neutrophil function from mechanism to disease

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Neutrophil Function:From Mechanisms to Diseas

Borko Amulic, Christel Cazalet,Garret L. Hayes, Kathleen D. Metzler,and Arturo Zychlinsky

Department of Cellular Microbiology, Max Planck Institute for Infection Biology,Charit eplatz 1, 10117 Berlin, Germany; email: [email protected],[email protected], [email protected], [email protected]@mpiib-berlin.mpg.de

Annu. Rev. Immunol. 2012. 30:45989

First published online as a Review in Advance onJanuary 3, 2012

TheAnnual Review of Immunologyis online atimmunol.annualreviews.org

This articles doi:10.1146/annurev-immunol-020711-074942

Copyright c2012 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 circula

and patients with congenital neutrophil deficiencies suffer from seinfections that are often fatal, underscoring the importance of t

cells in immune defense. In spite of neutrophils relevance in immuresearch on these cells has been hampered by their experimentall

tractable nature. Here, we present a survey of basic neutrophil bio

with an emphasis on examples that highlight the function of neutronot only as professional killers, but also as instructors of the immsystem in the context of infection and inflammatory disease. We f

on emerging issues in the field of neutrophil biology, address ques

in this area that remain unanswered, and critically examine the exmental basis for common assumptions found in neutrophil literatu

459

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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-staining

techniques to examine subpopulations of leuko-cytes.Hisexperimentationledtoanewappreci-

ation for the heterogeneity of white blood cellsand to the discovery of several novel leukocyte

subpopulations. 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 also

the 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 shuttles

for 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. Metchnikoffs seminal theory of cellularimmunity was based on comparative embryology and observa-

tions of phagocytes in various simpleorganisms, includingthe mi-croscopic crustacean Daphnia. Remarkably, even the slime mold

Dictyostelium discoideumhas phagocytic cells that protect it from

infection (200). The short-lived neutrophil with a lobulated nu-cleus andgranule-packed cytoplasm is 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 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 1520% of immune

cells. In chimpanzees, neutrophils account for more than 50% ofthe differential blood count (204).

early and enthusiastic evolutionary biologist i

terested in the phagocytic capacity of cells.Metchnikoff demonstrated that injury

starfish embryos resulted in recruitment phagocytic cells to the site of injury (3). H

theorized (correctly) that these cells migrate

injured sites and participate in microbe dige

tion. Remarkably, this prescient view of netrophil action still aptly summarizes, more tha century later, the basic role of neutroph

in immunity. The uniquely lobulated nucleof the neutrophil also inspired Metchnikoff

rename these cells: He called them polymophonuclear leukocytes (or PMNs), a title th

still enjoys frequent use and that is used intechangeably with neutrophil throughout this r

view. Together with two other developmentarelated cell types, the eosinophils and basoph

(also discovered by Ehrlich), PMNs form t

granulocyte family of white blood cells, a familywhosehallmarkisthepresenceofgranule

unique storage structures important in antimcrobial functions (see section on Granules an

Degranulation, below).Neutrophils were discovered at the daw

of the immunological sciences; consequentelucidation of their role in the immune r

sponse has been an ongoing process stretchinover more than a century. We now know th

they are key components of the innate immuresponse and vital in immune function; unfo

tunately, their importance has often been ove

shadowed by breakthroughs in the study of tadaptive immune response (4). Admittedly, th

situation is exacerbated by neutrophils notorous experimental intractability: They exhibi

short life span and are terminally differentiatepreventing growth in tissue culture. The sta

dard tools of molecular biology, such as tranfection and RNA interference, are of little u

when applied to these cells, and immortalizneutrophil-like cell lines rarely reflect t

functional diversification of neutrophils. Fu

thermore, neutrophil-like cells studied in tisolation of a culture dish most certainly do n

mimic the complex biological reality in tissuor circulation. Conclusions from in vitro stu

ies should, therefore, be carefully interprete

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Unfortunately, in vivo studies of neutrophil

function also raise concerns. Mouse neu-trophils, the preferred model for in vivo

studies, differ in important aspects from theirhuman equivalents. This is perhaps best

exemplified by the differences in the respectiveantimicrobial repertoires and the numbers of

PMNs in circulation (30% versus 70% in miceand humans, respectively).Despite these difficulties, no picture of the

immune response can be complete withouta comprehensive understanding of the neu-

trophil and its functions. The extensive natureof neutrophil research, however, precludes a

comprehensive review of the subject matter.In this review, we intend to provide a survey

of basic neutrophil biology and function, whileemphasizing recent advances in neutrophil re-

search and providing a critical assessment ofsome current reports on PMN action.

Our survey of the neutrophil begins in

adult bone marrow where, under the in-struction of growth factors and cytokines,

pluripotent hematopoietic cells differentiateinto myeloblasts, a developmental cell type

committed to becoming granulocytes. As theseprecursor cells mature to neutrophils, they syn-

thesize proteins that are sorted into differentgranules (5). Traditionally, granules have been

subdivided into three different classes basedon their resident cargo molecules: azurophilic,

specific, and gelatinase granules. Although this

subdivision is practical, these designations arelargely artificial. Granules areformedthrough a

continuous 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 at

the time of their formation. As the maturingneutrophil sequentially alters its transcriptional

profile, granule content changes, resulting in acontinuum of granule species with overlapping

cargoes (6).

The release of neutrophils from the bonemarrow is tightly regulated in healthy in-

dividuals: Chemokines control the passageof PMNs into circulation and maintain a

pool of cells ready for release in case of

infection. Indeed, the number of neutrophils

drastically increases during infection and somediseases. Interestingly, neutrophils circulate

for only approximately 68 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 for

eliminating neutrophils quickly as opposedto reducing leakage of their dangerous cargo

remains an unanswered and intriguing mystery.Mature neutrophils emerge from the bone

marrow intent on pursuing one simple, yetessential, question: Has host integrity been

compromised by potentially harmful invaders?

Should the answer prove to be yes, theneutrophil must swiftly enact a carefully

choreographed process to locate, attack, anddestroy the potential threat. At its disposal is

an impressive arsenal of antimicrobial weaponsthat are deadly, indiscriminate, and brutish in

their application. Although effective in theirdestructive capacity, these weapons can prove

to 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 locations

where they are both contained and effective.

How then does the neutrophil locate andidentify infections? How does it transition

at the correct time and place from an in-active cellular bystander to a fully activated

microbial killing machine? This 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 an

impressive multitude of cellular mechanisms:It will mobilize secretory vesicles and granules,

identify chemotactic gradients and traverse

them through destruction and reorganizationof the actin skeleton, penetrate the endothelial

barrier and navigate a course through thebasement membrane, and begin transcription

of 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 upon

neutrophil activationdue to production ofROS by the NADPHoxidase

cells. Ultimately, upon arriving at the infection

site, it will seek the insulting pathogens andunleash its extensive arsenal of antimicrobial

weapons. The initiation of these processes oc-curs in the bloodstream, where the neutrophil

acts as a monitor for host distress, patrollingvessels and vigilantly seeking out indications of

an incipient inflammatory response.

