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. 2004 Nov;63(11):1453–1459. doi: 10.1136/ard.2003.013011

Effects of RWJ 67657, a p38 mitogen activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts

J Westra 1, P Limburg 1, P de Boer 1, M H van Rijswijk 1
PMCID: PMC1754789  PMID: 15479895

Abstract

Objective: To investigate the effect of the p38 mitogen activated protein kinase (MAPK) inhibitor RWJ 67657 on inflammatory mediator production by rheumatoid synovial fibroblasts (RSF).

Methods: RSF were pretreated with RWJ 67657 and stimulated with TNFα and/or IL-1ß. Protein levels and mRNA expression of MMP-1, MMP-3, TIMP-1, IL-6, and IL-8 were determined, as was mRNA expression of COX-2 and ADAMTS-4.

Results: MMP-3 production was significantly inhibited at 1 µM RWJ 67657 and MMP-1 production at 10 µM, while TIMP-1 production was not inhibited. Inhibition of IL-6 and IL-8 protein production was seen at 0.1 µM RWJ 67657. Expression profiles of mRNA were in accordance with protein production. Inhibition of COX-2 mRNA expression occurred at 0.01 µM RWJ 67657.

Conclusions: RWJ 67657 inhibits major proinflammatory mediator production in stimulated RSF at pharmacologically relevant concentrations. These findings could have important relevance for the treatment of rheumatoid arthritis.

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Figure 1.

Figure 1

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 Protein production of MMP-1, MMP-3, TIMP-1, IL-6, and IL-8 by rheumatoid synovial fibroblasts (n = 5). Cells were stimulated with TNFα and/or IL-1ß for 48 hours and pretreated with a concentration range of RWJ 67657 (t = –1 h). Protein production was measured in supernatants by ELISA and expressed in ng/ml. unst, unstimulated; 0–10, concentration RWJ 67657 added. Bars show mean and SEM. *p<0.05, **p<0.001, Dunnett's post test, tested against the stimulated control. IL, interleukin; MMP, matrix-metalloproteinase; TIMP-1, tissue inhibitor of matrix-metalloproteinases; TNF, tumour necrosis factor.

Figure 2.

Figure 2

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 mRNA expression of MMP-1, MMP-3, TIMP-1, and ADAMTS-4 in rheumatoid synovial fibroblasts (n = 3). Cells were stimulated with TNFα and/or IL-1ß for six and 24 hours and pretreated with a concentration range of RWJ 67657. mRNA expression was determined with real time RT-PCR (reverse transcriptase polymerase chain reaction) and results were expressed as -fold induction compared with unstimulated cells (fold induction = 1). White bars represent values after TNFα stimulation (scale on left vertical axis); grey bars represent values after IL-1ß stimulation and black bars after IL-1ß+TNFα stimulation (scale on right vertical axis). Bars represent means and SEM. *p<0.05, Bonferroni multiple comparison test, tested against the stimulated control. ADAMTS, a disintegrin and metalloprotease with thrombospondin-1 motif; MMP, matrix-metalloproteinase; TIMP-1, tissue inhibitor of matrix-metalloproteinases.

Figure 3.

Figure 3

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 mRNA expression of IL-6, IL-8, and COX-2 in rheumatoid synovial fibroblasts (n = 3). Cells were stimulated with TNFα and/or IL-1ß for six hours and pretreated with a concentration range of RWJ 67657. mRNA expression was determined with real time RT-PCR (reverse transcriptase polymerase chain reaction) and results are expressed as -fold induction compared with unstimulated cells (-fold induction = 1). White bars represent values after TNFα stimulation (scale on left vertical axis); grey bars represent values after IL-1ß stimulation and black bars after IL-1ß+TNFα stimulation (scale on right vertical axis). Bars represent means and SEM. *p<0.05, Bonferroni multiple comparison test, tested against the stimulated control. COX, cyclo-oxygenase; IL, interleukin; TNF, tumour necrosis factor.

Figure 4.

Figure 4

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 (A) Representative presentation of phosphorylation of p38 MAPK in rheumatoid synovial fibroblasts after stimulation with TNFα and/or IL-1ß at different time points. (B) Phosphorylation was measured by Western blot using specific antibodies. Effect of RWJ 67657 on phosphorylation of the direct substrate of p38 MAPK, MAPKAPK-2, measured after 30 minutes of stimulation. IL, interleukin; MAPK, mitogen activated protein kinase; TNF, tumour necrosis factor.

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