Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase

Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase

Veterinary Immunology and Immunopathology 130 (2009) 243–250 Contents lists available at ScienceDirect Veterinary Immunology and Immunopathology jou...

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Veterinary Immunology and Immunopathology 130 (2009) 243–250

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm

Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase T. Franck a,b,*, S. Kohnen a,b, G. de la Rebie`re a, G. Deby-Dupont b, C. Deby b, A. Niesten b, D. Serteyn a,b a b

Department of Clinical Sciences, Large Animal Surgery, Faculty of Veterinary Medicine, B 41,University of Lie`ge, Sart Tilman, BE-4000 Lie`ge, Belgium Center for Oxygen, Research and Development, Institute of Chemistry B 6a, University of Lie`ge, Sart Tilman, BE-4000 Lie`ge, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 February 2008 Received in revised form 7 February 2009 Accepted 20 February 2009

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formylmethionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by a-keto-gmethylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CBfMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O2 ) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O2 generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Neutrophils Equine Stimulation Oxidative activity Myeloperoxidase

1. Introduction Polymorphonuclear leucocytes (neutrophils or PMNs) are highly specialized for their primary function of

* Corresponding author at: De´partement des Sciences Cliniques, Clinique Equine, Institut Ve´te´rinaire, B 41, Universite´ de Lie`ge, Sart Tilman, BE-4000 Lie`ge, Belgium. Tel.: +32 4 3663314; fax: +32 4 3662866. E-mail address: [email protected] (T. Franck). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.02.015

phagocytosis and destruction of microorganisms (Borregaard and Cowland, 1997; Klebanoff, 2005). Their stimulation by endogenous (cytokines, . . .) or exogenous agents (bacterial peptides, endotoxins) results in an increase of their oxygen consumption (respiratory burst) with a production of reactive oxygen species (ROS) starting with the production of the superoxide anion (O2 ) via the activation of NADPH oxidase (Lee et al., 2003) and its derived products, hydrogen peroxide (H2O2) (Klebanoff, 2005). Neutrophils also produce peroxynitrite from O2

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by reaction with nitric oxide (NO) (Carreras et al., 1994) and hydroxyl radical (OH) from H2O2 in the presence of transition metals ions bound to suitable ligands (Winterbourn, 1981). The potential of neutrophils to produce highly reactive oxidant species are important for host clearance of pathogen challenge, but may lead to cell or tissue damage upon activation (Deby-Dupont et al., 1999; Klebanoff, 2005). The stimulated neutrophils also discharge from their cytoplasmic granules hydrolytic and proteolytic enzymes as well as myeloperoxidase (MPO; EC 1.11.1.7), a hemic enzyme specific of azurophilic granules. MPO has a dual activity for peroxidase and chlorination which leads to the generation of other oxidant species and more particularly hypochlorous acid (HOCl), one of the more powerful oxidant molecules in vivo. A wide variety of stimuli activate directly or indirectly NADPH oxidase and the degranulation process of the neutrophils (Partrick et al., 1996). 12-Phorbol 13-myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are often used for in vitro studies with mammalian neutrophils. PMA is a receptor-independent stimulus, which enters the cell and directly activates protein kinase C leading to the assembly and activation of NADPH oxidase (Sheppard et al., 2005). The synthetic tripeptide, fMLP, which mimics the effect of various bacterial cell wall-derived peptides is commonly used as a G-protein receptor-dependent stimulus of the PMN and as a chemoattractant molecule (Brazil et al., 1998). The stimulation of neutrophils with fMLP often requires a pretreatment or a priming of the cells to initiate the stimulation process. In many studies, Cytochalasin B (CB), a cytoskeleton-disrupting compound that does not induce a neutrophil response by itself, is used as priming agent for post fMLP stimulation of neutrophils (Bylund et al., 2004). In horses, PMNs have been effectively stimulated in vitro with PMA and fMLP to enhance ROS production and degranulation (Bochsler et al., 1992; Benbarek et al., 1996). The efficacy of fMLP varies depending on the nature of the priming agent (lipopolysaccharide, CB. . .) and the exact technique used to measure ROS production or granules secretory activity (Snyderman and Pike, 1980; Bochsler et al., 1992; Brazil et al., 1998). Our group has recently demonstrated that neutrophil stimulation and MPO release played important roles in the inflammatory response of the horse (Art et al., 2006; de la Rebie`re et al., 2007; Riggs et al., 2007; Grulke et al., 2008). Therefore, we consider that lowering the oxidant activity of stimulated neutrophils and MPO is a useful therapeutic goal. In this perspective, we developed effective and safe tools for studying the modulating effect of selected compounds on the stimulation of equine neutrophils. Several modulators of equine neutrophils were compared for their effects on ROS production and degranulation. The ROS production was studied by two complementary methods: the lucigenin-enhanced CL response (Benbarek et al., 1996; Serteyn et al., 1999; Franck et al., 2008) and the ethylene production via the oxidation of KMB (MouithysMickalad et al., 2001; Deby-Dupont et al., 2005). The degranulation of equine neutrophils was studied by measuring MPO released in the supernatant. The total

