Objectives: To characterize the molecular events underlying cartilage injury in the early phase of mono-iodoacetate-induced osteoarthritis (OA) in rats.Methods: Experimental osteoarthritis was induced by intra-articular injection of 0.03 mg mono-iodoacetate (MIA) in Wistar rats. Animals were killed 2, 5, 10, 15 and 20 days post-injection. Synovial tissue and standardized biopsies from different areas of knee cartilage were examined. Proteoglycan synthesis (S-35 incorporation) and gelatinase activities (zymography), semi-quantitative RT-PCR and immunohistochemistry for IL1beta, iNOS, COX2 and PPARgamma, were performed on these samples.Results: Changes in proteoglycan synthesis and gelatinase activities were time and site-dependent. Proteoglycan synthesis inhibition was maximal by day 2 while the highest gelatinase activities were observed at day 5. Central part of patella and posterior plateaus and condyles, i.e. the weight-bearing cartilage areas, were the most affected.1L1beta and iNOS transcripts were induced early in cartilage at time of maximal proteoglycan synthesis inhibition, especially in weight-bearing areas. COX-2 was slightly up-regulated whereas PPARgamma gene expression remained unchanged. Gene expression profile in synovium paralleled that of cartilage, except for PPARgamma which was up-regulated at day 15 and 20.Immunostaining for IL1beta and iNOS showed that proteins were located in diseased cartilage areas at early stage of the experimental OA (day 2). At later time-points (day 20), IL1beta and iNOS were expressed in perilesional areas whereas immunostaining became below control level for COX-2 and PPARgamma.Conclusions: Time-dependent degradation of cartilage after injection of low dose of MIA (0.03mg) into rat knee joint can be related to early loss of proteoglycan anabolism, increased gelatinase activities and expression of IL1beta and downstream inflammatory genes. Increased susceptibility to MIA in weight-bearing areas of cartilage further indicate that MIA-induced experimental OA is a relevant model to study not only metabolical but also biomechanical aspects of human OA. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

OBJECTIVE:To compare the potency of 2 cytokines, interleukin 17 (IL-17) and IL-1beta, on rat cartilage proteoglycan synthesis with special attention to nitric oxide (NO) and peroxynitrite formation. METHODS:Chondrocytes in alginate beads were stimulated with human recombinant (rh) IL-17 (0.03 to 300.0 ng/ml) and/or rhIL-1beta (0.25 to 25.0 ng/ml) in the presence or not of L-NMMA or CuDips. Alternatively, rats were injected with either IL-17 (10.0 micro g) or IL-1beta (1.0 micro g) into each knee joint. NO concentrations were determined by a spectrofluorimetric assay, proteoglycan synthesis by 35SO4-2 incorporation, peroxynitrite generation by immunostaining for 3-nitrotyrosine, and IL-1beta mRNA expression by reverse transcription-polymerase chain reaction. RESULTS:IL-17 inhibited proteoglycan synthesis and increased NO production, both in vitro and in vivo, without inducing expression of IL-1beta mRNA in cartilage. Additive effects were observed when IL-17 was combined with low concentrations of IL-1. Surprisingly, a similar NO synthesis between IL-1 and IL-17 led to a less suppressive effect of IL-17 on cartilage anabolism than with IL-1. Both in vitro and in vivo, peroxynitrite formation was extensive with IL-1beta, but negligible or nonexistent with IL-17. L-NMMA and CuDips completely corrected the suppressive effect of IL-1beta on proteoglycan synthesis, unlike with IL-17. CONCLUSION:These data showed that NO is weakly involved in the IL-17 mediated inhibition of proteoglycan synthesis in rat. NO overload may not be predictive of any inhibitory effect on cartilage anabolism, but instead superoxide is a key regulator of NO contribution to chondrocyte dysfunction. Since IL-17 is a NO-producing cytokine with additive effects when combined with IL-1, it may play a pivotal role in cartilage destruction during rheumatoid arthritis, for which infiltrating cells produce high levels of superoxide and proinflammatory cytokines.

Nitric oxide (NO) is thought to mediate most effects of interleukin-1 (IL-1) on cartilage. In vitro evidence includes the decreased synthesis of extracellular matrix components, the abnormal cell renewal, the decreased production of IL-1 receptor antagonist, the induction of apoptosis and the enhanced sensitivity of chondrocytes to oxidative stress. Studies in NOS2(-/-) mice or administration of NO synthase inhibitors in animal models of joint disorders have confirmed its potent pathophysiological role in cartilage. Using L-NMMA (1 mM), as a NO synthase inhibitor, and CuDips (10 microM), as a SOD mimetic, we provide evidence that the inhibitory potency of IL-1beta on proteoglycan synthesis and its stimulating effect on COX-2 activity depend both on NO and O2-* production. Peroxynitrite formation is further demonstrated by the occurrence of 3-nitrotyrosines in chondrocytes stimulated in vitro with 2.5 ng/ml IL-1 and in femoral condyles of rats injected locally with 1 microg IL-1. Preliminary data suggest that such contribution of reactive oxygen species is not shared in common by IL-17, another NO-producing cytokine. We conclude that superoxide is a key modulator of NO-mediated effects in chondrocyte stimulated with IL-1 and that a combined therapy with NO synthase inhibitors and antioxidants may be promising for a full cartilage protection.