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.
Franck, Thierry ; ; de la Rebière de Pouyade, Geoffroy et al
in Veterinary Immunology and Immunopathology (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 ... [more ▼]
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-formyl-methionyl-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 alpha-keto-gamma-methylthiobutyric 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 CB-fMLP 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 (O(2)(-)) 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 O(2)(-) generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation. [less ▲]Detailed reference viewed: 95 (13 ULg)
In vivo administration of acepromazine or promethazine to horse decreases the reactive oxygen species production response of subsequently isolated neutrophils to stimulation with phorbol myristate acetate
; Franck, Thierry ; et al
in Journal of Veterinary Pharmacology & Therapeutics (2009)
The previous experiments have shown that some phenothiazines have antioxidant and anti-inflammatory properties in vitro. In this study the inhibition of the production of reactive oxygen species (ROS) by ... [more ▼]
The previous experiments have shown that some phenothiazines have antioxidant and anti-inflammatory properties in vitro. In this study the inhibition of the production of reactive oxygen species (ROS) by neutrophils was studied in two groups of horses, which received a dose of 0.1 mg⁄ kg of either acepromazine or promethazine intravenously. Blood samples were collected before (T0) and 0.5, 1, 3 and 5 h after drug administration. The chemiluminescence (CML) response of neutrophils was measured ex vivo in the presence of luminol for a period of 10 min and the maximum CML value (peak value) recorded. There was a significant inhibition of the ROS production in the acepromazine treated group (49% inhibition) at 5 h after administration and in the promethazine group (24% inhibition) at 3 h after administration (P < 0.05 vs. T0). These findings are of therapeutic relevance in the use of phenothiazines in equine patients with inflammatory diseases where neutrophil activation and ROS production are implicated. [less ▲]Detailed reference viewed: 67 (7 ULg)
Method for co-purification of equine neutrophil elastase and myeloperoxidase from a limited blood volume.
de la Rebière de Pouyade, Geoffroy ; Serteyn, Didier ; Deby, Ginette et al
in Research in Veterinary Science (2009)
Neutrophil myeloperoxidase (MPO) and elastase can be released in severe inflammatory diseases and cause tissue injuries. Equine enzymes have already been individually purified from large blood quantities ... [more ▼]
Neutrophil myeloperoxidase (MPO) and elastase can be released in severe inflammatory diseases and cause tissue injuries. Equine enzymes have already been individually purified from large blood quantities. We describe the isolation of both enzymes from a same limited blood volume. Both MPO and elastase were extracted by crushing PMN isolated by centrifugation on a percoll-gradient from a 460ml blood collection. MPO and elastase were separated by an ionic exchange chromatography phase and further purified by gel filtration chromatography on Superdex 200 and 75, respectively. Enzymes were identified in the collected fractions by specific enzymatic assays. The final purity was verified by electrophoresis. Specific activity was improved to 19.92 and 34.3x for elastase (final yield: 340mug) and MPO (final yield: 130mug), respectively, during the procedure. Results show the possibility of isolating both enzymes from the same blood sample with a sufficient yield and purity for future studies on their implication and interaction during inflammatory diseases. [less ▲]Detailed reference viewed: 32 (12 ULg)
Interleukin-1beta and Interleukin-6 Disturb the Antioxidant Enzyme System in Bovine Chondrocytes: A Possible Explanation for Oxidative Stress Generation
Mathy, Marianne ; Hogge, Laurence ; Sanchez, Christelle et al
in Osteoarthritis and Cartilage (2008), 16
OBJECTIVE: Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated ... [more ▼]
OBJECTIVE: Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated enzymatic antioxidant system formed principally by superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPX). This work was designed to assess the effects of interleukin (IL)-1beta and IL-6 on the enzymatic activity and gene expression of SODs, CAT and GPX in bovine chondrocytes. METHODS: Bovine chondrocytes were cultured in monolayer for 4-96h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20muM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10muM) and MG132 (0.1-10muM)] were used. SODs, CAT and GPX enzymatic activities were evaluated in cellular extract by using colorimetric enzymatic assays. Mn SODs, Cu/Zn SOD, extracellular SOD (EC SOD), CAT and GPX gene expressions were quantified by real-time and quantitative polymerase chain reaction (PCR). RESULTS: Mn SOD and GPX activities were dose and time-dependently increased by IL-1beta. In parallel, IL-1beta markedly enhanced Mn SOD and GPX gene expressions, but decreased Cu/Zn SOD, EC SOD and CAT gene expressions. Induction of SOD enzymatic activity and Mn SOD mRNA expression were inhibited by NF-kappaB inhibitors but not by MAPK inhibitors. IL-6 effects were similar but weaker than those of IL-1beta. CONCLUSIONS: In conclusion, IL-1beta, and to a lesser extend IL-6, dysregulates enzymatic antioxidant defenses in chondrocyte. These changes could lead to a transient accumulation of H(2)O(2) in mitochondria, and consequently to mitochondria damage. These changes contribute to explain the mitochondrial dysfunction observed in osteoarthritis chondrocytes. [less ▲]Detailed reference viewed: 43 (4 ULg)
