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Hypohalous acid-modified human serum albumin induces neutrophil NADPH-oxidase activation, degranulation and shape change Irina V. Gorudko, Daria V. Grigorieva, Ekaterina V. Shamova, Valeria A. Kostevich, Alexey V. Sokolov, Elena V. Mikhalchik, Sergey N. Cherenkevich, Jürgen Arnhold, Oleg M. Panasenko www.elsevier.com/locate/freeradbiomed

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S0891-5849(13)01564-5 http://dx.doi.org/10.1016/j.freeradbiomed.2013.12.023 FRB11856

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Free Radical Biology and Medicine

Received date: 22 May 2013 Revised date: 18 December 2013 Accepted date: 20 December 2013 Cite this article as: Irina V. Gorudko, Daria V. Grigorieva, Ekaterina V. Shamova, Valeria A. Kostevich, Alexey V. Sokolov, Elena V. Mikhalchik, Sergey N. Cherenkevich, Jürgen Arnhold, Oleg M. Panasenko, Hypohalous acidmodified human serum albumin induces neutrophil NADPH-oxidase activation, degranulation and shape change, Free Radical Biology and Medicine, http: //dx.doi.org/10.1016/j.freeradbiomed.2013.12.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hypohalous acid-modified human serum albumin induces neutrophil NADPH-oxidase activation, degranulation and shape change

Irina V. Gorudko1*, Daria V. Grigorieva1, Ekaterina V. Shamova1, Valeria A. Kostevich2,3, Alexey V. Sokolov2,3, Elena V. Mikhalchik3, Sergey N. Cherenkevich1, Jürgen Arnhold4, Oleg M. Panasenko3

1

Department of Biophysics, Belarusian State University, Nezavisimosti Avenue, 4, Minsk,

220050 Belarus 2

Institute of Experimental Medicine, ul. Akademika Pavlova 12, Saint-Petersburg, 197376 Russia

3

Research Institute of Physico-Chemical Medicine, ul. Malaya Pirogovskaya 1a, Moscow,

119435 Russia 4

Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig,

Härtelstrasse 16-18, 04107 Leipzig, Germany

*To whom correspondence should be addressed: Irina V. Gorudko, Department of Biophysics, Belarusian State University, Nezavisimosti Ave., 4, Minsk, 220030 Belarus (Email: [email protected]; tel. +375 17 209 54 37; fax: +375 17 209 54 45).

Key words: human serum albumin; myeloperoxidase; human neutrophil; degranulation; hypochlorous acid; hypobromous acid; oxidative stress

Abstract Halogenated lipids, proteins and lipoproteins formed in reactions with myeloperoxidase (MPO)derived hypochlorous acid (HOCl) and hypobromous acid (HOBr), can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum 

1

albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the ‘inflammatory loop’ under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the -subunit of 2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biological active substances which potentially able to contribute to activation of myeloid cells in the sites of inflammation and serve as inflammatory response modulators.

Abbreviations: AOPP – advanced oxidation protein products; cyt  – cytochrome c; HSA – human serum albumin; HOCl – hypochlorous acid; HOBr – hypobromous acid; HSA-Cl – HSA treated with HOCl; HSA-Br – HSA treated with HOBr; MPO – myeloperoxidase; mAb – monoclonal antibody; PI3K – phosphoinositide 3-kinase; PMA – 4-phorbol-12-myristate-13acetate; LF – lactoferrin; ROI – reactive oxygen intermediates; SOD – superoxide dismutase.



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Introduction The activation of polymorphonuclear leukocytes (also called neutrophils) at inflammatory loci is usually accompanied by degranulation and formation of a spectrum of aggressive oxidants [1]. The heme protein myeloperoxidase (MPO), found to a great extent in these cells, produces the potent oxidants hypochlorous acid (HOCl) and hypobromous acid (HOBr) [1-3]. Together with superoxide anion radicals (O2•) and hydrogen peroxide (H2O2), these oxidants are secreted both intracellularly into the forming phagosomes, and extracellularly to the neutrophil environment [1], resulting in microbial killing and tissue damage by halogenation and oxidation of a wide range of biological target molecules, including carbohydrates [4], nucleotides, DNA [5], lipids [6, 7], proteins [8] et al. Both acute and chronic diseases are associated with increased protein modification at sites of pathology. For example, elevated levels of advanced oxidation protein products (AOPP) were identied in subjects with coronary artery disease, diabetic patients, and patients with uremia [9-11]. Proteins are likely to be a major target for the hypohalous acids: HOCl and HOBr [10-17]. In vitro studies showed that HOCl generated via the MPO activity could represent one of the pathways for AOPP production in plasma proteins exposed to activated phagocytes [18]. Plasma MPO levels have been reported to correlate with AOPP levels in disease and MPOcatalyzed oxidation products are indicative for cardiovascular disease [19]. Capeillere-Blandin et al. [18] identied albumin as the main source for AOPP products in plasma. Accordingly, in uremic plasma the markers of protein oxidation [15, 20] are mainly associated with albumin. Moreover, HOCl-modified serum albumin acquires the same leukocyte-activating capability as that of AOPP generated in vivo [21]. Albumin is known to be an important plasma antioxidant [22, 23]. It is highly susceptible to oxidation and halogenation by HOCl [24]. Modification of eight amino acid residues of albumin, including methionine, tryptophan and tyrosine following incubation with the MPO-H2O2-chloride/bromide system was detected by fragment analysis of



