1 neutrófilos - primeira apresentação.pdf

IY30CH19-Zychlinsky ARI 27 December 2011 13:33

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Neutrophil Function:From Mechanisms to DiseaseBorko Amulic, Christel Cazalet,Garret L. Hayes, Kathleen D. Metzler,and Arturo Zychlinsky∗

Department of Cellular Microbiology, Max Planck Institute for Infection Biology,Chariteplatz 1, 10117 Berlin, Germany; email: [email protected],[email protected], [email protected], [email protected],[email protected]

Annu. Rev. Immunol. 2012. 30:459–89

The Annual Review of Immunology is online atimmunol.annualreviews.org

This article’s doi:10.1146/annurev-immunol-020711-074942

Copyright c© 2012 by Annual Reviews.All rights reserved

0732-0582/12/0423-0459$20.00

∗All authors contributed equally to the work andare listed alphabetically.

Keywords

inflammation, antimicrobial, granule, phagocytosis, NET

Abstract

Neutrophils are the most abundant white blood cells in circulation,and patients with congenital neutrophil deficiencies suffer from severeinfections that are often fatal, underscoring the importance of thesecells in immune defense. In spite of neutrophils’ relevance in immunity,research on these cells has been hampered by their experimentally in-tractable nature. Here, we present a survey of basic neutrophil biology,with an emphasis on examples that highlight the function of neutrophilsnot only as professional killers, but also as instructors of the immunesystem in the context of infection and inflammatory disease. We focuson emerging issues in the field of neutrophil biology, address questionsin this area that remain unanswered, and critically examine the experi-mental basis for common assumptions found in neutrophil literature.

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Review in Advance first posted online on January 3, 2012. (Changes may still occur before final publication online and in print.)

Changes may still occur before final publication online and in print

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INTRODUCTION

In the late nineteenth century, Paul Ehrlich,dissatisfied with what he considered an in-excusable disinterest in the white blood cell,began to utilize newly developed cell-stainingtechniques to examine subpopulations of leuko-cytes. His experimentation led to a new appreci-ation for the heterogeneity of white blood cellsand to the discovery of several novel leukocytesubpopulations. Ehrlich named one of thesenewly discovered cell types, characterized by a“polymorphous nucleus” and a tendency to re-tain neutral dyes, the “neutrophil” (1) (see alsothe sidebar, A Natural History of Neutrophils).

The function of neutrophils was initiallyshrouded in considerable mystery; their con-spicuous presence during infections led severalresearchers to arrive hastily at a rather ironicconclusion: They surmised that neutrophilspromote infection, serving as cellular shuttlesfor bacteria (2). Their actual function, that ofantimicrobial actors in the immune response,was eventually demonstrated conclusively by acontemporary of Ehrlich, Elie Metchnikoff, an

A NATURAL HISTORY OF NEUTROPHILS

Phagocytes are ancient cells that evolved to allow multicellularorganisms to thrive in the face of constant competition with mi-crobes for resources. Metchnikoff ’s seminal theory of cellularimmunity was based on comparative embryology and observa-tions of phagocytes in various simple organisms, including the mi-croscopic crustacean Daphnia. Remarkably, even the slime moldDictyostelium discoideum has phagocytic cells that protect it frominfection (200). The short-lived neutrophil with a lobulated nu-cleus and granule-packed cytoplasm is a more recent evolutionaryadaptation. In insects, phagocytes are long lived and have roundnuclei. They do, however, produce hydrogen peroxide and carrydistinct classes of granules (201). Bony fish and frogs have bonafide neutrophils that are functionally similar to mammalian ones(202, 203). In both zebrafish and rodents, neutrophils are lessabundant than in humans, comprising only 15–20% of immunecells. In chimpanzees, neutrophils account for more than 50% ofthe differential blood count (204).

early and enthusiastic evolutionary biologist in-terested in the phagocytic capacity of cells.

Metchnikoff demonstrated that injury ofstarfish embryos resulted in recruitment ofphagocytic cells to the site of injury (3). Hetheorized (correctly) that these cells migrate toinjured sites and participate in microbe diges-tion. Remarkably, this prescient view of neu-trophil action still aptly summarizes, more thana century later, the basic role of neutrophilsin immunity. The uniquely lobulated nucleusof the neutrophil also inspired Metchnikoff torename these cells: He called them polymor-phonuclear leukocytes (or PMNs), a title thatstill enjoys frequent use and that is used inter-changeably with neutrophil throughout this re-view. Together with two other developmentallyrelated cell types, the eosinophils and basophils(also discovered by Ehrlich), PMNs form thegranulocyte family of white blood cells, a fam-ily whose hallmark is the presence of “granules,”unique storage structures important in antimi-crobial functions (see section on Granules andDegranulation, below).

Neutrophils were discovered at the dawnof the immunological sciences; consequently,elucidation of their role in the immune re-sponse has been an ongoing process stretchingover more than a century. We now know thatthey are key components of the innate immuneresponse and vital in immune function; unfor-tunately, their importance has often been over-shadowed by breakthroughs in the study of theadaptive immune response (4). Admittedly, thissituation is exacerbated by neutrophils’ notori-ous experimental intractability: They exhibit ashort life span and are terminally differentiated,preventing growth in tissue culture. The stan-dard tools of molecular biology, such as trans-fection and RNA interference, are of little usewhen applied to these cells, and immortalized“neutrophil-like” cell lines rarely reflect thefunctional diversification of neutrophils. Fur-thermore, neutrophil-like cells studied in theisolation of a culture dish most certainly do notmimic the complex biological reality in tissuesor circulation. Conclusions from in vitro stud-ies should, therefore, be carefully interpreted.

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Unfortunately, in vivo studies of neutrophilfunction also raise concerns. Mouse neu-trophils, the preferred model for in vivostudies, differ in important aspects from theirhuman equivalents. This is perhaps bestexemplified by the differences in the respectiveantimicrobial repertoires and the numbers ofPMNs in circulation (30% versus 70% in miceand humans, respectively).

Despite these difficulties, no picture of theimmune response can be complete withouta comprehensive understanding of the neu-trophil and its functions. The extensive natureof neutrophil research, however, precludes acomprehensive review of the subject matter.In this review, we intend to provide a surveyof basic neutrophil biology and function, whileemphasizing recent advances in neutrophil re-search and providing a critical assessment ofsome current reports on PMN action.

Our survey of the neutrophil begins inadult bone marrow where, under the in-struction of growth factors and cytokines,pluripotent hematopoietic cells differentiateinto myeloblasts, a developmental cell typecommitted to becoming granulocytes. As theseprecursor cells mature to neutrophils, they syn-thesize proteins that are sorted into differentgranules (5). Traditionally, granules have beensubdivided into three different classes basedon their resident cargo molecules: azurophilic,specific, and gelatinase granules. Although thissubdivision is practical, these designations arelargely artificial. Granules are formed through acontinuous process; vesicles bud from the Golgiapparatus and fuse, producing granular struc-tures. The content of these structures is dic-tated by the transcriptional program active atthe time of their formation. As the maturingneutrophil sequentially alters its transcriptionalprofile, granule content changes, resulting in acontinuum of granule species with overlappingcargoes (6).

The release of neutrophils from the bonemarrow is tightly regulated in healthy in-dividuals: Chemokines control the passageof PMNs into circulation and maintain apool of cells ready for release in case of

infection. Indeed, the number of neutrophilsdrastically increases during infection and somediseases. Interestingly, neutrophils circulatefor only approximately 6–8 h and are amongthe shortest-lived cells in the human body.Although the reason for this short life is unclear,it may ensure neutrophil integrity; this hypoth-esis is bolstered by observations that apoptosisprevents the release of noxious molecules.Still, the question of why evolution opted foreliminating neutrophils quickly as opposedto reducing leakage of their dangerous cargoremains an unanswered and intriguing mystery.

Mature neutrophils emerge from the bonemarrow intent on pursuing one simple, yetessential, question: Has host integrity beencompromised by potentially harmful invaders?Should the answer prove to be “yes,” theneutrophil must swiftly enact a carefullychoreographed process to locate, attack, anddestroy the potential threat. At its disposal isan impressive arsenal of antimicrobial weaponsthat are deadly, indiscriminate, and brutish intheir application. Although effective in theirdestructive capacity, these weapons can proveto be just as dangerous to the host cells as totheir intended targets, the microbial invaders.Therefore, their deployment must be executedwith exquisite precision and timing, at locationswhere they are both contained and effective.

How then does the neutrophil locate andidentify infections? How does it transitionat the correct time and place from an in-active cellular bystander to a fully activatedmicrobial killing machine? This transitionprocess, during which the neutrophil inte-grates a complex barrage of environmentalcues and translates them into specific actions,is known as neutrophil “activation.” As itpursues microbes, the neutrophil will enact animpressive multitude of cellular mechanisms:It will mobilize secretory vesicles and granules,identify chemotactic gradients and traversethem through destruction and reorganizationof the actin skeleton, penetrate the endothelialbarrier and navigate a course through thebasement membrane, and begin transcriptionof cytokines for recruitment of new immune

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Selectins:transmembraneglycoproteins thatmediate cell adhesionvia binding to sugarmoieties

Integrins:transmembranereceptors that mediateattachment to theextracellular matrix, aswell as direct cell-cellinteraction andsignaling

Oxidative/respiratoryburst: a rapid increasein oxygenconsumption uponneutrophil activationdue to production ofROS by the NADPHoxidase

cells. Ultimately, upon arriving at the infectionsite, it will seek the insulting pathogens andunleash its extensive arsenal of antimicrobialweapons. The initiation of these processes oc-curs in the bloodstream, where the neutrophilacts as a monitor for host distress, patrollingvessels and vigilantly seeking out indications ofan incipient inflammatory response.

