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The Role of Neutrophil-Derived Myeloperoxidase in Organ Dysfunction and Sepsis

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in Organ Dysfunction and Sepsis

N.S. MacCallum, G.J. Quinlan, and T.W. Evans

Introduction: The Role of the Neutrophil in Sepsis

Neutrophils are the first cells to be activated in the host immune response to infec- tion or injury and are critical cellular effectors in both humoral and innate immu- nity, central to the pathogenesis of sepsis and multi-organ dysfunction [1]. However, the neutrophil capacity for bacterial killing lacks selectivity, despite stringent regula- tion, and thereby carries the potential to inflict collateral damage to, and destruc- tion of host tissue [2]. Host tissue damage characterizes both autoimmune and inflammatory conditions and may arise via a variety of mechanisms including pre- mature neutrophil activation during migration, extracellular release of cytotoxic molecules during microbial killing, removal of infected or damaged host cells or debris during host tissue remodeling, and failure to terminate acute inflammatory responses [3]. Sepsis-induced neutrophil mediated tissue injury has been demon- strated in a variety of organs including the lungs [4, 5], kidneys [6], and liver [7].

Innate Immunity

The principle function of the innate immune system is to effect a rapid response to microbial invasion. This is achieved via the complement system and anti-microbial peptides, as well as by phagocytes and antigen presenting cells. Initial microbial contact precipitates a series of simultaneous events including non-specific bacterial recognition via complement receptors (e.g., q 2-integrins, Fc-receptors interacting with ‘opsonizing’ serum components) and phagocytic sensing of pathogen-associ- ated molecular patterns (PAMPs), which are evolutionarily conserved molecules unique to microorganisms. Sensing occurs through a variety of pattern recognition receptors, of which Toll-like receptors (TLR) are the best defined within this context [8].

Neutrophil Biology

Neutrophils are terminally differentiated cells, released into the blood stream follow- ing maturation from bone marrow precursors under the influence of granulocyte-col- ony stimulating factor (G-CSF). Neutrophils have the shortest lifespan of all human cells, existing for only 6 – 10 hours in vivo. Prolongation of survival is an active pro- cess requiring new gene expression and protein synthesis. It is achieved following exposure to a variety of inflammatory mediators and is accompanied by markers of

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increased neutrophil activity. Resolution of the inflammatory process involves acti- vation of a constitutive program of apoptosis, or programmed cell death, with sub- sequent phagocytosis by hepatic cells and the reticulo-endothelial system [2].

Neutrophil Recruitment

Neutrophils are mobile cells, which interact with the microenvironment, migrating down a chemotactic gradient to inflammatory loci in response to signals released by infection and tissue injury [9]. Neutrophil recruitment is an active process involving distinct stages of neutrophil rolling, tight adhesion, and diapedesis [10]. Circulating blood neutrophils contact, and transiently interact, with endothelial cell molecules, resulting in slowing and a rolling/release-like motion. Rolling is the initial step in neutrophil tissue recruitment to inflammatory sites, is a prerequisite for imminent tight interactions with the endothelium, and is mediated by selectins and type I membrane glycoproteins. L-selectins on the neutrophil surface mediate interactions with endothelial cells and other neutrophils, facilitating leukocyte transient adhesive contacts with high tensile strength to permit rolling under shear stress. The L-selec- tin molecule is localized to the neutrophil microvilli and is cleaved during neutro- phil activation. Selectins, in addition to cell-cell adhesion may have a contributory role in cell signaling [10].

Chemo-attractants originating from tissues trigger neutrophil responses, replac- ing neutrophil rolling with a state of tight adhesion [10]. The q 2-integrins are cell surface molecules that are primarily responsible for tight adhesion to both the endo- thelium and extracellular matrix. They interact with the cytoskeleton allowing stabi- lization of cell adhesion, providing a framework for signaling proteins. Intercellular adhesion molecule (ICAM)-1 on the endothelium interacts with q 2-integrins on the neutrophil, producing changes in ligand binding. q 2-integrins are also necessary in facilitating neutrophil clearance following apoptosis [11].

In order to reach extravascular sites of infection, neutrophils must transmigrate between or directly through endothelial cells. Neutrophil derived granular enzymes including elastase and gelatinase facilitate the process of diapedesis.

