The Neutrophil in the Pathogenesis of Multiple Organ Dysfunction Syndrome

of Multiple Organ Dysfunction Syndrome

Z. Malam and J.C. Marshall

Introduction

Circulating neutrophils play a cardinal role in early host defenses against bacte- rial and viral pathogens. Originating from hematopoietic stem cells in the bone marrow, neutrophils – also known as polymorphonuclear neutrophils (PMNs) – mature to become terminally differentiated phagocytes that are incapable of cell division, and that synthesize only low levels of RNA and protein. They are recog- nized histologically by their multi-lobed nuclei and abundant cytoplasmic gran- ules. The first description of neutrophils was by Elie Metchnikoff. Upon inserting rose thorns into the larvae of starfish, he observed that wandering mesodermal cells aggregated at the insertion site. These cells revealed phagocytic abilities, with the larger phagocytes termed macrophagocytes, more commonly known today as macrophages. The smaller phagocytic cells were termed microphagocytes, and subsequently granulocytes; neutrophils were the predominant cell type [1].

Antimicrobial Defenses of the Neutrophil Neutrophil Localization at an Inflammatory Focus

The eradication of microbial pathogens is a complex and coordinated process.

Initiation of neutrophil defense requires their recruitment from the circulation

and from bone marrow reserves to the site of microbial invasion; this process is

facilitated by a combination of host- and pathogen-derived chemotactic signals

that include chemokines, cytokines, matrix metalloproteases, and products of the

invading microbe [2]. Activated vascular endothelial cells and fibroblasts produce

chemokines such as interleukin (IL)-8, which possess potent neutrophil chemo-

tactic ability [3]. A concentration gradient at the site of inflammation induces neu-

trophils to migrate toward the source of chemokine release. Expression of surface

chemokine receptors varies among neutrophils, and influences which neutrophil

subsets are recruited. Host-derived and microbial factors such as lipopolysaccha-

ride (LPS), tumor necrosis factor (TNF)- α , and platelet-activating factor (PAF)

also enhance the response of neutrophils to subsequent stimuli [2]. For example,

neutrophils primed with LPS produce significantly greater amounts of superoxide

after exposure to the bacterial peptide, N-formyl-methionyl-leucyl-phenylalanine

(fMLP) [4]. Furthermore, during inflammatory reactions, membrane receptor ex- pression is augmented by selective exocytosis of cellular vesicles and granules enriched in membrane proteins mediating neutrophil recruitment and phagocy- tosis [5].

Recruitment of neutrophils to the microenvironment of microbial challenge is followed by their extravasation through the vascular endothelium, and accu- mulation in targeted tissue. Adhesion to activated endothelium results from the co-ordinated activities of selectins, selectin ligands, integrins, and members of the immunoglobulin superfamily [6]. Selectins are composed of three carbohydrate- recognizing members: E- and P-selectin expressed on activated endothelium, and L-selectin constitutively found on the surface of neutrophils [7]. Selectin ligands are characterized by intense glycosylation through N-linked carbohydrates and O-linked side chains [8]. Integrins are heterodimers that recognize extracellular matrix and cell surface glycoproteins, in addition to soluble molecules such as complement factor C3bi [6]. The β 2 integrins (CD11/CD18) are of particular im- portance in the neutrophil. Following stimulation, they undergo a conformational change that optimizes their interaction with members of the immunoglobulin su- perfamily, including intercellular adhesion molecule (ICAM)-1 and -2, vascular cell adhesion molecule (VCAM)-1, and platelet-endothelial cell adhesion molecule (PECAM)-1 [9].

Leukocyte-endothelial cell adhesion occurs in a coordinated and sequential fashion. The initial attachment between the neutrophil and the endothelial surface entails a loose tethering mediated by cell-surface L-selectin [10]. The primary lig- and for selectins has been identified as the tetrasaccharide sialyl Lewis-X, although others include fucosylated and sulfated structures. During neutrophil activation, L- selectin is shed from the surface, facilitating extravascular passage: when shedding is blocked by a metalloprotease inhibitor, endothelial adherence and intravascular accumulation of neutrophils is increased [11]. Moreover, newly recruited neu- trophils arriving at microvascular endothelium covered with leukocytes can at- tach to already adherent neutrophils via L-selectin-dependent mechanisms [12], perhaps through cell-surface P-selectin glycoprotein ligand-1 (PSGL-1) on neu- trophils. Interestingly, L-selectin-deficient mice exhibited no defect in leukocyte adhesion and rolling following surgical tissue exteriorization [13], a process known to promote expression of P-selectin, suggesting that high P-selectin expression may provide an alternative to L-selectin-mediated adhesion.

Loosely attached neutrophils roll along the endothelial surface until firmer ad- hesion can take place. Endothelial P-selectin is necessary for early leukocyte rolling, since P-selectin antibodies can block constitutive [14] and trauma-induced [13]

rolling. A P-selectin-independent rolling mechanism also exists; mice lacking P-

selectin expression show no leukocyte rolling immediately following exterioriza-

tion of the mesentery, but do manifest rolling by 60-120 minutes following tissue

trauma. The P-selectin ligand, PSGL-1, on neutrophils is necessary for P-selectin-

dependent rolling; rolling was almost completely inhibited when neutrophils were

pretreated with monoclonal blocking antibody to PSGL-1 [15]. E-selectin and

P-selectin demonstrate redundant roles in neutrophil rolling. Mice deficient in E-selectin and wild type mice pretreated with P-selectin antibody exhibited no deficiency in neutrophil recruitment into the peritoneal cavity during the first 6 hours following thioglycollate injection, but recruitment was blocked when E- selectin deficiency was combined with anti-P-selectin pretreatment [16].

Selectins mediate more than cell-cell adhesion. Ligation and cross-linking of L-selectin on neutrophils primes them for increased superoxide production and calcium influx following exposure to a chemoattractant [17]. L-selectin is also associated with increased adhesive properties of CD11a and CD11b and L-selectin clustering triggers p38 MAPK-mediated signal transduction to effect neutrophil shape change and release of secondary, tertiary, and secretory granules [18]. Sim- ilarly, P-selectin on endothelial cells supports superoxide production, neutrophil degranulation, and polarization in response to inflammatory mediators and bac- terial peptides.

Firm adhesion of the neutrophil to the vascular endothelium is a prerequisite to extravascular migration into the tissues. Members of the β 2-integrin family, particularly LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), mediate this state of firm adhesion. Patients with leukocyte adhesion deficiency I (LAD-I), charac- terized by the absence of CD18 integrins, show an inability to sustain neutrophil recruitment and experience recurrent life-threatening bacterial infections [19].

