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    Neutrophil Function:From Mechanisms to DiseaBorko 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: [email protected],[email protected], [email protected], metzler@[email protected]

    Annu. Rev. Immunol. 2012. 30:45989

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

    The Annual Review of Immunology is online at immunol.annualreviews.org

    This articles 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.

    Keywordsinammation, antimicrobial, granule, phagocytosis, NET

    Abstract Neutrophils are the most abundant white blood cells in circuand patients with congenital neutrophil deciencies suffer from infections that are often fatal, underscoring the importance ofcells in immune defense. In spite of neutrophils relevance in immresearch on these cells has been hampered by their experimentatractable nature. Here, we present a survey of basic neutrophil b with an emphasis on examples that highlight the function of neutronot only as professional killers, but also as instructors of the isystem in the context of infection and inammatory disease. Won emerging issues in the eld of neutrophil biology, address quin this area that remain unanswered, and critically examine the mental basis for common assumptions found in neutrophil litera

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    INTRODUCTION In the late nineteenth century, Paul Ehrlich,dissatised with what he considered an in-excusable disinterest in the white blood cell,began to utilize newly developed cell-stainingtechniques to examine subpopulations of leuko-cytes.Hisexperimentationledtoanewappreci-

    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 apolymorphous 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 initially shrouded in considerable mystery; their con-spicuous presence during infections led severalresearchers to arrive hastily at a rather ironic

    conclusion: They surmised that neutrophilspromote infection, serving as cellular shuttlesfor bacteria (2). Their actual function, that of antimicrobial 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. Metchnikoffs seminal theory of cellularimmunity was based on comparative embryology and observa-tions ofphagocytes in various simpleorganisms, includingthe 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 andgranule-packed cytoplasmis a more recentevolutionary adaptation. In insects, phagocytes are long lived and have roundnuclei. They do, however, produce hydrogen peroxide and carry distinct classes of granules (201). Bony sh and frogs have bonade neutrophils that are functionally similar to mammalian ones(202, 203). In both zebrash and rodents, neutrophils are lessabundant than in humans, comprising only 1520% of immunecells. In chimpanzees, neutrophils account for more than 50% of the differential blood count (204).

    early and enthusiastic evolutionary biologistterested in the phagocytic capacity of cells.

    Metchnikoff demonstrated that injury starsh embryos resulted in recruitment phagocytic cells to the site of injury (3).theorized (correctly) that these cells migrateinjured sites and participate in microbe dig

    tion. Remarkably, this prescient view of ntrophil action still aptly summarizes, more tha century later, the basic role of neutrophin immunity. The uniquely lobulated nucleof the neutrophil also inspired Metchnikoffrename these cells: He called them polymphonuclear leukocytes (or PMNs), a title tstill enjoys frequent use and that is used inchangeably with neutrophil throughout this r view. Togetherwith two other developmentarelated cell types, the eosinophils and basop(also discovered by Ehrlich), PMNs form granulocyte family of white blood cells, a filywhosehallmarkisthepresenceofgranulunique storage structures important in antimcrobial functions (see section on Granules Degranulation, below).

    Neutrophils were discovered at the daof the immunological sciences; consequenelucidation of their role in the immune sponse has been an ongoing process stretchover more than a century. We now know ththey are key components of the innate immuresponse and vital in immune function; unftunately, their importance has often been ovshadowed by breakthroughs in the study ofadaptive immune response (4). Admittedly,situation is exacerbated by neutrophils notous experimental intractability: They exhibshort life span and are terminally differentiatpreventing growth in tissue culture. The stadard tools of molecular biology, such as trafection and RNA interference, are of little when applied to these cells, and immortalneutrophil-like cell lines rarely reect functional diversication of neutrophils. Fthermore, neutrophil-like cells studied in isolation of a culture dish most certainly domimic the complex biological reality in tisor circulation. Conclusions from in vitro sties should, therefore, be carefully interpre

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

    Once the endothelial barrier has beentraversed, the neutrophil nds itself in amuch different inammatory milieu: Here, theenvironment is awash in a soup of chemoat-tractants and inammatory stimulants, both

    host derived and of pathogenic origin. Thesecompounds will now be the primary dictatorsof neutrophil behavior and assume respon-sibility for initiating the concluding steps of neutrophil 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 proteincoupled receptors,as is the case with the fMLP receptor FPR1 orthe chemokine receptors), which initiate a sig-naling cascade dominated by the MAPK/ERK pathway (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 of specic 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, collectively called pathogen-associated molecular patterns(PAMPs), including LPS (TLR4), bacteriallipopeptides (TLR2), agellin (TLR5), andDNA (TLR9). In neutrophils, all but oneof these receptors (TLR3) are constitutively expressed, and their stimulation contributesto further activation, e.g., induction of theoxidative burst (25, 26). As the neutrophil nearsits target, continued activationby chemoattrac-tants further stimulates the oxidative responseand degranulation. Upon nally reaching a

    point of high chemoattractant concentration, where no discernible gradient exists, theneutrophil halts and begins the nal release of its antimicrobial arsenal; the neutrophil is now fully in an antimicrobial attack state.