Neutrophil Defenses

Neutrophil defenses incorporate indiscrete processes which may be classified as oxy- gen dependent and independent. Theses are activated simultaneously upon neutro- phil initiation of phagocytosis. Reactive oxygen species (ROS) are generated by means of the respiratory burst. Hypochlorous acid (HOCl), the most bactericidal oxidant produced by the neutrophil, is generated by the unique ability of myelopero- xidase (MPO), the main constituent of azurophilic granules, to oxidize chloride ions at physiological pH. HOCl is the major end product of the neutrophil respiratory burst. Neutrophil degranulation releases a plethora of preformed enzymes and anti- microbial factors from neutrophil granules into phagosomes leading to the forma- tion of phagolysosomes, in which bacteria are inhibited or killed.

Oxygen dependent mechanisms

NADPH oxidase generation of superoxide

The nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase enzyme com- plex is assembled on neutrophil activation. Once active, this enzyme complex con-

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sumes oxygen to generate superoxide (O2·–), metabolites of which play a part in anti- microbial defense strategy. NAPDH oxidase generation of O2·– controls the rate of production of these metabolites. The sequence of events which leads to the assembly of active NADPH oxidase is described in brief below.

NADPH oxidase is composed of at least six components: p47phox, p67phox, and p40phox found in resting neutrophils as a cytosolic complex, rac-2 a cytosolic ras- related protein, and membrane bound components p22phoxand gp91phox, comprising cytochrome b558(cyt b558). Translocation of cytosolic components to membrane and association with cyt b558renders the complex functional. Cyt b558then transfers elec- trons from NADPH to molecular oxygen generating O2·–(Equation I).

2O2+NADPH → 2 O2·–+NADP++H+ (Eqn 1)

Cyt b558is a membrane bound flavohemoprotein located predominantly in neutro- phil specific granules and secretory vesicles, and partially in the plasma membrane.

The membrane bound cytochrome components, p22phoxand gp91phox, closely inter- act with cyt b558. p47phox which has a vital role in oxidase function, chaperones p67phoxto the membrane. Membrane bound p22phoxbinds complexed p47phox/p67phox following translocation and phosphorylation by protein kinase C of the latter. Rac-2 performs a critical role in the activation of NADPH oxidase; activation frees it from its complex with rho-GDI and allows subsequent interaction with p67phox[10].

Although it is well established that O2·–is not a damaging oxidant and therefore ineffective as a direct bacterial cell killing agent (reviewed in [12, 13]) it nevertheless has an important antimicrobial role. This is aptly demonstrated in chronic granulo- matous disease, a group of inherited conditions, in which genetic deficiencies of NADPH oxidase components lead to diminished O2·–production, thereby providing an ineffectual inflammatory response to infection, contributing to other aspects of disease pathogenesis [10].

How then may O2·–help combat microbial infection? There are two dominant the- ories. First, O2·–may be converted via enzymatic and chemical reaction steps into an array of directly damaging oxidants. This can, for instance, be achieved by superox- ide dismutase (SOD) conversion of O2·– to hydrogen peroxide (H2O2). H2O2 is in turn, either reduced to O2and water by catalase (Equations 2 and 3) or converted to HOCl by the action of MPO. Phagosomal MPO release occurs concomitantly with NADPH oxidase/SOD generation of H2O2, therefore, providing substrate for this enzyme and hence the ability to generate HOCl, which is a powerful oxidant with known anti-microbial function. In addition O2·– generated by NADPH oxidase can potentially react with HOCl to produce the hydroxyl radical ((·OH) the most aggres- sive ROS known) and/or with nitric oxide (NO) to produce peroxynitrite (ONOO) another very reactive oxidant (vide infra). Thus, the end-products of these reactions have the ability to cause damage to and kill microbes (Fig. 1).

2 O2·–+2H+→ O2+H2O2 SOD (Eqn 2) 2H2O2→ 2H2O + O2 catalase (Eqn 3)

Second, release of O2·–into the phagosome of the neutrophil consumes H+ions dur- ing dismutation (Equation 2); the subsequent rise in pH within the phagosome is compensated for by a net influx of K+ions. The increased phagosomal ionic content signals the release of proteases, which together with the high pH provides ideal con- ditions for protease action (reviewed in [13]). There is evidence to support both oxi- dant, and pH mediated antimicrobial actions of O2·–(reviewed in [12]).

