G. Deby-Dupont

Effects of training on exercise-induced muscle damage and interleukin 6 production

To address the question of whether the increased plasma concentration of interleukin 6 (IL‐6) following strenuous muscular work could be related to exercise‐induced muscle damage, 5 moderately active male volunteers underwent two isokinetic exercise sessions in the eccentric mode, separated by a period of 3 weeks during which the subjects underwent five training sessions. Before training, exercise was followed by severe muscle pain (delayed‐onset muscle soreness; DOMS), and by significant increases in plasma IL‐6 level and serum myoglobin concentration (SMb) (P < 0.001). After training, postexercise DOMS and SMb values were significantly lower than those measured before training. There was no significant difference between plasma IL‐6 levels measured at the same time points before and after training. We conclude that the hypothetical relationship between exercise‐induced muscle damage and increased postexercise levels of circulating IL‐6 is not substantiated by the present results.

Interleukin-1β and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation

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 4-96 h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100 ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20 microM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10 microM) and MG132 (0.1-10 microM)] 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.

Are similar inflammatory factors involved in strenuous exercise and sepsis

An increasing body of data suggest that strenuous exercise triggers an inflammatory response having some similarity with those occurring in sepsis. Indices of this inflammatory response to exercise (IRE) especially include leukocytosis, release of inflammatory mediators and acute phase reactants, tissue damage, priming of various white blood cell lines, production of free radicals; activation of complement, coagulation and fibrinolytic cascades. Inflammatory responses to strenuous exercise and sepsis could in part be due to the release of endotoxin in blood as common triggering factor, but it seems that tissue damage and/or contact system activation are more important triggering mechanisms in exercising subjects. While the magnitude and duration of cellular and humoral changes associated with IRE are quite different from those observed in sepsis, recent human studies suggested that chronic and/or excessive IRE could have adverse effects. Among the possible consequences of acute and chronic IRE are delayed onset muscular soreness and loss of force, cardiovascular complications, intravascular hemolysis, hypoferraemia and increased susceptibility to infection.

Endotoxaemia, production of tumour necrosis factor α and polymorphonuclear neutrophil activation following strenuous exercise in humans

Abstract To examine whether endotoxaemia accompanying long-term, strenuous physical exercise is involved in exercise-induced increase in plasma tumour necrosis factor alpha (TNF-α) concentration and polymorphonuclear neutrophil (PMN) activation, 14 male recreational athletes [mean age 28 (SEM 1) years] were studied. Exercise consisted of a 1.5-km river swim, a 40-km bicycle race, and a 10-km road race. Mean time to complete the race was 149.8 (SEM 4.8) min. The plasma concentrations of granulocyte myeloperoxidase (MPO) and TNF-α were significantly higher than baseline values immediately and 1 h after exercise (P < 0.001). Both variables returned to pre-race levels the day after exercise. Marked, transient decreases in plasma concentrations of anti-lipopolysaccharide (LPS) immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies directed against a panel of selected smooth gram-negative LPS were observed after the race, reaching in most cases minimal values in the blood sample drawn immediately following the completion of the triathlon. There was no significant correlation between the magnitude of PMN activation, as assessed by the increase in plasma concentrations of MPO, and the humoral markers of endotoxaemia and TNF-α. An inverse, highly significant relationship between the increase in plasma TNF-α concentrations and the changes in circulating anti-LPS IgM antibodies concentrations was observed (r = −0.7; P < 0.01). These findings suggest that exercise-induced endotoxaemia was involved in the release of TNF-α, that the magnitude of the TNF-α response to exercise was down-regulated by anti-LPS antibodies of the IgM class, and that the production of TNF-α and endotoxaemia did not seem to play a role in the activation of circulating PMN in the exercising subjects.

Effects of propofol on endothelial cells subjected to a peroxynitrite donor (SIN-1)

We investigated the effect of propofol on endothelial cells subjected to the peroxynitrite (ONOO−) donor 3‐morpholino sydnonimine (SIN‐1). Cells were incubated overnight with 0.5, 1.0 or 2.0 mm SIN‐1, with or without 10−3 m propofol (Diprivan®). Cytotoxicity, assessed by measuring the release of pre‐incorporated 51Cr, increased when the concentration of SIN‐1 increased, and was significantly decreased by 10−3 m propofol (90%, 78% and 28% of protection against 0.5, 1.0 and 2.0 mm SIN‐1, respectively). Cell protection against 1 mm SIN‐1 was tested with 0.03–1.0 mm propofol and this was compared to tyrosine, a target molecule for peroxynitrite. Propofol protected cells in a dose‐dependent manner (r = 0.98; p < 0.001) and was as effective as tyrosine. Finally, using high‐performance liquid chromatography, we demonstrated that propofol reacted with ONOO− more rapidly than did tyrosine, inhibiting nitrotyrosine formation. In the absence of propofol, 3.5 mm ONOO− with 1 mm tyrosine yielded 39.6% nitrotyrosine, but nitrotyrosine was not produced when 5 mm propofol was added. We conclude that propofol protects endothelial cells against the toxicity of ONOO−. The anti‐oxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite, a property that might be relevant in pathological situations involving a significant contribution of peroxynitrite to tissue damage.