    The complex signaling cascade leading tonal neutrophil activation has several facets

    worthy 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 exemplied by one of the key neutrophil-recruiting chemokines and ac-tivators, IL-8. At low concentrations, IL-8stimulates L-selectin shedding and increasedexpression of 2 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 they stimulate the oxidative response only mildly,but they dramatically enhance the subsequent response to other stimuli. A notable example of this phenomenon is the strong priming effect of LPS on the fMLP response (28). In this case,exposure of the neutrophil to LPS inducesassembly of the NADPH oxidase machinery onthe membrane; fMLP stimulation then inducesactivation of this machinery (29). In contrast toreceptor priming, another critical feature of thestimulationprocessis thedesensitization topre- 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 input received by a neutrophil during this nal leg of the activation process is complex, and the exact effects of priming, desensitization, and signal-ing are incompletely understood. Regardless,the end result of this signaling cacophony 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 nal stage of the neutrophils journey through the activation process. How-ever, a prominent question remains largely

    unanswered by the preceding exposition: What exactly is meant by the (admittedly ambiguous)phrase neutrophil activation? A quick scanof the literature presents the inexperiencedreader with a sometimes rather conicting (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 assay for decades. This is, however, an oversimpli-ed view of a complex process. The myriadinteractions that occur during a neutrophils journey toward an inammatory site must be

    parsed by the complex neutrophilic signalmechanisms, a process that gradually leto complete activation and culminates in premiere killing functions of phagocytodegranulation, and NETosis. It is, therefomore insightful to view neutrophil activatas a continuum of processes, priming ste

    and signal cascades with varying effectsoutcomes, all focused on the realizationone goal: the transition of naive, circulatneutrophils to their microbe-eliminatintissue-resident counterparts (Figure 1 ).

    NEUTROPHILS AND THEELIMINATION OF MICROBES The basic instruction set of the activaneutrophil is both effective and ruthlessits simplicity: (1) kill microbes, (2) doharm to the host, and (3) when in doubt, rule 1. To fulll this antimicrobial agen

    Neutrophil

    Endothelial cell

    PSGL-1,L-selectin

    P-selectin andE-selectin

    IntegrinICAM

    Phagocytosis

    Degranulation

    Cytokine secretion

    NETs

    a Capture b Rolling c Firm adhesion

    Figure 1Neutrophil recruitment to sites of inammation. The circulating neutrophil must recognize signs of

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

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    Inammation:recruitment andactivation of imcells upon infecinjury; whenuncontrolled it ltissue damage

    neutrophils possess an array of toxic weaponsthat are carefully regulated through controlledmechanisms. These antimicrobial weapons vary considerably in their methods of actionand thus reect the neutrophils 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 speci-cally tuned mechanics that address the unique

    needs of neutrophils. Granules are, however,far more than just latent repository organellesfordangerous substances; they areactiveand in-dispensableparticipants in almostall neutrophilactivities during inammation.

    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 Min diameter, and are the rst 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 thedefensins,lysozyme, bactericidal/permeability-increasingprotein(BPI),anda numberof serineproteases:neutrophil elastase (NE), proteinase 3 (PR3),and cathepsin G (CG) (34). As such, thesegranules are brimming with antimicrobial

    Granule type Primary(azurophilic)

    Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cellStage of formation

    Myeloperoxidase

    Defensin

    Degranulationpropensity

    Lysozyme

    Elastase

    Lactoferrin

    Gelatinase

    Complement receptor 1Characteristicproteins

    Otherproteins

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

    Gp91phox/p22phox,CD11b, collagenase,hCAP18, NGAL, B12BP,SLPI, haptoglobin,pentraxin 3,oroscomucoid,2-microglobulin,heparanase, CRISP3

    Gp91phox/p22phox,CD11b, MMP25,arginase-1,2-microglobulin,CRISP3

    Gp91phox/p22phCD11b, MMP25, FPR, alkalinephosphatase, CD1CD13, CD14,plasma proteins

    FcRIII

    Secretoryvesicles

    Tertiary(gelatinase)

    Secondary(speci c)

    PMN

    Figure 2Neutrophil granules. Neutrophil granules carry a rich variety of antimicrobials and signaling molecules. They are typically divthree types (primary or azurophilic, secondary or specic, and tertiary or gelatinase). Additionally, structures called secretory vare also considered to be a granule subset. Considerable overlap exists in the cargo of the different granules, and their contentsdetermined by the timepoint during hematopoiesis at which they are produced (5). Granules also differ in their ability to mobilsecretory vesicles being the rst to fuse with the plasma membrane and the azurophilic granules demonstrating the least degrapropensity.

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    compounds and function as a primary reposi-tory for themolecularweaponry of neutrophils. The second class of granules, the specic (orsecondary) granules, are smaller (0.1 Mdiameter), do not contain MPO, and are char-acterized by the presence of the glycoproteinlactoferrin. These granules are formed after

    azurophilic granules; they also contain a widerange of antimicrobial compounds includingNGAL, hCAP-18, and lysozyme (33, 35). Thethird class, thegelatinase (tertiary)granules, arealso MPO-negative, are smaller than specicgranules, 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 set of 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 important membrane-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 inammatory signals: Azurophilicgranules are the most difcult to mobilize, fol-lowed by specic granules, gelatinase granules,and nally, secretory vesicles (3741). Theunderlying mechanisms for this differentialmobilization are not entirely understood, al-thoughregulationofintracellularcalciumlevelsappears to play a salient role (32, 39). Becauseof this varying mobilization propensity, each

    granule subset has been traditionally associ with a particular stage of neutrophil activat

    After neutrophils contact the endotheliustimulation through selectins and chemoattratants induces mobilization of secretory vcles, whose membranes are rich in key facnecessary for continued activation of the n

    trophil, including, among others, the

    2 intgrins, complement and fMLP receptors, as was the Fc RIII receptor CD16 (5, 38, 39, 4Fusion of the secretory vesicles with the plasmembrane exposes these components to theeternal environment. This results in the trantion to rm adhesion, mediated by 2 integrinteraction with the endothelium. As they pceed through the endothelium, neutrophils aexposed to further activationsignals that initimobilization of gelatinase granules, therebyleasing metalloproteases. The activity of thproteases may help neutrophils traverse basement membrane, although this has been conclusively demonstrated (43, 44).