NEUTROPHIL ACTIVATION

At inflammatory sites, bacterial-derived andhost-produced inflammatory signals areabundant; these compounds stimulate theendothelial cells near the inflammatory site.These stimulants, such as the bacterial-derivedlipopolysaccharide (LPS) and fMLP, as wellas the classical chemoattractants and cytokinestumor necrosis factor (TNF)-α, interleukin(IL)-1β, and IL-17, prompt endothelial cells toproduce adhesion molecules on their luminalside: the P-selectins, E-selectins, and severalmembers of the integrin superfamily, theICAMs (5). As neutrophils traverse the circu-latory system, they continuously and randomlyprobe the vessel wall; the postcapillary venules,where flow dynamics and the constricted spaceare particularly amenable to increased randomprobing, are often the best-suited locationfor neutrophils to encounter the stimulatedendothelial cells (7, 8).

On the surface of neutrophils, two constitu-tively expressed proteins are critical for recog-nition of the endothelial inflammatory signals:the glycoprotein P-selectin glycoproteinligand-1 (PSGL-1) and L-selectin (9, 10). Uponrandom contact with the endothelium, thesemolecules engage the P- and E-selectins ofendothelial cells, resulting in selectin-mediatedtethering of neutrophils to the vessel wall.This is followed by a characteristic “rolling” ofneutrophils along the endothelium. It is herethat the complex activation cascade beginsand the neutrophil commitment to microbialkilling commences. What changes occur in theneutrophil at this early time point? The engage-ment of PSGL-1 and L-selectin on neutrophilsactivates a variety of kinases, including Src

family kinases, Syk, phosphoinositide 3-kinase(PI3K), and p38 mitogen-activated proteinkinase (11–13). This cascade initiates a numberof changes in neutrophil biology and sets thestage for integrin activation and firm adhesion.

After selectin-mediated rolling, neutrophilsenter a “firm adhesion” state mediated by theβ2 integrin family of proteins (LFA-1 andMac-1 proteins on the neutrophil); firm adhe-sion is characterized by the arrest of neutrophilrolling in preparation for transendothelialmigration (13, 14). As the neutrophil rollsalong the endothelium, interaction withselectins, chemoattractants, cytokines, andbacterial products results in activation andclustering of the β2 integrins on the surface ofthe neutrophil (15, 16). The β2 integrins thenengage their endothelial ligands, members ofthe ICAM-1 immunoglobulin superfamily,resulting in arrest of neutrophil rolling andfirm adhesion. This integrin engagement, aswell as continuing input from inflammatorychemoattractants and cytokines, prepares theneutrophil for its final chemotactic pursuit: Thecell spreads, producing a leading-edge lamel-lipodium where chemokine and phagocyticreceptors are concentrated, the cytoskeleton isrebuilt and targeted toward movement alongchemotactic gradients, and initiation of theneutrophil oxidative burst begins (17, 18).

Now firmly adhered, the neutrophil mustnegotiate a path through the endothelium intothe underlying tissue. In a process dependenton β2 integrins and ICAMs, neutrophilscrawl along the vessel wall until a preferredsite of transmigration is reached (19–21).Upon arrival at an endothelial cell junction, acomplex interaction between (a) the neutrophilintegrins and their endothelial partners and(b) neutrophil surface proteins and variousendothelial junction molecules results in trans-migration through the endothelial junction(13). Once through the endothelial lining,the neutrophil must navigate the basementmembrane, a protein mesh consisting largelyof laminins and collagen type IV. Speculationabounds that granule proteases assist in thismigration by digesting the protein mesh

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subsequent to degranulation; however, conclu-sive experimental evidence for this is lacking.

Once the endothelial barrier has beentraversed, the neutrophil finds itself in amuch different inflammatory milieu: Here, theenvironment is awash in a soup of chemoat-tractants and inflammatory stimulants, bothhost derived and of pathogenic origin. Thesecompounds will now be the primary dictatorsof neutrophil behavior and assume respon-sibility for initiating the concluding steps ofneutrophil activation. In the interstitial space,the neutrophil follows chemotactic gradientstoward the invading microbes, pursuing host-produced cytokines (e.g., IL-8) and, in parallel,pathogen-derived chemoattractants (e.g.,fMLP). During this process, these chemoat-tractants bind to their respective neutrophilreceptors (often G protein–coupled receptors,as is the case with the fMLP receptor FPR1 orthe chemokine receptors), which initiate a sig-naling cascade dominated by the MAPK/ERKpathway (22, 23). Downstream moleculesprompt assembly of the oxidative burst ma-chinery, a hallmark of neutrophil activation.Furthermore, the stimulation of FPR1 triggersthe release of ATP, whose autocrine actionthrough activation of purinergic receptors iscritical for the initiation of effective functionalresponses in neutrophils (24). Concomitantly,a family of molecules, the pattern-recognitionreceptors, is activated through recognition ofspecific nonself patterns present on many mi-crobes (25). Perhaps the best-known exampleof this family is the Toll-like receptors (TLRs);they are responsible for recognizing a numberof pathogen-derived compounds, collectivelycalled pathogen-associated molecular patterns(PAMPs), including LPS (TLR4), bacteriallipopeptides (TLR2), flagellin (TLR5), andDNA (TLR9). In neutrophils, all but oneof these receptors (TLR3) are constitutivelyexpressed, and their stimulation contributesto further activation, e.g., induction of theoxidative burst (25, 26). As the neutrophil nearsits target, continued activation by chemoattrac-tants further stimulates the oxidative responseand degranulation. Upon finally reaching a

point of high chemoattractant concentration,where no discernible gradient exists, theneutrophil halts and begins the final release ofits antimicrobial arsenal; the neutrophil is nowfully in an antimicrobial attack state.

The complex signaling cascade leading tofinal neutrophil activation has several facetsworthy of note. The movement to ever-higherconcentrations of chemoattractant is key inthis process, as individual chemoattractantsmay have very different effects on neutrophilphysiology at different concentrations, aphenomenon exemplified by one of the keyneutrophil-recruiting chemokines and ac-tivators, IL-8. At low concentrations, IL-8stimulates L-selectin shedding and increasedexpression of β2 integrins; slightly higherconcentrations result in initiation of theoxidative burst. At the highest concentrations,IL-8 induces degranulation of neutrophils (27).In addition, many chemoattractant moleculesexert a “priming” effect. That is, alone theystimulate the oxidative response only mildly,but they dramatically enhance the subsequentresponse to other stimuli. A notable example ofthis phenomenon is the strong priming effectof LPS on the fMLP response (28). In this case,exposure of the neutrophil to LPS inducesassembly of the NADPH oxidase machinery onthe membrane; fMLP stimulation then inducesactivation of this machinery (29). In contrast toreceptor priming, another critical feature of thestimulation process is the desensitization to pre-viously encountered ligands. Stimulation of theneutrophil by a chemoattractant often resultsin endocytosis of the corresponding receptor,thus leading to a desensitization of the neu-trophil to repeated stimulation with the samemolecule (30, 31). The rich and varied inputreceived by a neutrophil during this final leg ofthe activation process is complex, and the exacteffects of priming, desensitization, and signal-ing are incompletely understood. Regardless,the end result of this signaling cacophony isunambiguous: The neutrophil begins to imple-ment its regime of microbial killing, executingprograms of phagocytosis, degranulation, andNETosis (i.e., the process of setting neutrophil



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extracellular traps) (see the section on Neu-trophils and the Elimination of Microbes,below).

The initiation of these microbicidal actionsindicates the final stage of the neutrophil’sjourney through the activation process. How-ever, a prominent question remains largelyunanswered by the preceding exposition: Whatexactly is meant by the (admittedly ambiguous)phrase “neutrophil activation”? A quick scanof the literature presents the inexperiencedreader with a sometimes rather conflicting (andoverwhelming) view of neutrophil activation.In fact, one could be (erroneously) led tobelieve that neutrophil activation refers only todirect stimulation of the oxidative burst, as thishas been the canonical in vitro activation assayfor decades. This is, however, an oversimpli-fied view of a complex process. The myriadinteractions that occur during a neutrophil’sjourney toward an inflammatory site must be

parsed by the complex neutrophilic signalingmechanisms, a process that gradually leadsto complete activation and culminates in thepremiere killing functions of phagocytosis,degranulation, and NETosis. It is, therefore,more insightful to view neutrophil activationas a continuum of processes, priming steps,and signal cascades with varying effects andoutcomes, all focused on the realization ofone goal: the transition of naive, circulatingneutrophils to their microbe-eliminating,tissue-resident counterparts (Figure 1).

NEUTROPHILS AND THEELIMINATION OF MICROBES

The basic instruction set of the activatedneutrophil is both effective and ruthless inits simplicity: (1) kill microbes, (2) do noharm to the host, and (3) when in doubt, seerule 1. To fulfill this antimicrobial agenda,

Neutrophil

Endothelial cell

PSGL-1,L-selectin

P-selectin andE-selection

IntegrinICAM

Phagocytosis

Degranulation

Cytokine secretion

NETs

a Capture b Rolling c Firm adhesion

Figure 1Neutrophil recruitment to sites of inflammation. The circulating neutrophil must recognize signs ofinflammation and migrate to areas where its antimicrobial arsenal is needed for the elimination of infection.(a) Close to the inflammatory sites, stimulated endothelial cells expose a class of molecules, the selectins,which serve to capture circulating neutrophils and tether them to the endothelium. (b) Selectin-mediatedrolling along chemoattractant gradients then ensues, followed by (c) integrin-mediated firm adhesion.Subsequently, the neutrophil traverses through the endothelium and arrives at the site of inflammation.Here, the neutrophil releases cytokines that recruit other immune cells, and it begins to implement itsantimicrobial agenda. Among the processes employed are engulfment of microbes via receptor-mediatedphagocytosis, release of granular antimicrobial molecules through degranulation, and formation ofneutrophil extracellular traps (NETs).