Development of an Enzyme-Linked Immunosorbent Assay for Specific Equine Neutrophil Myeloperoxidase Measurement in Blood

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex “primary antibody-MPO-secondary antibody” was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 ± 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.

Active oxygen species, articular inflammation and cartilage damage

Rheumatoid arthritis and osteoarthritis are age-related diseases, in which degenerative changes (arthrosis) and superimposed inflammatory reactions (arthritis) lead to progressive destruction of the joints. Active oxygen species derived from various sources play a role in this process, which may be influenced by appropriate treatment with antioxidants and free radical scavengers.

Exercise induces pentane production and neutrophil activation in humans. Effect of propranolol

SummaryThe effect of β-adrenergic receptor blockade on exercise-induced lipid peroxidation in man has been examined by measuring the production of pentane in expired air. For this purpose, five healthy male subjects were subjected to dynamic exercise of graded intensity on a cycle ergometer (10 min at 45%, 5 min at 60% and 75% maximal oxygen uptake 1 h after ingestion of either a placebo or 40-mg propranolol. At rest, mean pentane concentration ([pent]) with placebo was 4.13 pmol · l−1, SD 2.14. After exercise, this value significantly increased by 310% (17.1 pmol · l−1, SD 7.73, P < 0.01). Oral administration of 40-mg propranolol significantly lowered the mean resting [pent] to 1.75 pmol · l−1, SD 0.77, P < 0.05. After exercise, the increase of [pent] was much smaller (240%) and was less significant (P < 0.2) than with the placebo. The mechanism of this inhibitory effect of propranolol remains to be elucidated. However, as indicated by the measurement of plasma myeloperoxidase concentration, it can be concluded that the antioxidant property of propranolol cannot be attributed to the inhibition of neutrophil activation, a possible source of free radicals during exercise.

Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes.

Abstract:Objectives: To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1β, -6 and -8, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene in response to interleukin (IL)-1β 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 μM), 3-morpholinosydnonimine (SIN-1, 100 μM), with chemically synthesised peroxynitrite (ONOO-, 10 μM) or hydrogen peroxide (H2O2, 100 μM). After treatment by ROS, chondrocytes were washed and then cultured for the next 24 h with or without lipopolysaccharide LPS (10 μg/ml) or IL-1β (1.10-11M). IL-1β, 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-1β stimulated IL-1β, 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-1β, SIN-1 and ONOO- dramatically decreased all gene expressions while SNAP was inefficient. H2O2 treatment inhibited both LPS and IL-1β 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.

Protective activity of propofol, Diprivan® and intralipid against active oxygen species

We separately studied the antioxidant properties of propofol (PPF), Diprivan (the commercial form of PPF) and intralipid (IL) (the vehicle solution of PPF in Diprivan) on active oxygen species produced by phorbol myristate acetate (10(-6) M)-stimulated human polymorphonuclear leukocytes (PMN: 5 x 10(5) cells/assay), human endothelial cells (5 x 10(5) cells/assay) or cell-free systems (NaOCl or H2O2/peroxidase systems), using luminol (10(-4) M)-enhanced chemiluminescence (CL). We also studied the protective effects of Diprivan on endothelial cells submitted to an oxidant stress induced by H2O2/MPO system: cytotoxicity was assessed by the release of preincorporated 51Cr. Propofol inhibited the CL produced by stimulated PMN in a dose dependent manner (until 5 x 10(-5) M, a clinically relevant concentration), while Diprivan and IL were not dose-dependent inhibitors. The CL produced by endothelial cells was dose-dependently inhibited by Diprivan and PPF, and weakly by IL (not dose-dependent). In cell free systems, dose-dependent inhibitions were obtained for the three products with a lower effect for IL. Diprivan efficaciously protected endothelial cells submitted to an oxidant stress, while IL was ineffective. By HPLC, we demonstrated that PPF was not incorporated into the cells. The drug thus acted by scavenging the active oxygen species released in the extracellular medium. IL acted in the same manner, but was a less powerful antioxidant.