Anti-inflammatory effects of leflunomide on cultured synovial macrophages from patients with rheumatoid arthritis

• Published in: Annals of the rheumatic diseases Vol. 62; no. 4; pp. 297 - 302
• Main Authors: Cutolo, M, Sulli, A, Ghiorzo, P, Pizzorni, C, Craviotto, C, Villaggio, B
• Format: Journal Article
• Language: English
• Published: London BMJ Publishing Group Ltd and European League Against Rheumatism 01.04.2003; BMJ; BMJ Publishing Group LTD
• Subjects: Aniline Compounds - pharmacology; Anti-Inflammatory Agents, Non-Steroidal - pharmacology; Antirheumatic Agents - pharmacology; Arthritis, Rheumatoid - pathology; Biological and medical sciences; Bones, joints and connective tissue. Antiinflammatory agents; Cell adhesion & migration; Cell cycle; Cell Survival - drug effects; Cells, Cultured; COX-2; cyclo-oxygenase 2; Cyclooxygenase 2; Cytokines; Cytokines - biosynthesis; Cytoplasm - metabolism; Dose-Response Relationship, Drug; DRMRI; Drug dosages; dynamic gadolinium enhanced magnetic resonance imaging; Enzymes; Extended Report; Female; Humans; Hydroxybutyrates - pharmacology; ICAM-1; Immunoassay; Inflammation; intercellular adhesion molecule-1; Intercellular Adhesion Molecule-1 - metabolism; interleukin; Isoenzymes - biosynthesis; leflunomide; Lymphocytes; Macrophage Activation - drug effects; macrophages; Macrophages - drug effects; Macrophages - metabolism; Medical sciences; Membrane Proteins; methyl thiazolyl tetrazolium; Middle Aged; MTT; PBS; PBS/T; Pharmacology. Drug treatments; phosphate buffered saline; phosphate buffered saline/Tween 20; Prostaglandin-Endoperoxide Synthases - biosynthesis; Rheumatoid arthritis; Synovial Membrane - drug effects; Synovial Membrane - metabolism; Synovial Membrane - pathology; TNFα; Tumor Necrosis Factor-alpha - biosynthesis; Tumor necrosis factor-TNF; tumour necrosis factor α; Italy; Immunohistochemistry; Prostaglandin-endoperoxide synthase; Cell culture; Isoxazole derivatives; Metabolite; Diseases of the osteoarticular system; Autoimmune disease; Inflammatory joint disease; Leflunomide; Immunological method; Histopathology; Synovial membrane; Human; Immunopathology; Enzyme; Antiinflammatory agent; Cytokine; Cell adhesion molecule; Enzyme inhibitor; In vitro; Biological activity; Pathology; Chronic; Treatment; Rheumatoid arthritis; Oxidoreductases; Macrophage
• ISSN: 0003-4967; 1468-2060
• DOI: 10.1136/ard.62.4.297

Background: Leflunomide and its active metabolite A77 1726 reversibly inhibits the enzyme dihydro-orotate dehydrogenase, the rate limiting step in de novo synthesis of pyrimidines and progression of the cell cycle in different cell lines, mainly activated T lymphocytes. Objective: To analyse in vitro the possible anti-inflammatory effects exerted by A77 1726, on cultured macrophages, obtained from the synovial tissues of patients with rheumatoid arthritis (RA). Methods: The effects of different doses of A77 1726 on intracytoplasmic expression and extracellular concentration of inflammatory cytokines (tumour necrosis factor α (TNFα), interleukin (IL) 1β, IL6), as well as the influence on production and expression of intercellular adhesion molecule-1 (ICAM-1) and cyclo-oxygenase 2 (COX-2) by primary cultures of synovial macrophages from patients with RA, were evaluated by immunocytochemistry and western blot analysis. The observations were made at four and 24 hours. Results: A progressive and significant time and dose dependent decrease of the number of positive macrophages for intracellular TNFα and IL1β, treated with different doses of A77 1726, was found in comparison with untreated cells. The extracellular concentration of TNFα was found to be significantly decreased in media containing cultured macrophages at 24 hours for all tested doses of A77 1726. At 24 hours, a significant time and dose dependent decrease of ICAM-1 and COX-2 expression by cultured macrophages after A77 1726 treatment was found. Conclusions: In conclusion, the mechanism of antiproliferative activity exerted by leflunomide on activated T lymphocytes seems to be the same mechanism (alteration of the cell cycle progression) which interferes with the functions of other activated cells—namely, the monocytes/macrophages, which are strongly involved in the inflammatory reaction in RA synovial tissue. The positive clinical results seem to confirm that leflunomide exerts an anti-inflammatory action on phagocytic cells in short and long term treatment of RA.