OBJECTIVE: Rheumatoid arthritis (RA) is a chronic autoimmune disease. Macrophage migration inhibitory factor (MIF) has been shown to be an important pro-inflammatory cytokine in RA. The aim of this study was to determine if the engagement of toll-like receptor 3 (TLR3) induces the production of MIF in the fibroblast-like synoviocytes (FLS) of patients with RA. METHODS: The expression of inflammatory cytokines (e.g. MIF, IL-6, IL-1beta and TNFalpha) and toll-like receptors (e.g. TLR2, TLR3 and TLR4) in the synovial tissue were quantified by immunohistochemistry. FLS were isolated from the synovial tissues of patients with RA and stimulated with TLR-3 ligand polyI:C, in the presence of a neutralizing antibody against IL-6. The concentrations of MIF and IL-6 in the culture supernatants from the FLS were measured using sandwich ELISA. RESULTS: The engagement of TLR3 with PolyI:C increased the production of MIF in FLS. The stimulatory effect of these TLR ligands showed a dose-dependent trend. The combination of TLR3 and TLR4 synergistically increased the level of MIF and IL-6 production. The addition of neutralizing antibodies against IL-6 abrogated the stimulatory effect of the ligands of TLR3 and TLR4 on the production of MIF. CONCLUSION: These results show that TLR3 engagement stimulates the production of MIF and IL-6. Therefore, the TLRs help perpetuate of RA pathogenesis through production of MIF from the FLS in patients with RA, and might provide a new therapeutic approach for the treatment of rheumatoid arthritis.