H. Benbarek

High concentrations of histamine stimulate equine polymorphonuclear neutrophils to produce reactive oxygen species.

Abstract.Objective and Design: Because high concentrations of histamine are locally released in inflammation, we investigated the effects of supraphysiological doses of histamine on the production of reactive oxygen species (ROS) by neutrophils. ¶Materials and Methods: Isolated equine neutrophils were activated by 10-4 to 5 × 10-3 M histamine. The production of ROS and free radicals was estimated by luminol-enhanced chemiluminescence (CL) and electron spin resonance (ESR) with spin trapping technique. In this model of histamine-stimulated neutrophils, we tested the antagonists of H1 and H2 histamine receptors, the role of Ca2+ and Mg2+, the role of staurosporine and pertussis toxin (inhibitors of protein kinase C and proteins G) and the effects of superoxide dismutase, catalase, hydroxyl radical scavengers (phenylalanine and mannitol) and NG-monomethyl-L-arginine (L-NMMA), inhibitor of NO-synthase. ¶Results: Histamine (from 10-5 to 10-3 M) stimulated neutrophils to produce CL and ESR signals characterized by spin adducts of superoxide anion and/or hydroxyl radicals. The CL response was inhibited by 10-4 and 10-3 M H1 receptor antagonists (promethazine, pyrilamine, and diphenhydramine), by Ca2+ and Mg2+ depletion and by 10 nmoles staurosporine. CL was partially inhibited by pertussis toxin (4 μg/ mL). The ESR signals were practically suppressed by pyrilamine (an H1 receptor antagonist) and superoxide dismutase, and partially inhibited by catalase, hydroxyl radical scavengers and L-NMMA (respectively 59, ± 30% and 68% inhibition). ¶Conclusions: High concentrations of histamine stimulated the neutrophils to product ROS and free radicals via H1 receptors and the NADPH-oxidase pathway.

Interactions between lipopolysaccharides and blood factors on the stimulation of equine polymorphonuclear neutrophils

In horses, the mechanisms of lipopolysaccharide (LPS) stimulation of isolated neutrophils to produce reactive oxygen species remain unknown. We re-investigated this problem by monitoring the luminol-enhanced chemiluminescence (CL) produced by LPS-stimulated equine neutrophils. The neutrophils were isolated from horse blood by discontinuous density gradient centrifugation (> or = 99% neutrophils; viability > or = 98%). Increasing concentrations of Escherichia coli (E. coli) LPS (from 0.01-10 microg ml(-1)) were used to activate the neutrophils. When LPS was used directly, without another stimulator, the respiratory burst of neutrophils was not activated (N=12 horses; n=5 assays per horse). On the contrary, when LPS was added to whole blood, the neutrophils isolated from this blood were stimulated in a LPS dose-dependent manner, but polymyxin B added to whole blood suppressed this stimulation (N=2; n=6). LPS dissolved in autologous equine plasma stimulated the isolated neutrophils in a dose-dependent manner from 0.1-10 microg ml(-1) (N=5; n=12). Heat inactivation of the plasma abolished this CL increase (N=2; n=5). LPS added to equine albumin did not stimulate the isolated neutrophils (N=2; n=5). On the contrary, the addition of gamma-globulins (1 mg ml(-1)) to LPS (10 microg ml(-1)) led to the stimulation of neutrophils (N=2; n=5). We concluded that LPS did not directly stimulate the isolated equine neutrophils, but that plasmatic factors are needed for the stimulation of these cells by LPS.

Equine neutrophil myeloperoxidase in plasma: design of a radio-immunoassay and first results in septic pathologies.

The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). To estimate the possible spreading of this neutrophil activation to the systemic circulation, we designed a radioimmunoassay (RIA) for equine neutrophil myeloperoxidase (MPO) (EC 1.11.1.7) using a specific rabbit antiserum. MPO was labeled with 1 mCi 125I by a technique of self-labeling in the presence of 10(-4) M hydrogen peroxide. The RIA was performed by incubation of 100 microl diluted antiserum, 100 microl labeled MPO (+/-30,000 cpm) and 100 microl of the reference molecule (unlabeled MPO) solution or the unknown sample, at room temperature for 18 h. The antibody-antigen complexes were isolated by double antibody precipitation. The sensitivity of the RIA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coefficients of variation 6% and 8%, respectively, for MPO concentrations ranging from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measurement in plasma was to collect blood into EDTA, which allowed us to get a plasmatic value stable with time. The mean MPO value in normal horses was 69.5 +/- 19.4 ng/ml in EDTA anticoagulated plasma (n = 48). The stress of transport and anaesthesia did not modify the mean plasmatic value of MPO. No significant increase of plasma MPO was observed in 17 horses submitted to surgery for pathologies without systemic impact. But, in 25 horses with obstructive intestinal pathologies, persistent abnormal MPO concentrations were measured (until 740 ng/ml).