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tryptic digests with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [25]. It should be noted that HOBr formed in MPO-dependent reactions reacts with the majority of protein amino acid residues much faster than HOCl. For example the rate constant for the reaction of HOBr with tyrosine is about 5000-fold higher than those for HOCl [26]. Moreover, applying chloride and bromide together at physiological concentrations, MPO will apparently produce the interhalogen bromine chloride BrCl, a strong brominating species [27, 28]. As a result, the MPO-H2O2-halide system produces many specific changes in protein structures. For example, treatment of albumin by HOCl [29] or HOBr [30] may give rise to fragmentation of protein. Bovine serum albumin treated with MPO-H2O2-Cl¯ system yields derivatives with a decreased affinity to anti-albumin antibodies and increased electrophoretic mobility [31]. Oxidant-modified proteins are not only a sensitive marker of oxidative stress, but they can also participate in regulation of cell functions and serve as a mediator of inflammation [15, 16, 32]. HOCl-modied albumin is a potent inducer of the oxidative burst in vivo [33] and in vitro [21]. Thus, both HOCl-treated albumin (an in vitro source of AOPP) and in vivo-generated AOPP (isolated from uremic blood) were demonstrated to trigger the oxidative burst in neutrophils and monocytes, thereby acting as true inflammatory and oxidative stress mediator [21]. In hypercholesterolemic rabbits, intravenous infusion of HOCl-albumin signicantly increased macrophage inltration and smooth muscle cell proliferation in atherosclerotic plaques compared with animals treated with albumin [33]. In addition, HOCl-modied albumin acted as a ligand for the endothelial receptor for advanced glycation end products and promoted inammatory complications mediated by this receptor [34]. However, our knowledge about functional consequences of protein modification by hypohalous acids is limited. In particular, whether albumin modified by HOBr affect the activity of neutrophils or other cells remains unknown. 

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The aim of the present work was to further elucidate the molecular events in human neutrophils upon interaction with human serum albumin (HSA) treated by HOCl (HSA-Cl) or HOBr (HSA-Br). Both kinds of modification enable HSA to activate neutrophils. Special attention was also focused on the signaling pathways involved in neutrophil responses to HOCl/HOBr-modified HSA.

Materials and methods

Chemicals NaOCl, NaBr, polylysine, HSA, sodium citrate, scopoletin, -dianisidine, 22, triton X100, superoxide dismutase (SOD), cytochrome c (cyt ) (type VI from horse heart), luminol, 4phorbol-12-myristate-13-acetate (PMA), genistein, wortmannin, Krebs-Ringer buffer (Product No. K4002) were purchased from Sigma-Aldrich (St. Louis, MO, USA). BODIPY FL phalloidin was from Molecular Probes (Leiden, The Netherlands). Dextran T70 was from Roth (Karlsruhe, Germany). Purified monoclonal antibody (mAb) against CD18 (anti-CD18 mAb) (7E4; mouse IgG1) was purchased from Becton Dickinson (San Jose, CA).

Preparation of HSA-Cl/Br The concentration of hypochlorite/hypochlorous acid present in the diluted commercial NaOCl solution was determined spectrophotometrically using a molar absorption coefficient of 350 M-1cm-1 at 290 nm at pH 12 [35]. The working NaOCl solution was freshly prepared immediately before the experiment by solving the commercial solution in a phosphate buffered saline (PBS) (10 mM Na2HPO4/KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). NaOBr was obtained by mixing equal volumes of equimolar NaOCl and NaBr solutions in PBS [36]. The concentration of HOBr was determined using a molar absorption coefficient of 326 M-1cm-1 at 331 nm [37]. Hypochlorous acid (pKa = 7.5) [38] and hypobromous acid (pKa = 8.7) [39] exist 

5

both in the molecular form and as anions at physiological pH. In this paper, the terms HOCl or HOBr were used to describe the mixture of OC1¯ and HOCl or OBr¯ and HOBr, respectively. Modification of HSA by HOCl/HOBr was performed in PBS [34]. HSA was incubated with freshly prepared HOCl/HOBr solution (added as a single addition under gentle vortexing) at a molar ratio of HOCl/HOBr : HSA of 100 : 1 for at least 2 h at room temperature. Under these modification conditions HOCl/HOBr reacted completely with the protein. The absence of unreacted HOCl/HOBr was assessed by luminol-dependent chemiluminescence [40]. The conversion of native HSA to HSA-Cl/Br was monitored by the decrease of the intrinsic fluorescence (excitation at 285 nm and emission at 340 nm) due to the destruction of tryptophan residue in protein.