amount of MPO was measured by an enzyme immunoassay (ELISA) (Franck et al., 2005) and the active fraction of MPO by a recently developed method, the SIEFED (Franck et al., 2006). We report the effects of PMA and CB-fMLP on equine neutrophil stimulation and investigate the effects of a catalyser of O2 dismutation, superoxide dismutase (SOD), and an inhibitor of NADPH oxidase, diphenyliodonium (DPI) on PMA-stimulated cells. The purpose of this work was to investigate different stimulation models for equine neutrophil and to focus their effects on the enzymatic activity state of released MPO. 2. Methods 2.1. Reagents Analytical grade Na and K salts, dimethylsulfoxide (DMSO), hydrogen peroxide (30% v/v) and Tween 20 were from Merck (VWRI, Belgium). Bovine serum albumin (BSA), catalase, cytochalasin B (CB), diphenyleniodonium (DPI), EDTA, D-(+)-glucose, lucigenin (Bis-N-methylacridinium nitrate), N-formyl-methionyl-leucyl-phenylalanine (fMLP), a-keto-g-methylthiobutyric acid (KMB), percoll, phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) were purchased from Sigma–Aldrich (Bornem, Belgium). Microtitration plates (Cliniplate EB and White Combiplate 8) were from Thermo Labsystems (Finland). Amplex red substrate was purchased from Molecular Probes, Inc. (The Netherlands). Trypan blue was from MP Biomedicals (Illkirch, France). Monovette blood collection systems were from Sarstedt Aktiegesellschaft & Co (Germany). 2.2. Isolation of equine neutrophils Blood samples were drawn from healthy horses by jugular venipuncture in 9 ml vacutainer tubes with EDTA (1.6 mg ml 1 blood) as anticoagulant. The donors were healthy warmblood horses (age range: 5 –13 years) fed and bred in identical conditions and not under medical treatment (Faculty of Veterinary Medicine, University of Lie`ge, Belgium). The neutrophils were isolated from whole blood at room temperature (18–22 8C) by centrifugation (400  g, 30 min, 20 8C) on a discontinuous percoll density gradient according to the method of Pycock et al. (1987). The polymorphonuclear layer was gently collected and washed in 2 volumes of physiological saline solution. The cell pellets were suspended in 20 mM phosphate buffer saline (PBS) at pH 7.4 containing 137 mM NaCl and 2.7 mM KCl. The cell preparation was 96% neutrophils with a viability of 97% as measured by the trypan blue exclusion test. Each set of neutrophils was obtained from 60 ml of blood drawn from one horse, the cells were used immediately after isolation, the experiment was completed within 5 h and each assay was performed at least in duplicate. Each experiment was repeated at least 3 times with different cell batches (different horses). 2.3. Preparation of the neutrophil activator solutions The amounts of the stimulus used to activate equine neutrophils were previously determined (Benbarek et al.,