Severely compromised firm neutrophil adhesion and recruitment is also evident in mice with reduced CD18 expression [20]. Activated by mediators released from stimulated endothelium (for example IL-8 and PAF), the integrins bind to ICAM-1, their immunoglobulin ligand on the endothelial surface, although differing inte- grins bind different regions of the molecule. Circulating inflammatory mediators, such as IL-1, serve to upregulate ICAM-1 expression on activated endothelium.

Neutrophil adhesion to ICAM-1 occurs sequentially, with LFA-1 binding first fol- lowed by Mac-1-mediated stabilization [21]. Confocal microscopy studies show that Mac-1 rapidly accumulates at the neutrophil-endothelium interface during initial contact and subsequently redistributes away from the site as the neutrophil spreads. During migration, this redistribution is directed to the leading edge with rapid formation and dissociation of Mac-1-dense macroaggregates [22].

Following firm adhesion, neutrophils traverse the endothelium paracellu- larly [23] or transcellularly [24]. PECAM-1 is essential for transendothelial migra- tion, and is expressed at low levels on leukocytes but high levels (>106 molecules per cell) on the endothelium, localized in particular at endothelial intercellular junctions. Antibodies to PECAM-1 can block neutrophil transmigration through TNF- α -activated endothelial cell monolayers but exert no effect on adhesion. In vivo experiments demonstrate that PECAM-1 expression in mesenteric veins is critical for peritoneal neutrophil accumulation [25]. Recently, junctional adhesion molecule C (JAM-C) was identified as a novel receptor for Mac-1 on neutrophils.

JAM-C localizes within interendothelial junctions and is co-distributed with the

tight junction component, zonula occludens-1. Inhibiting JAM-C can significantly

reduce transendothelial neutrophil migration; simultaneous blockade of PECAM-

1 almost completely abolishes this effect in vitro. In an in vivo murine model of acute thioglycollate-induced peritonitis, inhibition of JAM-C with soluble mouse JAM-C results in 50% reduced neutrophil transmigration [26].

Pathogen Recognition and Uptake

Neutrophils recognize conserved molecules on the surface of invading microorgan- isms (for example, peptidoglycan on Gram-positive bacteria, and LPS on Gram- negative bacteria) through pattern recognition receptors that include CD14 and members of the Toll-like receptor (TLR) family [27]. Phagocytosis occurs through the extension of pseudopodia containing enzymes such as cathepsin G, myeloper- oxidase, lactoferrin, gelatinase, and elastase [28]. Pathogen binding and uptake are facilitated by opsonization of the pathogen with host serum antibody and complement; opsonization of Staphylococcus aureus with human serum results in an eightfold increase in neutrophil phagocytosis within 10 minutes. Activation of the complement cascade promotes the deposition of serum complement proteins C3b, iC3b, and C1q on the pathogen surface. Neutrophils, in turn, possess recep- tors such as C1qR, CR1, and Mac-1 that recognize complement and Fc ε ^{RI, Fc} ε ^RII,

Fc α ^{R, Fc} γ ^{RI, Fc} γ RIIa, and Fc γ RIIIb that recognize the Fc-region of antibody [2].

Engagement of these receptors triggers individual signaling cascades that result in actin polymerization and localized membrane remodeling for particle ingestion.

Together, they drive the formation of the phagosomal cup and its sealing following pathogen engulfment [29].

Phagosome maturation follows engulfment. The phagosome, through multi- ple and dynamic fusion events with potent secretory vesicles and granules, se- questers microbicidal peptides and proteolytic enzymes that facilitate microbial degradation. Neutrophil granules are classified into four categories: primary or azurophilic granules contain myeloperoxidase and membrane CD63; secondary or specific granules contain lactoferrin and membrane CD66b; tertiary granules contain gelatinase; and secretory vesicles contain albumin and express membrane alkaline phosphatase and CD35 (Table 1). For granular fusion with the phagosome to occur, changes in free cytosolic calcium must occur either by release from endo- plasmic reticulum stores which in turn triggers an extracellular calcium influx [30]

or by release from the phagosome itself [31]. Granules exhibit differential sensitiv-

ity to calcium, with secretory vesicles having the lowest threshold and azurophilic

granules the highest. Calcium may exert its permissive effect on granule fusion

through the rapid depolymerization of periphagosomal actin, which allows gran-

ules to access the phagosome [29]. Alternatively, calcium may catalyze coalescence

of the apposed phagosome and granule membrane bilayer [32]. In addition to

calcium, protein kinases such as protein kinase C isoforms [33] and members of

the Src-family kinases [34] have been implicated in membrane fusion; however,

their roles are still unclear.

Table 1. Neutrophil granule constituents

Protein Azurophil Specific Gelatinase Secretory

Membrane Proteins

CD11b/CD18 • • •

CD16 •

CD45 •

CD63 •

CD66, CD67 •

CR1 •

Vacuolar H+-ATPase • • •

Enzymes

Collagenase •

Elastase •

Gelatinase • •

Heparanase •

Myeloperoxidase •

Proteinase-3 •

Antimicrobial Peptides

Bactericidal permeability-increasing protein •

Cathepsins •

Defensins •

hCAP-18 •

Lysozyme • • •

Others

α 1-antitrypsin •

β 2-microglobulin • •

Lactoferrin •

Pathogen Killing

Neutrophil cytotoxic mechanisms can be broadly characterized as oxygen-depen-

dent and oxygen-independent. The former – effected through the generation of

highly reactive oxygen species (ROS) – is termed the ‘respiratory burst’. NADPH

oxidase, a multi-unit membrane protein, assembles at the phagosome membrane

following pathogen ingestion and neutrophil activation. Of the five glycoprotein

subunits that make up NADPH oxidase, cytochrome b558 is the most abundant, and

localizes in lipid rafts on the membrane. The remaining subunits translocate to lipid

rafts in response to phagocytic stimulation, with the lipid rafts serving to anchor

the NADPH oxidase subunits for optimal enzyme activation [35]. NADPH oxidase

transfers electrons from cytoplasmic NADPH to oxygen, generating the superoxide

anion (O

) which subsequently can be converted to other ROS, including hydrogen

peroxide (H

O

) and hypochlorous acid (HOCl) [29] (Fig. 1). HOCl oxidizes amino

acids and nucleotides and exerts the strongest bactericidal effects. The importance

of NADPH oxidase in antimicrobial activity is underlined by the observation that

Fig. 1. Generation of reactive oxygen species by the neutrophil occurs following assembly of the multi-complex NADPH oxidase, that catalyzes the reduction of molecular oxygen through a series of intermediates to water. These intermediates are highly reactive oxidants, capable of inducing irreversible damage to proteins and lipids. From [111] with permission

patients with chronic granulomatous disease in which oxidase function is defective, are highly susceptible to recurrent bacterial infection [36].

