Methotrexate, through adenosine release, downregulates tumor necrosis factor alpha-induced synovial fibroblast IL-15 expression and proliferation

Tumor necrosis factor alpha (TNF-α) is found at high concentrations in the rheumatoid joint and induces upregulation of synovial fibroblast IL-15, a cytokine known to induce fibroblast proliferation through an autocrine loop. The mechanism of action of low-dose oral methotrexate (MTX) is not well understood. By inhibiting AICAR transformylase, MTX has been described to induce the extra-cellular release of the potent anti-inflammatory autacoid adenosine.

To test the effect of MTX on TNF-α-induced synovial fibroblast IL-15 expression and proliferation.

Synovial fibroblasts (Sfib) isolated from surgical specimens of rheumatoid arthritis (RA) patients (n = 10) were cultured in six-well plates. Sfib were stimulated with 10 ng/ml TNF-α and pretreated for 2 hours with medium or MTX at varying doses (0, 0.01, 0.1, 1, 10 μM) in the presence or absence of adenosine deaminase, DPCPX (adenosine A1 receptor antagonist) or DMPX (adenosine A2 receptor antagonist). Surface and intracellular IL-15 protein were determined by immunofluorescence and flow cytometry. IL-15 mRNA was measured by real-time quantitative RT-PCR in a LightCycler instrument (Roche). Sfib proliferation was evaluated using the alamar blue bioassay.

TNF-α induced an upregulation of intracellular and surface IL-15 protein expression and of IL-15 mRNA, together with an increased Sfib proliferation rate. Sfib proliferation was significantly inhibited by a neutralizing anti-IL15 monoclonal antibody but not by an isotype control antibody. In the presence of MTX, TNF-α-induced IL-15 upregulation and Sfib proliferation were reduced by 40–65% in a dose-dependent manner. Adenosine deaminase reversed the effects of MTX as did addition of DMPX but not DPCPX 1 hour prior to MTX.

MTX interferes with TNF-α-induced synovial fibroblast IL-15 upregulation and proliferation. This effect seems to be mediated through adenosine release and adenosine A2 receptor engagement. This may be a mechanism by which MTX controls the aggressive behavior of synovial fibroblasts in RA.