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1996; Bochsler et al., 1992; Brazil et al., 1998). CB (1 mg ml 1) was dissolved in DMSO, and an adequate volume of this solution was added to the cell suspensions in PBS to reach the final concentration of 5 mg ml 1 CB and 0.5% DMSO. PMA and fMLP were dissolved in DMSO, and aliquots were kept refrigerated at 20 8C. Just prior to use, distilled water was added to the aliquots to obtain a stock solution of PMA (1.6  10 5 M) with 1% DMSO and a stock solution of fMLP (1  10 4 M) with 10% DMSO. Adequate volumes of these stock solutions were added to the neutrophil suspensions to reach the final concentration of 8  10 7 M PMA and 0.05% DMSO or 1  10 6 M fMLP and 0.1% DMSO. For each stimulus, the effect of the DMSO solution without stimulus (the diluent) was studied and compared to the control cell suspension in PBS buffer. Thus, when neutrophils were stimulated with PMA, parallel assays were made with cell suspensions in PBS buffer alone or with 0.05% DMSO. When neutrophils were stimulated with CB-fMLP, parallel assays were made with cell suspensions in PBS buffer alone or with 0.6% DMSO. 2.4. Measurement of the ROS production by activated neutrophils 2.4.1. Chemiluminescence The lucigenin enhanced chemiluminescence (CL) was measured according to the method of Benbarek et al. (1996) with minor modifications. The assays were performed on 96-well microtiter plates and CL was read with a Fluoroscan Ascent FL (Thermo Labsystems). Two hundred ml of the neutrophil suspensions (5  106 cells ml 1 PBS) were distributed in the wells (106 neutrophils per well) of the microtiter plate (White Combiplate 8, Thermo Labsystems) and incubated for 10 min at 37 8C. Prior to fMLP stimulation, a pre-incubation of the cells was performed for 10 min with 5 mg ml 1 CB. Thereafter, 25 ml CaCl2 (7.5 mM), 2 ml lucigenin (5 mM) and the adequate volume of the stimulus were added to the well, and CL was monitored during 30 min. PMA and fMLP final concentrations were 8  10 7 M and 1  10 6 M respectively. Control assays were made with unstimulated cells in PBS buffer alone or with the respective diluents of CB, PMA and fMLP. The CL response of the neutrophils was expressed as the integral value of the total CL emission. 2.4.2. Ethylene measurement by gas chromatography a-keto-g-methylthiobutyric acid (KMB) is oxidized by the ROS released from stimulated neutrophils in the extracellular medium. Oxidized KMB further decomposes to produce ethylene which is measured into the gaseous phase by gas chromatography (Mouithys-Mickalad et al., 2001; Deby-Dupont et al., 2005; Kohnen et al., 2007). Isolated equine neutrophils were suspended in calibrated vials at a concentration of 106 cells ml 1 in PBS added with glucose (1.5 mg ml 1 PBS). One ml of the neutrophil suspension was pre-incubated for 10 min at 37 8C. Prior to fMLP stimulation, a pre-incubation of the cells was performed during 10 min with 5 mg ml 1 CB. Thereafter, KMB was added at the final concentration of 10 3 M, and PMA or fMLP at the final concentration of 8  10 7 M or

