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Anti-inflammatory effects of leflunomide in combination with methotrexate on co-culture of T lymphocytes and synovial macrophages from rheumatoid arthritis patients

M Cutolo¹, S Capellino¹, P Montagna¹, A Sulli¹, B Seriolo¹, B Villaggio²

¹ Research Laboratory and Division of Rheumatology, Department of Internal Medicine, University of Genoa, Genoa, Italy
² Research Laboratory and Division of Nephrology, Department of Internal Medicine, University of Genoa, Genoa, Italy

Correspondence to:
Professor Maurizio Cutolo
Research Laboratory and Division of Rheumatology, Department of Internal Medicine, University of Genoa, Viale Benedetto XV, 6 - 16132 Genoa, Italy; mcutolo{at}unige.it

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 (TNF, IL1ß, IL6), adhesion molecule ICAM-1, cyclooxygenase isoenzymes (COX1, COX2), and the nuclear factor B (NF-B) 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 (TNF 29%, 37%, 49%, IL1ß 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 (TNF 40%, 41%, 44%; IL1ß 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-B 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.

Abbreviations: DHODH, dihydro-orotate dehydrogenase; DPBS, Dulbecco’s phosphate buffered saline; DRMRI, dynamic gadolinium enhanced magnetic resonance imaging; ICC, immunocytochemistry; Lef, leflunomide; Lef-M, leflunomide metabolite; MCP-1, monocyte derived chemokine; MDC-1, macrophage derived chemokine; MTX, methotrexate; NF-B, nuclear factor B; PBMC, peripheral blood mononuclear cells; RA, rheumatoid arthritis; RT-MPCR, reverse transcriptase-multiplex PCR; SD, standard deviation; SM, synovial macrophages

Keywords: leflunomide; macrophages; methotrexate; rheumatoid arthritis; TNF

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