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a

Fig. 1. Generation of oxidants by activated neutrophils, via NADPH oxidase and myeloperoxidase (MPO).

a Peroxidation cycle of MPO, generation of hypochlorous acid (HOCl) and radicals. Key reactive oxygen spe- cies (ROS) and free radicals marked in gray.

Myeloperoxidase generation of HOCl and secondary oxidants

MPO utilizes H2O2to oxidize chloride (Cl) to Cl+at physiological pH, thereby generating HOCl (Equation 4). The bactericidal properties of HOCl are attributable to its reactive nature and hence its ability to oxidize a variety of molecules including amino acids, nucleotides, lipids, and hemoproteins in much the same way as household bleach (sodium hypochlorite, NaClO) does. However, in addition to bacterial destruction, the non-specific nature of HOCl-mediated reactions also leads to associated host mediated tissue damage (vide infra), which may have both pro or anti-inflammatory consequences.

H2O2+2Cl→ 2HOCl MPO (Eqn 4)

The enzyme follows the normal peroxidase cycle, in which a single two-electron oxi- dation of native enzyme to compound I is followed by two successive one-electron reductions to native enzyme via compound II (Fig. 1a). Chloride, as well as other halides and pseudohalides (thiocyanate), are able to reduce compound I directly to native MPO by a two electron process (Equation 5) [14], this process yields HOCl and other hypohalous acids.

MPO+H2O2→ MPO-I+H2O MPO-I+AH2→ MPO-II+AH·

MPO-II+AH2→ MPO+AH·+H2O (Eqn 5)

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Table 1. Oxidant products generated as a result of neutrophil activation (see Figure 1) [14, 15, 46].

Oxidant Product Generated by Reaction

O2·–(superoxide) NADPH oxidase 2O2+ NADPH→ 2 O2·–+ NADP++ H+ H2O2(hydrogen peroxide) SOD 2 O2·–+ 2H+→ O2+ H2O2

HOCl (hypochlorous acid) MPO H2O2+ 2Cl → 2HOCl

1O2(singlet oxygen) HOCl HOCl + H2O21O2+ H2O + H++ Cl

·OH (hydroxyl radical) O2·–/ HOCl HOCl + O2·–→ ·OH+O2+ Cl

·OH (hydroxyl radical) Fe2+/ HOCl HOCl + Fe2+→ ·OH+Fe3++ Cl NO2Cl (nitryl chloride) HOCl HOCl + NO2·–→ NO2Cl +OH

(chlorinating / nitrating compound) ONOO(peroxynitrite) O2·– O2·–+ NO→ ONOO

RNHCl (chloramines) HOCL RNH2+ HOCl→ RNHCl+H2O

AH· (radical product) MPO MPO-I + AH2→ MPO-II+AH·

MPO-II + AH2→ MPO+AH· +H2O

(compound I & II form radical products by a one-electron subtraction)

MPO3+O2·–(compound III) O2·– MPO3++ O2·–→ MPO3+O2·–

MPO3+O2·–+ O2·–→ MPO2+O2·–+ O2

(involved in oxidation of aromatic substances, via MPO2+O2·–, an active hydroxylating intermediate)

In addition, the activated states of the enzyme, predominantly compound I, are also able to oxidize an array of different substrates which may have further implica- tions for pro and anti-inflammatory responses. Indeed, compound I, and to a lesser extent compound II, form radical products by a one-electron subtraction (AH·, Equation 5). Target molecules include tyrosine, tryptophan, sulfhydryls, phenol and indole derivatives, nitrite, H2O2, and xenobiotics [15]. Such reactions are favored as the couple compound I/II has one of the highest reduction potentials found in cellu- lar systems [16].

As one of the most potent oxidants produced by neutrophils, HOCl is directly damaging to bacteria as evidenced by chlorination of bacterial components within the phagosome [17]. However, further ROS with the potential for bacterial killing, as well as tissue damage may be produced by additional reactions involving HOCl. Oxi- dant products generated as a result of neutrophil activation are shown in Table 1.

Oxygen independent mechanisms

Oxygen independent mechanisms of neutrophil-mediated antimicrobial activity are centered on neutrophil granule enzymes and proteins, which form the foundation of the innate immune system. Neutrophil stimulation initiates membrane remodeling by means of exocytosis and phagocytosis. During exocytosis, cytoplasmic granules translocate and fuse to the plasma membrane, either expressing their contents on the cell surface (e.g., adhesion molecule) or discharging them into the extracellular space. Furthermore, exocytosis supplies stores of membrane for the purposes of phagoscytosis, which involves the internalization of receptor-target complexes by calcium triggered cytoskeletal remodeling. Fusion of azurophilic and specific gran-

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Table 2. Neutrophil proteins with antibacterial properties.

