immunity remains unclear. However. appendix infiltrates. Once neutrophils have executed their antimicrobial agenda. It is expressed more highly in immature neutrophils than in mature ones. neutrophil deployment must be tightly controlled.

This mechanism complements other anti-inflammatory processes in which chemokines are inactivated by neutrophil proteases. The inhibitory effect extends to all essential steps of neutrophil responses: migration. nose. a particularly prominent role is assumed by lipid mediators. resolvins. neutrophils synthesize proinflammatory lipid mediators. which correlates with an increase in caspase-3 and caspase-8 activities. All three lipid mediators reduce neutrophil recruitment. The successful progression of inflammation appears to hinge on a shift in the composition of secreted lipids. a process that involves the lipoxinA4 receptor and the leukotriene B4 receptor (BLT1) (161–167). endothelial cells. During apoptosis. lipoxins can inhibit the shedding of L-selectin and the upregulation of β2 integrins in response to proinflammatory stimuli. 170). For personal use by Harvard University on 09/10/13. respectively. This relies on the release of “find-me” signals at early stages of cell death. prosurvival effects were also attributed to proliferating cell nuclear antigen (PCNA).IY30CH19-Zychlinsky ARI Annu. their vicinity (epithelial cells. and protectins in clearing inflammatory sites.30:459-489. 160). These are derived from arachidonate precursor molecules and are synthesized through the cyclooxygenase and lipoxygenase pathways. (168) also proposed an interesting mechanism of action for lipoxins. In neutrophils. resolvins. adhesion. At early stages of inflammation. microbes also likely participate in synthesis of mediators with proresolving functions at the site of infection (159. Pharmacological inhibition of these cell cycle regulators induce caspase-dependent apoptosis and block life-span extension by survival factors (157). it associates with procaspases in the cytosol and is thought to prevent their activation. such as lipoxins. in the arachidonic acid pathway. Of these lipids. Failure to clear these apoptotic cells. resolvins. Immunol. such as prostaglandins and leukotrienes. and kidneys. During the later stages of the inflammatory response. and protectins. but in neutrophils. where it is involved in DNA replication. and protectins exert cell-type specific effects. PCNA is targeted for proteosomal degradation. governed by temporally regulated expression of different lipoxygenases and the mobilization of different fatty acid substrates. fibroblasts. How do lipid mediators contribute to the termination of inflammation? Lipoxins. Finally. Interestingly. platelets. Lipid Mediator Class Switch Soluble mediators play a crucial role in the resolution of inflammation. by contrast. The sequestration of these chemokines means they are unavailable to recruit neutrophils to inflamed sites (168) (Figure 4). and damage of vital organs Prostaglandins and leukotrienes: lipids synthesized by cyclooxygenases and 5-lipoxygenase. Thus. where stabilization of PCNA is associated with resistance of neutrophils to apoptosis (158). They showed that neutrophil exposure to these lipids increases expression of CCR5 on the surface of late apoptotic neutrophils. More recently. Wegener’s granulomatosis: vasculitis affecting the lungs. The different biosynthesis pathways of proresolving lipid mediators have been reviewed in detail elsewhere (118). This factor usually resides in the nucleus. cyclin-dependent kinases function as prosurvival factors in neutrophils. Equally important for the resolution of inflammation is the proper removal of apoptotic cells. In addition to neutrophil recruitment.annualreviews. Downloaded from www. 2012. thereby reducing adhesion of neutrophils to endothelial cells (169. lipoxins are the most completely understood. microorganisms are also a source of lipid precursors that can be used by neutrophils for resolvin synthesis. Likewise. leading to efficient sequestration of the chemoattractants CCL3 and CCL5. and leukocytes) and participate in the transcellular biosynthesis of lipid mediators with anti-inflammatory and proresolving activities. which attract phagocytes. This mechanism is relevant in Wegener’s granulomatosis and sepsis. A major lipid mediator class switch thus exists. promoting monocyte/macrophage recruitment and activation while inhibiting neutrophil functions. and activation. results in secondary necrosis and release of products that generate proinflammatory signals (Figure 4). inflammation leads to reduced blood flow. Ingestion of apoptotic cells by macrophages drives the production of the antiinflammatory cytokines tumor growth factor (TGF)-β and IL-10 (155). neutrophils interact with various cell types in 476 Amulic et al. tissue destruction. have proinflammatory functions including leukocyte recruitment 17 February 2012 13:38 Similarly. Ariel et al. distinct “eat me” signals are required for specific recognition of apoptotic cells. . Rev.

