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IntroductionHigh mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).MethodsHMGB1 was determined in human synovium using immunohistochemistry, comparing normal to OA. OA synoviocytes were incubated with HMGB1 at 15 or 25 ng/ml in the absence or presence of IL-1β (10 ng/ml). Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. Matrix metalloproteinase (MMP) activity was studied by fluorometric procedures and nuclear factor (NF)-κB activation by transient transfection with a NF-κB-luciferase plasmid.ResultsIn the normal synovium, HMGB1 was found in the synovial lining cells, sublining cells, and in the vascular wall cells. The distribution of HMGB1 in OA synovium was similar but the number of HMGB1 positive cells was higher and HMGB1 was also present in infiltrated cells. In normal synovial membrane cells, HMGB1 was found mostly in the nuclei, whereas in OA, HMGB1 was generally found mostly in the cytoplasm. In OA synoviocytes, HMGB1 alone at concentrations of 15 or 25 ng/ml did not affect the production of IL-6, IL-8, CCL2, CCL20, MMP-1 or MMP-3, but in the presence of IL-1β, a significant potentiation of protein and mRNA expression, as well as MMP activity was observed. HMGB1 also enhanced the phosphorylated ERK1/2 and p38 levels, with a lower effect on phosphorylated Akt. In contrast, JNK1/2 phosphorylation was not affected. In addition, HMGB1 at 25 ng/ml significantly potentiated NF-κB activation in the presence of IL-1β.ConclusionsOur results indicate that HMGB1 is overexpressed in OA synovium and mostly present in extracellular form. In OA synoviocytes, HMGB1 cooperates with IL-1β to amplify the inflammatory response leading to the production of a number of cytokines, chemokines and MMPs. Our data support a pro-inflammatory role for this protein contributing to synovitis and articular destruction in OA.

This study reveals the antiarthritic properties of CORM-3 in the CIA model and supports the notion that CO-RMs could be developed as a novel strategy for the treatment of inflammatory and arthritic conditions.

BackgroundOsteoarthritis (OA) is the most widespread degenerative joint disease. Inflamed synovial cells contribute to the release of inflammatory and catabolic mediators during OA leading to destruction of articular tissues. We have shown previously that CO-releasing molecules exert anti-inflammatory effects in animal models and OA chondrocytes. We have studied the ability of CORM-2 to modify the migration of human OA synoviocytes and the production of chemokines and other mediators sustaining inflammatory and catabolic processes in the OA joint.Methodology/Principal FindingsOA synoviocytes were stimulated with interleukin(IL)-1β in the absence or presence of CORM-2. Migration assay was performed using transwell chambers. Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. CORM-2 reduced the proliferation and migration of OA synoviocytes, the expression of IL-8, CCL2, CCL20, matrix metalloproteinase(MMP)-1 and MMP-3, and the production of oxidative stress. We found that CORM-2 reduced the phosphorylation of extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2 and to a lesser extent p38. Our results also showed that CORM-2 significantly decreased the activation of nuclear factor-κB and activator protein-1 regulating the transcription of chemokines and MMPs in OA synoviocytes.Conclusion/SignificanceA number of synoviocyte functions relevant in OA synovitis and articular degradation can be down-regulated by CORM-2. These results support the interest of this class of agents for the development of novel therapeutic strategies in inflammatory and degenerative conditions.

We have provided direct evidence that HO-1 down-regulates MMP-1, -3 and HMGB1 in osteoarthritic synoviocytes. HO-1 may be a potential strategy to control inflammatory and degradative processes in the progression of OA.