Protein Functions

Bactericidal permeability increasing protein (BPI)

allows binding to lipopolysaccharide, has opsonic effects and mediates bacterial attachment to, and phagocytosis by, neutrophils [47]

binding causes an increase in the permeability of the outer membrane of Gram-negative bacteria, hydrolysis of bacterial phospholipase and interruption of cell division [48]

Defensins major components of azurophilic granules disrupt target cell membranes [49]

act synergistically with BPI against Gram-negative bacteria Proteinase 3 found in azurophilic and secretory granules

role in the amplification of the inflammatory response.

Elastase a potent serine protease

degrades outer membrane protein (which is highly conserved among Gram-negative bacteria) [50]

PLA2

Cathelicidins

PLA2has potent bactericidal activity against Staphylococcus aureus bacterial killing is mediated by phospholipolysis of cell walls cathelicidins, found in the mobile specific granules

role in extracellular killing (upon release into inflammatory fluids), by means of bacterial phospholipid hydrolysis

both proteins synergize with BPI for bacterial killing [10].

Metalloproteinases (collagenase, gelatinase)

released in an inactive pro-enzyme form, require calcium, function at neutral pH

aid migration of neutrophils through basement membrane

collagenase, whose enzymatic activity depends upon oxidation by HOCl, has a preference for native collagen

gelatinase degrades denatured and native collagen types IV and V activation of pro-gelatinase occurs by both oxidative and non-oxidative

mechanisms [10]

ules with phagocytic vacuoles leads to the formation of phagolysosomes. These events culminate in the production of a highly toxic intravacuolar milieu, subject to oxidant metabolites formed by MPO and NADPH oxidase, cationic proteases inter- acting and disrupting negatively charged bacterial cell wall components, and other antimicrobial proteins.

Proteins with specific antibacterial properties are found primarily in azurophilic granules; these include bactericidal permeability increasing protein (BPI) and defen- sins (see Table 2). Further proteins have a supporting role in antibacterial defense;

these include lactoferrin, whose capacity to bind iron deprives bacteria of a co-fac- tor for proliferation.

Myeloperoxidase

MPO was first isolated from tuberculous empyema fluid by Agner in the first part of the 20thcentury [18]. Initially named verdoperoxidase due to its green color, it was subsequently renamed, as its distribution was limited to myeloid cell lines. MPO (donor: hydrogen peroxide oxidoreductase, EC 1.11.1.7) [19] is a lysosomal heme

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protein unique to neutrophils and monocytes. Monocytes contain one third of the MPO found in neutrophils.

MPO Structure

Among all the mammalian peroxidases, the three-dimensional structure is known only for MPO. It has been refined to 1.8 ˚A resolution by x-ray crystallography [20].

MPO is a strongly cationic glycosylated protein with a molecular weight of ' 146 kDa. The homodimer consists of two symmetrically related halves linked by a single disulfide bridge, each consisting of a central structure of 5 [ -helixes covalently attached to a central heme group, one from the 14.5 kDa light polypeptide chain and four from the large 58.5 kDa heavy chain, composed of 106 and 467 amino acids, respectively [21].

Each heme group is covalently attached via two ester and one sulfonium linkage.

The heme group is a derivative of protoporphyrin IX, modifications on pyrrole rings allowing for formation of two ester linkages. The sulfonium linkage between the sul- fur atom of Met243and the q -carbon of the vinyl group on pyrrole ring A, is a unique feature of MPO; other mammalian peroxidase lack methionine at this position [20].

The sulfonium ion linkage serves as an electron withdrawing substituent, and appears to be responsible for the lower symmetry of the heme group and distortion from planar conformation. The latter contributes to the unusual spectral properties of MPO, with a red shift in the Soret band (428 nm) and strong absorption bands in the visible spectrum being responsible for its green color. In addition, the redox properties of MPO, which differ from other mammalian peroxidases, seem to have their origin in the electron withdrawing sulfonium linkage and heme distortion [21]. Selective cleavage along the disulfide bridge linking the two halves of MPO yields hemi-enzyme, which exhibits spectral and catalytic properties indistinguish- able from the intact enzyme [20].