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Inflammation:recruitment andactivation of immunecells upon infection orinjury; whenuncontrolled it leads totissue damage

neutrophils possess an array of toxic weaponsthat are carefully regulated through controlledmechanisms. These antimicrobial weaponsvary considerably in their methods of actionand thus reflect the neutrophil’s attempt toexploit any and all weaknesses that microbesmight present during the course of infection.An understanding of these weapons, theiraction, and their method of release is criticalto understanding neutrophil function.

Granules and Degranulation

The neutrophil must safely transport a plethoraof dangerous substances through the blood-stream and then correctly deploy them at theappropriate time. Therefore, it comes as nosurprise that a specialty storage organelle hasevolved in neutrophils: the granule. Expect-edly, these structures are replete with specifi-cally tuned mechanics that address the unique

needs of neutrophils. Granules are, however,far more than just latent repository organellesfor dangerous substances; they are active and in-dispensable participants in almost all neutrophilactivities during inflammation.

As mentioned above, there are threefundamental types of granules in neutrophils(Figure 2). Azurophilic granules (also knownas peroxidase-positive or primary granules) arethe largest, measuring approximately 0.3 μMin diameter, and are the first formed duringneutrophil maturation. They are named fortheir ability to take up the basic dye azure A andcontain myeloperoxidase (MPO), an enzymecritical in the oxidative burst (32, 33). Othercargo of this granule class include the defensins,lysozyme, bactericidal/permeability-increasingprotein (BPI), and a number of serine proteases:neutrophil elastase (NE), proteinase 3 (PR3),and cathepsin G (CG) (34). As such, thesegranules are brimming with antimicrobial

Granule typePrimary

(azurophilic)

Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cellStage offormation

Myeloperoxidase

Defensin

Degranulationpropensity

Lysozyme

Elastase

Lactoferrin

Gelatinase

Complement receptor 1Characteristicproteins

Otherproteins

Cathepsin G, PR3,BPI, azurocidin,sialidase,β-glucuronidase

Gp91phox/p22phox,CD11b, collagenase,hCAP18, NGAL, B12BP,SLPI, haptoglobin,pentraxin 3,oroscomucoid,β2-microglobulin,heparanase, CRISP3

Gp91phox/p22phox,CD11b, MMP25,arginase-1,β2-microglobulin,CRISP3

Gp91phox/p22phox,CD11b, MMP25, C1q-R,FPR, alkalinephosphatase, CD10,CD13, CD14, plasma proteins

FcγRIII

Secretoryvesicles

Tertiary(gelatinase)

Secondary(specific)

PMN

Figure 2Neutrophil granules. Neutrophil granules carry a rich variety of antimicrobials and signaling molecules. They are typically divided intothree types (primary or azurophilic, secondary or specific, and tertiary or gelatinase). Additionally, structures called secretory vesiclesare also considered to be a granule subset. Considerable overlap exists in the cargo of the different granules, and their contents seemdetermined by the timepoint during hematopoiesis at which they are produced (5). Granules also differ in their ability to mobilize, withsecretory vesicles being the first to fuse with the plasma membrane and the azurophilic granules demonstrating the least degranulationpropensity.



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compounds and function as a primary reposi-tory for the molecular weaponry of neutrophils.The second class of granules, the specific (orsecondary) granules, are smaller (0.1 μMdiameter), do not contain MPO, and are char-acterized by the presence of the glycoproteinlactoferrin. These granules are formed afterazurophilic granules; they also contain a widerange of antimicrobial compounds includingNGAL, hCAP-18, and lysozyme (33, 35). Thethird class, the gelatinase (tertiary) granules, arealso MPO-negative, are smaller than specificgranules, and contain few antimicrobials,but they serve as a storage location for anumber of metalloproteases, such as gelatinaseand leukolysin. These granules are also thelast population of granules formed duringneutrophil maturation (5). Finally, a fourth setof structures, the secretory vesicles, are alsocommonly considered part of the neutrophilgranule family. In contrast to the classicalgranules, these do not bud from the Golgi,but instead are formed through endocytosisin the end stages of neutrophil maturation(36). Consequently, their cargo consists pre-dominantly of plasma-derived proteins such asalbumin. The membrane of secretory vesiclesserves as a reservoir for a number of importantmembrane-bound molecules employed duringneutrophil migration.

As a neutrophil proceeds through activation,granules are mobilized and fuse with either theplasma membrane or the phagosome, releasingtheir contents into the respective environment.In both cases, the membrane of the granulebecomes a permanent part of the target mem-brane, thus altering its molecular composition(6). The different classes of granules demon-strate varying propensities for mobilization inresponse to inflammatory signals: Azurophilicgranules are the most difficult to mobilize, fol-lowed by specific granules, gelatinase granules,and finally, secretory vesicles (37–41). Theunderlying mechanisms for this differentialmobilization are not entirely understood, al-though regulation of intracellular calcium levelsappears to play a salient role (32, 39). Becauseof this varying mobilization propensity, each

granule subset has been traditionally associatedwith a particular stage of neutrophil activation.

After neutrophils contact the endothelium,stimulation through selectins and chemoattrac-tants induces mobilization of secretory vesi-cles, whose membranes are rich in key factorsnecessary for continued activation of the neu-trophil, including, among others, the β2 inte-grins, complement and fMLP receptors, as wellas the FcγRIII receptor CD16 (5, 38, 39, 42).Fusion of the secretory vesicles with the plasmamembrane exposes these components to the ex-ternal environment. This results in the transi-tion to firm adhesion, mediated by β2 integrininteraction with the endothelium. As they pro-ceed through the endothelium, neutrophils areexposed to further activation signals that initiatemobilization of gelatinase granules, thereby re-leasing metalloproteases. The activity of theseproteases may help neutrophils traverse thebasement membrane, although this has notbeen conclusively demonstrated (43, 44).

At the inflammatory site, complete acti-vation of the neutrophil ensues, promptinginitiation of the oxidative burst and mobiliza-tion of the azurophilic and specific granules.These granules either fuse with the phagosome(see section on Phagocytosis, below), con-tributing to the antimicrobial activities of thiscompartment, or fuse with the plasma mem-brane, releasing their potent antimicrobialsinto the tissue. The fusion of specific granuleswith the plasma or phagosomal membrane is ofparticular importance for the oxidative burst,as flavocytochrome b558, a component of theNADPH oxidase machinery, resides in thespecific granule membrane (45). This fusionpermits assembly of the NADPH oxidase com-plex and allows reactive oxygen species (ROS)production both inside the phagolysosome andoutside of the cell. Degranulation of primaryand secondary granules contributes to thecreation of an antimicrobial milieu at the in-flammatory site and produces an environmentinhospitable to invading pathogens.

The release of granular proteins during de-granulation presents the astute observer witha tempting proposition: Could these granular

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components also serve as signaling moleculesfor subsequent inflammatory cell recruitment?Recent studies have provided experimental evi-dence suggesting this does seem to be the case:Granule proteins from neutrophils, includingPR3 and azurocidin, can induce monocyte re-cruitment. Furthermore, neutrophil granuleproteins may increase macrophage bacterialclearance by enhancing phagocytosis (46). Thiscould be advantageous in situations in which theextracellular concentration of released granuleproteins is insufficient to exert extensive micro-bicidal effects. In such cases, the granule pro-teins would instead operate as signaling and re-cruitment factors (see section on Neutrophilsin Immune Cell Cross Talk, below).

By necessity, most data on neutrophildegranulation and its effects on neutrophil ac-tivity have been acquired through biochemicalapproaches performed exclusively in vitro. Apertinent question therefore presents itself: Isthis process truly relevant during the in vivoinflammatory response? The data here aresparse, and understandably so: Historically, thepossibilities for such an in vivo observation havebeen restrained by technical limitations. Mostevidence for in vivo degranulation relies onobservation of increased levels of extracellulargranular proteins at inflammatory sites. Evenso, release of granular components could occurprimarily through other means, most notablythrough formation of neutrophil extracellulartraps, cell damage, or cell lysis. With theadvent of intravital microscopy techniques,direct observation of the degranulation processin vivo may soon be realized.

Antimicrobial Proteins

Neutrophils produce a plethora of peptides andproteins that directly or indirectly kill microbes(Table 1). Many of these antimicrobials wereidentified through biochemical fractionation ofneutrophil extracts, and their in vitro activityis easily demonstrated in optimized conditions;nonetheless, showing in vivo relevance is chal-lenging. The diversity of antimicrobials sug-gests that some of them evolved to act together,

whereas others may be redundant. One of thechallenges in understanding the neutrophil’santimicrobial mechanisms is to study theirfunction during concerted action and in con-ditions that mimic an infection site. Therefore,testing the relevance of antimicrobials in vivois essential. This is, however, particularly chal-lenging; ablation of a single antimicrobial genemay only subtly affect immune defense. In ad-dition, much biochemical identification of neu-trophil antimicrobials has been performed inrabbits and humans, species with abundant neu-trophils. Mice, which are genetically tractable,have neutrophils that function differently fromthose of other species. Indeed, as already men-tioned, mice lack the genes for some antimicro-bials identified in humans. Interestingly, thereare few clinically relevant innate immune de-ficiencies that directly link antimicrobial activ-ity with a particular mutation. Thus, with fewexceptions, evidence for clinical or biologicalrelevance of these molecules is still lacking.