Microbial killing also proceeds through oxygen-independent mechanisms involving anti-microbial peptides from azurophilic granules, proteolytic en- zymes, and acidification of the pathogen-containing endosome [37]. Antimicro- bial proteins and peptides such as defensins, permeability-increasing protein, and lysozyme increase bacterial permeability by disrupting anionic surfaces. Proteases such as neutrophil elastase and cathepsin G serve to breakdown bacterial proteins.

Vacuolar ATPase is responsible for proton transport that leads to acidification, which then activates hydrolytic enzymes that function optimally under conditions of low pH (4.5-6.0) [37].

Pathogens are degraded and fragments are transported from early endosomes

to late endosomes. Non-host peptides processed via the endosomal pathway be-

come antigens that, in turn, are presented to T-cells. The antigen-presenting nature

of macrophages and dendritic cells has been well characterized; however the role of

the neutrophil as an antigen-presenting cell has traditionally been dismissed. Rest- ing neutrophils harbor major histocompatibility complex (MHC) class I molecules on their surface and contain intracellular stores of both costimulatory and MHC class II molecules that bind to processed exogenous peptides, facilitating their traf- ficking to the cell surface, a key characteristic of a professional antigen-presenting cell [37]. Additional evidence for antigen-presenting function derives from the fact that neutrophils contain reserves of cathepsins B and D, lysosomal proteases necessary for antigen presentation [38]. Moreover antigen presentation during inflammation is facilitated by prolonged neutrophil survival resulting from the inhibition of apoptotic death by inflammatory cytokines that include IL-1, IL-6, TNF- α , and interferon (IFN)- γ [39, 40]. Antigen presentation is rendered substan- tially more efficient when neutrophils travel to regional lymph nodes [37].

Apoptosis and the Termination of Neutrophil-Mediated Inflammation

Clearance of neutrophils from an inflammatory focus occurs through apoptosis, or programmed cell death, of the neutrophil, followed by its uptake by fixed tissue macrophages [41]. Neutrophils are constitutively apoptotic cells, and the apoptotic program is activated within hours of their maturation and release from bone marrow stores [42].

Cellular Mechanisms of Neutrophil Apoptosis

There are two major pathways of apoptosis or programmed cell death, termed the extrinsic and intrinsic pathways, and mediated through signals from the exter- nal environment, and endogenous cell stress, respectively. Cell death is effected through the activity of a family of intracellular enzymes known as caspases, a fam- ily of intracellular proteases that cleave their target proteins at conserved sites adjacent to the amino acid, aspartic acid.

Apoptosis, in response to stimuli in the external environment of the cell, is effected through the CD95 family of death receptors that includes the transmem- brane proteins Fas and TNF receptor-1 having cysteine-rich extracellular domains and conserved cytoplasmic death domains [43]. Fas is activated by the bind- ing of Fas ligand which initiates cross-linking of three receptor molecules and subsequent clustering of intracellular death domains [44]. Fas-associated death domain-containing protein (FADD) is recruited and binds to the cluster; this com- plex interacts with procaspase-8 through its death effector domain (DED). The resultant union of the three molecules forms the death-inducing signaling complex (DISC). Likewise, TNF- α binding to TNF receptor 1 results in receptor trimeriza- tion, bringing death domains nearer, and allowing TNF receptor-associated death domain-containing proteins (TRADDs) to bind to the receptor cluster via their death domains; this complex can then associate with FADD and procaspase-8 [43].

Binding of procaspase-8 via Fas or TNF receptor activation induces autocleavage

and activation of caspase-8, which in turn, cleaves procaspase-3 to yield caspase-3.

Caspase-3, in turn, cleaves a large number of substrates, from nuclear proteins to cytosolic structures and cytoskeletal elements, and also catalyzes the degrada- tion of proteins crucial for neutrophil survival. Clustering of death receptors in ceramide-rich lipid rafts can also activate caspase-8 in the absence of ligand bind- ing [45]. Intriguingly, TRADD proteins can bind TNF receptor-associated factor-2 (TRAF2) and receptor interacting protein (RIP) to activate the anti-apoptotic tran- scription factors, nuclear factor-kappa B (NF- κ B) and activator protein (AP)-1 [43].

Since NF- κ B and AP-1 commonly promote the expression of survival proteins, TNF- α has the ability to either promote or delay apoptosis [46].

Caspase-9 is the apical caspase of the intrinsic pathway, and its activation also results in activation of caspase-3 [47]. Caspase-9 is activated by stimuli that induce the release of cytochrome c from the mitochondrion, where it complexes in the cytoplasm with pro-caspase-9, and cytosolic apoptotic protease-activating factor (APAF), resulting in catalytic cleavage and activation of caspase-9 [48]; formation of this complex results in progression of apoptosis. Cytochrome c release from the mitochondrion is regulated by the levels and activities of pro-and anti-apoptotic members of the Bcl-2 family. For example, anti-apoptotic members Bcl-X and Mcl-1 are down-regulated during apoptosis [49], while pro-apoptotic members Bax, Bid, Bak, and Bad are constitutively expressed [50]. The pro-apoptotic function of these members results from their redistribution from the cytosol to the mitochondrion, resulting in disruption of mitochondrial membrane integrity and an increase in pore formation.

Constitutive neutrophil apoptosis is associated with increased mitochondrial inner membrane permeability, a reduction in mitochondrial transmembrane po- tential, and opening of mitochondrial permeability transition pores [51]. As a con- sequence, cytochrome c is released from the mitochondrion. Activation of the membrane-bound caspase, caspase-8, is also implicated in the induction of apop- tosis [52].

Inhibition of Neutrophil Apoptosis During Inflammation

Neutrophil survival is prolonged through the active inhibition of apoptosis during acute inflammation. A number of host-derived cytokines including TNF- α ^{, IL-1} β ^,

IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) prolong neutrophil survival during acute inflammation (Table 2). Additionally, bacterial factors such as endotoxin, phenol-soluble modulins from S. epidermidis [53], lipotechoic acid from S. aureus [54], Escherichia coli verotoxin [55], Helicobacter pylori water-soluble surface proteins [56], and butyric acid and propionic acids from Gram-negative bacteria [57] all promote neutrophil survival. Furthermore, the process of endothelial transmigration can also signal delayed apoptosis. En- dotoxin from the invading pathogen has been prominently implicated in different facets of delayed neutrophil apoptosis. TLR-1, -2, -4, -5, and -6 are present on neu- trophils, and recognize a number of pathogen-specific molecular patterns [58].