NF-κB activation. These conditions lead to the accumulation of cytotoxic substances and are associated with severe pathologies. 171). as typically occurs during macrophage activation. In the presence of antiinflammatory lipids. including cystic fibrosis. Monocytes differentiated into macrophages ingest apoptotic neutrophils. and rheumatoid arthritis (RA). The contribution of macrophages to the clearance of NETotic neutrophils. engulfment of apoptotic neutrophils is not accompanied by the release of proinflammatory mediators. and IL-8 expression (170).annualreviews. Downloaded from www. which drive the lipid mediator class switch. resolvins. providing a means of scavenging chemokines. production of the anti-inflammatory cytokines TGF-β and IL-10 is increased (163.g. such as leukotrienes and prostaglandins. microbes trigger the production of proinflammatory lipid mediators. which also recruit neutrophils. e. Anti-inflammatory lipid mediators initiate the resolution of inflammation by blocking neutrophil and promoting monocyte recruitment. Disorders Associated with Nonresolved Inflammation The failure of neutrophils to apoptose or malfunctions in the removal of their apoptotic remains result in chronic inflammation. At the site of infection. In the early stages of inflammation. Rev. resident macrophages initiate an inflammatory response. inflammation leads to lung obstruction 477 . Proresolving lipid mediators also promote the expression of CCR5 on the surface of apoptotic neutrophils. Notably. is currently unknown.IY30CH19-Zychlinsky ARI Initiation of inflammation 17 February 2012 Leukotrienes 13:38 TNF-α Prostaglandins Lipoxins Resolvins Protectins IL-10 TGF-β Resolution of inflammation Platelets Monocyte Lipoxins • Neutrophil Functions Chronic obstructive pulmonary disease (COPD): lung disease caused by noxious particles or gas. chronic obstructive pulmonary disease (COPD). where smoking is a prime www.annualreviews. As inflammation progresses. driving the production of the anti-inflammatory cytokines tumor growth factor (TGF)-β and IL-10 and prostaglandin-E2 (PGE-2).org by Harvard University on 09/10/13. lipoxins also impact neutrophil activation by inhibiting ROS and peroxynitrite production. and protectins are produced. 2012. and how this could impact inflammation resolution. In addition to directly impacting neutrophil functions. Chemokine clearance upon phagocytosis of apoptotic neutrophils by macrophages further contributes to the reduction of neutrophil infiltration and the return to tissue homeostasis.. tobacco smoking. A timeline of the inflammation process from initiation to resolution is summarized in the upper part of the figure.30:459-489. interaction of neutrophils with platelets induces the production of lipoxins. and anti-inflammatory lipid mediators such as lipoxins. Neutrophil Lipoxin Resolvins Protectins Macrophage Apoptotic neutrophil Chemokines Chemokines CCR5 IL-10 TGF-β PGE-2 Chemokine clearance Leukotrienes Prostaglandins Microorganisms TNF-α IL-6 NETotic neutrophil ? Macrophage Figure 4 From inflammation to homeostasis: neutrophil apoptosis and lipid mediator class switching in the resolution of inflammation. COPD is a major cause of death in industrialized nations. Instead. Immunol. lipid mediators promote nonphlogistic (noninflammatory) phagocytosis of apoptotic neutrophils by monocytes and macrophages. a switch occurs. secreting proinflammatory cytokines and chemokines that alert the immune system and promote neutrophil recruitment. The severity of inflammation often directly correlates with poor clinical outcome. For personal use only.