MPO Biosynthesis: Genetics

MPO is encoded in a single 14 kb gene on the long arm chromosome 17 (17q23.1) [22]. MPO synthesis is restricted to late myeloblasts, and promyelocytes in the bone marrow, terminating as myeloid progenitors, enter the myelocyte stage of differenti- ation into neutrophil and related cell types. Monocyte precursors also synthesize MPO during bone marrow maturation, with normal expression limited to this stage of myeloid development. MPO is, therefore, present, but not actively synthesized, in circulating monocytes, Furthermore differentiation into macrophages is accompa- nied by downregulation of MPO synthesis [23]. However, MPO gene expression can potentially be reinitiated in certain disease states within a suitable tissue setting;

e.g., de novo macrophage synthesis of MPO in Alzheimer’s disease [24], peripheral mature neutrophil and monocyte MPO synthesis in anti-neutrophil cytoplasm anti- bodies (ANCA)-associated glomerulonephritis [25].

Transcription of MPO is regulated in a tissue and differentiation specific manner.

Progressive demethylation in the 5’ flanking region of the MPO gene is pre-requisite, providing the open chromatin structure required for transcription. Expression of the gene is regulated by the cell specific transcription factor, AML-1; integrity of the AML-1 binding site is essential for activity of MPO proximal enhancer [26]. Numer- ous allelic polymorphisms have been identified for MPO, the most studied of which is the promoter region polymorphism, –463G/A. This site contains an Alu receptor

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response element (AluRRE), which is recognized by a variety of nuclear receptors, including Sp1 [27], MPO transcription being enhanced by an intact Sp1 binding site (i.e., -463G). The functional significance related to this single nucleotide polymor- phism is unclear due in part to the strong gender influence and varied results from studies of different inflammatory diseases. In addition, peroxisome proliferator-acti- vated receptors also regulate MPO gene expression via AluRRE [28]. Furthermore, AluRRE has been implicated in the incidence of a variety of inflammatory disorders [23], the pathogenesis of which may in part be due to regulation of MPO expression.

At the phenotypic level in normal human bone marrow, cytokines such as tumor necrosis factor-[ (TNF- [ ) can decrease MPO transcription, whereas G-CSF induced differentiation of multi-potential progenitor cells results in activation and nuclear recruitment of proteins (Pu1 and C-EBP family) that bind to a distal MPO enhancer, which regulates MPO gene expression via the proximal enhancer and AML-1 [26].

A variety of MPO genetic mutations have been identified. Inherited MPO defi- ciency is more common in the USA and Europe (1 in 2000 – 4000) than in Japan (1 in 55,000). Deficiency is associated with an increased susceptibility to infection and incidence of malignancy. However, unlike NADPH oxidase deficiencies, reduced or absent MPO function often has a modest clinical phenotype [23]. So whilst MPO does have a role to play in terms of antimicrobial activity in humans, under many circumstances this does not seem to be an essential function.

MPO Biosynthesis: Assembly

During the initial stages of MPO biosynthesis, the 80 kDa primary translational product, preproMPO is converted into 90kDa apoproMPO, following co-translational cleavage of a signal peptide, N-linked glycosylation and limited deglucosylation of high mannose oligosaccharide side chains in the endoplasmic reticulum. Apo- proMPO has no peroxidase activity, as it lacks a prosthetic heme group. During its long half life within the endoplasmic reticulum, oligosaccharide side chains are added, which contribute to the transient associations with molecular chaperones calreticulin and calnexin. The latter are high capacity, low affinity calcium binding proteins, whose interactions with glycoproteins result in limited deglucosylation and modification during endoplasmic reticulum transit, promoting correct folding and

‘quality’ control. Calreticulin is a soluble protein within the endoplasmic reticulum;

calnexin is a transmembrane protein. Association of apoproMPO with calnexin leads to incorporation of heme forming the enzymatically active proMPO, and exit into the Golgi apparatus and downstream secretory pathway. Heme not only initiates peroxidase activity, but is also essential for the conformational change of proMPO required for export from the endoplasmic reticulum. The heme prosthetic group in MPO is derived from FeIIIprotoporphyrin IX.

