Abstract

Objectives: To investigate the anti-inflammatory effects of the active leflunomide metabolite A771726 (Lef-M) in combination with methotrexate (MTX) on synovial macrophages (SM) from rheumatoid arthritis (RA) patients co-cultured with an activated T cell line (Jurkat cell line).

Methods: Pro-inflammatory cytokines (TNFα, IL1β, IL6), adhesion molecule ICAM-1, cyclooxygenase isoenzymes (COX1, COX2), and the nuclear factor κB (NF-κB) complex were analysed on SM co-cultured with a T cell line, as intracellular protein expression by immunocytochemistry (ICC) and western blot analysis, as extracellular protein expression by ELISA assay, and as mRNA expression by reverse transcriptase-multiplex PCR (RT-MPCR) after treatment with Lef-M (1, 10, 30 μmol/l) alone or in combination with MTX (50 ng/ml).

Results: The most significant intracellular decrease in cytokines was observed by ICC in SM treated with the combination of Lef-M (1, 10, 30 μmol/l) and MTX (50 ng/ml) versus untreated SM (TNFα 29%, 37%, 49%, IL1β 56%, 43%, 50%, and IL6 59%, 62%, 71%, respectively). Furthermore, a significant decrease was confirmed concerning cytokine levels evaluated by ELISA in the medium of SM treated with the combination Lef-M+MTX (TNFα 40%, 41%, 44%; IL1β 10%, 20%, 60%; IL6 37%, 41%, 49%, respectively). Western blot and RT-PCR analysis confirmed these results. Concordant decreased expression was observed for ICAM-1, COX1, COX2, and the NF-κB complex after Lef-M+MTX treatment.

Conclusions: The combination of MTX and Lef-M shows additive inhibitory effects on the production of inflammatory mediators from SM co-cultured with a T cell line. These observations might support the positive results obtained in RA clinical studies by combination therapy.