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1  10 6 M respectively. Control assays were made with the unstimulated cells in the PBS-glucose buffer alone or with the diluents of CB, fMLP or PMA. Immediately after the stimulus addition, the vials were sealed and incubated for 1 h at 37 8C. The level of KMB oxidation was measured by quantifying the ethylene amount accumulated in the gaseous phase of the vial by gas chromatography on a Porapak T column (1 m length; ID 1/8 in.; supplied by Supelco, Belgium) at 50 8C using nitrogen as vector gas (40 ml min 1), with flame ionization detector at 120 8C. The gas samples (0.5 ml) were obtained by puncture through the sealing membrane of the vials with a 1 ml Hamilton gas syringe A-2 (Vici Precision Sampling Inc.). Column standardisation was performed with pure ethylene (C2H4, Air Liquide, Belgium). 2.5. Measurement of total and active MPO release by stimulated neutrophils The neutrophil suspensions (106 cells ml 1 PBS) were pre-incubated for 10 min at 37 8C and stimulated for 30 min at 37 8C with 8  10 7 M PMA or 1  10 6 M fMLP (final concentration). Prior to fMLP stimulation, the neutrophils were pre-incubated for 10 min with CB at the final concentration of 5 mg ml 1. Control assays were performed with unstimulated cells in PBS buffer alone or with the diluents of CB, fMLP or PMA. At the end of the stimulation period, the neutrophil suspensions were centrifuged (450  g, 10 min) and the supernatants were collected for the measurement of total and active MPO released by neutrophils. An ELISA assay was used to measure total equine MPO (Franck et al., 2005) (Equine MPO ELISA, BiopTis, Belgium). Before the assays, the samples were diluted 250 fold with 20 mM PBS buffer at pH 7.4, added with 5 g l 1 BSA and 0.1% Tween 20. The active MPO released by neutrophils was measured by a method called SIEFED (‘‘Specific Immunological Extraction Followed by Enzymatic Detection’’) recently developed for the specific detection of active equine neutrophil MPO (Franck et al., 2006). The method is a three steps procedure: firstly, the extraction of MPO out of a solution or a biological sample by its capture on specific immobilized antibodies, secondly washings to eliminate unspecifically bound compounds or interfering substances and, thirdly, the detection of MPO enzymatic activity by using H2O2 as substrate, Amplex Red as fluorogenic electron donor and nitrite as enhancer of the reaction (Franck et al., 2006). Before the assays, the samples were diluted 5-fold with the same PBS buffer as for ELISA. 2.6. Modulation assays of neutrophil stimulation These assays were performed with SOD and DPI on neutrophils stimulated with PMA. SOD and DPI were dissolved in PBS and added to the neutrophil suspensions to reach the final concentrations of 200, 20, 2, 0.2 U ml 1 and 10 4, 10 5, 10 6, 10 7 M respectively. Neutrophils were pre-incubated (37 8C) for 10 min with SOD and 20 min with DPI, and thereafter stimulated with 8  10 7 M PMA. ROS production was measured by chemiluminescence and

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Table 1 Effect of PMA (panel A) and CB + fMLP (panel B) on the ROS production and the release of total and active MPO by equine neutrophils. A CL KMB oxidation Total MPO Active MPO

Unstimulated cells (UNST) (%) 100.0  29.3 100.0  42.2 100.0  23.4 100.0  5.8

PMA-stimulated cells (ST) (%) *

965.2  303.7 616.4  200.0* 345.2  71.7* 15.3  7.9*

Increase factor ST vs UNST +9.7 +6.1 +3.5 6.5

B

Unstimulated cells (UNST) (%)

CB + fMLP-stimulated cells (ST) (%)

Increase factor ST vs UNST

CL KMB oxidation Total MPO Active MPO

100.0  21.8 100.0  23.3 100.0  7.2 100.0  9.3

121.9  86.7 75.2  9.2 163.5  46.6* 118.8  26.3

= = +1.6 =

ROS production by neutrophil was measured by chemiluminescence (CL) and KMB oxidation (KMB), and total and active MPO release by ELISA and SIEFED respectively. Data are presented in relative values (%) in reference to unstimulated cells (UNST) in the diluent of the stimulus taken as 100%. Data are means  SD of at least 3 independent experiments carried out at least in duplicate (n  12, except for KMB in panel B for which n = 6). * Significantly increased (p < 0.05) versus to the corresponding UNST. Increase factor was calculated for each experiment.

ethylene production and degranulation by total and active MPO measurements as described above. 2.7. Statistical analysis Data are presented in relative values (%) in reference to appropriate control groups defined as 100%. All data are expressed as mean  standard deviation (SD) of at least three independent experiments made with different blood batches and carried out at least in duplicate. An unpaired non-parametric Mann Whitney test was performed with the GraphPad Instat 3.05 (GraphPad Software, San Diego California, USA). A p value <0.05 was considered significant. 3. Results 3.1. Effect of the tested substances on the cell viability After isolation from blood, the neutrophil viability in PBS was 97.33  1.37%. CB, DMSO and SOD regardless of the concentration, had no significant effect on neutrophil viability. A slight but significant (p < 0.05) decrease of neutrophil viability was observed for all the tested concentrations of DPI: the percentages of viable cells were 92.33  1.86 at 10 6 M, 93.5  0.55 at 10 5 M and 89.5  4.18 at 10 4 M DPI. 3.2. Effect of PMA and CB-fMLP on the stimulation of neutrophils The diluents of CB-fMLP or PMA were checked for a possible stimulating effect on the neutrophils and no significant effects were observed in comparison to unstimulated cells in PBS buffer (results not show) except for the KMB experiments where the diluent of fMLP + CB induced a significant increasing effect (36%, p < 0.05) of KMB oxidation in comparison to the test made in PBS. For each experiment, the assay with the diluent alone was taken as the control value defined as 100%. The stimulation of neutrophils with PMA increased significantly the CL production (9.7), the KMB oxidation (6.1) and the release of total MPO (3.5) in comparison to non-activated cells (Table 1, panel A). However, the stimulation of neutrophils with PMA strongly inhibited (95.6%) the activity of the released MPO.