NEUTROPHIL ACTIVATION

At inflammatory sites, bacterial-derived and

host-produced inflammatory signals areabundant; these compounds stimulate the

endothelial cells near the inflammatory site.These stimulants, such as the bacterial-derived

lipopolysaccharide (LPS) and fMLP, as wellas the classical chemoattractants and cytokines

tumor necrosis factor (TNF)-, interleukin(IL)-1, and IL-17, prompt endothelial cells to

produce adhesion molecules on their luminal

side: the P-selectins, E-selectins, and severalmembers of the integrin superfamily, the

ICAMs (5). As neutrophils traverse the circu-latory system, they continuously and randomly

probe the vessel wall; the postcapillary venules,where flow dynamics and the constricted space

are particularly amenable to increased randomprobing, are often the best-suited location

for 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) andL-selectin(9, 10). Upon

random contact with the endothelium, thesemolecules engage the P- and E-selectins of

endothelial cells, resulting in selectin-mediatedtethering of neutrophils to the vessel wall.

This is followed by a characteristic rolling ofneutrophils along the endothelium. It is here

that the complex activation cascade begins

and the neutrophil commitment to microbialkilling commences. What changes occur in the

neutrophil at this early time point?The engage-ment of PSGL-1 and L-selectin on neutrophils

activates a variety of kinases, including Src

family kinases, Syk, phosphoinositide 3-kina

(PI3K), and p38 mitogen-activated protekinase (1113). This cascade initiates a numb

of changes in neutrophil biology and sets tstage for integrin activation and firm adhesio

After selectin-mediated rolling, neutroph

enter a firm adhesion state mediated by t

2 integrin family of proteins (LFA-1 anMac-1 proteins on the neutrophil); firm adh

sion is characterized by the arrest of neutroph

rolling in preparation for transendothelmigration (13, 14). As the neutrophil ro

along the endothelium, interaction wiselectins, chemoattractants, cytokines, a

bacterial products results in activation aclustering of the2 integrins on the surface

the neutrophil (15, 16). The 2 integrins th

engage their endothelial ligands, members the ICAM-1 immunoglobulin superfami

resulting in arrest of neutrophil rolling anfirm adhesion. This integrin engagement,

well as continuing input from inflammatochemoattractants and cytokines, prepares t

neutrophil for its final chemotactic pursuit: Tcell spreads, producing a leading-edge lame

lipodium where chemokine and phagocyreceptors are concentrated, the cytoskeleton

rebuilt and targeted toward movement alonchemotactic gradients, and initiation of t

neutrophil oxidative burst begins (17, 18).

Now firmly adhered, the neutrophil munegotiate a path through the endothelium in

the underlying tissue. In a process dependeon 2 integrins and ICAMs, neutroph

crawl along the vessel wall until a preferrsite of transmigration is reached (192

Upon arrival at an endothelial cell junctioncomplex interaction between (a) the neutroph

integrins and their endothelial partners a(b) neutrophil surface proteins and vario

endothelial junction molecules results in tranmigration through the endothelial juncti

(13). Once through the endothelial linin

the neutrophil must navigate the basememembrane, a protein mesh consisting large

of laminins and collagen type IV. Speculatioabounds that granule proteases assist in th

migration by digesting the protein me

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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 neutrophils

journey through the activation process. How-ever, a prominent question remains largely

unanswered by the preceding exposition: Whatexactly is meant by the (admittedly ambiguous)phrase neutrophil activation? A quick scan

of the literature presents the inexperiencedreader with a sometimes rather conflicting (and

overwhelming) view of neutrophil activation.In fact, one could be (erroneously) led to

believe that neutrophil activation refers only todirect stimulation of the oxidative burst, as this

has 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 neutrophils

journey toward an inflammatory site must be

parsed by the complex neutrophilic signalin

mechanisms, a process that gradually leato complete activation and culminates in th

premiere killing functions of phagocytosdegranulation, and NETosis. It is, therefor

more insightful to view neutrophil activatio

as a continuum of processes, priming step

and signal cascades with varying effects anoutcomes, all focused on the realization one goal: the transition of naive, circulatin

neutrophils to their microbe-eliminatintissue-resident counterparts (Figure 1).

NEUTROPHILS AND THEELIMINATION OF MICROBES

The basic instruction set of the activatneutrophil is both effective and ruthless

its simplicity: (1) kill microbes, (2) do nharm to the host, and (3) when in doubt, s

rule 1. To fulfill this antimicrobial agend

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 1

Neutrophil recruitment to sites of inflammation. The circulating neutrophil must recognize signs of

inflammation 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 imcells upon infecinjury; whenuncontrolled it

tissue damage

neutrophils possess an array of toxic weapons

that are carefully regulated through controlledmechanisms. These antimicrobial weapons

vary considerably in their methods of actionand thus reflect the neutrophils attempt to

exploit any and all weaknesses that microbesmight present during the course of infection.

An understanding of these weapons, their

action, and their method of release is criticalto understanding neutrophil function.

Granules and Degranulation

The neutrophil must safely transport a plethora

of dangerous substances through the blood-stream and then correctly deploy them at the

appropriate time. Therefore, it comes as nosurprise that a specialty storage organelle has

evolved 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 organellesfordangerous substances; they areactiveand in-

dispensable participants in almostall neutrophilactivities during inflammation.

As mentioned above, there are threefundamental types of granules in neutrophils

(Figure 2). Azurophilic granules (also known

as peroxidase-positive or primary granules) arethe largest, measuring approximately 0.3 M

in diameter, and are the first formed duringneutrophil maturation. They are named for

their ability to take up the basic dye azure A andcontain myeloperoxidase (MPO), an enzyme

critical in the oxidative burst (32, 33). Othercargo of this granule class include the defensins,

lysozyme, bactericidal/permeability-increasingprotein(BPI), anda number of serine proteases:

neutrophil elastase (NE), proteinase 3 (PR3),and cathepsin G (CG) (34). As such, these

granules are brimming with antimicrobial

Granule type Primary

(azurophilic)

Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cellStage offormation

Myeloperoxidase

Defensin

Degranulationpropensity

Lysozyme

Elastase

Lactoferrin

Gelatinase

Complement receptor Characteristicproteins

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/p22pCD11b, MMP25,FPR, alkalinephosphatase, CDCD13, CD14,plasma proteins

FcRIII

Secretovesicle

Tertiary(gelatinase)