Cytotoxicity of stimulated equine neutrophils on equine endothelial cells in culture

We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.

Failure of lipopolysaccharides to directly trigger the chemiluminescence response of isolated equine polymorphonuclear leukocytes.

Divergent results have been reported on the effects of lipopolysaccharides (LPS) on the activation of equine polymorphonuclear leukocytes (PMN). We therefore attempted to determine whether LPS alone can stimulate equine PMN or whether plasma factors are necessary. PMN were isolated from citrated blood on a discontinuous density gradient of Percoll. The luminol (10-3 mol/L)-enhanced chemiluminescence (CL) of 1.25 x 106 cells was measured after addition of Escherichia coli LPS (0.001-10 µg/ml) alone or after incubation in autologous plasma (1 h, 37°C). After direct stimulation with LPS, there were random variations of CL in 16 horses that were not reproducible from one sample to the next for the same horse. LPS which had been incubated in plasma gave a dose-dependent stimulation of the CL of the PMN, which did not occur if the plasma had been heat inactivated (1 h, 56°C). These results indicated a role for plasma factors, which were unlikely to be cytokines, as there were no monocytes or lymphocytes in the plasma incubated with LPS, but might have been complement fragments or LPS ligands, such as LPS binding protein. Studies using specific antibodies against these factors are needed to clarify this question.

Effect of honey on oxidation, chlorination and nitration by purified equine myeloperoxidase

Saad Aissat, Hama Benbarek, Thierry Franck, Stephan Kohnen, Didier Serteyn, Moussa Ahmed, Ange Mouithys-Mickalad Institute of Veterinary Sciences, Ibn Khaldoun University, Tiaret 14000, Algeria Pharmacognosy and Api-Phytotherapy Research Laboratory, Mostaganem University, Mostaganem, Algeria Faculty of Sciences of Nature and Life, Mascara University, Mascara 29000, Algeria Centre for Oxygen, Research and Development (CORD), Institute of Chemistry B6a, University of Liège, Sart Tilman, 4000 Liège 1, Belgium Department of Clinical Sciences, Large Animal Surgery, Faculty of Veterinary Medicine, B41, University of Liège, Sart Tilman, 4000 Liège 1, Belgium Celabor, Centre de Services Scientifique et Technique aux Entreprises, Zoning Industriel de Petit-Rechain, Avenue du Parc 38, 4650 Herve, Belgium Journal of Coastal Life Medicine 2017; 5(9): 398-402

Effect of honey on purified equine myeloperoxidase activity and superoxide radical production in activated Polymorphonuclear neutrophils

Three Algerian honey samples (nectar honey, mixed honey and honeydew honey) were analysed for their total phenolic content (TPC) using the Folin–Ciocalteu reagent and total flavonoid content (TFC) by the aluminium chloride method. The antioxidant activity was tested both against reactive oxygen species (ROS) produced by phorbol 12-myristate 13-acetate (PMA)-stimulated equine polymorphonuclear neutrophils (PMNs) and on purified equine myeloperoxidase (MPO) enzyme activity. The ROS production by stimulated PMNs was measured by lucigenin-enhanced chemiluminescence. Specific immunological extraction followed by enzymatic detection (SIEFED) was used to measure specifically the activity of equine MPO. Nectar honey, mixed honey and honeydew honey had TPCs of 59.52 ± 1.31, 68.36 ± 1.20 and 122.76 ± 4.20 mg gallic acid equivalents (GAE)/100 g honey, respectively (mean ± SD). As for the TPC, the highest TFC was observed for honeydew honey [20.55 ± 0.72 mg catechine equivalents (CE)/100 mg honey], followed by mixed honey (7.47 ± 0.07 CE/100 mg honey) and nectar honey (4.80 ± 0.08 CE/100 mg honey). All the tested honeys displayed a dose-dependent inhibitory effect on the oxidant activities of PMNs. By the SIEFED technique, it was shown that most of the honeys interact directly with MPO: the light honeys (nectar honey and mixed honey) were inhibitors of MPO activity, while the dark honey had less inhibiting effect and even behaved as an enhancer of MPO activity at high concentrations.