Myeloperoxidase Assay in Plasma and Peritoneal Fluid of Horses with Gastrointestinal Disease
Grulke, Sigrid ; Franck, Thierry ; et al
in Canadian Journal of Veterinary Research = Revue Canadienne de Recherche Vétérinaire (2008), 72(1), 37-42
Gastrointestinal disorders, especially strangulating intestinal obstructions, are still a major cause of illness and death in the horse. Circulating lipopolysaccharides may activate both neutrophils and ... [more ▼]
Gastrointestinal disorders, especially strangulating intestinal obstructions, are still a major cause of illness and death in the horse. Circulating lipopolysaccharides may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO concentrations in the plasma and peritoneal fluid (PF) of horses with colic and to check the hypothesis that these concentrations would be higher in a case of strangulating obstruction than in cases of nonstrangulating disease. By using a specific enzyme-linked immunosorbent assay for equine MPO, we determined the MPO concentrations in horses admitted to a clinic for colic. Horses with nonstrangulating or strangulating obstruction of the large intestine (NSLI or SLI), strangulating obstruction of the small intestine (SSI), or inflammatory bowel disease (IBD) were compared with healthy horses. The horses with SLI, SSI, or IBD had significantly higher MPO levels in plasma and PF than did those in the other 2 groups. The mean plasma level was significantly higher in the horses with NSLI than in the healthy horses. High MPO values in PF indicated necrotic bowel. These results show that neutrophil activation occurs during nonstrangulating and strangulating intestinal obstruction in horses and that the plasma and PF MPO concentrations may be a marker of the severity of the disease. [less ▲]Detailed reference viewed: 81 (12 ULg)
Inhibitory effect of curcuminoids and tetrahydrocurcuminoids on equine activated neutrophils and myeloperoxidase activity
Franck, Thierry ; ; et al
in Physiological Research (2008), 57Detailed reference viewed: 28 (9 ULg)
Acute neutrophil activation in direct stenting: comparison of stable and unstable angina patients.
Gach, Olivier ; Nys, Monique ; Deby, Ginette et al
in International Journal of Cardiology (2006), 112(1), 59-65
BACKGROUND: Polymorphonuclear neutrophils have been implicated in the pathophysiology of atherosclerosis. A substantial body of evidence has emerged to implicate the role of specific leucocyte derived ... [more ▼]
BACKGROUND: Polymorphonuclear neutrophils have been implicated in the pathophysiology of atherosclerosis. A substantial body of evidence has emerged to implicate the role of specific leucocyte derived enzyme myeloperoxidase in atherogenesis, since its initiation through progression until destabilization. The aim of the study was to determine the presence of polymorphonuclear neutrophils activation after coronary stenting, to compare this activation between stable and unstable setting and to evaluate the kinetic relation of this activation with inflammatory response following atherosclerotic plaque rupture. METHODS: Myeloperoxidase, lactoferrin, elastase, C-reactive protein and cytokine plasma levels were assessed in 15 patients undergoing direct coronary stenting for unstable angina (Group A) and compared to 11 patients undergoing this procedure for stable angina (Group B). Serial sampling starting before arteriography and continued for 24 h was carried out in all patients. RESULTS: A significant elevation in myeloperoxidase and lactoferrin levels was observed after stenting in both group A (p<0.0001) and group B (p<0.0001), but was higher in group A. Interleukin-8, interleukin-12 and interleukin-6 levels increased temporarily after stenting in the 2 groups. Baseline values of C-reactive protein were similar in the 2 groups and a progressive increase was observed after the intervention. CONCLUSIONS: Direct coronary artery stenting is associated with an early polymorphonuclear neutrophils activation followed by release of inflammatory cytokines (interleukin-6, interleukin-8, interleukin-12) and C-reactive protein elevation in both stable and unstable patients. We conclude that stenting by itself is associated with myeloperoxidase liberation with a significantly enhanced response in unstable patients. [less ▲]Detailed reference viewed: 21 (3 ULg)
Histology of two rice bodies isolated from the stifle of an adult draught horse stallion.