    At the inammatory site, complete a vation of the neutrophil ensues, promptinitiation of the oxidative burst and mobiltion of the azurophilic and specic granu These granules either fuse with the phagoso(see section on Phagocytosis, below), ctributing to the antimicrobial activities of compartment, or fuse with the plasma mebrane, releasing their potent antimicrobiinto the tissue. The fusion of specic granu with the plasma or phagosomal membrane iparticular importance for the oxidative buas avocytochrome b558, a component of NADPH oxidase machinery, resides in specic granule membrane (45). This fuspermits assembly of the NADPH oxidase coplex and allows reactive oxygen species (Rproduction both inside the phagolysosome aoutside of the cell. Degranulation of primand secondary granules contributes to creation of an antimicrobial milieu at the ammatory site and produces an environminhospitable to invading pathogens.

    The release of granular proteins during granulation presents the astute observer wa tempting proposition: Could these granu

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    Table 1 Mechanism of action of neutrophil antimicrobial proteins

    Antimicrobial peptide Antimicrobial mechanism a

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

    Permeabilize membrane bilayers containing negatively chargphospholipids

    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 bacterialphospholipids by binding to LPS

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

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

    Cleaves bacterial virulence factors and outer membraneproteins

    Mechanism independent of a proteolytic activity by bindingthe bacterial membrane

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

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

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

    the cell wall and an increase in membrane permeability Calprotectin 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 -defensins, aprotein family whose members possess multi-ple disulde bonds and whose structures may change under physiological conditions andincrease their activity (48). A surprising num-ber of functions are assigned to defensins, but none have been validated in vivo. Interestingly,inhibition of bacterial cell wall synthesis (49) was recently shown at low concentrations that may be more similar to those present at inam-matory sites. Cathelicidins, including the well-studied LL-37, are proteolytically processed

    from larger proteins, and in addition to thantimicrobial activity, they may potentDNA activation of dendritic cells (DCs) (5

    Neutrophils also contain a number full-length cationic antimicrobial proteiincluding BPI and histones. BPI is catioand binds LPS avidly, much like its structcousin the LPS binding protein. BPI bindingLPS results in increased bacterial permeabiand hydrolysis of bacterial phospholipids;death then follows (51). Interestingly, histoare extremely effective antimicrobials were one of the rst antimicrobials descri(52). The signicance of histones (and of peptides derived from them) as microbremains to be demonstrated in vivo (Given their dual role as an architectu

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    Chronicgranulomatousdisease (CGD):caused by mutarendering theNADPH oxidasnonfunctional,characterized bysusceptibility toinfection andautoinammatio

    scaffold for DNA and as antimicrobials, theirin vivo signicance is particularly difcult todemonstrate.

    The second class of neutrophil antimi-crobials encompasses a broad assortment of proteolytic enzymes that participate in microbedestruction. Lysozyme destroys the bacterial

    wall, making it an obvious antimicrobial, asshown in mice decient 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 specicities. They are tightly regulated intra- and extracellularly by serpins, indicating that their activity isdeployed under specic conditions. NE cleavesenterobacterial virulence factors with highspecicity (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, result in neutropenia. This can be rescued by the administration of recombinant granulocyte macrophage colony-stimulating factor (GM-CSF); however, thesepatients still exhibit signicant susceptibility to infections. Mice decient in NE or CG are 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 may all have antimicrobial activity independent of proteolysis, perhaps as a result of theircationicity. These serine proteases also play asalient role in autoimmunity (see discussion insection on Autoimmunity, below) (59).

    The nal class of neutrophil antimicrobialsconsists of a number of proteins that chelateessential metals from microbes and possibly impact bacterial growth. Two of these chela-tors are lactoferrin, rst identied in milk, which binds preferentially to iron, and cal-protectin (also called S100A and many othernames),whichsequesterszinc(60)andresultsinnutritional immunity (61).

    Reactive Oxygen SpeciesUpon 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 by reducing molecular oxygen to superoxide.Downstream of superoxide, many potentialreactions can occur (for details, see References6264). Superoxide, though not a strongoxidant, rapidly dismutates, forming hydrogenperoxide. Superoxide can also react with nitric

    oxide, which is produced at high levels at inammatory 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, it is assumed to have direct antimicrobial effectsin the phagosome. However, a theoretical

    model of the phagosome suggests that most of the hypochlorous acid produced would react with host proteins before reaching the bac-terium. This model predicts that chloramines,produced when hypochlorous acid reacts with amine groups, may be the most relevant antimicrobial actors in the phagosome (65).

    ROS are clearly important for neutrophilantimicrobial activity: Neutrophils fromchronic granulomatous disease (CGD) patientskill microbes poorly, making these patients

    susceptible 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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    inammation, which explains why CGDpatients often suffer from autoinammatory diseases (67).

    Paradoxically, although MPO is requiredfor neutrophil microbicidal activity in vitro, MPO-decient individuals do not have strikingclinical manifestations (68, 69). Some MPO-

    decient individuals suffer from frequent or se- vere infections, especially with Candida species,andafewhavebeenmistakenforCGDpatients.However, most MPO-decient individuals inthe developed world have apparently normalimmunity. The mild effects of MPO deciency suggest that MPOs 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 ispredictedtobepresentatantimicrobialconcen-trations (65). However, there may be a broaderreason for this discrepancy. Modern technolo-gies can distinguish between individuals whoare partially and completely MPO decient,and partial MPO deciency does not correlate with pathology (70). Residual activity of MPOmay be sufcient 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 deciency 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 nonspecic to be involved insignaling. However, specicity can be achievedon the submolecular level, by cellular redoxbuffering systems and by limited diffusion of ROS owing to their short half-lives (72). A well-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 generatileads to an ionic inux into the phagosomcompensate for charge; this may activate grule proteases by releasing them from their tative matrix (75). There is controversy arou which ions and which channel are responsfor charge compensation, but this theory

    protease activation is certainly intriguing (6Studies of ROS are hampered by varitechnical issues. Ideally, a probe for Rshould be specic, targetable to particuintracellular compartments, and capable being used in vivo. Traditional probes ROS do not meet these specications;addition, the probes often become radspecies (76). One promising new approfor ROS detection that meets these criteriathe use of redox-sensitive uorescent protebased probes, such as roGFP and HyP(76). Other methods that can be used in vinclude transcription proling of superoxor hydrogen peroxidesensitive genes as was the detection of relatively stable productreactive oxygen using mass spectrometry (7

    PhagocytosisPhagocytosis is the major mechanism to move pathogens and cell debris. It is an acreceptor-mediated process during which a pticle is internalized by the cell membrane ia vacuole called the phagosome. As with ophagocytes, the mechanistic details of internization depend on the type of interaction tween the neutrophil and the microorganisInteraction can be direct, through recognitiof PAMPs by pattern-recognition receptors,opsonin mediated.The lattermechanismis beter characterized and includes two prototypiexamples: Fc R-mediated phagocytosis, whrelieson theformationof pseudopodextensiofor engulfment of IgG-opsonized particles, acomplement receptor-mediated phagocytos which does not require membrane extensior pseudopods (77).

