An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid, and its application in finding inhibitors which interact directly with the enzyme.

Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme. The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content. To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation. The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.

Modulatory effects of non-steroidal anti-inflammatory drugs on the luminol and lucigenin amplified chemiluminescence of equine neutrophils.

The purpose of this study was to explore the potential modulation of equine neutrophil oxidative burst by a series of classical NSAIDs which was subsequently monitored by the luminol or lucigenin-enhanced chemiluminescence (CL) technique. A significant dose-dependent inhibition of the luminol CL was observed with the majority of investigated drugs. This inhibition was very significant for phenylbutazone and Indomethacin; while for aspirin, a higher concentration is required. The action of Ketoprofen was significant during the first 5 min and only when the concentration was above 1 mM. Indomethacin and acetylsalicylic acid result in an inhibition dose-dependent of luminol CL. On the other hand, the phenylbutazone showed an inhibiting effect when used either luminol or lucigenin though luminol is slightly better. When the ketoprofen is considered, an inhibiting effect of luminal CL was observed but less significant than the other NSAIDs investigated. The flunixin meglumine enhances strongly the CL.

Biochemical biomarkers of oxidative collagen damage.

Collagens are major constituents of connective tissues in the animal kingdom. During aging and inflammatory-related diseases, the collagen network undergoes oxidation that leads to structural and biochemical alterations within the collagen molecule. Collagen oxidation appears to be a key determinant of aging and a critical physiopathologic mechanism of numerous diseases. Further, the detection of oxidized-collagen peptides seems to be a promising approach for the diagnosis and the prognosis of inflammatory diseases. This chapter reviews the structural and biochemical changes to collagen induced by reactive oxygen and nitrogen species and discusses recent data on the use of collagen-derived biomarkers for measuring oxidative damage.

A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts.

The SIEFED ("Specific Immunological Extraction Followed by Enzymatic Detection") method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H(2)O(2) and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.

Method for co-purification of equine neutrophil elastase and myeloperoxidase from a limited blood volume.

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 460 ml 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 34.3 x for elastase (final yield: 340 microg) and MPO (final yield: 130 microg), 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.

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.

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 (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.

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.

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.

Interleukin-1beta 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.

Effects of unfractionated and fractionated heparins on myeloperoxidase activity and interactions with endothelial cells: possible effects on the pathophysiology of equine laminitis.

As heparins are sometimes used to prevent equine laminitis, the interactions between equine neutrophil myeloperoxidase (MPO), unfractionated (UFH) and fractionated low molecular weight (LMWH) heparins and digital endothelium have been investigated. The effects of the heparins on purified equine MPO activity were tested by immunocapture followed by enzymatic detection. Endothelium-MPO interactions were assessed by measuring total and active MPO uptake by arterial and venous digital endothelial cells in culture with or without the addition of heparins. A dose-dependent MPO inhibition by UFH and LMWH was seen, with the greatest reduction in MPO activity noted with the highest concentration of LMWH. The MPO capture was greater in arterial cells, but heparins better inhibited MPO capture in venous cells. The activity of cell-bound MPO was almost completely suppressed by the heparins, and no differences were observed between UFH and LMWH. The results confirm the anti-inflammatory properties of heparins and allow a better understanding of the potential role of MPO in laminitis.

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta.

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.

Inhibitory effect of curcuminoids and tetrahydrocurcuminoids on equine activated neutrophils and myeloperoxidase activity.

In the horse, the inflammation response to various pathologies (intestinal strangulations, laminitis, etc.) involves an excessive stimulation of the polymorphonuclear neutrophils releasing reactive oxygen species (ROS) and myeloperoxidase (MPO). The aim of the present work was to study the effect of natural polyphenols, curcuminoids and tetrahydrocurcuminoids (THC) on isolated stimulated equine neutrophils and on the activity of purified MPO. The ROS production and the release of MPO by activated neutrophils were measured by chemiluminescence and ELISA techniques, respectively. The activity of purified MPO was measured by studying its nitration, chlorination or oxidation capacity and by using an original method called SIEFED allowing the study of drug interaction with the enzyme without interferences of the medium. Curcuminoids and THC had dose-dependent inhibitory effects on ROS production and MPO release by activated neutrophils and on purified MPO activity. We suggest that the higher efficacy of curcuminoids versus THC could be explained, at least partially, by its chemical structure: the conjugated double bounds and the plane structure of curcuminoids made easier the neutralization of the radical species generated by activated neutrophils and the interaction of the drug with the active site of MPO. These inhibitory effects of curcuminoids on the oxidant activity of equine neutrophils and on MPO activity open therapeutic perspectives in equine pathologies with excessive inflammatory reactions.

Plasma concentration of insulin-like growth factor I (IGF-I) in growing Ardenner horses suffering from juvenile digital degenerative osteoarthropathy.

Degenerative osteoarthropathy resulting in a reduced active lifespan was observed in Ardenner horses. In the context of joint biology, insulin-like growth factor I (IGF-I) is a potential candidate to affect the anabolism of cartilage matrix molecules. A group of 30 Ardenner horses reared under standardized conditions from weaning were evaluated periodically from 15 to 28 months of age to detect the early manifestations of the disease. At the end of this period, horses were classified in two pathological groups related to the degree of interphalangeal degenerative osteoarthropathy based on clinical and radiographic evaluations: healthy (46.7%) and pathological (53.3%) horses. Seven sequential blood samples were taken from each horse (during the evaluation period) to study the variation of IGF-I plasma concentration. We tested the variations of the IGF-I plasma concentration during growth, and the effect of sex and of pathological classes. Significant variations were observed during the research period, with a maximum value corresponding to spring and a minimum in autumn. A significant reduction of the IGF-I plasma concentration was also observed in the pathological horses (433.5 +/- 19.5 ng/ml) compared to the healthy horses (493.9 +/- 18.2 ng/ml). An alteration in the level of this growth factor could induce a disregulation of the mechanisms involved in the local control of joint and bone tissue development.