Following transport from the endoplasmic reticulum to the Golgi apparatus, MPO precursors must reach their final intracellular destination or exit into the extracellular space. To achieve this, cells separate proteins destined for intracellular compartments or secretion. The conversion of apoproMPO to proMPO in the endo- plasmic reticulum is extremely slow; processing in the Golgi apparatus, granule tar- geting, and secretion are rapid. Like many glycoprotein enzymes, MPO precursors undergo a complex series of proteolytic processing to achieve final protein structure.

ProMPO is converted to a short lived 74 kDa intermediate (in the Golgi apparatus) following deletion of a 125 amino acid propeptide. In, or en route to, the primary granule this 74 kDa transient intermediate is cleaved into two subunits (59 kDa

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[ -subunit and 13.5 kDa q -subunit) linked by covalent bonds associated with the heme group. Mature MPO is formed by interaction of two heavy-light chain units forming a symmetrical homodimer linked by disulfide bond between heavy sub- units. Mature dimeric MPO is stored in azurophilic granules. It is the only member of the mammalian peroxidase family that is a dimer.

The secretory pathway is constitutive. Cells must tag and retrieve proteins from the secretory pathway and redirect them to the target organelle. This is achieved either by direct transport through the Golgi apparatus via late endosomes to lyso- somes, or by indirect targeting of the plasma membrane and later internalization into early and then late endosomes. During the course through the Golgi apparatus, proMPO destined for secretion is exposed to a variety of transferases and glucosi- dases, extensively modifying the oligosaccharide units forming complex oligosac- charides. The activity of a constitutive pathway is limited by the rate of synthesis of a given protein. Secreted MPO is monomeric, which suggests that dimer formation of mature MPO either takes place in the granule targeting pathway, or after proMPO has been compartmentalized into the granule. MPO species isolated from human plasma include both precursor and mature forms [23].

Neutrophil Granules and MPO Storage

Neutrophils exhibit four classes of granules. Primary or azurophilic granules contain preformed antimicrobial proteins: MPO, serine proteases and lysosyme hydrolases for release into phagosomes; they appear early at the promyelocyte stage. Specific or secondary granules appear later in neutrophil maturation at the metamyelocyte stage, containing proteins with antimicrobial activity, including collagenase, lacto- ferrin and gelatinase [10]. Tertiary or gelatinase granules resemble specific granules and are enriched with enzymes that are exocytosed, contributing to the degradation of intracellular junctions and extracellular matrix [2]. On reaching maturity, neutro- phils develop secretory vesicles which have cyt b558and adhesion molecules on the membrane surface. They contain proteins of endocytic origin and serve as a reser- voir of membrane constituents that can be rapidly mobilized for phagocytosis [2, 29].

Neutrophil stimulation causes intracellular granule secretion in the following order: secretory, gelatinase, specific, and azurophilic. Degranulation is triggered by changes in intracellular calcium; moderate increases of ‹0.25 µM are required for secretory granules, and 0.7 µM for azurophilic and specific granules[15].

Azurophilic granules contain glycosaminoglycans, molecules that may provide an anionic matrix, binding predominantly cationic cytotoxic granule proteins in a con- formation or state that renders them inactive [30].

Neutrophil-induced Host Tissue Damage

Host tissue damage occurs by a variety of mechanisms, including premature activa- tion of neutrophils, extracellular release of cytotoxic molecules during microbial killing and native tissue remodeling, failure of cessation of pro-inflammatory responses, and overwhelmed local anti-oxidant and anti-protease protection. Gener- ated ROS and granular antimicrobial proteins or enzymes are predominantly released into phagosomes, creating a controlled environment of extreme toxicity and, thus, preventing release into the extracellular space. However, in certain cir-

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cumstances, where targets are too large to be phagocytosed, these substances may be released into the extracellular environment. Indeed, neutrophil granular enzymes and proteins, in addition to end products of ROS damage have been detected in fluid and tissue isolated from inflammatory sites [31], implicating neutrophils in the pathogenesis of sepsis-induced organ dysfunction [5], chronic inflammatory condi- tions, and ischemia-reperfusion injury. The neutrophil contribution to the patho- genesis of organ dysfunction is perhaps most evident in the acute respiratory dis- tress syndrome (ARDS). Substantial pulmonary recruitment of neutrophils occurs in ARDS, with non-survivors having the highest numbers [32]. Alveolar and circulating neutrophils are activated, evidenced by increased expression of q 2-integrins and cytokine profiles. Activation correlates with an increased degree of lung injury [33].

