Background Synovial fibroblasts (SF) are activated in rheumatoid arthritis (RA) and show upregulation of enzymes of the PGE2 pathway. HMGB1 has been shown to promote inflammation together with ligands such as LPS and IL-1β. As HMGB1 is overexpressed extracellularly in synovitis, it might constitute a potential inducer of the mPGES-1/Cox/PGE2 axis in RASF. To verify this hypothesis, we investigated the cytokine and prostanoid-inducing capacity of HMGB1 alone or together with IL-1β in RASF cultures. We also studied receptor requirements for PGE2 induction.
Objectives To characterize the involvement of the PGE2 pathway in the response of SF to HMGB1 alone or in combination with IL-1β.
Methods SF obtained from RA patients were stimulated with recombinant HMGB1 or HMGB1 purified from thymus alone or together with low dose IL-1β (0.05 or 0.5 ng/ml). Pretreatment with IL-1RA was used to block IL-1RI-mediated signalling. IL-6, IL-8, RANTES and MCP-1 levels were measured by CBA and ELISA. Expression of enzymes of the PGE2 pathway (mPGES-1, Cox-1, Cox-2, 15-PGDH) were analysed by Western blot. Prostanoid levels were measured by EIA and LC-MS/MS.
Results HMGB1 alone did not induce either cytokine production or PGE2 pathway up-regulation in SF. Low doses of IL-1β (0.05-0.5 ng/ml) resulted in limited effects on PGE2 production and cytokines. In contrast, stimulation of RASF with HMGB1 in combination with trace amounts of IL-1β (0.05 or 0.5 ng/ml) exerted synergistic effects on the induction of mPGES-1 and COX-2 expression as well as PGE2, IL-6, IL-8, MCP-1 and RANTES production when compared to IL-1b stimulation alone. PGE2 and cytokine production induced by HMGB1/IL-1β were completely abolished by pretreatment with IL-1RA, indicating that the synergistic effects were mediated through IL-1RI. Investigating the kinetics of the SF response, we showed that the induction of PGE2 precedes that of IL-6 and IL-8. In line with this result, NS-398, a Cox-2 inhibitor, reduced the IL-6 and IL-8 production after a 24h incubation, indicating that the IL-1β/HMGB1 combination upregulates the IL-6 and IL-8 production in part through prostanoid synthesis.
Conclusions HMGB1 amplified the inflammatory response to suboptimal amounts of IL-1β resulting in the induction of the mPGES-1/Cox-2/PGE2 axis and the production of PGE2 and pro-inflammatory cytokines in SF. Therefore, upregulation of PGE2 synthesis constitutes an additional mechanism through which HMGB1 could perpetuate inflammatory and destructive activities in the arthritic joint.
Disclosure of Interest None Declared
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