Secondary(specic)

PMN

Figure 2

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



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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 (or

secondary) 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). The

third class, the gelatinase (tertiary) granules, arealso MPO-negative, are smaller than specific

granules, and contain few antimicrobials,but they serve as a storage location for a

number of metalloproteases, such as gelatinaseand leukolysin. These granules are also the

last population of granules formed duringneutrophil maturation (5). Finally, a fourth set

of structures, the secretory vesicles, are alsocommonly considered part of the neutrophil

granule family. In contrast to the classical

granules, these do not bud from the Golgi,but instead are formed through endocytosis

in the end stages of neutrophil maturation(36). Consequently, their cargo consists pre-

dominantly of plasma-derived proteins such asalbumin. The membrane of secretory vesicles

serves as a reservoir for a number of importantmembrane-bound molecules employed during

neutrophil migration.As a neutrophil proceeds through activation,

granules are mobilized and fuse with either the

plasma membrane or the phagosome, 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: Azurophilic

granules are the most difficult to mobilize, fol-lowed by specific granules, gelatinase granules,

and finally, secretory vesicles (3741). The

underlying mechanisms for this differentialmobilization are not entirely understood, al-

thoughregulationofintracellularcalciumlevelsappears to play a salient role (32, 39). Because

of this varying mobilization propensity, each

granule subset has been traditionally associat

with a particular stage of neutrophil activatioAfter neutrophils contact the endothelium

stimulation through selectins and chemoattratants induces mobilization of secretory ve

cles, whose membranes are rich in key facto

necessary for continued activation of the ne

trophil, including, among others, the

2 intgrins, complement and fMLP receptors, as was the FcRIII receptor CD16 (5, 38, 39, 42

Fusion of the secretory vesicles with the plasmmembrane exposes these components to the e

ternal environment. This results in the trantion to firm adhesion, mediated by2 integr

interaction with the endothelium. As they prceed through the endothelium, neutrophils a

exposed to further activationsignals that initia

mobilization of gelatinase granules, thereby rleasing metalloproteases. The activity of the

proteases may help neutrophils traverse tbasement membrane, although this has n

been conclusively demonstrated (43, 44).At the inflammatory site, complete ac

vation of the neutrophil ensues, promptiinitiation of the oxidative burst and mobiliz

tion of the azurophilic and specific granuleThese granules either fuse with the phagosom

(see section on Phagocytosis, below), cotributing to the antimicrobial activities of th

compartment, or fuse with the plasma mem

brane, releasing their potent antimicrobiinto the tissue. The fusion of specific granul

with the plasma or phagosomal membrane isparticular importance for the oxidative bur

as flavocytochrome b558, a component of thNADPH oxidase machinery, resides in t

specific granule membrane (45). This fusiopermits assembly of the NADPH oxidase com

plex and allows reactive oxygen species (ROproduction both inside the phagolysosome an

outside of the cell. Degranulation of primaand secondary granules contributes to t

creation of an antimicrobial milieu at the i

flammatory site and produces an environmeinhospitable to invading pathogens.

The release of granular proteins during dgranulation presents the astute observer wi

a tempting proposition: Could these granul

466 Amulic et al.


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Table 1 Mechanism of action of neutrophil antimicrobial proteins

Antimicrobial peptide Antimicrobial mechanisma

Cationic antimicrobial peptides

-defensins (HNP-1, HNP-2,

HNP-3, HNP-4)

Permeabilize membrane bilayers containing negatively charg

phospholipids 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 LPS

Histones Unknown mechanism

Proteolytic enzymes

Lysozyme Degrades bacterial cell wall

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

bacterial membrane

Neutrophil elastase (NE),

cathepsin G (CG)

Cleaves bacterial virulence factors and outer membrane

proteins Mechanism independent of a proteolytic activity by binding t

the bacterial membrane

Azurocidin Mechanism independent of a proteolytic activity by binding to the

bacterial membrane

Metal chelator proteins

Lactoferrin Alters bacterial growth by binding to iron, an essential bacteri

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

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 disulfide bonds and whose structures maychange under physiological conditions and

increase 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 that

may 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 the

antimicrobial activity, they may potentiaDNA activation of dendritic cells (DCs) (50)

Neutrophils also contain a number full-length cationic antimicrobial protein

including BPI and histones. BPI is cationand binds LPS avidly, much like its structur

cousin the LPS binding protein. BPI bindingLPS results in increased bacterial permeabili

and hydrolysis of bacterial phospholipids; c

death then follows (51). Interestingly, histonare extremely effective antimicrobials a

were one of the first antimicrobials describ(52). The significance of histones (and of th

peptides derived from them) as microbiremains to be demonstrated in vivo (5

Given their dual role as an architectu

468 Amulic et al.


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Chronicgranulomatousdisease (CGD)caused by mutatrendering theNADPH oxidas

nonfunctional,characterized bysusceptibility toinfection andautoinflammatio

scaffold for DNA and as antimicrobials, their

in vivo significance is particularly difficult 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 deficient in this enzyme (54).Surprisingly, this occurred independently of its

enzymatic activity (55). Neutrophils also con-tain several serine proteases (including PR3,

CG, and NE, collectively known as the serpro-cidins) that exhibit differing specificities. They

are tightly regulated intra- and extracellularlyby serpins, indicating that their activity is

deployed under specific conditions. NE cleavesenterobacterial virulence factors with high

specificity (56), indicating the possibility of thecoevolution of microbial virulence factors and

antimicrobial effectors. Of further interest, NE

mutations in humans, but not genetic ablationof this enzyme in mice, result in neutropenia.

This can be rescued by the administration ofrecombinant granulocyte macrophage colony-

stimulating factor (GM-CSF); however, thesepatients still exhibit significant susceptibility

to infections. Mice deficient in NE or CGare highly susceptible to bacterial and fungal

infections (57, 58). Another protein, azuro-cidin, is a member of the same family but lacks

protease activity. Unexpectedly, it still kills

microbes, suggesting that these proteins mayall have antimicrobial activity independent

of proteolysis, perhaps as a result of theircationicity. These serine proteases also play a

salient role in autoimmunity (see discussion insection on Autoimmunity, below) (59).

