Effect of P38 mapkinase inhibitor RWJ-67657 on proinflammatory mediators produced by IL-1β-and/or TNFα-stimulated rheumatoid synovial fibroblasts

Introduction

The p38-mitogen activated protein kinase (p38 MAPK) inhibitor RWJ-67657 has been shown to effectively suppress clinical and cytokine response to endotoxin in healthy human volunteers (Fijen JW: JCI 2001, 124:16). In patients with rheumatoid arthritis (RA) p38-MAPK activity is observed in the synovial lining layer (Schett G, Arthritis Rheum 2000, 43:2501). To further characterise the role of p38-MAPK in RA the effect of RWJ-67657 on IL-1β and/or TNFα induced mRNA expression and production of cytokines (IL-6 and IL-8) and matrixmetalloproteinases (MMP-1, MMP-3 and TIMP-1) as well as on mRNA expression of ADAMTS-4 (aggrecanase) and COX-2 was studied in cultured synovial fibroblasts.

Methods

Rheumatoid synovial fibroblasts were isolated from patients who underwent a total joint replacement. Cells at passage 3–8 were stimulated with 1 ng/ml IL-1β and/or TNFα with or without preincubation with 0.001–30 μM RWJ-67657. RNA isolation and RT-PCR was performed after 6 hour stimulation and protein levels in supernatants were measured by ELISA after 48 hour stimulation.

Results

RWJ-67657 induced a dose-related decrease in IL-6, IL-8 and MMP-3 production, both after IL-1β and TNF-α stimulation. Inhibition of MMP-1 was seen only at high levels of RWJ-67657. TIMP-1 was produced constitutively and was not affected by stimulation or inhibition. These findings were all confirmed by mRNA expression studies. COX-2 mRNA expression was induced both by IL-1β and TNFα and could be inhibited by RWJ-67657. ADAMTS-4 mRNA expression was only seen after IL-1β stimulation, which could be inhibited by RWJ-67657.

Conclusion

RWJ-67657 is a potent inhibitor of cytokine and MMP production, as well as of their mRNA expression in stimulated RA synovial fibroblasts. Also COX-2 and aggrecanase mRNA expression was inhibited by RWJ-67657. Thus, inhibition of the p38 MAPK pathway by RWJ-67657 effectively leads to inhibition of different inflammatory mediators produced by rheumatoid synovial fibroblasts. Therefore, RWJ-67657 could be of therapeutic significance in RA.