    After engulfment, the nascent phagosois relatively benign to microorganisms, acqing its lethal properties only after a dra

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    Autophagy: a in which cellulacontents are degin lysosomes,especially inconditions of nuscarcity and inf

    maturation process. Our understanding of this process is largely based on studies inmacrophages, and although these are certainly instructive, essential differences exist in neu-trophils. Macrophage phagocytosis follows anendocytic maturation pathway: In neutrophils,phagosome maturation happens upon fusion of

    granules to the phagosome, whereby delivery of antimicrobial molecules into the phagoso-mal lumen occurs. Simultaneously, assembly of the NADPH oxidase on the phagosomalmembrane allows ROSproduction, and jointly,these two mechanisms create an environment toxic to most pathogens. Neutrophil phago-somal pH regulation also differs signicantly from that observed in macrophages. While themacrophage phagosome gradually acidies,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 sustained via NADPH oxidase activity, despite contin-uing fusion of acidic granules. Key events of the 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 interfering with engulfment, modulating phagosomematuration, and creating a more hospitableintraphagosomal environment. The polysac-charide capsule expressed by Staphylococcus aureus confers antiphagocytic properties (81). Helicobacter pylori can disrupt targeting of NADPH oxidase to the phagosome so that superoxide anions accumulate extracellularly rather than in the phagosome (82). 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 efciently block gran-ule fusion with the phagosome (84, 85). The variety of mechanisms evolved by intracellularpathogens to resist killing and enable survival within the phagosome further emphasizes the

    importance of phagocytosis in the innateimmune defense.

    Neutrophil Extracellular TrapsUpon stimulation, neutrophils can undergoNETosis, an active form of cell death that

    leads to release of decondensed chromatin intothe extracellular space (86, 87). The brousstructures termed NETs contain histones as well 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 not completely understood. The reactive oxygenpathway is involved, as NADPH oxidase and MPO are required for NET formation in re-sponse to chemical and biological stimuli (87,90, 91). Nitric oxide donors can induce NETs via a mechanism that also requires ROS (90), anding that awaits geneticconrmation.All ac-tivators of NET formation tested so far requireROSproduction. S. aureus maybe an exception,although those experiments were done usingpharmacological inhibitors, not cells decient in ROSproduction(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 conrmedin primary human neutrophils (9597). Au-tophagy is also thought to be required for NETformation, but this has so far been shown only using a nonspecic 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 NETsDNA and histonesfrom an infection model. Moreover, the factors that are important for

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    Cystic brosis: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 disseminatemoreefciently in the host, which may point to evolutionary

    pressure to avoid entrapment by NETs (99,100). In addition, a persistent Aspergillus infection in a CGD patient was cleared aftergene therapy, which restored NADPH oxidaseactivity, NET formation, and NET-mediatedbut not phagocytosis-mediated killing by thepatientsneutrophils ex vivo(101). Ontheotherhand, the immune system has redundant mech-anisms to ght infection, and it may be that NETs 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 of autoantibodies, often against chromatin andneutrophil components (102106) (see sectionon Autoimmunity, below). Platelet-inducedNETs, formed during sepsis, are associated with 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 airway uids of cystic brosis patients, where they may 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 of immunity, issuing instructions to practically all other immune cells. As one of the rst celltypes to arrive at sites of infection, neutrophilssecrete cytokines and chemokines critical in theunfolding of the inammatory response and in

    establishing the correct environmental contions to launch the adaptive immune respon The cytokines released by PMNs are ofsynthesized de novo. Although neutroptranscribe little after leaving the bone marronce activated, these cells undergo a trscriptional burst that results in the synthe

    of signaling molecules (110, 111). Compa with other immune cells (e.g., macrophagneutrophils typically produce lower amouof cytokines per cell, but they are so abundat inammatory sites that their contributito total cytokine levels is signicant (4). Fthermore, neutrophil-secreted proteases cmodulate signaling networks in vivo throcytokine processing (112).

    The initial neutrophil cytokine responsean appeal for immunological reinforceme The most abundantly produced cytokine, ILprimarily serves to recruit other neutroph(113). Similarly, neutrophil-derived proinamatory IL-1 and TNF- induce other ceto produce neutrophil chemoattractants (1115) (for a comprehensive list of cytokproduced by neutrophils, please see Referen115, 116). In addition to cytokines, neutrophrelease other signaling mediators, includgranule contents (117), lipids (118), and Rsuch as hydrogen peroxide (119). They acommunicate via cell-cell contact (120). H we provide examples of how neutropinteract with other cells to shape the immuresponse (see Figure 3 ).

    Monocytes and Macrophages As they respond to infection or injuneutrophils and their relatives in the moncyte/macrophage lineage coordinate thactivities, leading to alternating waves ofcruitment of these two cell types. Macrophaand patrolling monocytes are among the inidetectors of PAMPsandendogenousactivatothe danger-associated molecular patterns (12and these cells work to summon large numbof neutrophils to the inammatory locus. Tinux of neutrophils is followed closely byarrival of monocytes, suggesting a causal

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    Neutrophil

    NeutrophilNeutrophil

    Neutrophil Monocyte

    T cell

    T cell

    Macrophage

    Lymph

    Blood

    Tissue

    Activation anddifferentiation

    ROS?Arginase?