Plasma concentrations of a type II collagen-derived peptide and its nitrated form in growing Ardenner sound horses and in horses suffering from juvenile digital degenerative osteoarthropathy.

Several breeds of draft horses suffer from degenerative digital osteoarthropathy, resulting in a reduced active lifespan. A group of 30 Ardenner horses was followed, in standardized conditions, from 15 to 28 months of age to detect the early manifestations of the disease. The severity of the disease was assessed according to a personal grading system including clinical and radiographic items. Coll 2-1, a peptide of the helical region of type II collagen, and its nitrated form (Coll 2-1 NO(2)) were assayed in blood plasma collected at 452 +/- 18 days, 504 +/- 20 days, 558 +/- 18 days, 613 +/- 19 days, 675 +/- 19 days, 752 +/- 21 days and 852 +/- 19 days of age. At the end of the follow-up period, 53.3% of Ardenner horses were affected by a degenerative digital osteoarthropathy. A significant effect (p<0.05) of time, sex and pathology was observed for Coll 2-1 NO(2). Variations of Coll 2-1 were not significant except for the time effect. The elevation of Coll 2-1 NO(2) in the pathological group could indicate an inflammatory process during the growth of the affected horses, as nitration of tyrosine is mediated through reactive oxygen/nitrogen species and/or myeloperoxidase activity. Coll 2-1 NO(2) appears to be an interesting early marker of cartilage degradation and oxidation in degenerative osteoarthropathy.

A type II-collagen derived peptide and its nitrated form as new markers of inflammation and cartilage degradation in equine osteochondral lesions.

Markers of cartilage breakdown enable studying the degradation of cartilage matrix in equine joint pathologies. This study was designed to determine the levels of Coll2-1, a peptide of the triple helix of type II collagen, and Coll2-1NO(2), its nitrated form in the plasma of healthy horses (controls; n=37) and horses suffering from osteochondrosis (n=34). Clinical and arthroscopic scores were attributed reflecting the severity of lesions and were related to the plasma levels of Coll2-1 and Coll2-1NO(2). The median of Coll2-1 was significantly higher in the control group, whereas the mean of Coll2-1NO(2) showed significant elevation in the pathological group. However, the measurement means of scoring classes did not vary significantly. The markers were able to differentiate the group of horses suffering from osteochondrosis from the group of healthy horses. The elevation of Coll2-1NO(2) in the pathological group indicates an inflammation, mediated through reactive oxygen species and/or increased myeloperoxidase activity.

A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests.

An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.

Plasma concentrations of myeloperoxidase in endurance and 3-day event horses after a competition.

REASON FOR PERFORMING STUDY: In man, exercise of any type has been shown to induce neutrophil degranulation and respiratory burst activity, as well as an increase in plasma myeloperoxidase (MPO), a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Until now, it is not known whether this is the same in horses. OBJECTIVES: To study whether degranulation of blood neutrophils may be induced by exercise by measuring the total concentration of MPO and assess the possible influence of type of competition on this exercise-induced adjustment. METHODS: Blood was sampled before, and 30 min after, the course, in 9 ponies performing the Phase D of a national 3-day event championship (CIC*), and in 7 endurance horses participating at the European endurance championship 2005. White blood cells and granulocytes, total plasma proteins, creatine phosphokinase (CK), and total MPO contents were determined from blood samples. In addition, blood was taken from all ponies and 4 of the endurance horses 5 min after completion of the course to give some idea of the intensity of exercise. RESULTS: The mean blood lactate was 15.8 +/- 5.8 mmol/l after the CIC* and 2.7 +/- 0.2 mmol/l after the 160 km course. Performing both competitions induced a significant increase in CK and MPO. After the endurance course, the number of granulocytes significantly increased. Whilst there was no significant correlation between the measurements in CIC* ponies, MPO was significantly correlated with granulocyte count (r2 = 0.776) and CK (r2 = 0.586) in endurance horses. CONCLUSIONS: Intense exercise induces an activation of blood granulocytes, with degranulation of neutrophils and release of MPO. The plasmatic MPO concentration after endurance was higher than the values reported in some inflammatory pathological conditions. POTENTIAL RELEVANCE: This phenomenon may partly contribute to the occurrence of an exercise-induced oxidative stress and to the alteration of muscular membrane permeability. Further studies should be conducted to assess the possible relationship between MPO concentration and markers of oxidative stress in performance horses.

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.

Nitric oxide-related products and myeloperoxidase in bronchoalveolar lavage fluids from patients with ALI activate NF-kappa B in alveolar cells and monocytes.

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.

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells.

Singlet oxygen (1O2), a reactive oxygen species, has been found to be implicated in many cellular events and pathological disorders. Herein, we investigated the reactivity of 1O2 towards the anaesthetic agent propofol (PPF) encapsulated within DMPC liposomes. By time resolved luminescence, the rate constant of 1O2 quenching by PPF was evaluated, depending on the location of the sensitizer, with following values: 1.35+/-0.05x10(7) M(-1) s(-1) for deuteroporphyrin (as embedded source) and 0.8+/-0.04x10(7) M(-1) s(-1) for uroporphyrin (as external source), respectively. The nature of the oxidation product, resulting from the reaction of 1O2 with PPF, was determined using absorption and HPLC techniques. Finally, the in vitro protective effect of PPF towards the 1O2-induced neuronal cell toxicity was evaluated in terms of cell viability.

Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes.

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.