Isolation of human neutrophils Venous blood samples were obtained from healthy donors at the Republic Scientic and Practical Centre of Hematology and Transfusion (Minsk, Belarus). The blood was collected in tubes containing 3.8% (w/v) trisodium citrate as anticoagulant at a ratio of 9:1. Neutrophils were isolated through centrifugation in the Lymphoprep density gradient as described elsewhere [41]. Cells were suspended in a PBS containing 5 mM D-glucose and stored at 4 °C. The percentage of neutrophils in the cell preparations was > 95%, and the viability was > 95% as determined by the ability to exclude trypan blue. The isolation procedure was performed at room temperature.

Measurements of H2O2 generation by neutrophils H2O2 generation by neutrophils was measured using the scopoletin/peroxidase fluorescent technique [41, 42]. Briefly, suspension of neutrophils (106 cells/ml in PBS, containing 1 mM CaCl2, 0.5 mM MgCl2) was supplemented with 1 PM scopoletin (a fluorescent substrate of peroxidase), 20 Pg/ml horseradish peroxidase, and 1 mM NaN3 (catalase and MPO inhibitor) and was challenged with native or modified HSA at various concentrations to activate plasma 

6

membrane NADPH oxidase. For inhibitor studies neutrophils were pre-incubated for 5 min at 37 °C with genistein, a tyrosine kinase inhibitor (100 μM), wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) (100 nM) or with anti-CD18 mAb (10 μg/ml) for 1 h at room temperature before the addition of stimuli. H2O2-mediated oxidation of scopoletin was recorded as a decrease of its fluorescence at 460 nm (excitation was at 350 nm) during 20 min at 37 °C using a LSF 1211A spectrofluorimeter (SOLAR). The maximal slope of the recorded traces was calculated and referred to as the rate of H2O2 generation by cells.

Assay of O2•  generation by neutrophils Production of O2• was continuously recorded by monitoring the SOD-inhibitable reduction of cyt  as previously described [41]. Neutrophils, suspended in PBS, containing 1 mM CaCl2, 0.5 mM MgCl2, were stimulated to produce O2• by adding native or modified HSA at various concentrations. Duplicate reaction mixtures containing neutrophils (106 cells/ml) and 75 μM cyt c were incubated at 37 °C in the presence or absence of SOD (25 μg/ml). The increase in absorption at 550 nm was monitored using a PB2201 spectrophotometer (SOLAR). Neutrophil O2• generation was finally expressed as nanomoles of O2• per 106 neutrophils for 15 min.

Investigation of F-actin by confocal microscopy Actin reorganization was analyzed using confocal laser scanning microscopy. Neutrophils (5 u 106 cells/ml) suspended in PBS, containing 1 mM CaCl2, 0.5 mM MgCl2, were stimulated with native or modified HSA (500 μg/ml) for 15 min at 37 °C. Stimulated cells were fixed with freshly prepared 4% paraformaldehyde in PBS while still in suspension for 10 min at room temperature. After being washed with PBS, fixed cells were adhered to poly-L-lysinecoated glass slides, permeabilized for 5 min at room temperature with 0.5% Triton X-100 in PBS and stained with 0.07 μM BODIPY FL phalloidin in the dark for 45 min at room temperature to detect F-actin. Images were acquired on laser scanning confocal microscope (Carl Zeiss LSM 

7

510 NLO, Germany) with an immersion lens -apochromat 40u/1.2 W (Carl Zeiss) and processed using Olympus Fluoview software.

Scanning electron microscopy Following the treatment with native or modified HS (500 μg/ml) for 15 min at 37 °C, cells were fixed with 2.5% glutaraldehyde overnight at 4 oC. To prepare samples for electron microscopy, fixed cells were rinsed once with PBS and twice with distilled H2O, added on the surface of glass slides to dry, and then coated with a thin layer (6 nm) of Au-Pd. The samples were analyzed using a Zeiss LEO1455 scanning electron microscope.

Chemiluminescence Neutrophil MPO-dependent oxygenation activity was measured by chemiluminescence using luminol as the chemoluminigenic substrate, which measures intracellular MPO-dependent formation of HOCl [43]. Briefly, suspension of neutrophils (final concentration 0.4 u 106 cells/ml) was distributed into polystyrene tubes containing Krebs-Ringer buffer (pH 7.4), luminol (0.2 ) and was challenged with native or modified HSA following the adding PMA. Luminescence response was measured using a single-photon luminometer (LKB Wallac Luminometer 1251, Finland).