TLR stimulation, in particular engagement of the LPS-receptor, TLR4 [59], delays

neutrophil apoptosis. LPS induces expression of cellular inhibitor of apoptosis protein-2 (cIAP-2) resulting in accelerated degradation of caspase-3 [60]. Inhibi- tion by LPS, or direct stimulation of TLR4, requires involvement of the PI3 kinase and Akt signaling pathways [61]; Erk and PI3 kinase/Akt signaling are necessary, but not sufficient for this delay. LPS and direct TLR4 stimulation also activate NF- κ B [61]. Under normal conditions, this transcription factor is sequestered in the cytoplasm under control of its inhibitor, I κ B. Stimuli that converge on the I κ ^B

kinase complex trigger phosphorylation and degradation of I κ B, and expose the NF- κ B nuclear localization sequence. This sequence promotes the translocation of activated NF- κ B into the nucleus where it binds to consensus sites of responsive genes including genes encoding the Bcl-2 family of survival proteins, and members of the inhibitor of apoptosis proteins (IAP) family [62].

GM-CSF activates JAK-2/STAT-3 signaling pathways which upregulate cIAP-2 to delay programmed cell death in neutrophils [63], through signaling pathways involving PI3 kinase, Akt, and Erk [64]. An observed increase in levels of survival protein Mcl-1 may occur through one of two mechanisms: increased stability of the normally short-lived protein [64], or activation of the NF- κ B transcription path- way [65]. GM-CSF may alternatively phosphorylate pro-apoptotic Bad at Ser-112

Table 2. Factors that delay neutrophil apoptosis

Microbial Products

Lipopolysaccharide (endotoxin) Mannan

Lipoteichoic acid

Modulins from S. epidermidis

H. pylori surface proteins

Butyric acid

Propionic acid Host-derived Mediators Interleukin-1 β

Interleukin-2 Interleukin-3 Interleukin-4 Interleukin-6 Interleukin-8 Tumor necrosis factor Interferons

G-CSF GM-CSF Leptin

Pre-B cell colony-enhancing factor (PBEF) Physiologic Processes

Integrin engagement and transendothelial migration

and Ser-136, inducing cytosolic translocation [66]. Phosphorylated Bad is unable to bind Bcl-2 and Bcl-X, with the result that apoptosis is inhibited [67]. Addition- ally granulocyte-colony stimulating factor (G-CSF) inhibits the translocation of pro-apoptotic Bax to mitochondria, with the result that cytochrome c release and caspase 3 activation are reduced [68]. An ROS-dependent mechanism of apoptotic delay caused by GM-CSF has also been proposed, but whether GM-CSF causes prolonged life by decreasing or increasing ROS generation is uncertain.

Other circulating factors from the inflammatory milieu that exert anti- apoptotic activity include type I and II IFNs. Both serve to upregulate cIAP-2, Mcl-1, and AP-1 via STAT-3 and JAK-2 signaling [63], and type I IFN-mediated delay occurs in a PI3 kinase-dependent manner necessitating PKC- δ ^{and NF-} κ ^B

activation [40]. Among the interleukins, IL-1 β , IL-2, IL-3, IL-6, and IL-8 [69] show substantial capacity to inhibit apoptosis. A novel inflammatory cytokine, pre-B cell colony-enhancing factor (PBEF), has also been shown to play a late, but req- uisite, role in delayed neutrophil apoptosis, by reducing activity of caspases-3 and -8 [70]. Other pro-survival mediators associated with neutrophil longevity include C5a, fMLP, leukotriene B4 [71], and TNF- α [72], although studies of TNF- α ^have

shown that it can both induce and inhibit apoptosis. Interestingly, downregulation of TNF- α receptors initiated during neutrophil transmigration is necessary for the delay. In fact, endothelial contact causes downregulation of all receptor-mediated apoptosis pathways, including Fas-activated cell death [73], implying that the neu- trophil lifespan is determined as early as cell recruitment to the inflammatory site.

Finally, neutrophil proteins directly participating in apoptosis are differentially regulated during inflammation. Activated neutrophils display increased mitochon- drial stability and diminished caspase-3 activity [74], and septic neutrophils main- tain mitochondrial membrane integrity and the organelle further accumulates el- evated levels of cytochrome c [75]. Caspase-3 transcription is downregulated, and caspase-9 transcription inhibited [75]. Since both mitochondria and caspases play pivotal roles in the control of cell fate, even minor perturbations of their abundance or activity in the cell can alter the expression of apoptosis.

Activation of Neutrophil Apoptosis by Microbial Phagocytosis

In contrast to the inhibitory influences described above, bacterial phagocytosis activates neutrophil apoptosis, and reverses the cytokine-induced delay [76]. Cell death by apoptosis rather than necrosis limits local host tissue damage [50]. During apoptosis, the capacity of the neutrophil for chemotaxis, oxidative burst potential, and degranulation is lost, and cells manifest such hallmarks of apoptosis as cell shrinkage, chromatin compaction, and loss of the multilobed nuclear morphol- ogy [49].

Induction of apoptosis by bacterial ingestion appears to occur through two

mechanisms: the engagement of surface death receptors [77], and the phagocy-

tosis of opsonized pathogen targets [78]. Phagocytosis of E. coli results in the

generation of ROS that have pro-apoptotic effects in neutrophils [79]. Apoptosis is also increased by immune complex or Fc-receptor-mediated phagocytosis, both of which generate ROS [79]. In fact, ROS production is crucial for both phagocytosis- induced cell death and the induction of apoptosis [80], and critical for normal clearance of neutrophils from inflammatory sites [81]. Further evidence support- ing a role for ROS in inducing apoptosis lies in the upregulated expression of the pro-apoptotic Bax proteins following phagocytosis [82]; ROS can directly or indi- rectly modulate Bax expression [83]. However, the mechanism of the pro-apoptotic effects of ROS has yet to be defined, and a correlation between ROS production and apoptosis in phagocytosing neutrophils has not been found [84].