Furthermore, these cells demonstrate reduced rates of apoptosis. In animal models of lung injury, neutrophil depletion or inhibition of localization attenuates histologi- cal injury and improves survival (reviewed in [4]).

Moreover, the array of oxidant molecules (Figure 1) produced by MPO are impli- cated in tissue damage, the outcome of which is dependent on the dose of the oxi- dant, higher doses causing necrosis and affecting signaling pathways, and apoptosis and growth arrest (endothelial cells) occurring at lower amounts (reviewed in [34]).

Neutrophils are able to generate long-lived oxidants, with half lives of up to 18 hours [35].

Pathways of HOCl-induced Tissue Damage

Numerous in vitro studies have demonstrated that HOCl can mediate tissue injury (reviewed in [34]), and HOCl has been detected in various pathological disease states [36]. Indeed HOCl can also halogenate cell constituents, chlorinating amines to chloramines (Equation 6). Chloramines have a longer half life than HOCl, retain two oxidizing equivalents allowing similar reactions to those of HOCl, and are able to cross plasma membranes thereby exporting or importing the potential bio-mole- cule modifications; in addition, said compounds also breakdown into aldehydes which are chemotactic and, at higher concentrations, cytotoxic [34].

RNH2+ HOCl→ RNHCl+H2O (Eqn 6)

In addition, HOCl reacts with nucleotides and DNA. NADH and NH-groups of pyrimidines are particularly susceptible, with DNA double stranded breaks occur- ring [37].

Moreover, chlorohydrin derivatives arise from HOCl-mediated chlorination of unsaturated fatty acids and cholesterol [38]. This, in conjunction with the cationic properties of MPO facilitating attachment to biological membranes, leads to the sus- ceptibility of lipid components of biological membranes to attack by HOCl.

Cellular proteins are targets of HOCl, producing several different oxidation prod- ucts. HOCl can act as a one or two-electron oxidizing agent. Thiol-groups, thioe- thers (mehionine), heme groups, and iron-sulfur centers are the most readily oxi- dized, at a rate 100 times that of amines [34]. Cysteine and methionine residues are readily oxidized. Amino groups of lysine and N-terminal amines react forming chlo- ramines, which can subsequently react with thiols. Chloramine formation may gen- erate radicals that result in protein fragmentation, lipid peroxidation, and protein carbonyl formation [34]. In addition, cell lysis has been demonstrated as a conse- quence of irreversible protein crosslink formation in membranes exposed to HOCl [39].

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Tyrosine, tryptophan, histidine, arginine, and amide peptide bonds are oxidant targets of HOCl [34], and as such the end product of the reaction of tyrosine with HOCl, 3-chloro-tyrosine, is used as a marker of neutrophil activation [40].

Although there are many potential biological targets for HOCl mediated reac- tions, low and high molecular mass thiols are among the most susceptible moieties.

As thiols, dependent on biological environment and setting, have numerous impor- tant functions including as antioxidants, as redox signaling switches, as ligand-bind- ing moieties, and as key determinants of tertiary structure, any perturbation in thiol oxidation state may have profound consequences.

Indeed, HOCl-mediated cellular necrosis, as demonstrated in rodent macrophage cell lines, has demonstrated disruption of plasma membrane ion transport channels that appears to be attributable to oxidation of membrane thiol groups as a key insti- gating factor. Furthermore, at lower concentrations of HOCl, apoptosis rather than necrosis ensues [41] indicating a thiol mediated cell signaling function. The fact that HOCl, like chloramines, can penetrate the cell membranes is important in this regard as this property enables this oxidant to instigate changes via reaction with intracellular constituents at sites distant from its zone of production [34].

Modulation of Neutrophil Function In Sepsis

Septic shock and multiple organ dysfunction are the most common causes of death in patients with sepsis, with associated mortalities of 25 – 30 and 40 – 70 %, respec- tively. The incidence is increasing; approximately one third of critical care admis- sions meeting the criteria for severe sepsis in the UK [42]. However, despite an observed decrease in mortality, the number of sepsis-related deaths is likely to rise still further due to an aging population, the use of increasingly sophisticated inter- ventions, and the rising incidence of treatment resistant organisms.