The final class of neutrophil antimicrobialsconsists of a number of proteins that chelate

essential 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 other

names),whichsequesterszinc(60)andresultsinnutritional 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, and

permeability 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 membranes

and begins the reactive oxygen cascade by

reducing molecular oxygen to superoxide.Downstream of superoxide, many potential

reactions can occur (for details, see References6264). Superoxide, though not a strong

oxidant, rapidly dismutates, forming hydrogenperoxide. Superoxide can also react with nitric

oxide, which is produced at high levels atinflammatory sites, to form peroxynitrite, a

strong oxidant. Upon degranulation into thephagosome, MPO can react with hydrogen

peroxide to produce various reactive species,including hypohalous acids. Hypochlorous

acid, thought to be the major product of MPO

in the phagosome, is more reactive than su-peroxide and is antimicrobial in vitro. Thus, it

is assumed to have direct antimicrobial effectsin the phagosome. However, a theoretical

model of the phagosome suggests that most ofthe hypochlorous acid produced would react

with host proteins before reaching the bac-terium. This model predicts that chloramines,

produced when hypochlorous acid reactswith amine groups, may be the most relevant

antimicrobial actors in the phagosome (65).ROS are clearly important for neutrophil

antimicrobial activity: Neutrophils from

chronic granulomatous disease (CGD) patientskill microbes poorly, making these patients

susceptible to many infections. Interestingly,CGD patients can control catalase-negative

bacteria, which produce, but do not degrade,their own hydrogen peroxide, thus providing

a substrate for reactions downstream in thereactive oxygen cascade (66). NADPH ox-

idase is also implicated in the regulation of



12/33

inflammation, which explains why CGD

patients 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 withCandidaspecies,andafewhavebeenmistakenforCGDpatients.

However, most MPO-deficient individuals inthe developed world have apparently normal

immunity. The mild effects of MPO deficiencysuggest that MPOs products are not essential

for antimicrobial action. Indeed, in the absenceof MPO, other reactive species (e.g., superox-

ide, hydrogen peroxide, hydroperoxyl radical,peroxynitrite) can still be produced in the

neutrophil phagosome; hydroperoxyl radical ispredictedtobepresentatantimicrobialconcen-

trations (65). However, there may be a broader

reason for this discrepancy. Modern technolo-gies can distinguish between individuals who

are partially and completely MPO deficient,and partial MPO deficiency does not correlate

with pathology (70). Residual activity of MPOmay be sufficient for antimicrobial activity: In

the case of CGD, even 1% of normal NADPHoxidase activity leads to an improved prognosis

(71). Epidemiological studies distinguishingthe degrees of MPO deficiency and their

correlation with clinical manifestations may be

necessary to understand the function of MPO.In addition to direct antimicrobial action,

ROS can modify host molecules. Becausethese species are highly reactive, they are often

thought to be too nonspecific to be involved insignaling. However, specificity can be achieved

on the submolecular level, by cellular redoxbuffering systems and by limited diffusion of

ROS owing to their short half-lives (72). Awell-studied example of ROS in signaling is

the reversible regulation of various targets

(including phosphatases, metalloproteinases,and caspases) by direct oxidation of cysteine

residues. In addition, neutrophil granuleproteases can be regulated by oxidative inacti-

vation of their inhibitors or by direct oxidation

(73, 74). Furthermore, superoxide generati

leads to an ionic influx into the phagosome compensate for charge; this may activate gra

ule proteases by releasing them from their ptative matrix (75). There is controversy aroun

which ions and which channel are responsib

for charge compensation, but this theory

protease activation is certainly intriguing (69Studies of ROS are hampered by variotechnical issues. Ideally, a probe for RO

should be specific, targetable to particuintracellular compartments, and capable

being used in vivo. Traditional probes fROS do not meet these specifications;

addition, the probes often become radicspecies (76). One promising new approa

for ROS detection that meets these criteria

the use of redox-sensitive fluorescent proteibased probes, such as roGFP and HyP

(76). Other methods that can be used in viinclude transcription profiling of superoxi

or hydrogen peroxidesensitive genes as was the detection of relatively stable products

reactive oxygen using mass spectrometry (76

Phagocytosis

Phagocytosis is the major mechanism to rmove pathogens and cell debris. It is an activ

receptor-mediated process during which a paticle is internalized by the cell membrane in

a vacuole called the phagosome. As with oth

phagocytes, the mechanistic details of internaization depend on the type of interaction b

tween the neutrophil and the microorganismInteraction can be direct, through recognitio

of PAMPs by pattern-recognition receptors, opsonin mediated. The latter mechanism is be

ter characterized and includes two prototypicexamples: FcR-mediated phagocytosis, whi

relieson theformationof pseudopodextensiofor engulfment of IgG-opsonized particles, a

complement receptor-mediated phagocytoswhich does not require membrane extensio

or pseudopods (77).

After engulfment, the nascent phagosomis relatively benign to microorganisms, acqu

ing its lethal properties only after a drast

470 Amulic et al.


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Autophagy: a pin which cellulacontents are degin lysosomes,especially inconditions of nu

scarcity and infe

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 an

endocytic maturation pathway: In neutrophils,phagosome maturation happens upon fusion of

granules to the phagosome, whereby deliveryof antimicrobial molecules into the phagoso-mal lumen occurs. Simultaneously, assembly

of 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 the

macrophage phagosome gradually acidifies,neutrophil phagosomal pH is initially alkaline

(78) and remains neutral for prolonged periodsof time (79). The maintenance of this alkaline

pH is essential for the activation of the major

serine 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 more

detail in Reference 80.Not all pathogens succumb to the hostile

environment of the phagosome. In fact, somehave evolved strategies to survive inside neu-

trophils. These strategies include interferingwith engulfment, modulating phagosome

maturation, and creating a more hospitable

intraphagosomal environment. The polysac-charide capsule expressed by Staphylococcus

aureusconfers antiphagocytic properties (81).Helicobacter pylori can disrupt targeting of

NADPH oxidase to the phagosome so thatsuperoxide anions accumulate extracellularly

rather than in the phagosome (82). Francisella

tularensisprevents triggering of the oxidative

burst and also inhibits ROS production inresponse to other stimuli (83). Finally, other

pathogens, such asSalmonella typhimuriumand

Streptococcus pyogenes, can efficiently 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 innate

immune defense.

Neutrophil Extracellular Traps

Upon stimulation, neutrophils can undergoNETosis, an active form of cell death that

leads to release of decondensed chromatin intothe extracellular space (86, 87). The fibrousstructures 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 to

kill microbes by exposing them to high localconcentrations of antimicrobials (89).

