Background The mechanism by which methotrexate (MTX), a well known antifolate inhibits inflammation is still unknown. The inflammation in rheumatoid arthritis is mediated by pro-inflammatory cytokines such as TNFα and IFNγ.

Objectives To assess whether cytokine production in blood of RA patients is inhibited after taking MTX.

Methods Blood was obtained from 25 RA patients at T = 0 h and T = 2 h after taking MTX (10 mg or) for the first time. Heparinized Whole Blood Cultures (WBC; dilution 1/4) using anti-CD3 and anti-CD28 as stimulus were performed, with and without addition of folinic acid (40 g/ml) to the culture. Because MTX has been reported to induce IL-10 production all experiments were also carried out with and without excess neutralising mAb to IL-10. After incubation for 72 h supernatants were measured by ELISA.

Results IFNγ production by WBC of 25 RA patients before (T = 0 h) and after (T = 2 h) MTX.

Conclusion MTX inhibits ex vivo IFNγ production in RA patients by a folate dependent mechanism. MTX does not act via induction of IL-10. Similar results were found for TNFα and GM-CSF. IL-8 production is not affected by MTX.