Degranulation assays Degranulation was measured by the release of MPO (an enzyme found exclusively in azurophilic granules) and lactoferrin (LF) (a component of specific granules) as previously described [44]. Neutrophils (3 u106 cells/ml) were suspended in PBS, containing 1 mM CaCl2, 0.5 mM MgCl2, and exposed to native or modified HSA at various concentrations for 15 min at 37 °C. When required, cells were pretreated with genistein (100 μM) or wortmannin (100 nM) for 5 min at 37 °C or with anti-CD18 for 1 h at room temperature before stimulation. 

8

Immediately after treatment, cells were chilled on ice and centrifuged for 10 min at 4000 g. The cell-free supernatant fluids were collected for determinations of MPO and LF. MPO activity in the supernatants was measured using -dianisidine and H2O2 as substrate. Activity of MPO released in the medium was expressed as the percentage of total MPO activity that was present in an equivalent number of cells lysed with 0.1% Triton X-100. MPO concentration in supernatants was measured by the previously developed original enzyme-linked immunosorbent assay (ELISA) method [45]. LF concentration in the supernatants was determined using a commercial ELISA kits ("Vector-Best", Russia).

Statistical analysis Data are expressed as mean±SEM. To analyze differences between the mean values of two groups, a Student t test was used. Differences between mean values of more than two groups were analyzed by ANOVA followed by Student–Newman–Keuls test. Statistical analysis was performed using Statistica software. A p value < 0.05 was considered to be significant.

Results

The effects of HSA-Cl/Br on neutrophil O2• and H2O2 generation The potency of native and modified HSA to effect neutrophil O2• and H2O2 generation was comparatively assessed. First, the kinetics of HSA-mediated scopoletin oxidation, a measure of H2O2 generation by cells, was examined. Scopoletin oxidation was minimal in suspension of neutrophils treated with native HSA (Fig. 1A). Modified HSA (HSA-Cl or HSA-Br) was found to induce H2O2 generation of different magnitude, as evident by decreasing the scopoletin fluorescence intensity (Fig. 1A). As shown in Fig. 1B, modified HSA stimulated the generation of H2O2 by neutrophils in a dose-dependent manner. HSA-Cl and HSA-Br also induced neutrophil O2• generation, which was dependent on concentrations of modified HSA (Fig. 2). It should be noted that HSA-Br had a greater activity 

9

than HSA-Cl to affect O2• and H2O2 generation by neutrophils. Decrease in the rate of scopoletin oxidation and especially O2• generation by neutrophils (Fig. 1B, Fig. 2) in the presence of modified albumin at a concentration of 1 mg/ml may be due to reactive oxygen intermediate (ROI) scavenging activity of protein.

Effects of HSA-Cl/Br on PMA-induced luminol-dependent chemiluminescence We also tested the effects of native and modified HSA on neutrophil MPO-dependent oxygenation

activity

which

was

measured

by

PMA-induced

luminol-dependent

chemiluminescence. In the presence of HSA-Cl/Br or unmodified HSA, PMA-induced neutrophil chemiluminescence was higher than in control cells (without HSA) (Fig. 3). But effect of HSA-Cl and HSA-Br on PMA-induced luminol-dependent neutrophil chemiluminescence was significantly higher than the effect of unmodified HSA (by 86 ± 20% for HSA-Cl and 91 ± 20% for HSA-Br). These results are consistent with the findings of the previous study [21] that HSACl stimulates chemiluminescence response of neutrophils and for the first time demonstrate that HSA-Br has also a stimulating effect on neutrophil chemiluminescence. Since neutrophil chemiluminescence in the presence of luminol is mainly due to its oxidation by HOCl [46, 47], generated by MPO to outside or inside of activated neutrophils, further we investigated the effect of native and modified HSA on the degranulation response of neutrophils.

Effects of HSA-Cl/Br on neutrophil degranulation Degranulation response of neutrophils to native or modified HSA was assessed by the peroxidase activity and concentration of MPO released from azurophilic granules and by concentration of LF released from specific granules. As shown in Fig. 4A, modified HSA (HSAl and HSA-Br) concentration-dependently induced an increase in MPO peroxidase activity in the extracellular medium which was assayed using the substrate o-dianisidine. The ability of HSA-Cl or HSA-Br to activate neutrophil degranulation was also confirmed by an increase in the 

10

concentration of MPO and LF, measured by ELISA, in the supernatants of activated cells (Fig. 4B, C). It should be noted that the ability of HSA-Br to induce a degranulation response of neutrophils was higher than those of HSA-Cl.