There are three main types of antimicro-bials: (a) cationic peptides and proteins thatbind to microbial membranes, (b) enzymes,and (c) proteins that deprive microorganismsof essential nutrients. Here we present anoverview of this rich field of investigation.There are more than 800 antimicrobialpeptides described in nature, some of themhighly conserved throughout evolution (47).These peptides are often charged, a feature thatprobably promotes their initial interaction withmicrobial surfaces. Under artificial conditions,many of these peptides disrupt the membraneintegrity. Because in vitro tests are often exe-cuted at high antimicrobial concentrations toobtain maximal microbial killing in the shortestpossible time, it is unclear whether this disrup-tion reflects their mechanism of action underphysiological conditions. Alternatively, someantimicrobials are thought to disrupt essentialmicrobial functions, such as DNA replication,transcription, or production of energy. Littleis known about antimicrobial concentrationsachieved at inflammatory sites or in the phago-some. This information, as well as informationabout the synergistic interactions of different



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Table 1 Mechanism of action of neutrophil antimicrobial proteins

Antimicrobial peptide Antimicrobial mechanisma

Cationic antimicrobial peptidesα-defensins (HNP-1, HNP-2,HNP-3, HNP-4)

� Permeabilize membrane bilayers containing negatively chargedphospholipids

� Inhibit DNA, RNA as well as protein biosynthesis� Inhibition of bacterial cell wall synthesis

LL-37 Transmembrane pore-formingBPI Increase bacterial permeability and hydrolysis of bacterial

phospholipids by binding to LPSHistones Unknown mechanismProteolytic enzymesLysozyme Degrades bacterial cell wallProteinase 3 (PR3) Mechanism independent of a proteolytic activity by binding to the

bacterial membraneNeutrophil elastase (NE),cathepsin G (CG)

� Cleaves bacterial virulence factors and outer membraneproteins

� Mechanism independent of a proteolytic activity by binding tothe bacterial membrane

Azurocidin Mechanism independent of a proteolytic activity by binding to thebacterial membrane

Metal chelator proteinsLactoferrin � Alters bacterial growth by binding to iron, an essential bacterial

nutrient� Binds to the lipid A part of LPS, causing a release of LPS from

the cell wall and an increase in membrane permeabilityCalprotectin Alters bacterial growth by sequestering manganese and zinc

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

antimicrobials, is essential for designing appro-priate in vitro conditions to probe mechanismsof action.

The neutrophil cationic antimicrobialpeptides include defensins and cathelicidins.Neutrophils mostly produce α-defensins, aprotein family whose members possess multi-ple disulfide bonds and whose structures maychange under physiological conditions andincrease their activity (48). A surprising num-ber of functions are assigned to defensins, butnone have been validated in vivo. Interestingly,inhibition of bacterial cell wall synthesis (49)was recently shown at low concentrations thatmay be more similar to those present at inflam-matory sites. Cathelicidins, including the well-studied LL-37, are proteolytically processed

from larger proteins, and in addition to theirantimicrobial activity, they may potentiateDNA activation of dendritic cells (DCs) (50).

Neutrophils also contain a number offull-length cationic antimicrobial proteins,including BPI and histones. BPI is cationicand binds LPS avidly, much like its structuralcousin the LPS binding protein. BPI binding toLPS results in increased bacterial permeabilityand hydrolysis of bacterial phospholipids; celldeath then follows (51). Interestingly, histonesare extremely effective antimicrobials andwere one of the first antimicrobials described(52). The significance of histones (and of thepeptides derived from them) as microbialsremains to be demonstrated in vivo (53).Given their dual role as an architectural

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Chronicgranulomatousdisease (CGD):caused by mutationsrendering theNADPH oxidasenonfunctional,characterized bysusceptibility toinfection andautoinflammation

scaffold for DNA and as antimicrobials, theirin vivo significance is particularly difficult todemonstrate.

The second class of neutrophil antimi-crobials encompasses a broad assortment ofproteolytic enzymes that participate in microbedestruction. Lysozyme destroys the bacterialwall, making it an obvious antimicrobial, asshown in mice deficient in this enzyme (54).Surprisingly, this occurred independently of itsenzymatic activity (55). Neutrophils also con-tain several serine proteases (including PR3,CG, and NE, collectively known as the serpro-cidins) that exhibit differing specificities. Theyare tightly regulated intra- and extracellularlyby serpins, indicating that their activity isdeployed under specific conditions. NE cleavesenterobacterial virulence factors with highspecificity (56), indicating the possibility of thecoevolution of microbial virulence factors andantimicrobial effectors. Of further interest, NEmutations in humans, but not genetic ablationof this enzyme in mice, results in neutropenia.This can be rescued by the administration ofrecombinant granulocyte macrophage colony-stimulating factor (GM-CSF); however, thesepatients still exhibit significant susceptibilityto infections. Mice deficient in NE or CGare highly susceptible to bacterial and fungalinfections (57, 58). Another protein, azuro-cidin, is a member of the same family but lacksprotease activity. Unexpectedly, it still killsmicrobes, suggesting that these proteins mayall have antimicrobial activity independentof proteolysis, perhaps as a result of theircationicity. These serine proteases also play asalient role in autoimmunity (see discussion insection on Autoimmunity, below) (59).

The final class of neutrophil antimicrobialsconsists of a number of proteins that chelateessential metals from microbes and possiblyimpact bacterial growth. Two of these chela-tors are lactoferrin, first identified in milk,which binds preferentially to iron, and cal-protectin (also called S100A and many othernames), which sequesters zinc (60) and results in“nutritional immunity” (61).

Reactive Oxygen Species

Upon activation, neutrophils produce ROS ina process called the respiratory burst. It is mis-leading to think of ROS as a single entity be-cause they differ in their stability, reactivity, andpermeability to membranes (62). However, allROS can modify and damage other molecules,properties exploited by the host cell for signal-ing and antimicrobial action.

The NADPH oxidase complex assembleson the phagosomal and plasma membranesand begins the reactive oxygen cascade byreducing molecular oxygen to superoxide.Downstream of superoxide, many potentialreactions can occur (for details, see References62–64). Superoxide, though not a strongoxidant, rapidly dismutates, forming hydrogenperoxide. Superoxide can also react with nitricoxide, which is produced at high levels atinflammatory sites, to form peroxynitrite, astrong oxidant. Upon degranulation into thephagosome, MPO can react with hydrogenperoxide to produce various reactive species,including hypohalous acids. Hypochlorousacid, thought to be the major product of MPOin the phagosome, is more reactive than su-peroxide and is antimicrobial in vitro. Thus, itis assumed to have direct antimicrobial effectsin the phagosome. However, a theoreticalmodel of the phagosome suggests that most ofthe hypochlorous acid produced would reactwith host proteins before reaching the bac-terium. This model predicts that chloramines,produced when hypochlorous acid reactswith amine groups, may be the most relevantantimicrobial actors in the phagosome (65).

ROS are clearly important for neutrophilantimicrobial activity: Neutrophils fromchronic granulomatous disease (CGD) patientskill microbes poorly, making these patientssusceptible to many infections. Interestingly,CGD patients can control catalase-negativebacteria, which produce, but do not degrade,their own hydrogen peroxide, thus providinga substrate for reactions downstream in thereactive oxygen cascade (66). NADPH ox-idase is also implicated in the regulation of



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inflammation, which explains why CGDpatients often suffer from autoinflammatorydiseases (67).

Paradoxically, although MPO is requiredfor neutrophil microbicidal activity in vitro,MPO-deficient individuals do not have strikingclinical manifestations (68, 69). Some MPO-deficient individuals suffer from frequent or se-vere infections, especially with Candida species,and a few have been mistaken for CGD patients.However, most MPO-deficient individuals inthe developed world have apparently normalimmunity. The mild effects of MPO deficiencysuggest that MPO’s products are not essentialfor antimicrobial action. Indeed, in the absenceof MPO, other reactive species (e.g., superox-ide, hydrogen peroxide, hydroperoxyl radical,peroxynitrite) can still be produced in theneutrophil phagosome; hydroperoxyl radical ispredicted to be present at antimicrobial concen-trations (65). However, there may be a broaderreason for this discrepancy. Modern technolo-gies can distinguish between individuals whoare partially and completely MPO deficient,and partial MPO deficiency does not correlatewith pathology (70). Residual activity of MPOmay be sufficient for antimicrobial activity: Inthe case of CGD, even 1% of normal NADPHoxidase activity leads to an improved prognosis(71). Epidemiological studies distinguishingthe degrees of MPO deficiency and theircorrelation with clinical manifestations may benecessary to understand the function of MPO.

In addition to direct antimicrobial action,ROS can modify host molecules. Becausethese species are highly reactive, they are oftenthought to be too nonspecific to be involved insignaling. However, specificity can be achievedon the submolecular level, by cellular redoxbuffering systems and by limited diffusion ofROS owing to their short half-lives (72). Awell-studied example of ROS in signaling isthe reversible regulation of various targets(including phosphatases, metalloproteinases,and caspases) by direct oxidation of cysteineresidues. In addition, neutrophil granuleproteases can be regulated by oxidative inacti-vation of their inhibitors or by direct oxidation

(73, 74). Furthermore, superoxide generationleads to an ionic influx into the phagosome tocompensate for charge; this may activate gran-ule proteases by releasing them from their pu-tative matrix (75). There is controversy aroundwhich ions and which channel are responsiblefor charge compensation, but this theory ofprotease activation is certainly intriguing (69).

