Differential effects of leflunomide and methotrexate on cytokine production in rheumatoid arthritis

Abstract

Background: T cells have a pivotal role in RA. Leflunomide inhibits pyrimidine biosynthesis, to which T cells are especially susceptible, and therefore may have a different cytokine profile than methotrexate.

Materials and methods: Serum samples of 100 patients with RA, treated with leflunomide (n = 50) or methotrexate (n = 50), were collected at baseline, after 16 weeks and after 1 year's treatment. Serum levels of interleukin 6 (IL6), and interferon (IFN) gamma were determined by ELISA. Additionally, peripheral blood mononuclear cells (PBMC) of five healthy volunteers and three patients with RA were isolated and the effects of the active metabolite of leflunomide (A77-1726, 0-200 mmol/l) on cell proliferation and on IL6 and IFNgamma production were determined by ELISA. In peripheral blood lymphocytes (PBL) and monocytes (PBM) from two healthy volunteers the effects of A77-1726 on IL6 production were measured by ELISA and PCR.

Results: Mean (SEM) serum levels of IFNgamma were significantly reduced after leflunomide treatment (baseline 43 (10) pg/ml; 1 year 29 (7) (p = 0.015), but there was no change in IL6 levels (baseline 158 (41), 1 year 151 (48)). Both IFNgamma and IL6 levels were significantly reduced after methotrexate treatment. This observation was supported by in vitro experiments. The production of IFNgamma by PBL was inhibited by A77-1726, but IL6 production by PBM was not inhibited.

Conclusion: The differential effect on IFNgamma and IL6 production supports the hypothesis that activated T cells are preferentially inhibited by leflunomide. An explanation may be either inhibition of uridine synthesis or effects on signal transduction pathways.

Figure 1

(A) Incorporation of [³H]thymidine by PBMC of healthy donors and patients with RA after stimulation with LPS. Graph depicts the number of counts as measured after 24 hours of incubation in the presence of various concentrations of leflunomide (0–100 µmol/l). (B) Production of IFNγ (pg/ml) by PBMC after 36 hours. Depicted are controls and cells stimulated with PHA in the presence of various concentrations of leflunomide (0–100 µmol/l). (C) Production of IL6 (pg/ml) by PBMC after 8 hours. Depicted are controls and cells stimulated with PHA in the presence of various concentrations of leflunomide (0–100 µmol/l).

Figure 2

Analysis of ß₂-microglobulin and IL6 mRNA levels in PHA stimulated monocytes. A constant volume of the ß₂-microglobulin and IL6 cellular cDNA products was mixed with graded amounts of a known concentration of pQA1 DNA (st-DNA) containing the specific sequences for the ß₂-microglobulin and IL6 PCR primers. PCR was performed, and the PCR products were separated by electrophoresis on a 1% agarose gel and visualised after ethidium bromide staining under ultraviolet light. The concentration of st-DNA that gave an amount of PCR product equal to that of the cellular DNA ß₂-microglobulin or IL6 PCR products, respectively, was determined. The intensity of the bands was quantified by densitometry. The density of the st-DNA was expressed as a percentage of the total density of st-DNA and cellular cDNA. Results are presented in the absence of A77-1726, and the presence of 10 µM and 100 µM A77-1726.