Effects of HSA-Cl/Br on shape change and cytoskeleton reorganization in neutrophils The activation of neutrophils is usually accompanied by changes in their shape and actin remodeling [47, 48]. The exposure of neutrophils to modified HSA (HSA-Cl or HSA-Br) for 15 min at 37 °C significantly changed the shape of the cells (Fig. 5A). Treated by HSA-Cl or HSABr cells became more polarized as compared to cells in the presence of native HSA, which had a more rounded shape. In the presence of modified HSA, a significant portion of cells (> 60% depending on experiments) showed a single large surface projection and superficially resembled polarized cells as induced by chemotactic peptides. We also investigated effects of modified HSA on F-actin filaments by confocal microscopy. F-actin as detected by BODIPY FL phalloidin binding showed a fairly diffusive and homogeneous distribution throughout the cytoplasm in spherical cells in the presence of native HSA (Fig. 5B, top). Modified HSA causes a dramatic rearrangement of actin microfilaments in neutrophils. At 15 min following exposure to HSA-Cl/Br F-actin was redistributed to focal regions within the increasingly polarized neutrophils and was found preferentially in projections, whereas the cell-body contained less F-actin compared to cells in the presence of native HSA. In fact, intensity profiling showed a relatively even distribution of phalloidin in resting cells (Fig. 5B, bottom). Phalloidin in neutrophils stimulated by modified HSA was observed as sharp peaks at the cell periphery giving further evidence for F-actin translocation to cell peripheries (Fig. 5B, bottom). Thus, modified HSA induces neutrophil ROI generation, exocytosis of azurophilic and specific granules and cytoskeleton reorganization.



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Effects of anti-CD18 mAb and EDTA on neutrophil MPO exocytosis, O2• and H2O2 generation in responses to modified HSA It was shown that denaturated proteins bind to leukocyte surface presumably via 2 integrins [49, 50]. To determine whether 2 integrin molecules contribute to neutrophil activation by modified HSA, O2• and H2O2 generation as well as degranulation in response to HSA-Cl/Br were measured in the presence of mAb against CD18, the -subunit of 2 integrins, and EDTA, which chelates extracellular divalent cations required for effective integrin binding to various ligands [51]. As shown in Fig. 6, anti-CD18 mAb caused a strong inhibition of HSA-Cl/Brinduced activation of neutrophils. In the presence of anti-CD18 mAb (10 μg/ml) exocytosis of MPO, H2O2 and O2• generation were only 56 ± 5% (Fig. 6A), 56 ± 9% (Fig. 6B) and 66 ± 14% (Fig. 6C), respectively, of the maximal neutrophil response to HSA-Cl. Anti-CD18 mAb caused also a strong inhibition of HSA-Br-induced neutrophil exocytosis of MPO (39 ± 2% inhibition) (Fig. 6A), H2O2 generation (38 ± 13% inhibition) (Fig. 6B) and O2• generation (80 ± 6% inhibition) (Fig. 6C). Like in other integrin-dependent cellular responses, HSA-Cl/Br-induced neutrophil degranulation, H2O2 and O2• generation were inhibited in the presence of EDTA (more than 40%) (Fig. 6). Anti-CD18 mAb and EDTA did not have any effect on neutrophil activation parameters in the absence of modified HSA.

Effects of tyrosine kinase and PI3K inhibitors on neutrophil MPO exocytosis, O2• and H2O2 generation in responses to modified HSA Because neutrophil responses to HSA-Cl/Br were strongly inhibited by anti-CD18 mAb, effect of inhibitors on intracellular signaling cascades was additionally investigated. We applied the fungal-derived, PI3K inhibitor, wortmannin [52] and the tyrosine kinase inhibitor, genistein. As shown in Fig. 7, 100 μM genistein strongly inhibited HSA-Cl/Br-induced neutrophil degranulation (42 ± 10% and 25 ± 10% inhibition for HSA-Cl and HSA-Br, respectively), H2O2 

12

generation (58 ± 6% and 34 ± 9% inhibition for HSA-Cl and HSA-Br, respectively) and O2• generation (59 ± 13% and 80 ± 5% inhibition for HSA-Cl and HSA-Br, respectively). Wortmannin also reduced HSA-Cl/Br-stimulated neutrophil responses but its effect was less pronounced in comparison to genistein. As shown in Fig. 7, PI3K inhibition diminished the HSA-Cl/Br-stimulated MPO exocytosis (32 ± 7% and 22 ± 8% inhibition for HSA-Cl and HSABr, respectively), neutrophil H2O2 generation (28 ± 14% and 20 ± 2% inhibition for HSA-Cl and HSA-Br, respectively) and O2• generation (84 ± 5% and 84 ± 5% inhibition for HSA-Cl and HSA-Br, respectively). None of the inhibitors used had any effect on neutrophil activation in the absence of modified HSA.

