Differential effects of leflunomide and methotrexate on cytokine production in RA

Background

T cells are considered to be pivotal cells in the pathogenesis of rheumatoid arthritis (RA) and therefore represent a potential target for treatment. The novel disease-modifying antirheumatic drug (DMARD) leflunomide inhibits pyrimidine biosynthesis. T cells are especially susceptible to inhibition of this enzyme due to increased demand for pyrimidines after activation, together with the absence of a salvage pathway.

Objective

We investigated the effects of leflunomide on cytokine profiles in vivo and in vitro to provide more insight into the mechanism of action of leflunomide in RA.

Methods

Serum samples from 100 RA patients, treated with either leflunomide (n = 50) or methotrexate (n = 50), were collected at baseline, after 12 weeks and after 1 year of treatment. In these samples, serum levels of interleukin-6 (IL-6) and interferon gamma (IFN-γ) were determined by ELISA. The effects of the active metabolite of leflunomide (A77-1726; 0–200 μM) on IL-6 and IFN-γ production by peripheral blood mononuclear cells (PBMCs) from healthy volunteers (n = 6) and RA patients (n = 3) were studied by ELISA after activation (with phytohemagglutinin, lipopolysaccharide, and αCD3/αCD28) by ELISA. In addition, monocytes and lymphocytes were isolated from two healthy volunteers by density-gradient centrifugation methods, and effects of A77-1726 on IL-6 production after activation (with phytohemagglutinin or lipopolysaccharide) were measured by ELISA and PCR. Effects on cell proliferation (³H-thymidine incorporation), were measured as well.

Results

Serum levels of IFN-γ were significantly reduced after leflunomide treatment (baseline 43 pg/ml ± 10 [mean ± SEM]; 1 year 29 ± 7 [P = 0.015]), whereas we did not observe a change in IL-6 concentrations in serum (baseline 158 ± 41, 4 months 151 ± 48). In contrast, both IFN-γ and IL-6 serum levels were significantly reduced after methotrexate treatment. Consistent with these data, in vitro experiments revealed a dose-dependent inhibition of IFN-γ production by activated PBMCs in both healthy volunteers and RA patients in the presence of A77-1726, without a clear-cut effect on IL-6 production. IL-6 production by monocytes was not inhibited (measured by ELISA and PCR). Production of IFN-γ by lymphocytes was inhibited by A77-1726.

Conclusion

The results presented here show inhibition of IFN-γ production by leflunomide without a clear-cut effect on IL-6 production. This differential effect supports the hypothesis that leflunomide preferentially affects activated T cells. The effects on T cells could be explained by both DHODH inhibition and effects on signal transduction pathways.