Hypochlorous acid (HOCl) is produced in the human body by the family of mammalian heme peroxidases, mainly by myeloperoxidase, which is secreted by neutrophils and monocytes at sites of inflammation. This review discusses the reactions that occur between HOCl and the major classes of biologically important molecules (amino acids, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and inorganic substances) to form free radicals. The generation of such free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, neurodegenerative, infectious, and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress. Investigations aimed at elucidating the mechanisms regulating the activity of enzyme systems that are responsible for the production of reactive halogen species are a crucial step in opening possibilities for control of the development of the body's inflammatory response.

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Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca, showed that dimeric MPO induces Ca mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca whereas the effect of monomeric MPO on Ca increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

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IntroductionTwo major responses are central to neutrophil leukocyte (polymorphonuclear neutrophil [PMN]) functioning: chemotaxis and phagocytosis. The migration of leukocytes is governed by chemotactic cytokines called chemokines. Chemokines are a large family of small chemotactic proteins divided into 4 subfamilies according to the positioning of cysteines in their primary sequences. Those whose first 2 cysteines are separated by one amino acid belong to the CXC (or ␣) subfamily and regulate the responses of PMNs. 1,2 Human CXC chemokines interact with the specific G-proteincoupled receptors CXCR1 and CXCR2. CXCR1 has high affinity for interleukin-8 (IL-8) and low affinity for other ␣-chemokines, neutrophil-activating peptide-2 (NAP-2), melanoma growthstimulatory activity (growth-regulated oncogene), and others, whereas CXCR2 shows a high affinity for all CXC chemokines that contain an N-terminal Glu-Leu-Arg motif. 3 Because it has long been shown that after phagocytosis the chemotactic responses of neutrophils are impaired, 4 we investigated the effect of phagocytic stimulation of human neutrophils on the expression of IL-8 receptors CXCR1 and CXCR2. Study design NeutrophilsNeutrophils were isolated from heparinized blood of healthy volunteers by dextran sedimentation of erythrocytes followed by centrifugation on Lymphoprep (Nycomed, Oslo, Norway) and lysis of residual erythrocytes with distilled water. 5,6 Approval was obtained from the institutional review board at the Institute of Hematology and Blood Transfusion, Minsk, Belarus, for these studies. Informed volunteer consent was obtained according to the Declaration of Helsinki. Neutrophils were 95% to 96% pure and more than 98% viable. Purified neutrophils were incubated at 37°C in RPMI containing 2% fetal calf serum (Hyclone, Logan, UT) at 2 to 3 ϫ 10 6 cells per milliliter in polypropylene tubes (Corning, NY) as described. 6 The cells were stimulated with heat-killed and opsonized 7 Saccharomyces cerevisiae (OSC; OSC/PMN ratio, 2:1), opsonized sheep red blood cells (SRBCs; 5:1), or opsonized zymosan particles (30 mg/mL; Sigma, St Louis, MO). All reagents and solutions were controlled for endotoxin contamination by means of Limulus amebocyte assay (E-toxate; Sigma). Phagocytic activity of PMNs was determined as described previously 8 in cell smears by means of Wright stain (Accustain; Sigma). Flow cytometry analysisNeutrophils were stained as described 6 with the use of our mouse monoclonal antibody (mAb) 6 E3 recognizing N-terminal amino acids 10 through 12 (Trp-Asp-Phe) of human CXCR1, mAb R115 to CXCR2, 9 kindly provided by Dr Ernst Brandt (Forschungszentrum, Borstel, Germany), mAb to receptor for complement fragment C5a (C5aR, CD88 10 ; all mAbs were immunoglobulin G1 [IgG1]), kindly donated by Prof Otto Goetze (Georg-August-University, Goettingen, Germany), or isotype control mAb and were analyzed by FACScan (Becton Dickinson, Mountain View, CA) by means of Lysis II software (Becton Dickinson). Confocal microscopySmears were fixed in acetone, permeab...

Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

We performed a comparative analysis of functional activity of neutrophils in patients with type 2 diabetes mellitus with and without symptoms of CHD. Enhanced H2O2 production by neutrophils in response to N-formyl-Met-Leu-Phe (fMLP) was found in patients with type 2 diabetes mellitus. In patients with type 2 diabetes mellitus associated with CHD, fMLP-induced release of myeloperoxidase from azurophilic granules of neutrophils was reduced and plasma myeloperoxidase level was elevated. Increased peroxidase activity of myeloperoxidase, reduced plasma catalase activity, and increased levels of TBA-reactive lipid peroxidation products and oxidized glutathione were detected in patients of both groups. Since myeloperoxidase is an important neutrophilic mediator of oxidative stress, its increased activity in the blood can be an additional marker of oxidative stress and cardiovascular risk in patients with diabetes mellitus.