The majority of findings support a “protumor” and “antihost” effect of these cells. and their role in pathogenesis has been demonstrated in several animal models. Later. Indeed.annualreviews. Rheumatoid arthritis (RA): chronic inflammatory disease that affects many tissues and organs but primarily synovial joints. Although this has been demonstrated in vitro (178. Immunol. These studies exemplify the complex regulation cascades involving lipids. First. Second. Cancer The link between cancer and inflammation was noted as early as 1863 by Rudolf Virchow (177). It is. LTA4H is an aminopeptidase that inactivates a specific neutrophil chemoattractant. including production of angiogenic factors (185). immune complexes are essential for stimulating infiltrating neutrophils to deliver IL-1β into the joint. certain malignant cancers are also prime examples of illnesses in which neutrophils play a salient role. This enzyme has two opposing activities. and . The protumor function of neutrophils operates at multiple levels.30:459-489. the proline-glycine-proline tripeptide (PGP). tobacco smoke selectively inhibits only the aminopeptidase activity of LTA4H. neutrophil action can also support disease progression in other illnesses. Neutrophils do. NEUTROPHILS IN DISEASE Neutrophils are prominent players in the innate immune response and the clearance of infection.IY30CH19-Zychlinsky ARI Annu. clinical studies indicate that neutrophil infiltration of tumors is associated with poorer prognosis (181. a subject addressed in several prominent reviews. However. 179). In addition. convincing evidence for PMN-mediated DNA mutagenesis in vivo is still lacking. ROS. Downloaded from www. however. the recruitment of a second wave of neutrophils is independent of this leukotriene B4–BLT1 pathway. A chronic neutrophil infiltration in the lungs of COPD patients promotes tissue damage and organ dysfunction. 2012. Rev. This in turn induces the production of chemokines by synovial tissue cells and sustains neutrophil recruitment (175. Interestingly. antibody depletion of neutrophils reduces tumor growth (184). At this stage. 176). In one model. it has been proposed that neutrophil-derived ROS have the potential to initiate tumor formation by genotoxic stress and induction of genomic instability. A host of autoimmune disorders belong to this category. The relevance of this model in human disease remains to be established. the previous section. severe inflammation causes deformity 478 17 February 2012 13:38 instigator of this disease. some cancers seem to actively recruit neutrophils through production of IL-8 (183).org by Harvard University on 09/10/13. enhancement of metastasis (186). synthesis of leukotriene B4 by neutrophils in joints is essential for disease development (174). 182). For personal use only. They also demonstrate the cross talk between neutrophils and other immune cells discussed in Amulic et al. thus conferring the enzyme with anti-inflammatory properties. Another prime example of a disease linked to nonresolving inflammation is RA. its hydrolase activity converts leukotriene A4 into leukotriene B4. Neutrophils are the most abundant leukocytes present in the synovial fluid of RA patients. promoting the accumulation of both leukotriene B4 and PGP. cytokines. including cytokines. Leukotriene B4 can act in an autocrine manner via the neutrophil receptor BLT1 to promote the recruitment of a first wave of neutrophils into the joint. although the clinical similarities between this mouse model and human RA are encouraging. impact cancer progression. In agreement with this. One of the key molecules controlling the inflammatory response in the lung is leukotriene A4 hydrolase (LTA4H). a potent neutrophil chemoattractant and proinflammatory agent. These models primarily used neutrophil depletion or adoptive transfer of wild-type neutrophils in leukotriene-deficient mice (173–175). and matrix-degrading proteases (reviewed in Reference 180). unknown whether all neutrophils are capable of adapting to the changing chemoattractant environment or if different subsets of neutrophils are successively involved. They are abundant in tumors and influence tumor development through several secreted mediators. Since then. This in turn promotes neutrophil recruitment and fuels chronic lung inflammation (172). however. and chemokines that orchestrate neutrophil recruitment in chronic inflammation.

Neutrophil products act as both targets and mediators of autoimmunity. promoting degranulation and release of chemoattractants and ROS. In cancer and some autoimmune and infectious diseases. ANCA bind MPO and PR3 expressed on the surface of activated neutrophils. For personal use only. the extent of organ damage in patients with Wegener’s granulomatosis correlates with the PMN infiltrate rather than with traditional autoimmunity parameters such as T cell activation or autoantibody titers (196). 192). SLE is another chronic autoimmune disease affecting multiple tissues and organs. leading to accumulation of these precursors. 2012. Another major mechanism of tumor escape from immune control has recently been attributed to a heterogeneous category of immature myeloid cells. In human renal cell carcinoma. 