Discussion

The heme enzyme myeloperoxidase (MPO; EC 1.11.1.7), released from activated polymorphonuclear leukocytes and monocytes at sites of inflammation, plays a key role in the generation of oxidants by the human immune system [1-3]. MPO catalyzes oxidation of halogenides, primarily of chloride and bromide, by H2O2 according to the reaction (Hal – halogen): Hal¯ + H2O2 + H+ HOHal + H2O. Hypohalous acids (HOHal), e.g. HOCl and HOBr, are most important products of the halogenating activity of MPO. These reactive oxidants play a key role in host defense as well as in a number of pathological, inflammatory conditions, targeting a wide range of biological substrates [2]. Besides, in recent years, accumulating evidence shows that lipids, proteins and lipoproteins modified by MPO-derived oxidants (halogenated molecules) can also modulate signaling pathways in cells and participate in the regulation of their functional activity. For example, chloro- and bromohydrins formed in the reactions of hypohalous acids with phosphatidylcholine [44, 53], HOCl-oxidized HSA [9, 21] as well as HOCl-oxidized low-density 

13

lipoproteins [54] have been shown to stimulate various functions of human leukocytes. In general, halogenated molecules are considered as a new class of biologically active substances that are potentially able to contribute to the regulation of functional activity of myeloid cells at sites of inflammation and serve as inflammatory response modulators. The results obtained in the present work are in support of this hypothesis. In our study we demonstrated that HSA, the main plasma protein, modified by HOCl and HOBr, stimulates neutrophils for NADPH oxidase activation, exocytosis of LF (a marker of specific granules) and MPO (a marker of azurophilic granules), as well as enhances PMAinduced luminol-dependent chemiluminescence. Furthermore, we have shown that HSA-Cl/Br induces neutrophil cell shape change and cytoskeleton reorganization. The obtained results are in agreements with findings of Witko-Sarsat et al. [9, 21], who demonstrated that HSA treated with HOCl induces activation of human neutrophil and monocyte oxidative metabolism. At the same time our study demonstrates for the first time that not only HSA-Cl, but also HSA modified with HOBr, serves as a modulator of neutrophil activity. It should be noted that at physiological concentrations of chloride and bromide (140 mM and 100 μM, respectively), not only chlorination was observed but also bromination of albumin fragments [55]. The level of 3bromotyrosine (a marker of the effect of HOBr) was 16-fold greater than that of 3-chlorotyrosine (a marker of the effect of HOCl). This means that under physiological conditions, in spite of the significant difference in concentrations between Cl¯ and Br¯, bromination of tyrosyl residues was considerably more facile than chlorination. In numerous disease conditions, proteins modified by HOCl as well as by HOBr were detected [19, 56-58]. Thus, MPO-dependent modification of HSA may represent a general autocrine and paracrine pro-inammatory enhancing mechanism for human neutrophil activation. The signaling mechanisms through which modified HSA stimulates neutrophil responses have not yet been defined and were probed in this work. In this study, we demonstrated that HSA-Cl/Br activates neutrophil H2O2 generation and MPO exocytosis through 2 integrin 

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signaling. Neutrophil responses to HSA-Cl/Br were inhibited in the presence of antibodies against D18, a common -subunit of neutrophil 2 integrin family, and in the presence of EDTA, which inhibits ligand binding to integrins due to the chelation of extracellular divalent cations. These results are in agreements with findings of Körmöczi and coworkers [49] who proposed that recognition by integrins of C4, C5, and HSA modified by ROI could serve as an explanation for induction of protein-dependent neutrophils responses. Previously, it was demonstrated that 2 integrins, namely CD11b/CD18 and CD11c/CD18, provide cell adhesion to a variety of denatured protein substrates, recognizing common epitopes on these proteins [50]. It seems that the main mechanism of the increased availability of integrins to denatured protein is a conformational change of the latter, leading to exposure of negatively charged amino acid [59]. Protein oxidation produces widespread conformational alterations that lead to changes in overall charge, folding, hydrophobicity, specific functional activity and binding property [14]. It is known that modication of albumin by HOCl/HOBr leads to an increase in negative charge and enhanced electrophoretic mobility mainly due to halogenation of the amino groups and the formation of carbonyl compounds [34]. Given this fact, it can be assumed that MPO-dependent modification of HSA serve as a signal for recognition by integrin and neutrophil NADPH oxidase activation, degranulation and cytoskeleton reorganization. We propose that HSA modified by HOCl/HOBr undergoes conformational alterations, acquires new binding properties for neutrophils and serves as a agonist that triggers activation of signaling in neutrophils. As activation of tyrosine kinase is essential for integrin-dependent functions [60, 61], in the present study we revealed that HSA-Cl/Br-induced neutrophil degranulation and O2• and H2O2 generation were inhibited in the presence of genistein, a tyrosine kinase inhibitor. The main signaling proteins downstream of activation of tyrosine kinases include Cbl, PI3K, Vav, cytoskeletal proteins, mitogen activated protein kinase et al. [60, 61]. Tyrosine kinases may activate PI3K, directly or indirectly, via activation of Ras or phosphorylation of c-Cbl [60, 61]. As noted in our results, wortmannin, a PI3K inhibitor, showed an inhibitory effect on HSA-Cl