Studies of ROS are hampered by varioustechnical issues. Ideally, a probe for ROSshould be specific, targetable to particularintracellular compartments, and capable ofbeing used in vivo. Traditional probes forROS do not meet these specifications; inaddition, the probes often become radicalspecies (76). One promising new approachfor ROS detection that meets these criteria isthe use of redox-sensitive fluorescent protein-based probes, such as roGFP and HyPer(76). Other methods that can be used in vivoinclude transcription profiling of superoxideor hydrogen peroxide–sensitive genes as wellas the detection of relatively stable products ofreactive oxygen using mass spectrometry (76).

Phagocytosis

Phagocytosis is the major mechanism to re-move pathogens and cell debris. It is an active,receptor-mediated process during which a par-ticle is internalized by the cell membrane intoa vacuole called the phagosome. As with otherphagocytes, the mechanistic details of internal-ization depend on the type of interaction be-tween the neutrophil and the microorganism.Interaction can be direct, through recognitionof PAMPs by pattern-recognition receptors, oropsonin mediated. The latter mechanism is bet-ter characterized and includes two prototypicalexamples: FcγR-mediated phagocytosis, whichrelies on the formation of pseudopod extensionsfor engulfment of IgG-opsonized particles, andcomplement receptor-mediated phagocytosis,which does not require membrane extensionsor pseudopods (77).

After engulfment, the nascent phagosomeis relatively benign to microorganisms, acquir-ing its lethal properties only after a drastic

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Autophagy: a processin which cellularcontents are degradedin lysosomes,especially inconditions of nutrientscarcity and infection

maturation process. Our understanding ofthis process is largely based on studies inmacrophages, and although these are certainlyinstructive, essential differences exist in neu-trophils. Macrophage phagocytosis follows anendocytic maturation pathway: In neutrophils,phagosome maturation happens upon fusion ofgranules to the phagosome, whereby deliveryof antimicrobial molecules into the phagoso-mal lumen occurs. Simultaneously, assemblyof the NADPH oxidase on the phagosomalmembrane allows ROS production, and jointly,these two mechanisms create an environmenttoxic to most pathogens. Neutrophil phago-somal pH regulation also differs significantlyfrom that observed in macrophages. While themacrophage phagosome gradually acidifies,neutrophil phagosomal pH is initially alkaline(78) and remains neutral for prolonged periodsof time (79). The maintenance of this alkalinepH is essential for the activation of the majorserine proteases NE and CG, and it is sustainedvia NADPH oxidase activity, despite contin-uing fusion of acidic granules. Key events ofthe maturation process are described in moredetail in Reference 80.

Not all pathogens succumb to the hostileenvironment of the phagosome. In fact, somehave evolved strategies to survive inside neu-trophils. These strategies include interferingwith engulfment, modulating phagosomematuration, and creating a more hospitableintraphagosomal environment. The polysac-charide capsule expressed by Staphylococcusaureus confers antiphagocytic properties (81).Helicobacter pylori can disrupt targeting ofNADPH oxidase to the phagosome so thatsuperoxide anions accumulate extracellularlyrather than in the phagosome (82). Francisellatularensis prevents triggering of the oxidativeburst and also inhibits ROS production inresponse to other stimuli (83). Finally, otherpathogens, such as Salmonella typhimurium andStreptococcus pyogenes, can efficiently block gran-ule fusion with the phagosome (84, 85). Thevariety of mechanisms evolved by intracellularpathogens to resist killing and enable survivalwithin the phagosome further emphasizes the

importance of phagocytosis in the innateimmune defense.

Neutrophil Extracellular Traps

Upon stimulation, neutrophils can undergoNETosis, an active form of cell death thatleads to release of decondensed chromatin intothe extracellular space (86, 87). The fibrousstructures termed NETs contain histones aswell as antimicrobial granular and cytoplasmicproteins (88). NETs trap many types of mi-crobes ex vivo and have been found in variousdisease models in vivo; they are thought tokill microbes by exposing them to high localconcentrations of antimicrobials (89).

The mechanism of NET formation is notcompletely understood. The reactive oxygenpathway is involved, as NADPH oxidase andMPO are required for NET formation in re-sponse to chemical and biological stimuli (87,90, 91). Nitric oxide donors can induce NETsvia a mechanism that also requires ROS (90), afinding that awaits genetic confirmation. All ac-tivators of NET formation tested so far requireROS production. S. aureus may be an exception,although those experiments were done usingpharmacological inhibitors, not cells deficientin ROS production (92). Upstream of NADPHoxidase, the Raf-MEK-ERK pathway is impli-cated in NET formation (93), but further alongin the process, NE translocates from the gran-ules to the nucleus and degrades histones, lead-ing to chromatin decondensation (94). Histonecitrullination may also play a role in NET for-mation, although this has not been confirmedin primary human neutrophils (95–97). Au-tophagy is also thought to be required for NETformation, but this has so far been shown onlyusing a nonspecific inhibitor of autophagy (98).

The majority of research on NETs has beenconducted ex vivo. Ideally, to test the relevanceof NETs, a “NETs knockout” organism shouldbe generated to investigate its response topathogens. Unfortunately, it is not possible toeliminate the main components of NETs—DNA and histones—from an infection model.Moreover, the factors that are important for



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Cystic fibrosis:caused by defects inthe CFTR iontransporter,characterized by thick,sticky mucus anddecreases in lung anddigestive function

NET formation, such as NADPH oxidase,MPO, and NE, are also critical for other an-timicrobial neutrophil functions. For now, theevidence for the relevance of NETs is indirect.On the one hand, bacteria that express DNasesas virulence factors disseminate more efficientlyin the host, which may point to evolutionarypressure to avoid entrapment by NETs (99,100). In addition, a persistent Aspergillusinfection in a CGD patient was cleared aftergene therapy, which restored NADPH oxidaseactivity, NET formation, and NET-mediatedbut not phagocytosis-mediated killing by thepatient’s neutrophils ex vivo (101). On the otherhand, the immune system has redundant mech-anisms to fight infection, and it may be thatNETs are especially important under certainconditions, such as during infections with largepathogens that are not readily phagocytosed.

NETs can also have detrimental effects onthe host. Because NETs expose self moleculesextracellularly, they lead to autoimmunity:NETs have been implicated in systemiclupus erythematosus (SLE), an autoimmunedisease characterized by the formation ofautoantibodies, often against chromatin andneutrophil components (102–106) (see sectionon Autoimmunity, below). Platelet-inducedNETs, formed during sepsis, are associatedwith hepatotoxicity due to tissue damage(107). Platelets also bind to NETs, raising thepossibility that NETs nucleate blood clots inthe context of deep vein thrombosis (108).NETs have also been observed in the airwayfluids of cystic fibrosis patients, where theymay increase the viscosity of the sputum anddecrease lung function (109).

NEUTROPHILS IN IMMUNECELL CROSS TALK

Neutrophils participate in the communica-tion networks that form the foundations ofimmunity, issuing instructions to practicallyall other immune cells. As one of the first celltypes to arrive at sites of infection, neutrophilssecrete cytokines and chemokines critical in theunfolding of the inflammatory response and in

establishing the correct environmental condi-tions to launch the adaptive immune response.The cytokines released by PMNs are oftensynthesized de novo. Although neutrophilstranscribe little after leaving the bone marrow,once activated, these cells undergo a tran-scriptional burst that results in the synthesisof signaling molecules (110, 111). Comparedwith other immune cells (e.g., macrophages),neutrophils typically produce lower amountsof cytokines per cell, but they are so abundantat inflammatory sites that their contributionto total cytokine levels is significant (4). Fur-thermore, neutrophil-secreted proteases canmodulate signaling networks in vivo throughcytokine processing (112).

The initial neutrophil cytokine response isan appeal for immunological reinforcement.The most abundantly produced cytokine, IL-8,primarily serves to recruit other neutrophils(113). Similarly, neutrophil-derived proinflam-matory IL-1β and TNF-α induce other cellsto produce neutrophil chemoattractants (114,115) (for a comprehensive list of cytokinesproduced by neutrophils, please see References115, 116). In addition to cytokines, neutrophilsrelease other signaling mediators, includinggranule contents (117), lipids (118), and ROSsuch as hydrogen peroxide (119). They alsocommunicate via cell-cell contact (120). Herewe provide examples of how neutrophilsinteract with other cells to shape the immuneresponse (see Figure 3).

Monocytes and Macrophages

As they respond to infection or injury,neutrophils and their relatives in the mono-cyte/macrophage lineage coordinate theiractivities, leading to alternating waves of re-cruitment of these two cell types. Macrophagesand patrolling monocytes are among the initialdetectors of PAMPs and endogenous activators,the danger-associated molecular patterns (121),and these cells work to summon large numbersof neutrophils to the inflammatory locus. Theinflux of neutrophils is followed closely by thearrival of monocytes, suggesting a causal link

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Neutrophil

Neutrophil

Neutrophil

Neutrophil Monocyte

T cell

T cell

Macrophage

Lymph node

Blood

Tissue

Activation anddifferentiation

ROS?

Arginase?

