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

Abstract

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 (TNF)driven inflammatory program in human FLS. Methods FLS obtained from the synovial tissues of patients with RA or osteoarthritis were stimulated with TNF and assayed for gene expression and cytokine production by real-time quantitative reverse transcriptionpolymerase chain reaction analysis and enzyme-linked immunosorbent assay. NF-B signaling was evaluated by Western blotting. Histone acetylation, chromatin accessibility, and NF-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 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 stimulation was sustained for several days. This contrasted with the macrophage response to TNF, which characteristically involved a transient increase in the expression of proinflammatory genes. In FLS, TNF induced prolonged activation of NF-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-B activation 3 (ABIN-3), IL-1 receptorassociated 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 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 contribute to the persistence of synovial inflammation in RA.