β 2-integrins have also been implicated in phagocytosis-induced neutrophil death. Although β 2-integrin engagement during endothelial transmigration de- lays apoptosis, β 2-integrin-dependent phagocytosis promotes apoptosis by re- ducing activation of Akt. Patients with β 2-integrin deficiency exhibit neutrophilia, suggesting that these receptors play a critical role in maintaining neutrophil home- ostasis [85]. In murine peritonitis models, Mac-1 deficiency results in neutrophil accumulation and delayed neutrophil apoptosis; circulating neutrophil counts and their rates of apoptosis are normal, however, implying that Mac-1 regulation of neutrophil apoptosis is limited to inflamed tissue [80]. ROS have been associated with Mac-1-mediated phagocytosis as well, since this phagocytic pathway results in ROS production and a specific ROS threshold must be met for phagocytosis- induced apoptosis to occur [86]. Patients with chronic granulomatous disease (CGD) who lack functional NADPH oxidase show impaired phagocytosis-induced cell death [86]. Finally, caspases-8 and -3 are activated by Mac-1-dependent phago- cytosis, and ROS are established requisites in caspase-8 cleavage and activation.

Neutrophil apoptosis following microbial phagocytosis is fundamental to the resolution of inflammation [2]. Oligonucleotide microarrays have identi- fied transcriptional responses to the phagocytosis of microbes by neutrophils.

Phagocytosis-induced apoptosis commences soon after pathogen ingestion [2].

Within 90 minutes of receptor-mediated phagocytosis, 256 genes undergo induc-

tion or repression; more than 30 of these genes encode proteins participating in

three distinct pathways of apoptosis [87]. At 3 hours and 6 hours, gene expression

analysis identified differential expression of 94 genes involved in cell fate, includ-

ing upregulation of BAX, TLR2, and CASP-1 [87]; more than 20 pro-apoptotic

molecules are upregulated, whereas genes encoding anti-apoptotic factors show

either downregulation or no change in expression [87]. In contrast, genes encoding

133 important pro-inflammatory mediators or signal transduction molecules are

downregulated during the induction of phagocytosis-induced neutrophil apopto-

sis, including IL-8, IL-10, C1q, and TLR-6 [2]. Thus, differential expression of genes

that are central to the regulation of apoptosis plays a critical role in the resolution

of inflammation, modulating both programmed cell death and pro-inflammatory

capacity [2].

The Role of the Neutrophil in Clinical Inflammation

A balance between neutrophil recruitment and removal at the site of injury or infection supports the divergent biologic imperatives of optimizing host defenses whilst minimizing host cytotoxicity. In vivo, disruption of apoptotic clearance contributes to sustained inflammation [88, 89], while deficiencies in apoptosis- promoting molecules such as NADPH oxidase and Mac-1 result in sustained neu- trophil accumulation [80, 90, 91]. Neutrophils harvested from the systemic circu- lation [75] (Fig. 2), or lungs [92], of patients with sepsis show profound inhibition of apoptosis.

Neutrophils have been implicated in injury of the lung [93], liver [94] intes- tine [95], and kidney [96] following experimental infectious insult as well as in clinical sepsis, a state of prolonged systemic inflammation and the leading cause of death for patients in intensive care units (ICUs). Patients with sepsis exhibit widespread neutrophil infiltration of the lung [97] (Fig. 3) and distant organs [98].

Neutrophil apoptosis is profoundly inhibited in patients with sepsis [99,100], mul- tiple trauma and burn injury [101, 102], pancreatitis, and the acute respiratory distress syndrome (ARDS) [92]. The extent to which inappropriately prolonged neutrophil survival contributes to the expression of the multiple organ dysfunction

Fig. 2. Neutrophils harvested from the systemic circulation of patients with sepsis show profound

inhibition of the constitutive apoptotic program, with the result that survival after 24 hours of in

Fig. 3. Photomicrograph of the lung of a patient dying with acute respiratory distress syndrome (ARDS). Massive neutrophil infiltration is evident

Fig. 4. Induction of neutrophil apoptosis improves survival in experimental intestinal is- chemia/reperfusion (I/R) injury. Rats underwent laparotomy and intestinal I/R injury by oc- clusion of the superior mesenteric artery. Survival was 100% in sham-treated animals, but only 40% in those with intestinal I/R; pulmonary neutrophilia was prominent in these animals. In- tratracheal instillation of heat-killed E. coli prior to intestinal I/R resulted in attenuation of neutrophilia, and a significant improvement in survival. * p < 0.05 vs sham-treated animals;

** p < 0.05 vs animals with I/R alone. From [103] with permission

syndrome is unknown. In animal models of intestinal ischemia/reperfusion injury, however, acceleration of apoptosis of neutrophils infiltrating the lung by the intra- tracheal instillation of heat-killed E. coli results in improved rates of survival [103]

(Fig. 4).

Conclusion

The neutrophil is a key contributor to the innate immune response. The anti- microbial activity of the neutrophil is non-selective, and so the resolution of neutrophil-mediated inflammation must be regulated to prevent tissue injury. The execution of a controlled cell death program after phagocytosis serves this dual role – resolving inflammation, and preventing damage to healthy host tissue [2].

While the process of neutrophil apoptosis is increasingly well understood, the molecular pathways involved remain incompletely characterized. A better under- standing of the processes underlying programmed cell death in neutrophils will aid in the development of therapies for treatment of neutrophil-mediated inflam- matory disease states.

A variety of therapeutic interventions have been proposed that target differing aspects of the neutrophil response to infection. Preventing neutrophil recruitment across the vascular endothelium may potentially involve the selectins, since P- and E-selectins are solely expressed on endothelial cells after induction by cy- tokines [104]. Small sized inhibitors of selectin-mediated capture and rolling are under development, and studies in a rat model show promising results with the inhibitor molecule, bimosiamose [105]. Downregulating the activation of neu- trophils through manipulation of intracellular signaling molecules, such as NF- κ ^B

or PI3 kinase, has also been proposed although, to date, no specific inhibitors have yielded efficacy in this regard [104]. Studies targeting the cytotoxic products of neutrophil activation suggest that the antioxidants vitamin C and E can reduce su- peroxide production by neutrophils in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis, though further studies are necessary [106]. Finally, manipulating neutrophil apoptosis for safe disposal of neutrophils is paramount in resolving inflammation, and compounds that promote this have been identified including lipoxins [107], annexin 1 [108], and prostaglandin D2 [109]. Specifically, lipoxin B4 and 15-epi-lipoxin B4 can stimulate the removal of apoptotic neutrophils by macrophages [110].