IFN-γ

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IL-12

NK cellDC

DC

DC

Activation

Activation

Crosspriming

Bacteria

Th1

Antigenpresentation

CD4+

T cell

CD8+

T cell

DC

DC

Figure 3Neutrophil communication with other immune cells. Neutrophils interact with a variety of cell types. They are important both forrecruitment of monocytes and dendritic cells (DCs) to infected tissues and for enhancement of macrophage and DC activity. Incontrast, in the lymph nodes, neutrophils impede DC function by inhibiting antigen presentation to CD4+ cells. Neutrophils alsointeract with the adaptive arm of the immune system: They can act as antigen-presenting cells by cross-presenting antigen to CD8+ Tcells; they also secrete IL-12, which activates T cells. T cells, in turn, activate neutrophils by secreting IFN-γ. Finally, neutrophils,DCs and natural killer (NK) cells colocalize and enhance each other’s activity via receptor-receptor interactions and soluble mediators.

behind these temporal dynamics. Indeed, neu-trophils recruit monocytes via several differentmechanisms. They express classical monocytechemoattractants such as CCL2 (MCP-1)(122), CCL3 (MIP-1α) (123), CCL20 (MIP-3α), and CCL19 (MIP-3β) (124). Additionally,and perhaps more unexpectedly, neutrophilsuse granule proteins to induce extravasationof monocytes in vivo, as shown for LL-37,azurocidin (HBP/CAP37), and CG (125–127).Monocyte recruitment is also affected indirectlyby neutrophils: via upregulation of endothelialadhesion factors, increase of transendothelialpermeability, enhancement of production ofchemoattractants by other cell types, and mod-ulation of the activities of these chemokinesvia proteolytic processing (reviewed in 128).In addition to recruitment, neutrophils mod-ulate monocyte and macrophage cytokineproduction (128), directly enhancing their

microbicidal activity (129). The circuitousnature of the cross talk of these two cell typesbecomes obvious during inflammation abate-ment: Monocytes, recruited by neutrophilsand differentiated into macrophages, repressfurther neutrophil chemotaxis and ensurethe appropriate removal of their postmortemremains (see section on Neutrophils andResolution of Inflammation, below).

Dendritic Cells

Neutrophils can also recruit and activateDCs in vivo. This was recently illustratedin a mouse model of Leishmaniasis, wheresubcutaneous inoculation of Leishmania majortriggered a massive and rapid infiltration ofneutrophils (130). These cells secrete thechemokine CCL3, recruiting DCs to thesite of inoculation and initiating a protective



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DC-SIGN:dendritic cell–specificintercellular adhesionmolecule-3-grabbingnonintegrin

Granulocytereceptor 1 (Gr1):the anti-Gr1 antibodyRB6-8C5 reacts withboth Ly6G (specificfor neutrophils) andLy6C (present onmany immune celltypes)

Th17 cells: subset ofT helper cells thatproduce IL-17,important ininflammation andimplicated inautoimmunity

Th1 response (131). Interestingly, activatedneutrophils can induce the maturation of DCsin vitro through specific receptor-receptorinteractions between Mac-1 and DC-SIGN,leading to local secretion of TNF-α (120).In this case, the reduced levels of cytokineproduction foster specificity, as only proximalDCs receive the maturation signal. A similaractivation model was earlier proposed for Tox-oplasma gondii (132). Neutrophil-activated DCsproduce the proinflammatory cytokine IL-12and induce proliferation of T cells (120, 132).However, some of these experiments shouldbe interpreted cautiously because they arebased on the injection of the anti-Gr1 antibody(RB6), which depletes neutrophils but may alsoresult in depletion of many other cell types inmice. The anti-Ly6G monoclonal antibody ismore specific and hence a better reagent for thistype of experiment (133). The crucial role ofneutrophils in DC activation was recently con-firmed using anti-Ly6G antibody depletion: InMycobacterium tuberculosis infection, timely traf-ficking of DCs to lymph nodes and activation ofCD4+ T cells were both dependent on PMNs.Furthermore, this study demonstrated thatDCs presented bacterial antigens when theyingested infected neutrophils just as efficientlyas they did via direct uptake of Mycobacterium(134). In sharp contrast to the above findings,a separate study using an immunization modelshowed that neutrophils recruited to lymphnodes compete for antigen with DCs andmacrophages and that these neutrophils inhibittheir interactions with T cells (135). It is possi-ble that neutrophils have site-specific effects onDCs and can be stimulatory at peripheral sitesand inhibitory in the lymph nodes. Neutrophilsexhibit fascinating and somewhat enigmatic be-havior in the lymph nodes, where they engagein swarming activity in response to parasiticinfection (136). The functions and mechanisticdetails of these swarms are unknown andrepresent questions of immense interest.

Natural Killer Cells

Studies of interactions between neutrophil andnatural killer (NK) cells have historically been

performed in vitro, and their interpretation isfrustratingly difficult owing to the question-able purity of cell preparations. Recently, itwas shown that neutrophils, NK cells, and DCsinteract in a menage a trois involving bothcytokine signaling and direct cell-cell contact(137, 138). In one report, infection of micewith Legionella pneumophila triggered produc-tion of IFN-γ by NK cells; this was dependenton both PMN-derived IL-18 and DC-derivedIL-12 (137). Similarly, human neutrophils, NKcells, and DCs colocalize at inflammatory sites,and a positive feedback loop has been proposedon the basis of in vitro data. In this scheme, neu-trophils interact with a specific subset of DCs,(via CD18-ICAM-1 interactions), promptingthe DCs to produce IL-12p70, which in turnstimulates IFN-γ production by NK cells andfurther activates neutrophils. Simultaneously,neutrophils also activate NK cells by direct con-tact (139). Additional in vitro interactions be-tween neutrophils and NK cells are extensivelyreviewed in Reference 138.

Lymphocytes

A surprising finding in recent years is the exten-sive cross talk between cells located at oppositeends of the immune spectrum. Previouslythought to belong to isolated compartments,neutrophils and T cells shape and impacteach other’s functions, both qualitatively andquantitatively (140). Neutrophils affect T cellfunction indirectly via DCs, as outlined above,but can also influence T cell function directly.PMNs secrete IL-12, which may be crucial forTh1 cell differentiation (141, 142). They alsoexpress several T cell chemoattractants (116)as well as B cell development and maturationfactors (143, 144). Cytokine communicationoccurs in both directions: For instance, IFN-γ,which is secreted by T cells, prolongs neu-trophil life span, induces gene expression, andincreases phagocytic capacity (145). The Thelper 17 (Th17) cell subset secretes IL-17,a key cytokine in the control of neutrophildynamics, which acts by upregulating expres-sion of CXCL8 (IL-8), G-CSF, and TNF-α

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Ulcerative colitis: atype of inflammatorybowel diseasecharacterized by ulcersand tissue erosion inthe colon and rectum

by epithelial, endothelial, and stromal cells(146). Collectively, these Th17-associatedcytokines increase granulopoeisis as well as therecruitment and life span of neutrophils.

Neutrophils potentially have suppressive ef-fects on T cells via two proposed mechanisms:(a) L-arginine depletion by release of arginase,which inhibits T cell responses in vitro (147),and (b) hydrogen peroxide–mediated suppres-sion, as proposed in a cancer model (119) (seesection on Cancer, below). Direct evidence ofsuch interactions in vivo is still missing.

Interestingly, neutrophils influence CD8+

T cell responses by cross-presenting exogenousantigens in vivo. Using mice in which profes-sional antigen-presenting cells do not expressfunctional MHC class I, Beauvillain et al. (148)showed that antigen-pulsed neutrophils caninduce differentiation of cytotoxic T cells.These striking findings imply that neutrophilshave characteristics of antigen-presenting cells.Neutrophils also appear capable of expressingMHC class II and costimulatory moleculesunder inflammatory conditions (149–151),and they can present antigen to CD4+ T cellsin vitro (152–154). However, the functionalsignificance for protective immunity remainsunclear, especially in light of the finding thatmouse neutrophils that migrate to the lymphnode have a negative effect on CD4 responsesin an immunization system (135). In humans,there are large variations in the ability ofdonors to express MHC class II (149, 151),suggesting concomitant variations in the abilityto activate T cells, a finding that could haveimplications for susceptibility to autoimmunediseases. Therefore, neutrophil modulation ofadaptive immunity seems to be highly complexand is only now starting to be unraveled.

NEUTROPHILS ANDRESOLUTION OFINFLAMMATION

The lethal cargo of neutrophils is not onlydestructive toward invading microbes, butalso harmful to host cells. Thus, neutrophildeployment must be tightly controlled.

Although some collateral damage to hosttissues is inevitable during infection, neu-trophils must be removed before they haveserious, detrimental effects on inflamed tissues.Resolution of inflammation is an active processthat limits further leukocyte infiltration andremoves apoptotic cells from inflamed sites.This process is essential for maintenance oftissue homeostasis and, if impeded, leads to“nonresolving inflammation,” a problematiccondition that contributes to many diseases.

Apoptosis and Clearance

Apoptosis is a central aspect of inflammationresolution. Once neutrophils have executedtheir antimicrobial agenda, they die via a built-in cell-death program. However, not only doesapoptosis reduce the number of neutrophilspresent, it also produces signals that abro-gate further neutrophil recruitment. Phagocy-tosis of apoptotic neutrophils also reprogramsmacrophages to adopt an anti-inflammatoryphenotype.

Neutrophil death is influenced by inflamma-tory mediators such as GM-CSF and LPS andby environmental conditions such as hypoxia,all of which prolong neutrophil survival. Thesignaling networks that regulate survival havealso been well characterized. These networksalso control the expression of known antiapop-totic (Mcl-1 and A1) or proapoptotic proteins(Bad, Bax, Bak, and Bid), and they also activatecaspases (for an extensive review, see Reference155). Given that neutrophils are terminallydifferentiated, it is unexpected that moleculescontrolling cell proliferation regulate survival.Proposed to have prosurvival effects, one suchprotein is survivin. It is expressed more highlyin immature neutrophils than in mature ones,but its expression can be restored in maturecells by inflammatory signals such as G-CSF orGM-CSF. In line with these findings, survivinis also highly expressed in neutrophils at sitesof inflammation, such as cystic fibrosis sputum,appendix infiltrates, and intestines of patientswith ulcerative colitis (156).