15

induced neutrophil degranulation and H2O2 generation. Since neutrophil responses to modified HSA depend on both tyrosine kinase and PI3K activation which is a key event in the regulation of cytoskeletal changes it can be assumed that exactly these signaling pathways are involved in HSA-Cl/Br-induced neutrophil shape change and cytoskeleton reorganization. Our findings and current understanding of neutrophil activation by HSA-Cl/Br are summarized in a schematic (Fig. 8). We proposed that modified HSA is able to trigger an activating signal in neutrophils through an integrin-dependent mechanism. Tyrosine kinases and PI3K as well as remodeling of the actin cytoskeleton are activated in response to HSA modified by MPO-derived oxidants. This activation results in neutrophil MPO exocytosis and H2O2 generation. MPO uses H2O2 to oxidize Cl¯/Br¯ ions to HOCl/HOBr. Then HOCl/HOBr reacts with HSA to produce HSA-Cl/HSA-Br. Moreover, cationic MPO is known to form a tight complex with acidic plasma proteins such as HSA [62]. Thus, HSA-Cl/Br can initially act as a switch and then as an amplifier of the ‘inflammatory loop’ under oxidative stress. Recently we described a relationship between the increase in PMA-induced luminoldependent chemiluminescence response of neutrophils from healthy donors upon incubation with an albumin fraction from plasma of children with severe burns and the MPO activity in these plasma samples [63]. From these results and the fact that in blood and lesions of patients with inflammatory diseases MPO activity, MPO protein concentration [4, 64-67], biomarkers for MPO (3-chlortyrosine, 2-chlorhexadecanal, chlorohydrins of lipids, 5-chlorouracil, glutathione sulfonamide) [38, 68-71] as well as neutrophil susceptibility to activators [67, 72-74] are increased, it can be expected that mechanisms schematically presented in Fig. 8 are most likely exist in vivo. In conclusion, this paper provides novel insight into the mechanism of neutrophil activation in response to HSA modified by MPO-derived oxidants, which may be important to identify potential novel targets for anti-inflammatory therapy. In general, oxidative modification of



16

various molecules may represent a general autocrine and paracrine pro-inammatory enhancing mechanism for neutrophil activation and accumulation during inammation.

Acknowledgements

This work was supported by RFBR (grants 12-04-90003) and BRFBR (grant B12R-036). We also thank Luda Baran and Helena Golubeva (Belarusian State University) for the help with the scanning electron microscopy and with laser confocal microscopy, respectively. References [1] Hampton, M. B.; Kettle, A. J.; Winterbourn, C. C. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92:3007-3017; 1998. [2] Klebanoff, S. J. Myeloperoxidase: friend and foe. J. Leukoc. Biol. 77:598-625; 2005. [3] Arnhold, J.; Flemmig, J. Human myeloperoxidase in innate and acquired immunity. Arch. Biochem. Biophys. 500:92-106; 2010. [4] Schiller, J.; Arnhold, J.; Sonntag, K, Arnold K. NMR studies on human, pathologically changed synovial fluids: role of hypochlorous acid. Magn. Reson. Med. 35:848-853; 1996. [5] Hawkins, C. L.; Davies, M. J. Hypochlorite-induced damage to DNA, RNA, and polynucleotides: formation of chloramines and nitrogen-centered radicals. Chem. Res. Toxicol. 15:83-92; 2002. [6] Panasenko, O. M.; Vakhrusheva, T.; Tretyakov, V.; Spalteholz, H.; Arnhold, J. Influence of chloride on modification of unsaturated phosphatidylcholines by the myeloperoxidase/hydrogen peroxide/bromide system. Chem. Phys. Lipids 149:40-51; 2007. [7] Skaff, O.; Pattison, D. I.; Davies, M. J. Kinetics of hypobromous acid-mediated oxidation of lipid components and antioxidants. Chem. Res. Toxicol. 20:1980-1988; 2007. [8] Hawkins, C. L.; Pattison, D. I.; Davies, M. J. Hypochlorite-induced oxidation of amino acids, peptides and proteins. Amino Acids 25:259-274; 2003. 

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Fig. 1. Effects of HSA, HSA-Cl and HSA-Br on oxidation of scopoletin by human neutrophils as a measure of H2O2 generation. (A) Scopoletin oxidation by human neutrophils in response to native HSA (1), HSA-Cl (2) and HSA-Br (3) at a HSA concentration of 500 μg/ml. Measurements were performed at 37 ° in PBS, containing 1 mM CaCl2 and 0.5 mM MgCl2. The suspension of neutrophils (106 cells/ml) contained 1 M scopoletin, 20 g/ml horseradish peroxidase, and 1 mM NaN3. Scopoletin fluorescence intensity was measured at 460 nm; excitation wavelength was 350 nm. Typical kinetic curves of at least 3-5 experiments in each case are shown. (B) Dose-dependent effect of HSA, HSA-Cl and HSA-Br on scopoletin oxidation by human neutrophils.