References

1. Segal AW (2005) How neutrophils kill microbes. Annu Rev Immunol 23:197–223

2. Kobayashi SD, Voyich JM, DeLeo FR (2003) Regulation of the neutrophil-mediated inflam-

matory response to infection. Microbes Infect 5:1337–1344

3. Zlotnik A, Yoshie O (2000) Chemokines: a new classification system and their role in immunity. Immunity 12:121–127

4. DeLeo FR, Renee J, McCormick S, et al (1998) Neutrophils exposed to bacterial lipopolysac- charide upregulate NADPH oxidase assembly. J Clin Invest 101:455–463

5. Witko-Sarsat V, Rieu P, Scamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L (2000) Neu- trophils: molecules, functions and pathophysiological aspects. Lab Invest 80:617–653 6. Ley K (1996) Molecular mechanisms of leukocyte recruitment in the inflammatory process.

Cardiovasc Res 32:733–742

7. McEver RP (1994) Selectins. Curr Opin Immunol 6:75–84

8. Varki A (1994) Selectin ligands. Proc Natl Acad Sci U S A 91:7390–7397

9. Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11–25

10. Ley K, Tedder TF (1995) Leukocyte interactions with vascular endothelium. New insights into selectin-mediated attachment and rolling. J Immunol 155:525–528

11. Walcheck B, Kahn J, Fisher JM, et al (1996) Neutrophil rolling altered by inhibition of L-selectin shedding in vitro. Nature 380:720–723

12. Bargatze RF, Kurk S, Butcher EC, Jutila MA (1994) Neutrophils roll on adherent neutrophils bound to cytokine- induced endothelial cells via L-selectin on the rolling cells. J Exp Med 180:1785–1792

13. Ley K, Bullard DC, Arbones ML, et al (1995) Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J Exp Med 181:669–675

14. Nolte D, Schmid P, Jager U, et al (1994) Leukocyte rolling in venules of striated muscle and skin is mediated by P-selectin, not by L-selectin. Am J Physiol 267:H1637–H1642

15. Norman KE, Moore KL, McEver RP, Ley K (1995) Leukocyte rolling in vivo is mediated by P-selectin glycoprotein ligand-1. Blood 86:4417–4421

16. Labow MA, Norton CR, Rumberger JM, et al (1994) Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins. Immunity 1:709–

720

17. Waddell TK, Fialkow L, Chan CK, Kishimoto TK, Downey GP (1995) Signaling functions of L-selectin. Enhancement of tyrosine phosphorylation and activation of MAP kinase. J Biol Chem 270:15403–15411

18. Smolen JE, Petersen TK, Koch C, et al (2000) L- selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase. J Biol Chem 275:15876–15884 19. Anderson DC, Springer TA (1987) Leukocyte adhesion deficiency: an inherited defect in

the Mac-1, LFA-1, and p150,95 glycoproteins. Annu Rev Med 38:175–194

20. Wilson RW, Ballantyne CM, Smith CW, et al (1993) Gene targeting yields a CD18-mutant mouse for study of inflammation. J Immunol 151:1571–1578

21. Hentzen ER, Neelamegham S, Kansas GS, et al (2000) Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule-1. Blood 95:911–920

22. Rochon YP, Kavanagh TJ, Harlan JM (2000) Analysis of integrin (CD11b/CD18) movement during neutrophil adhesion and migration on endothelial cells. J Microsc 197 ( Pt 1):15–24 23. Del MA, Zanetti A, Corada M, et al (1996) Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions. J Cell Biol 135:497–510 24. Feng D, Nagy JA, Pyne K, Dvorak HF, Dvorak AM (1998) Neutrophils emigrate from venules

by a transendothelial cell pathway in response to FMLP. J Exp Med 187:903–915

25. Chosay JG, Fisher MA, Farhood A, Ready KA, Dunn CJ, Jaeschke H (1998) Role of PECAM-1 (CD31) in neutrophil transmigration in murine models of liver and peritoneal inflammation.

Am J Physiol 274:G776–G782

26. Chavakis T, Keiper T, Matz-Westphal R, et al (2004) The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo. J Biol Chem 279:55602–

55608

27. Kurt-Jones EA, Mandell L, Whitney C, et al (2002) Role of toll-like receptor 2 (TLR2) in neutrophil activation: GM- CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils. Blood 100:1860–1868

28. Brinkmann V, Reichard U, Goosmann C, et al (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535

29. Lee WL, Harrison RE, Grinstein S (2003) Phagocytosis by neutrophils. Microbes Infect 5:1299–1306

30. Stendahl O, Krause KH, Krischer J, et al (1994) Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils. Science 265:1439–1441

31. Lundqvist-Gustafsson H, Gustafsson M, Dahlgren C (2000) Dynamic ca(2+)changes in neutrophil phagosomes A source for intracellular ca(2+)during phagolysosome formation?

Cell Calcium 27:353–362

32. Peters C, Mayer A (1998) Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion. Nature 396:575–580

33. Korchak HM, Rossi MW, Kilpatrick LE (1998) Selective role for beta-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells. J Biol Chem 273:27292–27299

34. Mohn H, Le C, V, Fischer S, Maridonneau-Parini I (1995) The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation. Biochem J 309 ( Pt 2):657–665

35. Shao D, Segal AW, Dekker LV (2003) Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils. FEBS Lett 550:101–106

36. Burg ND, Pillinger MH (2001) The neutrophil: function and regulation in innate and humoral immunity. Clin Immunol 99:7–17

37. Ishikawa F, Miyazaki S (2005) New biodefense strategies by neutrophils. Arch Immunol Ther Exp (Warsz ) 53:226–233

38. Kimura Y, Yokoi-Hayashi K (1996) Polymorphonuclear leukocyte lysosomal proteases, cathepsins B and D affect the fibrinolytic system in human umbilical vein endothelial cells. Biochim Biophys Acta 1310:1–4

39. Cowburn AS, Deighton J, Walmsley SR, Chilvers ER (2004) The survival effect of TNF-alpha in human neutrophils is mediated via NF-kappa B-dependent IL-8 release. Eur J Immunol 34:1733–1743

40. Wang K, Scheel-Toellner D, Wong SH, et al (2003) Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase, protein kinase C-delta, and NF-kappa B signaling pathways. J Immunol 171:1035–1041

41. Fadeel B, Kagan VE (2003) Apoptosis and macrophage clearance of neutrophils: regulation by reactive oxygen species. Redox Rep 8:143–150

42. Haslett C (1992) Resolution of acute inflammation and the role of apoptosis in the tissue fate of granulocytes. Clin Sci (Lond) 83:639–648

43. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305–

1308

44. Akgul C, Edwards SW (2003) Regulation of neutrophil apoptosis via death receptors. Cell Mol Life Sci 60:2402- 2408

45. Scheel-Toellner D, Wang K, Assi LK, et al (2004) Clustering of death receptors in lipid rafts initiates neutrophil spontaneous apoptosis. Biochem Soc Trans 32:679- 681