Schneider, Nicole ; ; Lejeune, Jean-Philippe et al
in Journal of Veterinary Science (Suwon-si, Korea) (2006), 7(1), 83-5
In the human and equine species, different kinds of free floating intra-articular particles are related to certain disorders. Osteochondral fragments formed during osteochondrosis dissecans are the most ... [more ▼]
In the human and equine species, different kinds of free floating intra-articular particles are related to certain disorders. Osteochondral fragments formed during osteochondrosis dissecans are the most common finding in the equine species, whereas in humans rice bodies due to rheumatoid arthritis are more frequent. Herein we report a third type of floating body inside the stifle of an adult draught horse stallion, in macroscopic appearance similar to articular rice bodies known in humans. As revealed by histologic examination, the two particles consist of polypoid degenerated structures derived from synovial villi. Their formation was probably induced by ischemia. [less ▲]Detailed reference viewed: 85 (11 ULg)
Interleukin-1beta and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation
Mathy, Marianne ; ; Sanchez, Christelle et al
in Osteoarthritis and Cartilage (2006), 14(Suppl B), 80Detailed reference viewed: 15 (2 ULg)
L’interleukine-1beta et l’interleukine-6 dérèglent le système antioxydant enzymatique chez les chondrocytes bovins : une explication possible au stress oxydant.
Mathy, Marianne ; ; Sanchez, Christelle et al
in Revue du Rhumatisme (2006), 73(10-11), 1077Detailed reference viewed: 39 (1 ULg)
Myeloperoxidase assay in peritoneal fluid and plasma of horses with small and large intestinal pathologies.
Grulke, Sigrid ; Franck, Thierry ; Deby, Ginette et al
Conference (2005, August 04)Detailed reference viewed: 9 (0 ULg)
Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes.
Mathy, Marianne ; ; Deby, Ginette et al
in Osteoarthritis and Cartilage (2005), 13(1), 74-9
OBJECTIVES: To determine the in vitro effects of oxygen tension on interleukin (IL)-1beta induced nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) production by bovine chondrocytes. DESIGN ... [more ▼]
OBJECTIVES: To determine the in vitro effects of oxygen tension on interleukin (IL)-1beta induced nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) production by bovine chondrocytes. DESIGN: Enzymatically isolated bovine chondrocytes were cultured for different periods in suspension in 21 (atmospheric), 5 or 1% (low) oxygen tension and in the absence or in the presence of increased amounts (0.01 to 1nM) of IL-1beta. Nitrite and nitrate concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. PGE(2) production was quantified by a specific radioimmunoassay (RIA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA steady state levels were also quantified by real-time polymerase chain reaction (PCR). RESULTS: In the absence of IL-1beta, ()NO production remained stable whatever the oxygen tension used. IL-1beta dose-dependently increased *NO production in both atmospheric and low oxygen conditions but the effect was more pronounced in low (1 and 5%) than in atmospheric (21%) oxygen tension (P<0.001). Under low and atmospheric oxygen tension, iNOS gene expression was increased by IL-1beta, but to a lesser extent in 21% than in 1 or 5% oxygen (P<0.01). In the basal condition, bovine chondrocytes spontaneously produced PGE(2) whatever the oxygen tension used. At 21% oxygen, IL-1beta dose-dependently increased PGE(2) production while no significant effect was observed at 1 or 5% oxygen. COX-2 gene expression was significantly upregulated by IL-1beta in both low and atmospheric oxygen tension. No significant difference between oxygen tension conditions was observed. CONCLUSIONS: This study demonstrates that a hypoxic environment fully blocks COX-2 activity but favours iNOS gene expression in chondrocytes culture. These findings indicate that O(2) tension modulates cellular behaviour in culture and supports the concept of chondrocyte culture in low oxygen tension to reproduce in vitro the life conditions of chondrocytes. [less ▲]Detailed reference viewed: 26 (12 ULg)
Design of an ELISA specific for the measurement of equine neutrophil myeloperoxidase in biological fluids
; Franck, Thierry ; et al
in Clinical Chemistry (2005), 51(Suppl. 6), 211-212Detailed reference viewed: 16 (0 ULg)
Nitric oxide-related products and myeloperoxidase in bronchoalveolar lavage fluids from patients with ALI activate NF-kappa B in alveolar cells and monocytes.
Nys, Monique ; Preiser, Jean-Charles ; Deby, Ginette et al
in Vascular Pharmacology (2005), 43(6), 425-33
An increased production of NO* and peroxynitrite in lungs has been suspected during acute lung injury (ALI) in humans, and recent studies provided evidence for an alveolar production of nitrated compounds ... [more ▼]
An increased production of NO* and peroxynitrite in lungs has been suspected during acute lung injury (ALI) in humans, and recent studies provided evidence for an alveolar production of nitrated compounds. We observed increased concentrations of nitrites/nitrates, nitrated proteins and markers of neutrophil degranulation (myeloperoxidase, elastase and lactoferrine) in the fluids recovered from bronchoalveolar lavage fluids (BALF) of patients with ALI and correlated these changes to the number of neutrophils and the severity of the ALI. We also observed that BALFs stimulated the DNA-binding activity of the nuclear transcription factor kappa B (NF-kappaB) as detected by electrophoretic mobility shift assay in human alveolar cells (A549) and monocytes (THP1). The level of activation of the NF-kappaB-binding activity was correlated to the concentration of nitrated proteins and myeloperoxidase. Furthermore, in vitro studies confirmed that NO*-derived species (peroxynitrite and nitrites) and the neutrophil enzyme myeloperoxidase by themselves increased the activation of NF-kappaB, thereby arguing for an in vivo pathogenetic role of NO*-related products and neutrophil enzymes to human ALI. [less ▲]Detailed reference viewed: 24 (2 ULg)
Effects of glucocorticoids on the respiratory burst of Chlamydia-primed THP-1 cells.