Fig. 2. Dose-dependent effects of HSA, HSA-Cl and HSA-Br on O2• generation by human neutrophils as assessed by SOD-inhibitable reduction of cyt c at 550 nm. The final concentration



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of cyt c was 75 μM. Measurements were performed at 37 ° in PBS, containing 1 mM CaCl2 and 0.5 mM MgCl2. Data represent the mean ± SEM of results from 6-7 experiments.

Fig. 3. Effects of HSA, HSA-Cl and HSA-Br on PMA-induced luminol-dependent chemiluminescence response of neutrophils. Neutrophils were exposed to native or modified HSA (500 μg/ml) at 37 ° for 3 min in Krebs-Ringer buffer,  7.4, following addition of PMA (0.156 μM). Typical kinetic curves of 3-5 experiments in each case are shown.

Fig. 4. Effects of HSA, HSA-Cl and HSA-Br on neutrophil degranulation. Neutrophils were exposed to native or modified HSA for 15 min in PBS, contained 1 mM CaCl2 and 0.5 mM MgCl2, following cell-free supernatants were analyzed for enzyme release. (A) MPO activity was measured spectophotometrically in supernatants from cells incubated with increasing concentrations of native or modified HSA (0-1000 μg/ml) and expressed in % of total activity in cells treated with triton X-100. (B) MPO and (C) LF concentration were analyzed by ELISA in supernatants from neutrophils stimulated by native or modified HSA at a concentration of 500 μg/ml. Effects of HSA-Cl and HSA-Br on MPO and LF release were normalized by setting the enzyme release in neutrophils exposed to native HSA to 100%. Data represent the mean ± SEM of results from 5-6 experiments. *, Signicantly different (p < 0.05) from the corresponding release in the presence of native HSA.

Fig. 5. Surface morphology of human neutrophils in the presence of native or modified HSA. (A) Representative micrographs of human neutrophils incubated with HSA, HSA-Cl and HSABr obtained using scanning electron microscopy. Following the treatment with HSA, HSA-Cl or HSA-Br (15 min, 37 °C) cells were fixed in 2.5% glutaraldehyde overnight at 4 °C and then coated with a thin layer (6 nm) of Au–Pd. Scale bars represent 2 μm. 

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(B) Distribution of F-actin in permeabilized neutrophils stimulated by native or modified HSA (500 μg/ml) for 15 min at 37 °C. Cells were xed while still in suspension and mounted on polyL-lysine-coated slides for confocal microscopy. F-actin was stained with BIODIPY FL phalloidin (top). Cross-sectional intensity proles for F-actin taken from the middle left to right are shown below. Scale bars represent 2 μm.

Fig. 6. Effect of anti-CD18 mAb and EDTA on neutrophil degranulation (MPO exocytosis) (A), 22 generation (B) and O2• generation (C). Human neutrophils were pre-incubated for 1 h with anti-CD18 (10 μg/ml) at room temperature or for 5 min at 37 C with EDTA (1 mM) before addition of 500 μg/ml HSA-Cl or HSA-Br. Neutrophil responses were expressed as a percentage of HSA-Cl or HSA-Br-induced neutrophil activation in the absence of inhibitors. Data represent the mean ± SEM of results from 3-7 experiments. *, Signicantly different (p < 0.05) from the corresponding response in the absence of inhibitor.

Fig. 7. Effect of genistein (100 μM) and wortmannin (100 nM) on neutrophil degranulation (MPO exocytosis) (A), 22 generation (B) and O2• generation (C). Human neutrophils were pre-incubated with inhibitors for 5 min at 37 C before addition of 500 μg/ml HSA-Cl or HSABr. Neutrophil responses were expressed as a percentage of HSA-Cl or HSA-Br-induced neutrophil activation in the absence of inhibitors. Data represent the mean ± SEM of results from 3-7 experiments. *, Signicantly different (p < 0.05) from the corresponding response in the absence of inhibitor.

Fig. 8. Hypothetical model of the ‘inflammatory loop’ sustained by integrin-dependent recognition of HSA, modified in reactions involving MPO under oxidative stress. Further details are given in the text.



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Highlights Hypohalousacidmodifiedhumanserumalbumininducesneutrophilactivation. •

ModifiedHSAstimulatesneutrophildegranulationandO2 andH2O2generation.

IntegrinscontributetoneutrophilactivationbymodifiedHSA. BothtyrosinekinaseandPI3KareinvolvedinneutrophilactivationbymodifiedHSA. ModifiedHSAinducesneutrophilshapechangeandactincytoskeletonreorganization.



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B 14

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