Tumor Necrosis Factor [alpha] Induces Sustained Signaling and a Prolonged and Unremitting Inflammatory Response in Rheumatoid Arthritis Synovial Fibroblasts

Objective The nonresolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the tumor necrosis factor [alpha] (TNF[alpha])-driven inflammatory program in human FLS. Methods FLS obtained from the synovial tissues of patients with RA or osteoarthritis were stimulated with TNF[alpha] and assayed for gene expression and cytokine production by real-time quantitative reverse transcription-polymerase chain reaction analysis and enzyme-linked immunosorbent assay. NF-[kappa]B signaling was evaluated by Western blotting. Histone acetylation, chromatin accessibility, and NF-[kappa]B p65 and RNA polymerase II (Pol II) occupancy at the interleukin-6 (IL-6) promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays. Results In FLS, TNF[alpha] induced prolonged transcription of messenger RNA (mRNA) for IL-6 and progressive accumulation of IL-6 protein over 4 days. Similarly, induction of mRNA for CXCL8/IL-8, CCL5/RANTES, matrix metalloproteinase 1 (MMP-1), and MMP-3 after TNF[alpha] stimulation was sustained for several days. This contrasted with the macrophage response to TNF[alpha], which characteristically involved a transient increase in the expression of proinflammatory genes. In FLS, TNF[alpha] induced prolonged activation of NF-[kappa]B signaling and sustained transcriptional activity, as indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL-6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors A20-binding inhibitor of NF-[kappa]B activation 3 (ABIN-3), IL-1 receptor-associated kinase M (IRAK-M), suppressor of cytokine signaling 3 (SOCS-3), and activating transcription factor 3 (ATF-3), which terminate inflammatory responses in macrophages. Conclusion TNF[alpha] signaling is not effectively terminated in FLS, which leads to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS in response to synovial TNF[alpha] contribute to the persistence of synovial inflammation in RA. [PUBLICATION ABSTRACT]