FIGURE 3.

Synovial fibroblast products suppress IFN-β expression and responsiveness in TNF-stimulated macrophages. (A) Macrophages stimulated by TNF for 16 h with synovial fibroblasts in standard co-cultures, transwell cultures or with synovial fibroblast supernatants were analyzed for mRNA levels by qPCR. The supernatant media (Fib^TNF Supe) was collected from independent fibroblast cultures stimulated with TNF for 16 h. (B) qPCR mRNA analysis of macrophages cultured with control media (media) or supernatant media (Fib^TNF Supe) from fibroblasts independently cultured with TNF for 16h. Supernatants obtained from TNF-induced fibroblast cultures were first treated with Infliximab (Ifx) to eliminate TNF activity or isotype control immunoglobulin (IgG). Following the 16h supernatant incubation, the macrophages were stimulated with IFN-β for 3h. mRNA levels were normalized to GAPDH and made relative to the TNF-induced macrophage alone sample (part A) or IFN-induced macrophage with control media and IgG (part B), which were set to 100. The mean level of IFNB mRNA relative to GAPDH mRNA (% GAPDH) in part (A) was: coculture, 0.1%; transwell, 0.1%; supernatant, 0.1%; and the IFN-β-induced responses in part (B): CXCL10, 800%; IFIT1, 140%. Mean of n=4 donor experiments; error bars represent standard error (SE); ***<p=0.0001; **<p=0.001; *<p=0.05.