The mechanism of NET formation is notcompletely understood. The reactive oxygen

pathway 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), a

finding that awaits genetic confirmation. All ac-tivators of NET formation tested so far require

ROS production. S. aureusmaybe an exception,although those experiments were done using

pharmacological inhibitors, not cells deficientin ROS production (92). Upstream of NADPH

oxidase, the Raf-MEK-ERK pathway is impli-cated in NET formation (93), but further along

in the process, NE translocates from the gran-

ules to the nucleus and degrades histones, lead-ing to chromatin decondensation (94). Histone

citrullination may also play a role in NET for-mation, although this has not been confirmed

in primary human neutrophils (9597). Au-tophagy is also thought to be required for NET

formation, 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 relevance

of NETs, a NETs knockout organism should

be generated to investigate its response topathogens. Unfortunately, it is not possible to

eliminate the main components of NETsDNA and histonesfrom 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 and

decreases in lung anddigestive function

NET formation, such as NADPH oxidase,

MPO, and NE, are also critical for other an-timicrobial neutrophil functions. For now, the

evidence for the relevance of NETs is indirect.On the one hand, bacteria that express DNases

as virulence factors disseminate more efficientlyin the host, which may point to evolutionary

pressure to avoid entrapment by NETs (99,100). In addition, a persistent Aspergillusinfection in a CGD patient was cleared after

gene therapy, which restored NADPH oxidaseactivity, NET formation, and NET-mediated

but not phagocytosis-mediated killing by thepatients neutrophils ex vivo(101). On the other

hand, the immune system has redundant mech-anisms to fight infection, and it may be that

NETs are especially important under certainconditions, such as during infections with large

pathogens that are not readily phagocytosed.NETs can also have detrimental effects on

the host. Because NETs expose self molecules

extracellularly, they lead to autoimmunity:NETs have been implicated in systemic

lupus erythematosus (SLE), an autoimmunedisease characterized by the formation of

autoantibodies, often against chromatin andneutrophil components (102106) (see section

on Autoimmunity, below). Platelet-inducedNETs, formed during sepsis, are associated

with hepatotoxicity due to tissue damage(107). Platelets also bind to NETs, raising the

possibility that NETs nucleate blood clots in

the context of deep vein thrombosis (108).NETs have also been observed in the airway

fluids of cystic fibrosis patients, where theymay increase the viscosity of the sputum and

decrease lung function (109).

NEUTROPHILS IN IMMUNECELL CROSS TALK

Neutrophils participate in the communica-

tion networks that form the foundations ofimmunity, issuing instructions to practically

all other immune cells. As one of the first celltypes to arrive at sites of infection, neutrophils

secrete cytokines and chemokines critical in the

unfolding of the inflammatory response and in

establishing the correct environmental cond

tions to launch the adaptive immune responThe cytokines released by PMNs are oft

synthesized de novo. Although neutrophtranscribe little after leaving the bone marro

once activated, these cells undergo a trascriptional burst that results in the synthe

of signaling molecules (110, 111). Comparwith other immune cells (e.g., macrophageneutrophils typically produce lower amoun

of cytokines per cell, but they are so abundaat inflammatory sites that their contributi

to total cytokine levels is significant (4). Futhermore, neutrophil-secreted proteases c

modulate signaling networks in vivo throucytokine processing (112).

The initial neutrophil cytokine responsean appeal for immunological reinforcemen

The most abundantly produced cytokine, IL-primarily serves to recruit other neutroph

(113). Similarly, neutrophil-derived proinflam

matory IL-1 and TNF-induce other ceto produce neutrophil chemoattractants (11

115) (for a comprehensive list of cytokinproduced by neutrophils, please see Referenc

115, 116). In addition to cytokines, neutrophrelease other signaling mediators, includi

granule contents (117), lipids (118), and ROsuch as hydrogen peroxide (119). They al

communicate via cell-cell contact (120). Hewe provide examples of how neutroph

interact with other cells to shape the immun

response (seeFigure 3).

Monocytes and Macrophages

As they respond to infection or injurneutrophils and their relatives in the mon

cyte/macrophage lineage coordinate thactivities, leading to alternating waves of r

cruitment of these two cell types. Macrophagand patrolling monocytes are among the init

detectors of PAMPsand endogenousactivatothe danger-associated molecular patterns (12

and these cells work to summon large numbe

of neutrophils to the inflammatory locus. Tinflux of neutrophils is followed closely by t

arrival of monocytes, suggesting a causal lin

472 Amulic et al.


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Neutrophil

Neutrophil

Neutrophil

Neutrophil Monocyte

T cell

T cell

Macrophage

Lymph

Blood

Tissue

Activation anddifferentiation

ROS?

Arginase?

IFN-

IFN-

IL-12

NK cell

DC

DC

DC

Activation

Activation

Crosspriming

Bacteria

Th1

Antigenpresentation

CD4+

T cell

CD8+

T cell

DC

DC

Figure 3

Neutrophil 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 CDcells; 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 med

behind these temporal dynamics. Indeed, neu-

trophils recruit monocytes via several differentmechanisms. They express classical monocyte

chemoattractants such as CCL2 (MCP-1)(122), CCL3 (MIP-1) (123), CCL20 (MIP-

3), and CCL19 (MIP-3) (124). Additionally,

and perhaps more unexpectedly, neutrophilsuse granule proteins to induce extravasation

of monocytes in vivo, as shown for LL-37,azurocidin (HBP/CAP37), and CG (125127).

Monocyte recruitment is also affectedindirectlyby neutrophils: via upregulation of endothelial

adhesion 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 cytokine

production (128), directly enhancing their

microbicidal activity (129). The circuitous

nature of the cross talk of these two cell typesbecomes obvious during inflammation abate-

ment: Monocytes, recruited by neutrophils

and differentiated into macrophages, repressfurther neutrophil chemotaxis and ensure

the appropriate removal of their postmortemremains (see section on Neutrophils and

Resolution of Inflammation, below).

Dendritic Cells

Neutrophils can also recruit and activateDCs in vivo. This was recently illustrated

in a mouse model of Leishmaniasis, wheresubcutaneous inoculation of Leishmania majortriggered a massive and rapid infiltration ofneutrophils (130). These cells secrete the

chemokine CCL3, recruiting DCs to the

site of inoculation and initiating a protective



16/33

DC-SIGN:dendritic cellspecificintercellular 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 in

inflammation andimplicated inautoimmunity

Th1 response (131). Interestingly, activated

neutrophils can induce the maturation of DCsin vitro through specific receptor-receptor

interactions between Mac-1 and DC-SIGN,leading to local secretion of TNF- (120).

In this case, the reduced levels of cytokineproduction foster specificity, as only proximal

DCs receive the maturation signal. A similaractivation model was earlier proposed forTox-oplasma gondii(132). Neutrophil-activated DCs

produce the proinflammatory cytokine IL-12and induce proliferation of T cells (120, 132).