The stimulation of PMN by CB-fMLP had no significant effect on the ROS production neither on the CL response nor on the KMB oxidation in comparison to non-activated cells (Table 1, panel B). However, this stimulus significantly increased the release of MPO (1.6) while retaining most (73%) of its enzymatic activity (Table 1, panel B). 3.3. Effects of SOD and DPI on the ROS production by stimulated neutrophils: chemiluminescence (CL) response and KMB oxidation SOD had a dose dependent inhibitory effect on the chemiluminescence response of stimulated neutrophils and significantly inhibited the neutrophils response at all the tested concentrations (from 200 to 0.2 IU ml 1) (Fig. 1). DPI inhibited the response at a dose of 10 4 M. SOD had also an inhibitory effect on ethylene oxidation measurements, except at 0.2 IU ml 1 (Fig. 2). However, the inhibitory effect of SOD was less important on KMB oxidation than on CL response: at 200 IU ml 1 of SOD, 91% inhibition for CL against 77% for KMB oxidation. DPI had a significant inhibitory effect only at 10 4 M. At lower concentrations, DPI slightly, but not significantly increased the oxidant response of the stimulated neutrophils: 34%, 25% and 15% increase at 10 5, 10 6, and 10 7 M respectively. 3.4. Effects of SOD and DPI on the degranulation of stimulated neutrophils: release of total and active MPO SOD and DPI did not influence the release of total MPO by activated neutrophils (Fig. 3). When neutrophils were activated with PMA, the activity of released MPO was strongly inhibited (95.7%) but the addition of SOD induced a protective effect on the MPO activity in a dose dependent manner, with a plateau value at 20 U. This protective effect was significant at 2, 20 and 200 U SOD (p < 0.05) compared to neutrophil stimulated with PMA alone (ST) but, the protection was not complete probably because SOD does not readily penetrate cells (Szeto, 2006). At 20 U SOD, 78.6% of the MPO released by PMAactivated neutrophil was still inhibited (Fig. 3). DPI did not impact on PMA’s ability to diminish the released of active MPO.

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Fig. 1. Effect of SOD (white columns) and DPI (hatched grey columns) on the CL response of 1  106 equine neutrophils stimulated with 8  10 7 M PMA. Results are expressed in relative values (%) in reference to PMA stimulated cells (ST) taken as 100% (black column). Data are means  SD of at least 3 independent experiments carried out at least in duplicate (n  6). * the asterisk indicates that the CL was significantly decreased (*p < 0.05) compared to stimulated cells (ST) and the decrease percentages are indicated on the top of the column. UNST: unstimulated cells in the diluent of PMA.

4. Discussion Horse pathologies such as gastro-intestinal diseases, laminitis, and recurrent airway obstruction involve recruitment and stimulation of neutrophils with release of ROS and MPO of which the oxidant activities can lead to tissue damage (Art et al., 2006; Riggs et al., 2007; de la Rebie`re et al., 2007; Grulke et al., 2008). In this context, we were interested to study the modulation of neutrophil activity by natural molecules such as curcumin and resveratrol and demonstrated that these molecules are inhibitors of the oxidant activity of equine neutrophils stimulated with PMA (Franck et al., 2008; Kohnen et al.,

