title: Anti-inflammatory effects of leflunomide in combination with methotrexate on co-culture of T lymphocytes and synovial macrophages from rheumatoid arthritis patients.
abstract: OBJECTIVE
  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 (TNFalpha, IL1beta, IL6), adhesion molecule ICAM-1, cyclooxygenase isoenzymes (COX1, COX2), and the nuclear factor kappaB (NF-kappaB) 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 micromol/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 micromol/l) and MTX (50 ng/ml) versus untreated SM (TNFalpha 29%, 37%, 49%, IL1beta 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 (TNFalpha 40%, 41%, 44%; IL1beta 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-kappaB 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.
authors.affiliation: Research Laboratory and Division of Rheumatology, Department of Internal Medicine, University of Genoa, Viale Benedetto XV, 6-16132 Genoa, Italy. mcutolo@unige.it
authors.forename: M
authors.initials: M
authors.lastname: Cutolo
authors.forename: S
authors.initials: S
authors.lastname: Capellino
authors.forename: P
authors.initials: P
authors.lastname: Montagna
authors.forename: A
authors.initials: A
authors.lastname: Sulli
authors.forename: B
authors.initials: B
authors.lastname: Seriolo
authors.forename: B
authors.initials: B
authors.lastname: Villaggio
mesh_terms: D000704:Analysis of Variance; D000893:Anti-Inflammatory Agents / Q000627:therapeutic use*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy* / Q000276:immunology; D015153:Blotting, Western / Q000379:methods; D018920:Coculture Techniques; D051545:Cyclooxygenase 1 / Q000235:genetics; D051546:Cyclooxygenase 2 / Q000235:genetics; D016207:Cytokines / Q000235:genetics / Q000276:immunology*; D004359:Drug Therapy, Combination; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis; D007555:Isoxazoles / Q000627:therapeutic use*; D019169:Jurkat Cells; D000077339:Leflunomide; D008264:Macrophages / Q000276:immunology; D008565:Membrane Proteins / Q000235:genetics; D008727:Methotrexate / Q000627:therapeutic use*; D016328:NF-kappa B / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000893:Anti-Inflammatory Agents; D016207:Cytokines; D007555:Isoxazoles; D008565:Membrane Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D018799:Intercellular Adhesion Molecule-1; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D000077339:Leflunomide; D008727:Methotrexate