    IFN-

    IFN-

    IL-12

    NK cellDC

    DC

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    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 recruitment 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 alinteract with the adaptive arm of the immune system: They can act as antigen-presenting cells by cross-presenting antigen to Ccells; they also secrete IL-12, which activates T cells. T cells, in turn, activate neutrophils by secreting IFN- . Finally, neutrophDCs and natural killer (NK) cells colocalize and enhance each others activity via receptor-receptor interactions and soluble m

    behind these temporal dynamics. Indeed, neu-trophils recruit monocytes via several different mechanisms. They express classical monocytechemoattractants such as CCL2 (MCP-1)(122), CCL3 (MIP-1 ) (123), CCL20 (MIP-3 ), and CCL19 (MIP-3 ) (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 (125127). Monocyte recruitment is also affectedindirectly by neutrophils: via upregulation of endothelialadhesion factors, increase of transendothelialpermeability, enhancement of production of chemoattractants by other cell types, and mod-ulation of the activities of these chemokines via proteolytic processing (reviewed in 128).In addition to recruitment, neutrophils mod-ulate monocyte and macrophage cytokineproduction (128), directly enhancing their

    microbicidal activity (129). The circuitousnature of the cross talk of these two cell typesbecomes obvious during inammation 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 Inammation, below).

    Dendritic CellsNeutrophils can also recruit and activateDCs in vivo. This was recently illustratedin a mouse model of Leishmaniasis, wheresubcutaneous inoculation of Leishmania major triggered a massive and rapid inltration of neutrophils (130). These cells secrete thechemokine CCL3, recruiting DCs to thesite of inoculation and initiating a protective

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    DC-SIGN:dendritic cellspecicintercellular adhesionmolecule-3-grabbingnonintegrin

    Granulocytereceptor 1 (Gr1):the anti-Gr1 antibody RB6-8C5 reacts withboth Ly6G (specicfor neutrophils) andLy6C (present onmany immune celltypes)

    Th17 cells: subset of T helper cells that produce IL-17,important in

    inammation andimplicated inautoimmunity

    Th1 response (131). Interestingly, activatedneutrophils can induce the maturation of DCsin vitro through specic receptor-receptorinteractions between Mac-1 and DC-SIGN,leading to local secretion of TNF- (120).In this case, the reduced levels of cytokineproduction foster specicity, as only proximal

    DCs receive the maturation signal. A similaractivation model was earlier proposed for Tox-oplasma gondii (132). Neutrophil-activated DCsproduce the proinammatory 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 ismorespecic and hence a betterreagent for thistype of experiment (133). The crucial role of neutrophils in DC activation was recently con-rmed using anti-Ly6G antibody depletion: In Mycobacterium tuberculosis infection, timely traf-cking of DCs to lymph nodes and activation of CD4 + T cells were both dependent on PMNs.Furthermore, this study demonstrated that DCs presented bacterial antigens when they ingested infected neutrophils just as efciently as they did via direct uptake of Mycobacterium(134). In sharp contrast to the above ndings,a separate study using an immunization modelshowed that neutrophils recruited to lymphnodes compete for antigen with DCs andmacrophages and that these neutrophils inhibit their interactions with T cells (135). It is possi-ble that neutrophils have site-specic effects onDCs and can be stimulatory at peripheral sitesand inhibitory in the lymph nodes. Neutrophilsexhibit fascinating andsomewhat 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 CellsStudies of interactions between neutrophil andnatural killer (NK) cells have historically been

    performed in vitro, and their interpretationfrustratingly difcult owing to the questiable purity of cell preparations. Recently was shown that neutrophils, NK cells, and Dinteract in a m enage a trois involving bcytokine signaling and direct cell-cell con(137, 138). In one report, infection of m

    with Legionella pneumophila triggered prodution of IFN- by NK cells; this was dependon both PMN-derived IL-18 and DC-deriveIL-12 (137). Similarly, human neutrophils, Ncells, and DCs colocalize at inammatory sand a positive feedback loop has been propoon the basis of invitro data. In this scheme, netrophils interact with a specic subset of D(via CD18-ICAM-1 interactions), promptithe DCs to produce IL-12p70, which in tustimulates IFN- production by NK cells afurther activates neutrophils. Simultaneouneutrophils alsoactivate NK cells by directctact (139). Additional in vitro interactionstween neutrophils and NK cells are extensivreviewed in Reference 138.

    Lymphocytes A surprising nding in recent years is the extsive cross talk between cells located at oppends of the immune spectrum. Previouthought to belong to isolated compartmenneutrophils and T cells shape and impeach others functions, both qualitatively quantitatively (140). Neutrophils affect T function indirectly via DCs, as outlined abobut can also inuence T cell function direcPMNs secrete IL-12, which may be crucial Th1 cell differentiation (141, 142). They aexpress several T cell chemoattractants (1as well as B cell development and maturafactors (143, 144). Cytokine communicatoccurs in both directions: For instance, IFN which is secreted by T cells, prolongs ntrophil life span, induces gene expression, increases phagocytic capacity (145). Thehelper 17 (Th17) cell subset secretes IL-a key cytokine in the control of neutropdynamics, which acts by upregulating expsion of CXCL8 (IL-8), G-CSF, and TNF-

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    Ulcerative colititype of inammbowel diseasecharacterized byand tissue erosithe colon and re

    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 peroxidemediated suppres-sion, as proposed in a cancer model (119) (seesection on Cancer, below). Direct evidence of such interactions in vivo is still missing.