Mouithys-Mickalad, Ange ; Deby, Ginette ; Mathy, Marianne et al
in Biochemical and Biophysical Research Communications (2004), 318(4), 941-8
We previously observed that the respiratory burst of human monocytes (THP-1 cell line) triggered by phorbol myristate acetate was strongly enhanced by a priming of the cells by Chlamydia pneumoniae ... [more ▼]
We previously observed that the respiratory burst of human monocytes (THP-1 cell line) triggered by phorbol myristate acetate was strongly enhanced by a priming of the cells by Chlamydia pneumoniae [Biochem. Biophys. Res. Commun. 287 (2001) 781]. We describe here the modifications of the responses of Chlamydia-primed THP-1 cells to hydrocortisone (HCT) and methylprednisolone (MPL). HCT and MPL inhibited the production of the cytokines TNFalpha and IL-8. But HCT, which inhibited the respiratory burst in LPS-primed monocytes, paradoxically stimulated the phenomenon in Chlamydia-primed cells; MPL exerted no significant effect. Both glucocorticoids did not significantly modify the triggering effect of Chlamydia on NF-kappaB binding activity. On the expression of p22(phox), a protein subunit of the NADPH oxidase, HCT had an increasing and MPL a decreasing effect. Glucocorticoids thus had unexpected effects on the inflammatory response of Chlamydia-primed monocytes. [less ▲]Detailed reference viewed: 72 (31 ULg)
Effects of interleukin-1 beta and dexamethasone on the expression by chondrocytes of antioxidant enzymes
Mathy, Marianne ; ; Sanchez, Christelle et al
in Osteoarthritis and Cartilage (2004), 12(Suppl. B), 51-52Detailed reference viewed: 20 (2 ULg)
Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1)
Mouithys-Mickalad, Ange ; Deby, Ginette ; Dogné, Jean-Michel et al
in Biochemical and Biophysical Research Communications (2004), 325Detailed reference viewed: 30 (11 ULg)
Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes.
Mathy, Marianne ; ; et al
in Inflammation Research (2003), 52(3), 111-8
OBJECTIVES: To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1beta, -6 and -8, inducible nitric oxide ... [more ▼]
OBJECTIVES: To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1beta, -6 and -8, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene in response to interleukin (IL)-1beta or lipopolysaccharide (LPS). METHODS: Human chondrocytes in monolayer culture were incubated for 3 h with ROS generating molecules such as S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 microM), 3-morpholinosydnonimine (SIN-1, 100 microM), with chemically synthesised peroxynitrite (ONOO-, 10 microM) or hydrogen peroxide (H2O2, 100 microM). After treatment by ROS, chondrocytes were washed and then cultured for the next 24 h with or without lipopolysaccharide LPS (10 microg/ml) or IL-1beta (1.10(-11) M). IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression was analysed by real time and quantitative RT PCR. IL-6, IL-8 and prostaglandin (PG) E2 productions were assayed by specific immunoassays. Nitrite was measured in the culture supernatants by the Griess procedure. RESULTS: LPS and IL-1beta stimulated IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression. SNAP significantly downregulated LPS induced overall gene expressions, whereas SIN-1 had no effect. ONOO- inhibited iNOS and COX-2 gene expression but not that of the cytokine genes. When chondrocytes were incubated with IL-1beta, SIN-1 and ONOO dramatically decreased all gene expressions while SNAP was inefficient. H2O2 treatment inhibited both LPS and IL-1beta induced gene expressions. CONCLUSIONS: These data provide an evidence that ROS may have anti-inflammatory properties by depressing inflammatory gene expression. Further, we demonstrate that ROS effects are dependent on the nature of radical species and the signalling pathway that is activated. These findings should be taken into consideration for the management of antioxidant therapy in treatment of inflammatory joint diseases. [less ▲]Detailed reference viewed: 30 (6 ULg)
Bronchoalveolar lavage fluids of ventilated patients with acute lung injury activate NF-kB in alveolar epithelial cell line: role of reactive oxygen/nitrogen species and cytokines
Nys, Monique ; Deby, Ginette ; et al
in Nitric Oxide (2003), 9(1), 33-43Detailed reference viewed: 29 (10 ULg)