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Ann Rheum Dis 65:728-735 doi:10.1136/ard.2005.045641
  • Extended report

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

  1. M Cutolo1,
  2. S Capellino1,
  3. P Montagna1,
  4. A Sulli1,
  5. B Seriolo1,
  6. B Villaggio2
  1. 1Research Laboratory and Division of Rheumatology, Department of Internal Medicine, University of Genoa, Genoa, Italy
  2. 2Research Laboratory and Division of Nephrology, Department of Internal Medicine, University of Genoa, Genoa, Italy
  1. 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
  • Accepted 24 October 2005
  • Published Online First 3 November 2005

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.

Footnotes

  • Published Online First 3 November 2005

  • This study was supported by a bursary for a young scientist from Aventis

  • Competing interests: none declared