    Interestingly, neutrophils inuence 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 ndings imply that neutrophilshave characteristics of antigen-presenting cells.Neutrophils also appear capable of expressing MHC class II and costimulatory moleculesunder inammatory conditions (149151),and they can present antigen to CD4+ T cellsin vitro (152154). However, the functionalsignicance for protective immunity remainsunclear, especially in light of the nding that mouse 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 of donors to express MHC class II (149, 151),suggesting concomitant variations in the ability to activate T cells, a nding that could haveimplications for susceptibility to autoimmunediseases. Therefore, neutrophil modulation of adaptive immunity seems to be highly complexand is only now starting to be unraveled.

    NEUTROPHILS ANDRESOLUTION OF INFLAMMATION The lethal cargo of neutrophils is not only destructive toward invading microbes, but also harmful to host cells. Thus, neutrophildeployment must be tightly controlled.

    Although some collateral damage to host tissues is inevitable during infection, neu-trophils must be removed before they haveserious, detrimental effects on inamed tissues.Resolution of inammation is an active processthat limits further leukocyte inltration andremoves apoptotic cells from inamed sites.

    This process is essential for maintenance of tissue homeostasis and, if impeded, leads tononresolving inammation, a problematiccondition that contributes to many diseases.

    Apoptosis and Clearance Apoptosis is a central aspect of inammationresolution. 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-inammatory phenotype.

    Neutrophil death is inuencedby inamma-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 antiapo-ptotic (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 terminally differentiated, it is unexpected that moleculescontrolling cell proliferation regulate survival.Proposed to have prosurvival effects, one suchprotein is survivin. It is expressed more highly in immature neutrophils than in mature ones,but its expression can be restored in maturecells by inammatory signals such as G-CSF orGM-CSF. In line with these ndings, survivinis also highly expressed in neutrophils at sitesof inammation, such as cystic brosis sputum,appendix inltrates, and intestines of patients with ulcerative colitis (156).

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    Wegenersgranulomatosis: vasculitis affecting thelungs, nose, andkidneys; inammationleads to reduced bloodow, tissuedestruction, anddamage of vital organs

    Prostaglandins andleukotrienes: lipidssynthesized by cyclooxygenases and5-lipoxygenase,respectively, in thearachidonic acidpathway; haveproinammatory functions includingleukocyte recruitment

    Similarly, cyclin-dependent kinases func-tion as prosurvival factors in neutrophils.Pharmacological inhibition of these cell cycleregulators induce caspase-dependent apoptosisandblock life-span extension by survival factors(157). More recently, prosurvival effects werealso attributed to proliferating cell nuclear

    antigen (PCNA). This factor usually residesin the nucleus, where it is involved in DNA replication, but in neutrophils, it associates with procaspases in the cytosol and is thought to prevent their activation. During apoptosis,PCNA is targeted forproteosomal degradation, which correlates with an increase in caspase-3and caspase-8 activities. This mechanism is rel-evant in Wegeners granulomatosis and sepsis, where stabilization of PCNA is associated withresistance of neutrophils to apoptosis (158).

    Equally important for the resolution of in-ammation is the proper removal of apoptoticcells. This relies on the release of nd-mesignals at early stages of cell death, which at-tract phagocytes. Likewise, distinct eat mesignals are required for specic recognition of apoptotic cells. Ingestion of apoptotic cells by macrophages drives the production of the anti-inammatory cytokines tumor growth factor(TGF)- andIL-10 (155).Failure to clear theseapoptotic cells, by contrast, results in secondary necrosis and release of products that generateproinammatory signals (Figure 4 ).

    Lipid Mediator Class Switch Soluble mediators play a crucial role in theresolution of inammation. In neutrophils,a particularly prominent role is assumed by lipid mediators. The successful progressionof inammation appears to hinge on a shift in the composition of secreted lipids. At early stages of inammation, neutrophils synthesizeproinammatory 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 inammatory response,neutrophils interact with various cell types in

    their vicinity (epithelial cells, endothelial cbroblasts, platelets, and leukocytes) and ticipate in the transcellular biosynthesis of lmediators with anti-inammatory and prosolving activities, such as lipoxins,resolvinsprotectins. A major lipid mediator class swthus exists, governed by temporally regula

    expression of different lipoxygenases andmobilization of different fatty acid substra The different biosynthesis pathways of prosolving lipid mediators have been reviewedetail elsewhere (118). Interestingly, microganisms are also a source of lipid precurthat can be used by neutrophils for resolsynthesis. Thus,microbes also likely participin synthesis of mediators with proresolvfunctions at the site of infection (159, 160)

    How do lipid mediators contribute the termination of inammation? Lipoxiresolvins, and protectins exert cell-type speeffects, promoting monocyte/macropharecruitment and activation while inhibitneutrophil functions. The inhibitory effextends to all essential steps of neutroresponses: migration, adhesion, and activat All three lipid mediators reduce neutroprecruitment, a process that involves the lipox A4 receptor and the leukotriene B4 recep(BLT1) (161167). Ariel et al. (168) also pposed an interesting mechanism of action lipoxins,resolvins, andprotectins in clearingammatory sites. They showed that neutropexposure to these lipids increases expresof CCR5 on the surface of late apoptotic ntrophils, leading to efcientsequestrationof tchemoattractants CCL3 and CCL5. The squestration of these chemokines means theyunavailable to recruit neutrophils to inamsites (168) (Figure 4 ). This mechanism coplements other anti-inammatory procesin which chemokines are inactivated by ntrophil proteases. Of these lipids, lipoxinsthe most completely understood. In additionneutrophil recruitment, lipoxins can inhibit tshedding of L-selectin and the upregulation 2 integrins in response to proinammatostimuli, thereby reducing adhesion of ntrophils to endothelial cells (169, 170). Fina

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    TNF-IL-6

    IL-10 TGF-PGE-

    Neutrophil

    Monocyte

    Platelets

    Lipoxins

    Macrophage

    Macrophage

    ChemokinesApoptoticneutrophil

    NEToticneutrophil

    LeukotrienesProstaglandins

    ?Microorganisms

    LipoxinResolvinsProtectins

    Chemokines

    CCR5

    Initiationof in ammation

    Resolutionof in ammationLeukotrienes Prostaglandins TNF- TGF-Lipoxins Resolvins Protectins IL-10