However, some of these experiments shouldbe interpreted cautiously because they are

based on the injection of the anti-Gr1 antibody(RB6), which depletes neutrophils but may also

result in depletion of many other cell types inmice. The anti-Ly6G monoclonal antibody is

more specific and hence a betterreagent for thistype of experiment (133). The crucial role of

neutrophils in DC activation was recently con-

firmed using anti-Ly6G antibody depletion: InMycobacterium tuberculosisinfection, 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 they

ingested infected neutrophils just as efficientlyas they did via direct uptake ofMycobacterium(134). In sharp contrast to the above findings,a separate study using an immunization model

showed that neutrophils recruited to lymph

nodes 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-specific effects on

DCs and can be stimulatory at peripheral sitesand inhibitory in the lymph nodes. Neutrophils

exhibit fascinating and somewhat enigmatic be-havior in the lymph nodes, where they engage

in swarming activity in response to parasiticinfection (136). The functions and mechanistic

details of these swarms are unknown andrepresent questions of immense interest.

Natural Killer Cells

Studies of interactions between neutrophil and

natural killer (NK) cells have historically been

performed in vitro, and their interpretation

frustratingly difficult owing to the questioable purity of cell preparations. Recently,

was shown that neutrophils, NK cells, and DCinteract in a menage a trois involving bo

cytokine signaling and direct cell-cell conta(137, 138). In one report, infection of mi

with Legionella pneumophila triggered prodution of IFN-by NK cells; this was dependeon both PMN-derived IL-18 and DC-deriv

IL-12 (137). Similarly, human neutrophils, Ncells, and DCs colocalize at inflammatory sit

and a positive feedback loop has been proposon the basis of in vitro data. In this scheme, ne

trophils interact with a specific subset of DC(via CD18-ICAM-1 interactions), promptin

the DCs to produce IL-12p70, which in tustimulates IFN-production by NK cells an

further activates neutrophils. Simultaneousneutrophils alsoactivate NK cells by direct co

tact (139). Additional in vitro interactions b

tween neutrophils and NK cells are extensivereviewed in Reference 138.

Lymphocytes

A surprising finding in recent years is the exte

sive cross talk between cells located at opposiends of the immune spectrum. Previous

thought to belong to isolated compartmenneutrophils and T cells shape and impa

each others functions, both qualitatively an

quantitatively (140). Neutrophils affect T cfunction indirectly via DCs, as outlined abov

but can also influence T cell function directlPMNs secrete IL-12, which may be crucial f

Th1 cell differentiation (141, 142). They alexpress several T cell chemoattractants (11

as well as B cell development and maturatifactors (143, 144). Cytokine communicati

occurs in both directions: For instance, IFN-which is secreted by T cells, prolongs ne

trophil life span, induces gene expression, anincreases phagocytic capacity (145). The

helper 17 (Th17) cell subset secretes IL-1

a key cytokine in the control of neutrophdynamics, which acts by upregulating expre

sion of CXCL8 (IL-8), G-CSF, and TNF

474 Amulic et al.


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Ulcerative colitype of inflammbowel diseasecharacterized byand tissue erosiothe colon and re

by epithelial, endothelial, and stromal cells

(146). Collectively, these Th17-associatedcytokines increase granulopoeisis as well as the

recruitment and life span of neutrophils.Neutrophils potentially have suppressive ef-

fects on T cells via two proposed mechanisms:(a) L-arginine depletion by release of arginase,

which inhibits T cell responses in vitro (147),and (b) hydrogen peroxidemediated suppres-sion, as proposed in a cancer model (119) (see

section on Cancer, below). Direct evidence ofsuch interactions in vivo is still missing.

Interestingly, neutrophils influence CD8+

T cell responses by cross-presenting exogenous

antigens in vivo. Using mice in which profes-sional antigen-presenting cells do not express

functional MHC class I, Beauvillain et al. (148)showed that antigen-pulsed neutrophils can

induce differentiation of cytotoxic T cells.These striking findings imply that neutrophils

have characteristics of antigen-presenting cells.

Neutrophils also appear capable of expressingMHC class II and costimulatory molecules

under inflammatory conditions (149151),and they can present antigen to CD4+ T cells

in vitro (152154). However, the functionalsignificance for protective immunity remains

unclear, especially in light of the finding thatmouse neutrophils that migrate to the lymph

node 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 finding that could haveimplications for susceptibility to autoimmune

diseases. Therefore, neutrophil modulation ofadaptive immunity seems to be highly complex

and is only now starting to be unraveled.

NEUTROPHILS ANDRESOLUTION OFINFLAMMATION

The lethal cargo of neutrophils is not onlydestructive toward invading microbes, but

also harmful to host cells. Thus, neutrophil

deployment must be tightly controlled.

Although some collateral damage to host

tissues is inevitable during infection, neu-trophils must be removed before they have

serious, detrimental effects on inflamed tissues.Resolution of inflammation is an active process

that limits further leukocyte infiltration andremoves apoptotic cells from inflamed sites.

This process is essential for maintenance oftissue homeostasis and, if impeded, leads tononresolving inflammation, a problematic

condition that contributes to many diseases.

Apoptosis and Clearance

Apoptosis is a central aspect of inflammation

resolution. Once neutrophils have executedtheir antimicrobial agenda, they die via a built-

in cell-death program. However, not only doesapoptosis reduce the number of neutrophils

present, it also produces signals that abro-

gate further neutrophil recruitment. Phagocy-tosis of apoptotic neutrophils also reprograms

macrophages to adopt an anti-inflammatoryphenotype.

Neutrophil death is influencedby inflamma-tory mediators such as GM-CSF and LPS and

by environmental conditions such as hypoxia,all of which prolong neutrophil survival. The

signaling networks that regulate survival havealso been well characterized. These networks

also control the expression of known antiapo-

ptotic (Mcl-1 and A1) or proapoptotic proteins(Bad, Bax, Bak, and Bid), and they also activate

caspases (for an extensive review, see 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 mature

cells by inflammatory signals such as G-CSF or

GM-CSF. In line with these findings, survivinis also highly expressed in neutrophils at sites

of inflammation, such as cystic fibrosis sputum,appendix infiltrates, and intestines of patients

with ulcerative colitis (156).