2007). However, when we started to study the effects of curcumin and resveratrol on the activity of MPO released by PMA stimulated neutrophils, we observed that the neutrophils stimulated with PMA released mainly inactive MPO. We were thus interested to use other neutrophil stimulators to compare with PMA and started with fMLP in combination with CB, a well known stimulus used as alternative to PMA to study the neutrophil stimulation especially in human (Gougerot-Podicalo et al., 1996; Abdel-Latif et al., 2004). In equine research, CB was used as priming agent before a subsequent stimulation with C5a (Bochsler et al., 1992; Rickards et al., 2001). N-formylated peptides were described as very poor chemotactic agents

Fig. 2. Effect of SOD (white columns) and DPI (hatched grey columns) on the KMB oxidation resulting from the ROS production by 1  106 equine neutrophils stimulated with PMA. Results are expressed in relative values (%) in reference to PMA stimulated cells (ST) taken as 100% (black column). Data are means  SD of at least 3 independent experiments carried out at least in duplicate (n  6). * the asterisk indicates that the KMB oxidation was significantly decreased (p < 0.05) compared to stimulated cells (ST) and the decrease percentages are indicated on the top of the column. UNST: unstimulated cells in the diluent of PMA.

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Fig. 3. Effect of SOD and DPI on the release of total (white column) and active (hatched grey column) MPO by PMA-stimulated neutrophils. Results are expressed in relative values (%) in reference to unstimulated cells (UNST) in the diluent of PMA taken as 100% (grey column). Data are means  SD of at least 3 independent experiments carried out at least in duplicate (n  6). The percentages of active MPO versus total MPO are indicated on the top of the column. ST: PMA-stimulated cells. * the asterisk indicates that the MPO activity was significantly increased (p < 0.05) compared to stimulated cells (ST). UNST: unstimulated cells in the diluent of PMA.

for equine neutrophils but induced lysosomal enzyme secretion (Snyderman and Pike, 1980) and priming of neutrophils (e.g. by LPS, TNF-a) was a prerequisite for an efficient stimulation of ROS production (Brazil et al., 1998). Dagleish et al. (2003) showed that a combination of two bacterial cell wall components (LPS and fMLP) was required for efficient release of elastase and ROS generation, but to our knowledge the combination of fMLP and CB has not been used previously to stimulate equine neutrophils. We observed that contrary to PMA, the CBfMLP system was poorly active on the ROS production measured by chemiluminescence and KMB oxidation, but modestly increased the release of total MPO without affecting the activity of the enzyme. While PMA directly stimulates the PKC pathway leading to the assembly of NADPH oxidase subunits and enzyme activation, fMLP acts by an indirect process via receptors in relation with G proteins and the cytoskeleton leading to the stimulation of chemotaxis, exocytosis and multiple signalling pathways including the production of second messengers and the activation of PKC (Sheppard et al., 2005). CB by itself is not able to activate neutrophils and many studies performed in a wide variety of species have shown that CB ‘‘primes’’ neutrophils for superior responsiveness to fMLP or other stimuli. The effect of CB on phagocytes signalling pathway is not fully understood and diverse regulatory mechanism on cytoskeleton and receptor activity have been suggested (Packman and Lichtman, 1990; Saeki et al., 2001; Bylund et al., 2004). Contrary to PMA, the stimulation of equine neutrophils with CB-fMLP did not increase ROS production and did not strongly inhibit the activity of released MPO. These results suggested that ROS released by PMA stimulated neutrophils were responsible of the inhibition of released MPO. The CB-fMLP stimulus would rather favour the secretory activity of the primary granules of neutrophils probably by acting on the cytoskeleton while

