Synovial fibroblasts display an uncontrolled inflammatory and tissue destructive response to TNF-α

Background

Synovial fibroblasts are key players in the pathogenesis of Rheumatoid Arthritis (RA) and potentially attractive treatment targets. Upon activation within the joint's inflammatory milieu, they gain a transformed phenotype and produce pro-inflammatory cytokines (mainly IL-6) and tissue destructive enzymes [1].

Materials and methods

Synovial fibroblasts were isolated via enzymatic processing from synovial tissues obtained from patients with RA or Osteoarthritis (OA). Synovial fibroblasts (passages 2-4) were stimulated with TNF-α (10 ng/ml) only on day 1. The expression of TNF-α-target genes was measured by qPCR in time course experiments (1, 3, 6, 24, 48, 72, 96 and 120 hours after TNF-α stimulation).

Human macrophages (Mϕ) generated in vitro (blood derived CD14+ cells stimulated for 48 h with M-CSF) were used in similar time course experiments as controls.

Results

In Mϕ it was observed a rapid (within 1-3 hours) induction of TNF-α-target genes (including TNF-a, IL-1β, IL-6 and IL-8) that was restrained back to the baseline within a few hours (3-24 hours depending on the gene). In stark contrast, synovial fibroblasts displayed a remarkably more sustained response to TNF-α. IL-6 mRNA expression was induced within a few hours by TNF-α, and induction increased continuously for 72-96 h despite the absence of any further exogenous TNF-α stimulation. The levels of IL-6 mRNA induced by TNF-α in synovial fibroblasts were substantially higher compared to human Mϕ, suggesting that within the joint microenvironment, synovial fibroblasts and not Mϕ are the main source of IL-6. By adding the supernatants from 96 h TNF-α-stimulated fibroblast cultures on unstimulated synovial fibroblasts, a similar robust induction of IL-6 mRNA was observed, suggesting that there is a TNF-α-induced soluble factor that mediates the sustained response. A similar pattern of sustained expression was observed for other TNF-α-target genes including IL-1β, IL-8 and MMPs. Interestingly, there was no difference between OA- and RA-derived synovial fibroblasts in their response to TNF-α.

Conclusions

In contrast to human Mϕ, synovial fibroblasts display a sustained inflammatory and tissue destructive response to TNF-α. Our observations suggest that synovial fibroblasts may lack the homeostatic mechanisms that control and terminate the effects of TNF-α on human Mϕ [2]. To support this hypothesis, further investigation is needed at the level of proximal and distal TNF-α signaling events and at the level of epigenetic regulation of TNF-α-target genes in synovial fibroblasts.