18/33

Wegenersgranulomatosis:vasculitis affecting thelungs, nose, andkidneys; inflammationleads to reduced blood

flow, tissuedestruction, anddamage of vital organs

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

leukocyte recruitment

Similarly, cyclin-dependent kinases func-

tion as prosurvival factors in neutrophils.Pharmacological inhibition of these cell cycle

regulators induce caspase-dependent apoptosisand block 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 DNAreplication, but in neutrophils, it associates

with 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-3

and 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-flammation is the proper removal of apoptotic

cells. 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 by

macrophages drives the production of the anti-inflammatory cytokines tumor growth factor

(TGF)- and IL-10 (155). Failure to clear theseapoptotic cells, by contrast, results in secondary

necrosis 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 progression

of inflammation appears to hinge on a shiftin the composition of secreted lipids. At early

stages of inflammation, neutrophils synthesizeproinflammatory lipid mediators, such as

prostaglandins and leukotrienes. These arederived from arachidonate precursor molecules

and 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 cel

fibroblasts, platelets, and leukocytes) and paticipate in the transcellular biosynthesis of lip

mediators with anti-inflammatory and prorsolving activities, such as lipoxins,resolvins, a

protectins. A major lipid mediator class swit

thus exists, governed by temporally regulat

expression of different lipoxygenases and tmobilization of different fatty acid substrateThe different biosynthesis pathways of pror

solving lipid mediators have been reviewed detail elsewhere (118). Interestingly, microo

ganisms are also a source of lipid precursothat can be used by neutrophils for resolv

synthesis. Thus, microbes also likely participain synthesis of mediators with proresolvi

functions at the site of infection (159, 160).How do lipid mediators contribute

the termination of inflammation? Lipoxin

resolvins, and protectins exert cell-type specieffects, promoting monocyte/macropha

recruitment and activation while inhibitineutrophil functions. The inhibitory effe

extends to all essential steps of neutropresponses: migration, adhesion, and activatio

All three lipid mediators reduce neutrophrecruitment, a process that involves the lipoxi

A4 receptor and the leukotriene B4 recept(BLT1) (161167). Ariel et al. (168) also pr

posed an interesting mechanism of action flipoxins,resolvins, andprotectinsin clearing i

flammatory sites. They showed that neutroph

exposure to these lipids increases expressiof CCR5 on the surface of late apoptotic ne

trophils, leading to efficientsequestration of tchemoattractants CCL3 and CCL5. The s

questration of these chemokines means they aunavailable to recruit neutrophils to inflam

sites (168) (Figure 4). This mechanism complements other anti-inflammatory process

in which chemokines are inactivated by netrophil proteases. Of these lipids, lipoxins a

the most completely understood. In addition

neutrophil recruitment, lipoxins can inhibit tshedding of L-selectin and the upregulation

2 integrins in response to proinflammatostimuli, thereby reducing adhesion of ne

trophils to endothelial cells (169, 170). Final

476 Amulic et al.


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TNF-IL-6

IL-10TGF-PGE-2

Neutrophil

Monocyte

Platelets

Lipoxins

Macrophage

Macrophage

ChemokinesApoptotic

neutrophil

NEToticneutrophil

LeukotrienesProstaglandins

?Microorganisms

LipoxinResolvinsProtectins

Chemokines

CCR5

Initiationof inammation

Resolutionof inammationLeukotrienes Prostaglandins TNF- TGF-Lipoxins Resolvins Protectins IL-10

Chemokine clear

Figure 4

From inflammation to homeostasis: neutrophil apoptosis and lipid mediator class switching in the resolution of inflammation. Asite of infection, resident macrophages initiate an inflammatory response, secreting proinflammatory cytokines and chemokines alert the immune system and promote neutrophil recruitment. In the early stages of inflammation, microbes trigger the productproinflammatory lipid mediators, such as leukotrienes and prostaglandins, which also recruit neutrophils. As inflammation progrswitch occurs, and anti-inflammatory lipid mediators such as lipoxins, resolvins, and protectins are produced. Notably, interactioneutrophils with platelets induces the production of lipoxins. Anti-inflammatory lipid mediators initiate the resolution of inflammby blocking neutrophil and promoting monocyte recruitment. Monocytes differentiated into macrophages ingest apoptotic neutdriving the production of the anti-inflammatory cytokines tumor growth factor (TGF)- and IL-10 and prostaglandin-E2 (PGEwhich drive the lipid mediator class switch. Proresolving lipid mediators also promote the expression of CCR5 on the surface of

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

Chronic obstrupulmonary dise(COPD): lung caused by noxioparticles or gas,tobacco smokininflammation lelung obstruction

lipoxins also impact neutrophil activation byinhibiting ROS and peroxynitrite production,

NF-B activation, and IL-8 expression (170).In addition to directly impacting neu-

trophil functions, lipid mediators promotenonphlogistic (noninflammatory) phagocyto-

sis of apoptotic neutrophils by monocytes

and macrophages. In the presence of anti-inflammatory lipids, engulfment of apoptoticneutrophils is notaccompanied by thereleaseof

proinflammatory mediators, as typically occurs

during macrophage activation.Instead, produc-tionof theanti-inflammatory cytokines TGF-and 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. These

conditions lead to the accumulation of cyto-toxic substances and are associated with severe

pathologies, including cystic fibrosis, chronic

obstructive 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



20/33

Rheumatoid arthritis(RA): chronicinflammatory diseasethat affects manytissues and organs butprimarily synovial

joints; severeinflammation causesdeformity

instigator of this disease. A chronic neutrophil

infiltration in the lungs of COPD patientspromotes tissue damage and organ dysfunc-

tion. One of the key molecules controllingthe inflammatory response in the lung is

leukotriene A4 hydrolase (LTA4H). Thisenzyme has two opposing activities. First, its

hydrolase activity converts leukotriene A4 intoleukotriene B4, a potent neutrophil chemoat-tractant and proinflammatory agent. Second,

LTA4H is an aminopeptidase that inactivatesa specific neutrophil chemoattractant, the

proline-glycine-proline tripeptide (PGP), thusconferring the enzyme with anti-inflammatory

properties. Interestingly, tobacco smoke selec-tively inhibits only the aminopeptidase activity

of LTA4H, promoting the accumulation ofboth leukotriene B4 and PGP. This in turn

promotes neutrophil recruitment and fuelschronic lung inflammation (172).

Another prime example of a disease linked to

nonresolving inflammation is RA. Neutrophilsare the most abundant leukocytes present in the

synovial fluid of RA patients, and their role inpathogenesis has been demonstrated in several

animal models. These models primarily usedneutrophil depletion or adoptive transfer of

wild-type neutrophils in leukotriene-deficientmice (173175). In one model, synthesis

of 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 first wave of neutrophils

into the joint. Later, the recruitment of asecond wave of neutrophils is independent of

this leukotriene B4BLT1 pathway. At thisstage, immune complexes are essential for

stimulating infiltrating neutrophils to deliverIL-1into the joint. This in turn induces the

production of chemokines by synovial tissuecells and sustains neutrophil recruitment (175,

176). These studies exemplify the complex

regulation cascades involving lipids, cytokines,and chemokines that orchestrate neutrophil

recruitment in chronic inflammation. Theyalso demonstrate the cross talk between neu-

trophils and other immune cells discussed in

the previous section. It is, however, unknow

whether all neutrophils are capable of adaptinto the changing chemoattractant environme

or if different subsets of neutrophils are sucessively involved. The relevance of this mod

in human disease remains to be establishe

although the clinical similarities between th

mouse model and human RA are encouragin

NEUTROPHILS IN DISEASE

Neutrophils areprominent players in theinna

immune response and the clearance of infetion, a subject addressed in several promine

reviews. However, neutrophil action can al

support disease progression in other illnesseA host of autoimmune disorders belong to th

category. In addition, certain malignant canceare also prime examples of illnesses in whi

neutrophils play a salient role.