the oxidative response of the neutrophils would start later (following a different kinetics) without triggering an immediate and strong oxidative response as does PMA. With human neutrophils, King et al. (1997) observed that most of the secreted MPO was inactivated when the cells were stimulated with PMA, and that the addition of SOD blocked the loss of MPO activity. They concluded that  O2 was mainly responsible for the MPO inactivation. We also observed that SOD protected the activity of the released enzyme during the stimulation of equine neutrophils with PMA but the protection was not complete, perhaps due to the lack of penetration of SOD into the cells (Szeto, 2006). On the other hand, we observed that SOD inhibited ROS production in a dose dependent manner as demonstrated by two different techniques. Lucigenin is considered as a good chemiluminescence probe for measuring extracellular superoxide anions, as it does not enter the cells (Li et al., 1998; Caldefie-Che´zet et al., 2002). Nevertheless, the specificity of lucigenin to measure ROS is controversial (Tarpey and Fridovich, 2001). Therefore to consolidate our results, we used the method of KMB oxidation yielding ethylene in the gaseous phase of the reaction vial which can be measured without interferences of colour or lack of homogenicity of the reaction medium. (i.e., cell suspensions or tissue samples) (Von Kruedener et al., 1995; Mouithys-Mickalad et al., 2001; Deby-Dupont et al., 2005). KMB oxidation is mainly attributed to OHradical-type oxidants essentially confined to a few type of reactions: reduction of hydrogen peroxide by Fe2+ or Cu+ ions (Fenton-like reactions), reaction of hypochlorite with  O2 , formation of peroxynitrite (ONOO ) from O2 and NO and its decay (Hippeli et al., 1997). In our assays, SOD inhibited the CL response, probably because it accelerated the dismutation of O2 that became unavailable for reacting with lucigenin. SOD also inhibited the secondary OH-radical type reactions implicating O2 and the ONOO-

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production, but it favoured H2O2 formation more available for Fenton-like reactions, increasing KMB oxidation. This can explain the less efficient effect of SOD on KMB oxidation compared to the CL response. The involvement of superoxide anion in the activity cycle of MPO has already been demonstrated (Kettle and Winterbourn, 1988). During the reaction with H2O2, ferric MPO (resting state) is oxidized into compound I (CpI) characterized by a cation radical state. When MPO acts as a peroxidase, the CpI is reduced back into ferric MPO by 2 monoelectronic oxidations of an electron donor via the formation of an intermediate non-radical state (CpII) of the enzyme (Deby-Dupont et al., 1999; Klebanoff, 2005). Kettle and Winterbourn (1988) showed that O2 plays a role in the reduction of CpII into the native form of the enzyme favouring the peroxidasic reaction to the detriment of the chlorination one. But O2 can also compete with the substrate used to measure the peroxidasic activity of MPO. In the SIEFED technique, MPO was captured by immobilized antibodies and a wash eliminated all the other compounds before the revelation of the peroxidasic activity. Thus, the inhibition of the MPO activity that we observed by using the SIEFED did not reveal a competitive reaction but an inhibition due to an alteration of the active site of the enzyme. It was also reported that the native ferric MPO can be reduced to inactive ferrous MPO (CpIII) by O2 (see Deby-Dupont et al., 1999 for review). Our SIEFED results perhaps also emphasized this way of MPO inactivation: the destruction of iron-containing prosthetic group of MPO by O2 . We also tested the effects of DPI, a widely used noncompetitive inhibitor of the flavoenzymes. DPI had only a significant inhibitory effect on the ROS production by equine neutrophils at 10 4 M as measured by CL and KMB oxidation. With this last technique, we even observed an increase, but not significant, of the ethylene production at 10 5 M and 10 6 M DPI. The effect of DPI has not been tested hitherto on stimulated equine neutrophils. With human neutrophils, an incubation time of 5 or 10 min with 1 mM of DPI was sufficient to obtain an inhibitory effect higher than 50% on the ROS production (O’Donnell et al., 1993; Hampton and Winterbourn, 1995). In our experimental conditions (20 min incubation with DPI), we did not observe a 50% inhibition of the ROS release even at a concentration of 100 mM of DPI. The efficiency of DPI on equine NADPH oxidase and NO synthase is thus questionable. One of the factors affecting the inactivation of flavins is the redox potential and the accessibility to the active site, which could be different in horse neutrophils compared to human neutrophils. Moreover, it was reported that DPI was not only a binding agent for flavin but could also induce oxidative stress (Riganti et al., 2004). We cannot exclude that the reaction pathway of DPI involving a radical state could be a source of pro-oxidant mechanisms (Laguerre et al., 2007). These potential oxidant effects of DPI could explain the increase of KMB oxidation that we observed at 10 5 and 10 6 M DPI, but further studies are needed to understand the paradoxical results obtained with DPI on stimulated equine neutrophils. As for human neutrophils, the study of the mechanisms of equine neutrophil stimulation is essential in the

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