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Wegener’sgranulomatosis:vasculitis affecting thelungs, nose, andkidneys; inflammationleads to reduced bloodflow, tissuedestruction, anddamage of vital organs

Prostaglandins andleukotrienes: lipidssynthesized bycyclooxygenases and5-lipoxygenase,respectively, in thearachidonic acidpathway; haveproinflammatoryfunctions includingleukocyte recruitment

Similarly, cyclin-dependent kinases func-tion as prosurvival factors in neutrophils.Pharmacological inhibition of these cell cycleregulators induce caspase-dependent apoptosisand block life-span extension by survival factors(157). More recently, prosurvival effects werealso attributed to proliferating cell nuclearantigen (PCNA). This factor usually residesin the nucleus, where it is involved in DNAreplication, but in neutrophils, it associateswith procaspases in the cytosol and is thoughtto prevent their activation. During apoptosis,PCNA is targeted for proteosomal degradation,which correlates with an increase in caspase-3and caspase-8 activities. This mechanism is rel-evant in Wegener’s granulomatosis and sepsis,where stabilization of PCNA is associated withresistance of neutrophils to apoptosis (158).

Equally important for the resolution of in-flammation is the proper removal of apoptoticcells. This relies on the release of “find-me”signals at early stages of cell death, which at-tract phagocytes. Likewise, distinct “eat me”signals are required for specific recognition ofapoptotic cells. Ingestion of apoptotic cells bymacrophages drives the production of the anti-inflammatory cytokines tumor growth factor(TGF)-β and IL-10 (155). Failure to clear theseapoptotic cells, by contrast, results in secondarynecrosis and release of products that generateproinflammatory signals (Figure 4).

Lipid Mediator Class Switch

Soluble mediators play a crucial role in theresolution of inflammation. In neutrophils,a particularly prominent role is assumed bylipid mediators. The successful progressionof inflammation appears to hinge on a shiftin the composition of secreted lipids. At earlystages of inflammation, neutrophils synthesizeproinflammatory lipid mediators, such asprostaglandins and leukotrienes. These arederived from arachidonate precursor moleculesand are synthesized through the cyclooxy-genase and lipoxygenase pathways. Duringthe later stages of the inflammatory response,neutrophils interact with various cell types in

their vicinity (epithelial cells, endothelial cells,fibroblasts, platelets, and leukocytes) and par-ticipate in the transcellular biosynthesis of lipidmediators with anti-inflammatory and prore-solving activities, such as lipoxins, resolvins, andprotectins. A major lipid mediator class switchthus exists, governed by temporally regulatedexpression of different lipoxygenases and themobilization of different fatty acid substrates.The different biosynthesis pathways of prore-solving lipid mediators have been reviewed indetail elsewhere (118). Interestingly, microor-ganisms are also a source of lipid precursorsthat can be used by neutrophils for resolvinsynthesis. Thus, microbes also likely participatein synthesis of mediators with proresolvingfunctions at the site of infection (159, 160).

How do lipid mediators contribute tothe termination of inflammation? Lipoxins,resolvins, and protectins exert cell-type specificeffects, promoting monocyte/macrophagerecruitment and activation while inhibitingneutrophil functions. The inhibitory effectextends to all essential steps of neutrophilresponses: migration, adhesion, and activation.All three lipid mediators reduce neutrophilrecruitment, a process that involves the lipoxin-A4 receptor and the leukotriene B4 receptor(BLT1) (161–167). Ariel et al. (168) also pro-posed an interesting mechanism of action forlipoxins, resolvins, and protectins in clearing in-flammatory sites. They showed that neutrophilexposure to these lipids increases expressionof CCR5 on the surface of late apoptotic neu-trophils, leading to efficient sequestration of thechemoattractants CCL3 and CCL5. The se-questration of these chemokines means they areunavailable to recruit neutrophils to inflamedsites (168) (Figure 4). This mechanism com-plements other anti-inflammatory processesin which chemokines are inactivated by neu-trophil proteases. Of these lipids, lipoxins arethe most completely understood. In addition toneutrophil recruitment, lipoxins can inhibit theshedding of L-selectin and the upregulation ofβ2 integrins in response to proinflammatorystimuli, thereby reducing adhesion of neu-trophils to endothelial cells (169, 170). Finally,

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TNF-αIL-6

IL-10TGF-βPGE-2

Neutrophil

Monocyte

Platelets

Lipoxins

Macrophage

Macrophage

ChemokinesApoptotic

neutrophil

NEToticneutrophil

LeukotrienesProstaglandins

?Microorganisms

LipoxinResolvinsProtectins

Chemokines

CCR5

Initiationof inflammation

Resolutionof inflammationLeukotrienes Prostaglandins TNF-α TGF-βLipoxins Resolvins Protectins IL-10

Chemokine clearance

Figure 4From inflammation to homeostasis: neutrophil apoptosis and lipid mediator class switching in the resolution of inflammation. At thesite of infection, resident macrophages initiate an inflammatory response, secreting proinflammatory cytokines and chemokines thatalert the immune system and promote neutrophil recruitment. In the early stages of inflammation, microbes trigger the production ofproinflammatory lipid mediators, such as leukotrienes and prostaglandins, which also recruit neutrophils. As inflammation progresses, aswitch occurs, and anti-inflammatory lipid mediators such as lipoxins, resolvins, and protectins are produced. Notably, interaction ofneutrophils with platelets induces the production of lipoxins. Anti-inflammatory lipid mediators initiate the resolution of inflammationby blocking neutrophil and promoting monocyte recruitment. Monocytes differentiated into macrophages ingest apoptotic neutrophils,driving the production of the anti-inflammatory cytokines tumor growth factor (TGF)-β and IL-10 and prostaglandin-E2 (PGE-2),which drive the lipid mediator class switch. Pro-resolving lipid mediators also promote the expression of CCR5 on the surface ofapoptotic neutrophils, providing a means of scavenging chemokines. Chemokine clearance upon phagocytosis of apoptotic neutrophilsby macrophages further contributes to the reduction of neutrophil infiltration and the return to tissue homeostasis. The contribution ofmacrophages to the clearance of NETotic neutrophils, and how this could impact inflammation resolution, is currently unknown. Atimeline of the inflammation process from initiation to resolution is summarized in the upper part of the figure.

Chronic obstructivepulmonary disease(COPD): lung diseasecaused by noxiousparticles or gas, e.g.,tobacco smoking;inflammation leads tolung obstruction

lipoxins also impact neutrophil activation byinhibiting ROS and peroxynitrite production,NF-κB activation, and IL-8 expression (170).

In addition to directly impacting neu-trophil functions, lipid mediators promotenonphlogistic (noninflammatory) phagocyto-sis of apoptotic neutrophils by monocytesand macrophages. In the presence of anti-inflammatory lipids, engulfment of apoptoticneutrophils is not accompanied by the release ofproinflammatory mediators, as typically occursduring macrophage activation. Instead, produc-tion of the anti-inflammatory cytokines TGF-βand IL-10 is increased (163, 171).

Disorders Associated withNonresolved Inflammation

The failure of neutrophils to apoptose or mal-functions in the removal of their apoptotic re-mains result in chronic inflammation. Theseconditions lead to the accumulation of cyto-toxic substances and are associated with severepathologies, including cystic fibrosis, chronicobstructive pulmonary disease (COPD), andrheumatoid arthritis (RA). The severity of in-flammation often directly correlates with poorclinical outcome.

COPD is a major cause of death in indus-trialized nations, where smoking is a prime



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Rheumatoid arthritis(RA): chronicinflammatory diseasethat affects manytissues and organs butprimarily synovialjoints; severeinflammation causesdeformity

instigator of this disease. A chronic neutrophilinfiltration in the lungs of COPD patientspromotes tissue damage and organ dysfunc-tion. One of the key molecules controllingthe inflammatory response in the lung isleukotriene A4 hydrolase (LTA4H). Thisenzyme has two opposing activities. First, itshydrolase activity converts leukotriene A4 intoleukotriene B4, a potent neutrophil chemoat-tractant and proinflammatory agent. Second,LTA4H is an aminopeptidase that inactivatesa specific neutrophil chemoattractant, theproline-glycine-proline tripeptide (PGP), thusconferring the enzyme with anti-inflammatoryproperties. Interestingly, tobacco smoke selec-tively inhibits only the aminopeptidase activityof LTA4H, promoting the accumulation ofboth leukotriene B4 and PGP. This in turnpromotes neutrophil recruitment and fuelschronic lung inflammation (172).

Another prime example of a disease linked tononresolving inflammation is RA. Neutrophilsare the most abundant leukocytes present in thesynovial fluid of RA patients, and their role inpathogenesis has been demonstrated in severalanimal models. These models primarily usedneutrophil depletion or adoptive transfer ofwild-type neutrophils in leukotriene-deficientmice (173–175). In one model, synthesisof leukotriene B4 by neutrophils in jointsis essential for disease development (174).Leukotriene B4 can act in an autocrine mannervia the neutrophil receptor BLT1 to promotethe recruitment of a first wave of neutrophilsinto the joint. Later, the recruitment of asecond wave of neutrophils is independent ofthis leukotriene B4–BLT1 pathway. At thisstage, immune complexes are essential forstimulating infiltrating neutrophils to deliverIL-1β into the joint. This in turn induces theproduction of chemokines by synovial tissuecells and sustains neutrophil recruitment (175,176). These studies exemplify the complexregulation cascades involving lipids, cytokines,and chemokines that orchestrate neutrophilrecruitment in chronic inflammation. Theyalso demonstrate the cross talk between neu-trophils and other immune cells discussed in

the previous section. It is, however, unknownwhether all neutrophils are capable of adaptingto the changing chemoattractant environmentor if different subsets of neutrophils are suc-cessively involved. The relevance of this modelin human disease remains to be established,although the clinical similarities between thismouse model and human RA are encouraging.