Cancer

The link between cancer and inflammati

was noted as early as 1863 by Rudolf Vircho(177). Since then, it has been proposed th

neutrophil-derived ROS have the potential initiate tumor formation by genotoxic stre

and induction of genomic instability. Althouthis has been demonstrated in vitro (178, 179

convincing evidence for PMN-mediated DNmutagenesis in vivo is still lacking. Neutroph

do, however, impact cancer progressio

They are abundant in tumors and influentumor development through several secret

mediators, including cytokines, ROS, amatrix-degrading proteases (reviewed in Re

erence 180). The majority of findings suppoa protumor and antihost effect of the

cells; clinical studies indicate that neutrophinfiltration of tumors is associated with poor

prognosis (181, 182). Indeed, some canceseem to actively recruit neutrophils throug

production of IL-8 (183). In agreement withis, antibody depletion of neutrophils reduc

tumor growth (184). The protumor functio

of neutrophils operates at multiple leveincluding production of angiogenic facto

(185), enhancement of metastasis (186), an

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Acute-phaseproteins: secreliver, concentraplasma changes 25% or more duinflammation

suppression 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 in

the 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 heightenedproinflammatory state, causing a reduction in

tumor growth. These alternatively activatedneutrophils underwent a complete reversal in

their effect on CD8+T cells, serving to activaterather than suppress these cells. Differential

neutrophil responses were also demonstrated ina melanoma study. In this instance, increased

systemic levels of the acute-phase proteinserum amyloid A (SAA-1) induced neutrophils

to secrete the anti-inflammatory cytokine IL-

10, which also inhibited T cell responses. Crosstalk with invariant NKT cells could counter

this response, restoring a proinflammatoryactivation status (188). Thus, investigation of

neutrophils in cancer has revealed considerableplasticity in their responses. Although little

evidence currently supports the existence ofdifferent populations, it is likely that neutrophil

responses are more flexible and less stereotypedthan previously thought.

Another major mechanism of tumor escape

from 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 mature

neutrophils and monocytes. In cancer andsome autoimmune and infectious diseases,

differentiation is partially blocked, leading toaccumulation of these precursors, which act as

powerful 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). In

human renal cell carcinoma, MDSCs have

identical 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 and

NOS 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 drug

interventions 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 patientswith Wegeners granulomatosis correlates with

the PMN infiltrate rather than with traditional

autoimmunity parameters such as T cell acti-vation or autoantibody titers (196). Likewise,

ANCA bind MPO and PR3 expressed on thesurface of activated neutrophils, promoting

degranulation and release of chemoattractantsand ROS, which together lead to a vicious

cycle of tissue damage and inflammation. Earlyreportsalsosuggestthat,inaninflammatoryen-

vironment, ANCA accelerate ROS-dependentneutrophilapoptosis,suggestingafeed-forward

cycle 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.

Although neutrophils had long been suspected



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Vasculitis:inflammation of bloodvessels

to be causative agents, their role in SLE patho-

genesis remained elusive. The recent discoveryof a link between SLE and NET formation

has helped to shed light on this quandary.It was proposed that TNF- and IFN-prime cells for NET formation in response toanti-PR3, antiribonucleoprotein, anti-HNP,

or anti-LL-37 autoantibodies (103, 104, 106).Thus, high levels of inflammatory cytokines inautoimmune patients are believed to sensitize

neutrophils to NETosis, whereas autoanti-bodies may trigger a switch from apoptosis to

NETosis. Additional evidence suggesting arole for NETs in autoimmune pathology was

obtained when NETs were identified in renaland/or skin biopsies from patients with SLE

and small vessel vasculitis (103106). Severalstudies have reported the presence of a particu-

lar subset of neutrophils in PBMC preparationsfrom pediatric and adult SLE patients. These

low-density granulocytes display phenotypic

characteristics of immature neutrophils withnonsegmented nuclei and higher expression

of MPO, NE, and defensin-3, and they maybe related to the MDSCs discussed previously

(see section on Cancer, above) (197, 198).An increased capacity to form NETs and a

heightened cytotoxicity toward endothelialcells could bestow them with pathogenic

properties in lupus (105).Because NETs appear to be formed during

autoimmune disease, their timely removal may

be an essential mechanism for maintainingtissue homeostasis. Human serum contains the

nuclease DNase I, which degrades NETs invitro. Notably, a familial form of SLE is linked

to a mutation in DNase I (199). Furthermore,in a cohort of SLE patients, 36% exhibited

either elevated titers of autoantibodies directedagainst NET components or inhibitors of

DNase I, both of which may protect NETsfrom degradation. Most notably, impaired

NET 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? We

know that NETs induce plasmacytoid DCs

to produce IFN-, a central cytokine in SL

pathogenesis (103, 104). However, it remainsbe determined if DCs can present NET com

ponents or if they contribute to autoreactivecell activation. It is also possible that NETs a

involved in other autoimmune diseases. Shou

this prove to be the case, understanding t

role of NETs may provide critical insights inthe role of microbial infections as a trigger autoimmunity.

CONCLUDING REMARKS

Neutrophils are specialized phagocytes th

arose as an evolutionary adaptation in vertbratestocoordinateandexecuteoneofthemo

fundamental physiological responses: inflammation. They are endowed with antimicrob

mechanisms that make them the preeminemicrobe exterminators of the immune system

In addition to this important role, PMNs al

network with many other immune cells anhelp regulate the initiation of specific T an

B cell immunity. However, neutrophils do nalwaysactin ways beneficialto thehost: Unco

trolled neutrophil responses can exacerbate aeven cause autoimmune and inflammatory d

eases. Many challenges remain in understaning neutrophil function: Is there specializatio

among PMNs? Are they more plastic than wsuspect? How do they make decisions befo

deploying their armamentaria? How do th

kill microbes? How specific are their instrutions to other cells? Answering these questio

will better define neutrophils role in defenand disease and will provide a rational path f

pursuing new therapies. Moreover, neutrophcan potentially provide insights into sever

unique aspects of basic cell biology. Their striingly short life spans make them excellent mo

els for investigating cell death, whereas thereliance on ROS as biochemical effectors m

reveal novel ways for relaying intracellul

signals. The uniquely lobulated neutropnucleus is a feat of higher-order nucle

architecture that is just beginning to yieits secrets. In short, exciting times await t

humble neutrophil.

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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that

might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

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

help. G.H. is an Alexander von Humboldt Foundation Scholar, andB.A. is supported by an EMBOLong-Term Fellowship.

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