Evidence from numerous observational and in vivo studies (Table 3) indicates that MPO-derived oxidants do contribute to tissue modification and damage. How- ever, although therapies that target the neutrophil and reduce either its activation or its accumulation in the tissues can reduce tissue injury in animal models of sepsis, anti-neutrophil therapies have not conferred benefit in clinical trials. Conversely, the cytokine G-CSF, which increases neutrophil release from the bone marrow and delays neutrophil apoptosis, has failed to show convincing evidence of either harm or benefit in human sepsis, although either effect can be reproduced in animal mod- els [2]. There are several known inhibitors of NADPH oxidase and MPO, but their use to date has been confined to in vivo animal and in vitro studies.

Several clinical trials have been conducted, using interventions targeting path- ways or mediators of sepsis, aiming to either directly or indirectly affect neutrophil function. These include anti-lipopolysaccharide, anti-TNF-[ , interleukin-1-receptor antagonist (IL-1ra), anti-inflammatory drugs (ibuprofen, corticosteroids), bradyki- nin antagonist, platelet activating factor acetyl hydrolase, elastase, and NO synthase inhibitors (reviewed in [43]). All have proved ineffective in terms of a mortality ben- efit. New therapeutic targets are being investigated, with particular focus on bacte- rial products (TLR) and the coagulation pathway.

An alternative approach, which may be acceptable for use in human studies, relates to certain properties of the plasma protein, ceruloplasmin. Ceruloplasmin, an acute phase protein, and the major copper containing protein of plasma, has several antioxidant functions ascribed to it, including the recently discovered ability to

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Table 3. Additional functional changes attributed to MPO and HOCl Cellular function

homeostasis

HOCl decreases ATP at sublethal doses in vitro HOCl can react directly with ATP, limiting its availability

HOCl causes inhibition of GAPDH, mitochondrial respiration and glucose transport in vitro

HOCl decreases NAD at high doses in vitro (reviewed in [34]) Cellular integrity HOCl mediated increased cell permeability and oxidative damage to

cytoskeletal proteins in vitro, via mobilization of zinc and loss cell thiols (reviewed in [34])

Serine protease inhibitor inactivation

HOCl inactivates [ 1-anti-proteinase, the major circulating inhibitor of serine proteases

Metalloproteinase activation

HOCl mediates the activation of pro-collagenase and pro-gelatinase [10]

Vessel tone: nitric oxide bioavailability

MPO regulates the availability of nitric oxide (NO) in inflammation (rodent model), localising around the endothelium, thereby impairing NO-dependent blood vessel relaxation[51]

NO serves as a substrate for MPO, potentially influencing bioavailability [52]

Cardiac myocyte HOCl impairs contractility in rodent models via oxidation of thiol groups inducing loss of ATPase activity and inhibition of Na+K+ATPase (reviewed in [34])

Neutrophil responses MPO modulates inflammatory responses, by inactivation of granular con- tents and decreased binding to chemotactic receptors

MPO contributes to the physiological feedback by termination of neutro- phil recruitment [15]

bind MPO [44, 45], a process that decreases generation of HOCl by MPO. Indeed, a binding deficit between this protein and MPO may contribute to, or perpetuate, pro- inflammatory responses related to HOCl under certain defined circumstances. The ability to manipulate ceruloplasmin levels in plasma may afford some protection against the unwanted side effects of extracellular MPO activity. However, studies in the research area are limited, hence more investigations need to be undertaken before such a therapeutic approach can be considered.

Conclusion

Oxidative modification of bio-molecules including that attributable to MPO-derived oxidants seems to be an inevitable consequence of tissue injury. There seems little doubt that this is also the case in patients with sepsis. Moreover, there is ample evi- dence that neutrophil MPO-derived HOCl can cause biological damage and alter pro-inflammatory cell signaling responses with the potential to modulate inflamma- tion and, hence, the onset of the sepsis syndromes. There are, however, confounding issues including the failure of antioxidant therapy (in humans) to limit sepsis and critical illness. The possible explanations for this discrepancy include the complex nature of in vivo antioxidant function, the timing of administration, limited focus on pro-inflammatory pathways, which may in themselves prove critical to host defense. Finally, as oxidants, including HOCl, are cell-signaling agents that may up-

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regulate beneficial and self regulating responses as well as those of pro-inflamma- tory origin, modulation of such signaling events by antioxidant intervention may actually prove counterproductive. Thus, although oxidants derived from neutrophil MPO may contribute to the onset of the sepsis syndromes via numerous mecha- nisms the extent to which the production of these species contribute to, or are a con- sequence of, the disease process remains unclear.

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