Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
A Novel Role for the Jak-Stat Pathway in Rheumatoid Arthritis: Inhibition of TNF-Induced T Cell Attracting Chemokine Synthesis in Fibroblast-Like Synoviocytes (FLS) by Tasocitinib.
Rosengren2, Sanna, Firestein2, Gary S., Boyle1, David L.
The pan-Janus kinase (Jak) inhibitor, tasocitinib (CP690,550) is currently in Phase III clinical trials for rheumatoid arthritis. Whereas the Jak-Stat signaling in lymphoid and myeloid lineage cells is well understood, less is known about its involvement in FLS signaling in the context of synovitis. Therefore, we measured the effect of tasocitinib on expression and secretion of inflammatory mediators by cytokine stimulated FLS.
Passage 38 RA FLS isolated from tissue obtained at the time of arthroplasty were serum-starved 48 h prior to stimulation. mRNA levels were determined by qPCR, and protein levels in supernatants determined by ELISA or multiplex bead assay. Phosphorylation of Stat proteins was determined by Western blot analysis.
Stimulating FLS with IL-6, but not TNF, induced phosphorylation of Stat1 and Stat3, downstream indicators of Jak activation. Stat activation was inhibited by tasocitinib with IC50 values of approximately 21 and 72 nM, respectively. IL-6 also induced expression of MCP1 mRNA and protein secretion but not other markers of FLS activation such as MMP3. MCP-1 expression was also completely prevented by tasocitinib (200 nM-1 mM). Unexpectedly, tasocitinib also inhibited TNF-induced gene expression and production of several other pro-inflammatory proteins. Time-course studies showed induction of IP-10, RANTES, and MCP1 mRNA expression and protein secretion after 7h but no inhibition by tasocitinib. However, tasocitinib significantly decreased IP-10, RANTES, and MCP-1 mRNA expression after 16 hours of TNF stimulation (96%, 67%, and 55% inhibition, respectively; p<0.001 for all). TNF-induced secretion of RANTES and MCP-1 protein at 18h was inhibited by 53% and 38%, respectively (p<0.005 for both). A smaller effect was observed on TNF-induced IL-6 and MMP1 expression, whereas other TNF-induced genes like IL-8 and MMP3 were unaffected by tasocitinib. mRNA stability studies showed that the effect of the Jak inhibitor was independent of mRNA half life. However, blocking de novo protein synthesis with cycloheximide abolished the inhibitory effect of tasocitinib on TNF induced IP-10 and RANTES mRNA expression.
TNF induces FLS expression of several chemokines involved in T cell recruitment in a manner that indirectly depends on Jak-Stat activation. TNF-induced FLS expression of these chemokines requires de novo protein synthesis and could involve disrupting late autocrine cytokine networks mediated by Jak-Stat signaling. These findings illuminate a potential novel mechanism of tasocitinib in rheumatoid arthritis: not only does the compound directly inhibit cytokine signaling in T cells, but it also diminishes the production of chemokines by synovial fibroblasts and could limit recruitment of T cells and other infiltrating leukocytes to the synovium.
To cite this abstract, please use the following information:
Rosengren, Sanna, Firestein, Gary S., Boyle, David L.; A Novel Role for the Jak-Stat Pathway in Rheumatoid Arthritis: Inhibition of TNF-Induced T Cell Attracting Chemokine Synthesis in Fibroblast-Like Synoviocytes (FLS) by Tasocitinib. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1240