    Chemokine clearan

    Figure 4From inammation to homeostasis: neutrophil apoptosis and lipid mediator class switching in the resolution of inammation. site of infection, resident macrophages initiate an inammatory response, secreting proinammatory cytokines and chemokinealert the immune system and promote neutrophil recruitment. In the early stages of inammation, microbes trigger the productproinammatory lipid mediators, such as leukotrienes and prostaglandins, which also recruit neutrophils. As inammation progswitch occurs, and anti-inammatory lipid mediators such as lipoxins, resolvins, and protectins are produced. Notably, interacneutrophils with platelets induces the production of lipoxins. Anti-inammatory lipid mediators initiate the resolution of inamby blocking neutrophil and promoting monocyte recruitment. Monocytes differentiated into macrophages ingest apoptotic neutdriving the production of the anti-inammatory cytokines tumor growth factor (TGF)- and IL-10 and prostaglandin-E2 (PGE which drive the lipid mediator class switch. Proresolving lipid mediators also promote the expression of CCR5 on the surface

    apoptotic neutrophils, providing a means of scavenging chemokines. Chemokine clearance upon phagocytosis of apoptotic neuby macrophages further contributes to the reduction of neutrophil inltration and the return to tissue homeostasis. The contribumacrophages to the clearance of NETotic neutrophils, and how this could impact inammation resolution, is currently unknowtimeline of the inammation process from initiation to resolution is summarized in the upper part of the gure.

    Chronic obstrucpulmonary disea(COPD): lungcaused by noxioparticles or gas,tobacco smokininammation lelung obstruction

    lipoxins also impact neutrophil activation by inhibiting ROS and peroxynitrite production,NF- B activation, and IL-8 expression (170).

    In addition to directly impacting neu-trophil functions, lipid mediators promotenonphlogistic (noninammatory) phagocyto-sis of apoptotic neutrophils by monocytes

    and macrophages. In the presence of anti-inammatory lipids, engulfment of apoptoticneutrophils is notaccompanied by thereleaseof proinammatory mediators, as typically occursduring macrophage activation.Instead, produc-tionof theanti-inammatory cytokines TGF-and IL-10 is increased (163, 171).

    Disorders Associated with Nonresolved Inammation The failure of neutrophils to apoptose or mal-functions in the removal of their apoptotic re-mains result in chronic inammation. Theseconditions lead to the accumulation of cyto-toxic substances and are associated with severepathologies, including cystic brosis, chronicobstructive pulmonary disease (COPD), andrheumatoid arthritis (RA). The severity of in-ammation 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): chronicinammatory diseasethat affects many tissues and organs but primarily synovial joints; severeinammation causesdeformity

    instigator of this disease. A chronic neutrophilinltration in the lungs of COPD patientspromotes tissue damage and organ dysfunc-tion. One of the key molecules controllingthe inammatory response in the lung isleukotriene A4 hydrolase (LTA4H). Thisenzyme has two opposing activities. First, its

    hydrolase activity converts leukotriene A4 intoleukotriene B4, a potent neutrophil chemoat-tractant and proinammatory agent. Second,LTA4H is an aminopeptidase that inactivatesa specic neutrophil chemoattractant, theproline-glycine-proline tripeptide (PGP), thusconferring the enzyme with anti-inammatory properties. Interestingly, tobacco smoke selec-tively inhibits only the aminopeptidase activity of LTA4H, promoting the accumulation of both leukotriene B4 and PGP. This in turnpromotes neutrophil recruitment and fuelschronic lung inammation (172).

    Another prime example of a disease linked tononresolving inammation is RA. Neutrophilsare themost abundant leukocytes present in thesynovial uid of RA patients, and their role inpathogenesis has been demonstrated in severalanimal models. These models primarily usedneutrophil depletion or adoptive transfer of wild-type neutrophils in leukotriene-decient mice (173175). In one model, synthesisof leukotriene B4 by neutrophils in jointsis essential for disease development (174).Leukotriene B4 can act in an autocrine manner via the neutrophil receptor BLT1 to promotethe recruitment of a rst wave of neutrophilsinto the joint. Later, the recruitment of asecond wave of neutrophils is independent of this leukotriene B4BLT1 pathway. At thisstage, immune complexes are essential forstimulating inltrating neutrophils to deliverIL-1 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 inammation. They also demonstrate the cross talk between neu-trophils and other immune cells discussed in

    the previous section. It is, however, unkno whether all neutrophils are capable of adaptto the changing chemoattractant environmeor if different subsets of neutrophils are scessively involved. The relevance of this min human disease remains to be establishalthough the clinical similarities between

    mouse model and human RA are encouragi

    NEUTROPHILS IN DISEASENeutrophils areprominentplayers in theinnaimmune response and the clearance of inftion, a subject addressed in several prominreviews. However, neutrophil action can asupport disease progression in other illnes A host of autoimmune disorders belong to category. In addition, certain malignantcanceare also prime examples of illnesses in w

    neutrophils play a salient role.