46. Ward C, Chilvers ER, Lawson MF, et al (1999) NF-kappaB activation is a critical regulator of human granulocyte apoptosis in vitro. J Biol Chem 274:4309–4318

47. Weinmann P, Gaehtgens P, Walzog B (1999) Bcl-Xl- and Bax-alpha-mediated regulation of apoptosis of human neutrophils via caspase-3. Blood 93:3106–3115

48. Li P, Nijhawan D, Budihardjo I, et al (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase- 9 complex initiates an apoptotic protease cascade. Cell 91:479–489 49. Moulding DA, Quayle JA, Hart CA, Edwards SW (1998) Mcl- 1 expression in human neu-

trophils: regulation by cytokines and correlation with cell survival. Blood 92:2495–2502

50. Akgul C, Moulding DA, Edwards SW (2001) Molecular control of neutrophil apoptosis.

FEBS Lett 487:318–322

51. Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309–1312

52. Liles WC, Kiener PA, Ledbetter JA, Aruffo A, Klebanoff SJ (1996) Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: Implications for the regulation of apoptosis in neutrophils. J Exp Med 184:429- 440

53. Liles WC, Thomsen AR, O’Mahony DS, Klebanoff SJ (2001) Stimulation of human neu- trophils and monocytes by staphylococcal phenol-soluble modulin. J Leukoc Biol 70:96- 102

54. Lotz S, Aga E, Wilde I, et al (2004) Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2. J Leukoc Biol 75:467–477

55. Liu J, Akahoshi T, Sasahana T, et al (1999) Inhibition of neutrophil apoptosis by verotoxin 2 derived from Escherichia coli O157:H7. Infect Immun 67:6203–6205

56. Kim JS, Kim JM, Jung HC, Song IS, Kim CY (2001) Inhibition of apoptosis in human neutrophils by Helicobacter pylori water-soluble surface proteins. Scand J Gastroenterol 36:589–600

57. Stehle HW, Leblebicioglu B, Walters JD (2001) Short- chain carboxylic acids produced by gram-negative anaerobic bacteria can accelerate or delay polymorphonuclear leukocyte apoptosis in vitro. J Periodontol 72:1059–1063

58. Takeda K, Akira S (2004) Microbial recognition by Toll- like receptors. J Dermatol Sci 34:73–82

59. Sabroe I, Prince LR, Jones EC, et al (2003) Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation of neutrophil activation and life span. J Immunol 170:5268–5275 60. Mica L, Harter L, Trentz O, Keel M (2004) Endotoxin reduces CD95-induced neutrophil

apoptosis by cIAP-2- mediated caspase-3 degradation. J Am Coll Surg 199:595–602 61. Francois S, El BJ, Dang PM, Pedruzzi E, Gougerot- Pocidalo MA, Elbim C (2005) Inhibition of

neutrophil apoptosis by TLR agonists in whole blood: involvement of the phosphoinositide 3-kinase/Akt and NF-kappaB signaling pathways, leading to increased levels of Mcl-1, A1, and phosphorylated Bad. J Immunol 174:3633–3642

62. Ward C, Walker A, Dransfield I, Haslett C, Rossi AG (2004) Regulation of granulocyte apoptosis by NF-kappaB. Biochem Soc Trans 32:465–467

63. Sakamoto E, Hato F, Kato T, et al (2005) Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2. J Leukoc Biol 78:301–309

64. Derouet M, Thomas L, Cross A, Moots RJ, Edwards SW (2004) Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1. J Biol Chem 279:26915–26921

65. Watson RW, Rotstein OD, Parodo J, Bitar R, Marshall JC (1998) The IL-1 beta-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1 beta. J Immunol 161:957–962

66. Cowburn AS, Cadwallader KA, Reed BJ, Farahi N, Chilvers ER (2002) Role of PI3-kinase- dependent Bad phosphorylation and altered transcription in cytokine- mediated neutrophil survival. Blood 100:2607–2616

67. Downward J (1999) How BAD phosphorylation is good for survival. Nat Cell Biol 1:E33–E35 68. Maianski NA, Mul FP, van Buul JD, Roos D, Kuijpers TW (2002) Granulocyte colony- stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neu- trophils. Blood 99:672–679

69. Hofman P (2004) Molecular regulation of neutrophil apoptosis and potential targets for therapeutic strategy against the inflammatory process. Curr Drug Targets Inflamm Allergy 3:1–9

70. Jia SH, Li Y, Parodo J, et al (2004) Pre-B cell colony- enhancing factor inhibits neutrophil

apoptosis in experimental inflammation and clinical sepsis. J Clin Invest 113:1318–1327

71. Lee E, Lindo T, Jackson N, et al (1999) Reversal of human neutrophil survival by leukotriene B(4) receptor blockade and 5-lipoxygenase and 5-lipoxygenase activating protein inhibitors.

Am J Respir Crit Care Med 160:2079–2085

72. van den Berg JM, Weyer S, Weening JJ, Roos D, Kuijpers TW (2001) Divergent effects of tumor necrosis factor alpha on apoptosis of human neutrophils. J Leukoc Biol 69:467–473 73. Tennenberg SD, Finkenauer R, Wang T (2002) Endothelium down-regulates Fas, TNF, and

TRAIL-induced neutrophil apoptosis. Surg Infect (Larchmt ) 3:351–357

74. Watson RW, O’Neill A, Brannigen AE, et al (1999) Regulation of Fas antibody induced neutrophil apoptosis is both caspase and mitochondrial dependent. FEBS Lett 453:67- 71 75. Taneja R, Parodo J, Jia SH, Kapus A, Rotstein OD, Marshall JC (2004) Delayed neutrophil

apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane po- tential and reduced caspase-9 activity. Crit Care Med 32:1460–1469

76. Watson RW, Redmond HP, Wang JH, Bouchier-Hayes D (1996) Bacterial ingestion, tumor necrosis factor-alpha, and heat induce programmed cell death in activated neutrophils.