191. Using the anti-Ly6G antibody. IY30CH19-Zychlinsky ARI 17 February 2012 13:38 suppression of the antitumor immune response (119. autoantibodies directed against antigens present in the cytoplasm of neutrophils. be induced to target their cytotoxic arsenal at tumor cells. MDSCs have identical morphology and express the same surface markers as do activated neutrophils (190. which also inhibited T cell responses. Although neutrophils had long been suspected www. Furthermore. Interfering with the release of MDSCs or using drug interventions to polarize neutrophil responses in the tumor microenvironment could represent novel therapeutic strategies against cancer. ANCA accelerate ROS-dependent neutrophil apoptosis. called myeloid-derived suppressor cells (MDSCs) (189).annualreviews. they are typically detected using the neutrophil surface markers CD11b+ and Gr-1+ . Furthermore. Early reports also suggest that. under certain circumstances. Downloaded from • Neutrophil Functions 479 . 192). restoring a proinflammatory activation status (188). MPO and PR3 are the main targets of antineutrophil cytoplasmic antibodies (ANCA). 187). investigation of neutrophils in cancer has revealed considerable plasticity in their responses. MDSCs inhibit T cell proliferation by limiting L-arginine availability via arginase and NOS activities. although they consist of variable proportions of monocytic and granulocytic cells (189). Pharmacological inhibition of TGF-β signaling led tumorassociated neutrophils to assume a heightened proinflammatory state. which act as powerful suppressors of T cell functions. suggesting a feed-forward cycle culminating in organ damage (194. 195).Annu. Acute-phase proteins: secreted by liver. whose growth they usually help to fuel. where they differentiate into mature neutrophils and monocytes. Likewise. differentiation is partially blocked. Differential neutrophil responses were also demonstrated in a melanoma study. Wegener’s granulomatosis is consistently associated with the presence of ANCA. Although little evidence currently supports the existence of different populations.annualreviews. 191). the study showed that neutrophils in the tumor microenvironment could. MDSCs are found in the bone marrow. Moreover. both of which use this amino acid as a substrate (189. which suppresses T cell responses (119. given that release of proteolytic and cytotoxic molecules from neutrophils can trigger organ damage. and in mice. These alternatively activated neutrophils underwent a complete reversal in their effect on CD8+ T cells. which together lead to a vicious cycle of tissue damage and inflammation. Fridlender and colleagues (187) depleted neutrophils and confirmed their tumorigenic role. Cross talk with invariant NKT cells could counter this response. concentration in plasma changes by 25% or more during inflammation Autoimmunity Deregulated neutrophil cell death and/or clearance often accompanies autoimmune syndromes (193–195) and may play a major role in disease pathogenesis. Thus. in an inflammatory environment. MDSCs are strong producers of ROS. Autoantibodies produced in SLE are predominantly either ANCA or directed against chromatin. In this instance. Rev. MDSCs have characteristics of neutrophils.30:459-489. it is likely that neutrophil responses are more flexible and less stereotyped than previously thought. In healthy individuals. Immunol. serving to activate rather than suppress these cells. causing a reduction in tumor by Harvard University on 09/10/13. increased systemic levels of the acute-phase protein serum amyloid A (SAA-1) induced neutrophils to secrete the anti-inflammatory cytokine IL10.

Several studies have reported the presence of a particular subset of neutrophils in PBMC preparations from pediatric and adult SLE patients. 36% exhibited either elevated titers of autoantibodies directed against NET components or inhibitors of DNase I. They are endowed with antimicrobial mechanisms that make them the preeminent microbe exterminators of the immune system. 2012. to produce IFN-α. PMNs also network with many other immune cells and help regulate the initiation of specific T and B cell immunity. 104). above) (197. Should this prove to be the case. . anti-HNP. However. neutrophils can potentially provide insights into several unique aspects of basic cell biology. Notably. For personal use only. a central cytokine in SLE pathogenesis (103. their role in SLE pathogenesis remained elusive. Vasculitis: inflammation of blood vessels 480 17 February 2012 13:38 to be causative agents. Immunol. Downloaded from www. An increased capacity to form NETs and a heightened cytotoxicity toward endothelial cells could bestow them with pathogenic properties in lupus (105). whereas their reliance on ROS as biochemical effectors may reveal novel ways for relaying intracellular signals. It was proposed that TNF-α and IFN-α prime cells for NET formation in response to anti-PR3. neutrophils do not always act in ways beneficial to the host: Uncontrolled neutrophil responses can exacerbate and even cause autoimmune and inflammatory diseases. whereas autoantibodies may trigger a switch from apoptosis to NETosis. their timely removal may be an essential mechanism for maintaining tissue homeostasis. in a cohort of SLE patients. which degrades NETs in vitro. Moreover. high levels of inflammatory cytokines in autoimmune patients are believed to sensitize neutrophils to NETosis. Human serum contains the nuclease DNase I. In short. However. one of the most severe manifestations of SLE (102). These low-density granulocytes display phenotypic characteristics of immature neutrophils with nonsegmented nuclei and higher expression of MPO.annualreviews. Most notably. impaired NET degradation correlates with development of lupus nephritis. Their strikingly short life spans make them excellent models for investigating cell death. and defensin-3. antiribonucleoprotein. Furthermore. CONCLUDING REMARKS Neutrophils are specialized phagocytes that arose as an evolutionary adaptation in vertebrates to coordinate and execute one of the most fundamental physiological responses: inflammation. understanding the role of NETs may provide critical insights into the role of microbial infections as a trigger of autoimmunity. 