NEUTROPHILS IN DISEASE

Neutrophils are prominent players in the innateimmune response and the clearance of infec-tion, a subject addressed in several prominentreviews. However, neutrophil action can alsosupport disease progression in other illnesses.A host of autoimmune disorders belong to thiscategory. In addition, certain malignant cancersare also prime examples of illnesses in whichneutrophils play a salient role.

Cancer

The link between cancer and inflammationwas noted as early as 1863 by Rudolf Virchow(177). Since then, it has been proposed thatneutrophil-derived ROS have the potential toinitiate tumor formation by genotoxic stressand induction of genomic instability. Althoughthis has been demonstrated in vitro (178, 179),convincing evidence for PMN-mediated DNAmutagenesis in vivo is still lacking. Neutrophilsdo, however, impact cancer progression.They are abundant in tumors and influencetumor development through several secretedmediators, including cytokines, ROS, andmatrix-degrading proteases (reviewed in Ref-erence 180). The majority of findings supporta “protumor” and “antihost” effect of thesecells; clinical studies indicate that neutrophilinfiltration of tumors is associated with poorerprognosis (181, 182). Indeed, some cancersseem to actively recruit neutrophils throughproduction of IL-8 (183). In agreement withthis, antibody depletion of neutrophils reducestumor growth (184). The protumor functionof neutrophils operates at multiple levels,including production of angiogenic factors

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Acute-phaseproteins: secreted byliver, concentration inplasma changes by25% or more duringinflammation

(185), enhancement of metastasis (186), andsuppression of the antitumor immune response(119, 187). Using the anti-Ly6G antibody,Fridlender and colleagues (187) depleted neu-trophils and confirmed their tumorigenic role.Moreover, the study showed that neutrophils inthe tumor microenvironment could, under cer-tain circumstances, be induced to target theircytotoxic arsenal at tumor cells, whose growththey usually help to fuel. Pharmacologicalinhibition of TGF-β signaling led tumor-associated neutrophils to assume a heightenedproinflammatory state, causing a reduction intumor growth. These alternatively activatedneutrophils underwent a complete reversal intheir effect on CD8+ T cells, serving to activaterather than suppress these cells. Differentialneutrophil responses were also demonstrated ina melanoma study. In this instance, increasedsystemic levels of the acute-phase proteinserum amyloid A (SAA-1) induced neutrophilsto secrete the anti-inflammatory cytokine IL-10, which also inhibited T cell responses. Crosstalk with invariant NKT cells could counterthis response, restoring a proinflammatoryactivation status (188). Thus, investigation ofneutrophils in cancer has revealed considerableplasticity in their responses. Although littleevidence currently supports the existence ofdifferent populations, it is likely that neutrophilresponses are more flexible and less stereotypedthan previously thought.

Another major mechanism of tumor escapefrom immune control has recently beenattributed to a heterogeneous category of im-mature myeloid cells, called myeloid-derivedsuppressor cells (MDSCs) (189). In healthyindividuals, MDSCs are found in the bonemarrow, where they differentiate into matureneutrophils and monocytes. In cancer andsome autoimmune and infectious diseases,differentiation is partially blocked, leading toaccumulation of these precursors, which act aspowerful suppressors of T cell functions. MD-SCs have characteristics of neutrophils, and inmice, they are typically detected using the neu-trophil surface markers CD11b+ and Gr-1+,although they consist of variable proportions

of monocytic and granulocytic cells (189). Inhuman renal cell carcinoma, MDSCs haveidentical morphology and express the same sur-face markers as do activated neutrophils (190,191). MDSCs inhibit T cell proliferation bylimiting L-arginine availability via arginase andNOS activities, both of which use this aminoacid as a substrate (189, 191, 192). Furthermore,MDSCs are strong producers of ROS, whichsuppresses T cell responses (119, 192). Inter-fering with the release of MDSCs or using druginterventions to polarize neutrophil responsesin the tumor microenvironment could repre-sent novel therapeutic strategies against cancer.

Autoimmunity

Deregulated neutrophil cell death and/orclearance often accompanies autoimmune syn-dromes (193–195) and may play a major rolein disease pathogenesis, given that release ofproteolytic and cytotoxic molecules from neu-trophils can trigger organ damage. Neutrophilproducts act as both targets and mediators ofautoimmunity. MPO and PR3 are the main tar-gets of antineutrophil cytoplasmic antibodies(ANCA), autoantibodies directed against anti-gens present in the cytoplasm of neutrophils.Wegener’s granulomatosis is consistently as-sociated with the presence of ANCA. Further-more, the extent of organ damage in patientswith Wegener’s granulomatosis correlates withthe PMN infiltrate rather than with traditionalautoimmunity parameters such as T cell acti-vation or autoantibody titers (196). Likewise,ANCA bind MPO and PR3 expressed on thesurface of activated neutrophils, promotingdegranulation and release of chemoattractantsand ROS, which together lead to a viciouscycle of tissue damage and inflammation. Earlyreports also suggest that, in an inflammatory en-vironment, ANCA accelerate ROS-dependentneutrophil apoptosis, suggesting a feed-forwardcycle culminating in organ damage (194, 195).

SLE is another chronic autoimmune diseaseaffecting multiple tissues and organs. Autoan-tibodies produced in SLE are predominantlyeither ANCA or directed against chromatin.



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Vasculitis:inflammation of bloodvessels

Although neutrophils had long been suspectedto be causative agents, their role in SLE patho-genesis remained elusive. The recent discoveryof a link between SLE and NET formationhas helped to shed light on this quandary.It was proposed that TNF-α and IFN-αprime cells for NET formation in response toanti-PR3, antiribonucleoprotein, anti-HNP,or anti-LL-37 autoantibodies (103, 104, 106).Thus, high levels of inflammatory cytokines inautoimmune patients are believed to sensitizeneutrophils to NETosis, whereas autoantibod-ies may trigger a switch from apoptosis to NE-Tosis. Additional evidence suggesting a role forNETs in autoimmune pathology was obtainedwhen NETs were identified in renal and/orskin biopsies from patients with SLE and smallvessel vasculitis (103–106). Several studies havereported the presence of a particular subset ofneutrophils in PBMC preparations from pedi-atric and adult SLE patients. These low-densitygranulocytes display phenotypic characteristicsof immature neutrophils with nonsegmentednuclei and higher expression of MPO, NE,and defensin-3, and they may be related to theMDSCs discussed previously (see section onCancer, above) (197, 198). An increased capac-ity to form NETs and a heightened cytotoxicitytoward endothelial cells could bestow themwith pathogenic properties in lupus (105).

Because NETs appear to be formed duringautoimmune disease, their timely removal maybe an essential mechanism for maintainingtissue homeostasis. Human serum contains thenuclease DNase I, which degrades NETs invitro. Notably, a familial form of SLE is linkedto a mutation in DNase I (199). Furthermore,in a cohort of SLE patients, 36% exhibitedeither elevated titers of autoantibodies directedagainst NET components or inhibitors ofDNase I, both of which may protect NETsfrom degradation. Most notably, impairedNET degradation correlates with developmentof lupus nephritis, one of the most severemanifestations of SLE (102).

Can it be that NETs play a general rolein modulation of autoimmune responses? Weknow that NETs induce plasmacytoid DCs

to produce IFN-α, a central cytokine in SLEpathogenesis (103, 104). However, it remains tobe determined if DCs can present NET com-ponents or if they contribute to autoreactive Bcell activation. It is also possible that NETs areinvolved in other autoimmune diseases. Shouldthis prove to be the case, understanding therole of NETs may provide critical insights intothe role of microbial infections as a trigger ofautoimmunity.

CONCLUDING REMARKS

Neutrophils are specialized phagocytes thatarose as an evolutionary adaptation in verte-brates to coordinate and execute one of the mostfundamental physiological responses: inflam-mation. They are endowed with antimicrobialmechanisms that make them the preeminentmicrobe exterminators of the immune system.In addition to this important role, PMNs alsonetwork with many other immune cells andhelp regulate the initiation of specific T andB cell immunity. However, neutrophils do notalways act in ways beneficial to the host: Uncon-trolled neutrophil responses can exacerbate andeven cause autoimmune and inflammatory dis-eases. Many challenges remain in understand-ing neutrophil function: Is there specializationamong PMNs? Are they more plastic than wesuspect? How do they make decisions beforedeploying their armamentaria? How do theykill microbes? How specific are their instruc-tions to other cells? Answering these questionswill better define neutrophils’ role in defenseand disease and will provide a rational path forpursuing new therapies. Moreover, neutrophilscan potentially provide insights into severalunique aspects of basic cell biology. Their strik-ingly short life spans make them excellent mod-els for investigating cell death, whereas theirreliance on ROS as biochemical effectors mayreveal novel ways for relaying intracellularsignals. The uniquely lobulated neutrophilnucleus is a feat of higher-order nucleararchitecture that is just beginning to yieldits secrets. In short, exciting times await thehumble neutrophil.

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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Diane Schad for assistance with graphic design and Cornelia Heinz for administrativehelp. G.H. is an Alexander von Humboldt Foundation Scholar, and B.A. is supported by an EMBOLong-Term Fellowship.

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