Shock 5:47–51

77. Ward C, Dransfield I, Chilvers ER, Haslett C, Rossi AG (1999) Pharmacological manipulation of granulocyte apoptosis: potential therapeutic targets. Trends Pharmacol Sci 20:503–509 78. Watson RW, Redmond HP, Wang JH, Condron C, Bouchier-Hayes D (1996) Neutrophils

undergo apoptosis following ingestion of Escherichia coli. J Immunol 156:3986–3992 79. Schettini J, Salamone G, Trevani A, et al (2002) Stimulation of neutrophil apoptosis by

immobilized IgA. J Leukoc Biol 72:685–691

80. Coxon A, Rieu P, Barkalow FJ, et al (1996) A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5:653–666 81. Hampton MB, Vissers MC, Keenan JI, Winterbourn CC (2002) Oxidant-mediated phos-

phatidylserine exposure and macrophage uptake of activated neutrophils: possible impair- ment in chronic granulomatous disease. J Leukoc Biol 71:775–781

82. Kobayashi SD, Braughton KR, Whitney AR, et al (2003) Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils. Proc Natl Acad Sci U S A 100:10948–10953

83. Kobayashi SD, Voyich JM, Braughton KR, et al (2004) Gene expression profiling provides insight into the pathophysiology of chronic granulomatous disease. J Immunol 172:636–643 84. Yamamoto A, Taniuchi S, Tsuji S, Hasui M, Kobayashi Y (2002) Role of reactive oxygen species in neutrophil apoptosis following ingestion of heat-killed Staphylococcus aureus.

Clin Exp Immunol 129:479–484

85. Wehrle-Haller B, Imhof BA (2003) Integrin-dependent pathologies. J Pathol 200:481–487 86. Zhang B, Hirahashi J, Cullere X, Mayadas TN (2003) Elucidation of molecular events leading

to neutrophil apoptosis following phagocytosis: cross-talk between caspase 8, reactive oxygen species, and MAPK/ERK activation. J Biol Chem 278:28443–28454

87. Kobayashi SD, Voyich JM, Buhl CL, Stahl RM, DeLeo FR (2002) Global changes in gene ex- pression by human polymorphonuclear leukocytes during receptor-mediated phagocytosis:

cell fate is regulated at the level of gene expression. Proc Natl Acad Sci U S A 99:6901–6906 88. Vandivier RW, Fadok VA, Hoffmann PR, et al (2002) Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis.

J Clin Invest 109:661–670

89. Teder P, Vandivier RW, Jiang D, et al (2002) Resolution of lung inflammation by CD44.

Science 296:155- 158

90. Rowe SJ, Allen L, Ridger VC, Hellewell PG, Whyte MK (2002) Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury. J Immunol 169:6401–6407

91. Segal BH, Kuhns DB, Ding L, Gallin JI, Holland SM (2002) Thioglycollate peritonitis in mice lacking C5, 5- lipoxygenase, or p47(phox): complement, leukotrienes, and reactive oxidants in acute inflammation. J Leukoc Biol 71:410–416

92. Matute-Bello G, Liles WC, Radella F, et al (1997) Neutrophil apoptosis in the acute respiratory

distress syndrome. Am J Respir Crit Care Med 156:1969–1977

93. Abraham E (2003) Neutrophils and acute lung injury. Crit Care Med 31:S195–S199 94. Ho JS, Buchweitz JP, Roth RA, Ganey PE (1996) Identification of factors from rat neutrophils

responsible for cytotoxicity to isolated hepatocytes. J Leukoc Biol 59:716–724

95. Kubes P, Hunter J, Granger DN (1992) Ischemia/reperfusion-induced feline intestinal dys- function: importance of granulocyte recruitment. Gastroenterology 103:807–812

96. Lowell CA, Berton G (1998) Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr. Proc Natl Acad Sci U S A 95:7580–

7584

97. Steinberg KP, Milberg JA, Martin TR, Maunder RJ, Cockrill BA, Hudson LD (1994) Evolution of bronchoalveolar cell populations in the adult respiratory distress syndrome. Am J Respir Crit Care Med 150:113–122

98. Goris RJ, te Boekhorst TP, Nuytinck JK, Gimbrere JS (1985) Multiple-organ failure. Gener- alized autodestructive inflammation? Arch Surg 120:1109–1115

99. Jimenez MF, Watson RW, Parodo J, et al (1997) Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg 132:1263- 1269 100. Keel M, Ungethum U, Steckholzer U, et al (1997) Interleukin-10 counterregulates proinflam-

matory cytokine- induced inhibition of neutrophil apoptosis during severe sepsis. Blood 90:3356–3363

101. Chitnis D, Dickerson C, Munster AM, Winchurch RA (1996) Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients. J Leukoc Biol 59:835–839

102. Ertel W, Keel M, Infanger M, Ungethum U, Steckholzer U, Trentz O (1998) Circulating me- diators in serum of injured patients with septic complications inhibit neutrophil apoptosis through up-regulation of protein- tyrosine phosphorylation. J Trauma 44:767–775 103. Sookhai S, Wang JJ, McCourt M, Kirwan W, Bouchier-Hayes D, Redmond HP (2002) A novel

therapeutic strategy for attenuating neutrophil-mediated lung injury in vivo. Ann Surg 235:285–291

104. Morgan MD, Harper L, Lu X, Nash G, Williams J, Savage CO (2005) Can neutrophils be manipulated in vivo? Rheumatology (Oxford) 44:597–601

105. Onai Y, Suzuki J, Nishiwaki Y, et al (2003) Blockade of cell adhesion by a small molecule selectin antagonist attenuates myocardial ischemia/reperfusion injury. Eur J Pharmacol 481:217–225

106. Harper L, Nuttall SL, Martin U, Savage CO (2002) Adjuvant treatment of patients with antineutrophil cytoplasmic antibody-associated vasculitis with vitamins E and C reduces superoxide production by neutrophils. Rheumatology (Oxford) 41:274–278

107. Goh J, Godson C, Brady HR, Macmathuna P (2003) Lipoxins: pro-resolution lipid mediators in intestinal inflammation. Gastroenterology 124:1043–1054

108. Perretti M, Flower RJ (2004) Annexin 1 and the biology of the neutrophil. J Leukoc Biol 76:25–29

109. Gilroy DW, Colville-Nash PR, McMaster S, Sawatzky DA, Willoughby DA, Lawrence T (2003) Inducible cyclooxygenase- derived 15-deoxy(Delta)12-14PGJ2 brings about acute inflammatory resolution in rat pleurisy by inducing neutrophil and macrophage apoptosis.

FASEB J 17:2269–2271

110. Mitchell S, Thomas G, Harvey K, et al (2002) Lipoxins, aspirin-triggered epi-lipoxins, lipoxin stable analogues, and the resolution of inflammation: stimulation of macrophage phagocytosis of apoptotic neutrophils in vivo. J Am Soc Nephrol 13:2497–2507

111. Marshall JC (2005) Neutrophils in the pathogenesis of sepsis. Crit Care Med 33:S502–S505