106). Many challenges remain in understanding neutrophil function: Is there specialization among PMNs? Are they more plastic than we suspect? How do they make decisions before deploying their armamentaria? How do they kill microbes? How specific are their instructions to other cells? Answering these questions will better define neutrophils’ role in defense and disease and will provide a rational path for pursuing new therapies. NE.IY30CH19-Zychlinsky ARI Annu. Can it be that NETs play a general role in modulation of autoimmune responses? We know that NETs induce plasmacytoid DCs Amulic et al. Because NETs appear to be formed during autoimmune disease. or anti-LL-37 autoantibodies (103. a familial form of SLE is linked to a mutation in DNase I (199). Additional evidence suggesting a role for NETs in autoimmune pathology was obtained when NETs were identified in renal and/or skin biopsies from patients with SLE and small vessel vasculitis (103–106).30:459-489. The recent discovery of a link between SLE and NET formation has helped to shed light on this quandary. both of which may protect NETs from degradation. and they may be related to the MDSCs discussed previously (see section on Cancer. by Harvard University on 09/10/13. The uniquely lobulated neutrophil nucleus is a feat of higher-order nuclear architecture that is just beginning to yield its secrets. it remains to be determined if DCs can present NET components or if they contribute to autoreactive B cell activation. exciting times await the humble neutrophil. It is also possible that NETs are involved in other autoimmune diseases. Thus. 198). In addition to this important role. Rev.

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Ayres and David S. 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. Michael Schorpp. 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 Inflammatory Responses Ryan M. Immunol. Jr.IY30-Frontmatter ARI 17 February 2012 11:21 Annual Review of Immunology Contents Volume 30. Nathanael McCurley. 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 . and Maureen R. 2012 Annu. Dinesh S. Heikamp. O’Connell. Stritesky. Kristen N. and Future Ralph M. 2012. ¨ George S. Yoichi Sutoh. Helmink and Barry P. Powell. 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 by Harvard University on 09/10/13. Stephen C. Brenchley and Daniel C. 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. 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. Steinman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 The Basel Institute for Immunology Fritz Melchers p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p23 Regulation of Immune Responses by mTOR Jonathan D. Emily B. Downloaded from www. 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. Jameson. Rev. For personal use only. Decisions About Dendritic Cells: Past. 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.30:459-489. Masanori Kasahara. Cyster and Susan R. Present.annualreviews. 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. 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. Rao. Pollizzi. and Kristin A. Deepe. and Max D.

Victora and Michel C. Lineage Relationships. 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. 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. Metzler. and Louis M. Cheng and Mark S. Hayes. Annaiah Cariappa. Kiyoshi Hirahara. Whitney M. Golnaz Vahedi. Shaffer III. Adam B. 2012. Nolte. For personal use only. and Alexander Y. Xavier p p p p p p p 611 Innate Lymphoid Cells: Emerging Insights in Development. Castoreno. and Hamid Mattoo p p p p p p p p p p p p p 357 Monogenic Autoimmunity Mickie H.IY30-Frontmatter ARI 17 February 2012 11:21 Reflex Principles of Immunological Homeostasis Ulf Andersson and Kevin J. Li-Fan Lu. Kathleen D. and Arturo Zychlinsky p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 459 Signaling by Myeloid C-Type Lectin Receptors in Immunity and Homeostasis David Sancho and Caetano Reis e Sousa p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 491 Regulatory T Cells: Mechanisms of Differentiation and Function Steven Z. Ryan M. Nussenzweig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429 Neutrophil Function: From Mechanisms to Disease Borko Amulic. Josefowicz. Downloaded from www. Rev. Ilka Arun Netravali.30:459-489. Robert Mansson. Bilate and Juan J. O’Shea p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 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. Garret L. 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. Kentner Singleton. 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 . and Ramnik J. Young. by Harvard University on 09/10/13. 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 Inflammation Kenya Honda and Dan R. 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.annualreviews. Germinal Centers Gabriel D. Christel Cazalet. and John J. 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 Inflammation: 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 Specification: Molecular Mechanisms Underlying Commitment and Plasticity Yuka Kanno. 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 Annu.