    Cancer The link between cancer and inammat was noted as early as 1863 by Rudolf Virc(177). Since then, it has been proposed tneutrophil-derived ROS have the potentialinitiate tumor formation by genotoxic strand induction of genomic instability. Althouthis has been demonstrated in vitro (178, 1convincing evidence for PMN-mediated DNmutagenesis in vivo is still lacking. Neutropdo, however, impact cancer progressi They are abundant in tumors and inuentumor development through several secremediators, including cytokines, ROS, matrix-degrading proteases (reviewed in Rerence 180). The majority of ndings suppa protumor and antihost effect of thcells; clinical studies indicate that neutroinltration of tumors is associated with pooprognosis (181, 182). Indeed, some canseem to actively recruit neutrophils throuproduction of IL-8 (183). In agreement wthis, antibody depletion of neutrophils redutumor growth (184). The protumor functiof neutrophils operates at multiple levincluding production of angiogenic fact(185), enhancement of metastasis (186),

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    Acute-phaseproteins: secreliver, concentraplasma changes25% or more duinammation

    suppression of the antitumor immune response(119, 187). Using the anti-Ly6G antibody,Fridlender and colleagues (187) depleted neu-trophils and conrmed their tumorigenic role. Moreover, the study showed that neutrophils inthe tumor microenvironment could, under cer-tain circumstances, be induced to target their

    cytotoxic arsenal at tumor cells, whose growththey usually help to fuel. Pharmacologicalinhibition of TGF- signaling led tumor-associated neutrophils to assume a heightenedproinammatory 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-inammatory cytokine IL-10, which also inhibited T cell responses. Crosstalk with invariant NKT cells could counterthis response, restoring a proinammatory activation status (188). Thus, investigation of neutrophils in cancer has revealed considerableplasticity in their responses. Although littleevidence currently supports the existence of different populations, it is likely that neutrophilresponses aremore exible 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 healthy individuals, 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 proportionsof monocytic and granulocytic cells (189). In

    human renal cell carcinoma, MDSCs haveidentical morphology andexpress the same sur-face markers as do activated neutrophils (190,191). MDSCs inhibit T cell proliferation by limiting 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 (193195) and may play a major rolein disease pathogenesis, given that release of proteolytic and cytotoxic molecules from neu-trophils can trigger organ damage. Neutrophilproducts act as both targets and mediators of autoimmunity. MPO and PR3 are the main tar-gets of antineutrophil cytoplasmic antibodies(ANCA), autoantibodies directed against anti-gens present in the cytoplasm of neutrophils. Wegeners granulomatosis is consistently as-sociated with the presence of ANCA. Further-more, the extent of organ damage in patients with Wegeners granulomatosis correlates withthe PMN inltrate 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 inammation. Early reportsalsosuggestthat,inaninammatoryen- vironment, ANCA accelerate ROS-dependent neutrophilapoptosis,suggestingafeed-forwardcycle culminating in organ damage (194, 195).

    SLE is another chronic autoimmune diseaseaffecting multiple tissues and organs. Autoan-tibodies produced in SLE are predominantly either ANCA or directed against chromatin. Although neutrophils had long been suspected

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    Vasculitis:inammation of blood vessels

    to be causative agents, their role in SLE patho-genesis remained elusive. The recent discovery of a link between SLE and NET formationhas helped to shed light on this quandary.It was proposed that TNF- and IFN- prime cells for NET formation in response toanti-PR3, antiribonucleoprotein, anti-HNP,

    or anti-LL-37 autoantibodies (103, 104, 106). Thus, high levels of inammatory cytokines inautoimmune patients are believed to sensitizeneutrophils to NETosis, whereas autoanti-bodies may trigger a switch from apoptosis toNETosis. Additional evidence suggesting arole for NETs in autoimmune pathology wasobtained when NETs were identied in renaland/or skin biopsies from patients with SLEand small vessel vasculitis (103106). Severalstudies have reported the presence of a particu-lar subset of neutrophils in PBMC preparationsfrom pediatric and adult SLE patients. Theselow-density granulocytes display phenotypiccharacteristics of immature neutrophils withnonsegmented nuclei and higher expressionof MPO, NE, and defensin-3, and they may be related to the MDSCs discussed previously (see section on Cancer, above) (197, 198). An increased capacity to form NETs and aheightened cytotoxicity toward endothelialcells could bestow them with pathogenicproperties in lupus (105).

    Because NETs appear to be formed duringautoimmune disease, their timely removal may be an essential mechanism for maintainingtissue homeostasis. Human serum contains thenuclease DNase I, which degrades NETs in vitro. 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 of DNase I, both of which may protect NETsfrom degradation. Most notably, impairedNET degradation correlates with development of 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- , a central cytokine in Spathogenesis (103,104).However, it remainsbe determined if DCs can present NET components or if they contribute to autoreactivcell activation. It is also possible that NETsinvolved in other autoimmune diseases. Shothis prove to be the case, understanding

    role of NETs may provide critical insights ithe role of microbial infections as a triggeautoimmunity.

    CONCLUDING REMARKSNeutrophils are specialized phagocytes tarose as an evolutionary adaptation in vebratestocoordinateandexecuteoneofthemofundamental physiological responses: inmation. They are endowed with antimicrobmechanisms that make them the preeminemicrobe exterminators of the immune systeIn addition to this important role, PMNs anetwork with many other immune cells ahelp regulate the initiation of specic T aB cell immunity. However, neutrophils do alwaysact inways benecial to thehost: Unctrolled neutrophil responses canexacerbate aeven cause autoimmune and inammatory deases. Many challenges remain in understaing neutrophil function: Is there specializatamong PMNs? Are they more plastic than suspect? How do they make decisions bedeploying their armamentaria? How do thkill microbes? How specic are their instrtions to other cells? Answering these questi will better dene neutrophils role in defeand disease and will provide a rational pathpursuing new therapies. Moreover, neutrophcan potentially provide insights into sevuniqueaspects of basic cellbiology. Their stringly short life spans make them excellent mels for investigating cell death, whereas treliance on ROS as biochemical effectors mreveal novel ways for relaying intracelsignals. The uniquely lobulated neutropnucleus is a feat of higher-order nucarchitecture that is just beginning to yiits secrets. In short, exciting times awaithumble neutrophil.

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    DISCLOSURE STATEMENT The authors are not aware of any afliations, memberships, funding, or nancial holdings that might be perceived as affecting the objectivity of this review.

    ACKNOWLEDGMENTS We thank Diane Schad for assistance with graphic design and Cornelia Heinz for administrative

    help. G.H. is an Alexander vonHumboldt FoundationScholar, andB.A. is supportedby an EMBOLong-Term Fellowship.

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