Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes

Abstract

Objective To examine the potential of methotrexate (MTX) to acts as a differentiation‐stimulating factor for monocytes, which could explain the antiinflammatory properties of this agent in the treatment of rheumatoid arthritis (RA). Methods Fluorescence‐activated cell sorter analysis was used to measure the changes in antigen expression (CD11b/c, CD16, CD64, CD14, CD68, and CD95) in response to MTX, 1,25‐OH‐cholecalciferol (1,25‐OH‐CCF), and granulocyte‐macrophage colony‐stimulating factor in the human monoblastic leukemia cell line U937, bone marrow mononuclear cells (BMMC), and peripheral blood mononuclear cells (PBMC). Release of interleukin‐1β (IL‐1β), IL‐1 receptor antagonist (IL‐1Ra), tumor necrosis factor α, and soluble tumor necrosis factor receptors (sTNFR) p55 and p75 during the differentiation in vitro was assessed by immunoassay in the culture supernatants. Results MTX alone and in combination with 1,25‐OH‐CCF markedly stimulated the differentiation of the monocytic U937 cells and simultaneously increased Fas‐antigen expression. Differentiation was associated with enhanced IL‐1Ra and sTNFR p75 release from U937 cells. MTX had fewer effects on phenotypic differentiation of human BMMC and PBMC, but did stimulate IL‐1Ra release and inhibit IL‐1β synthesis in BMMC. Conclusion MTX acts as a strong differentiation factor for immature and undifferentiated monocytic cells. Differentiation in vitro is associated with an increase in natural cytokine inhibitor release and a simultaneous down‐regulation of IL‐1β. These findings may explain the marked clinical antiinflammatory effects of MTX when used in the treatment of RA.

The anti‐inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species

“…Treatment of U937 monocytes with MTX at low (30 n M ) and high (1 μ M ) induced a differentiated like phenotype with the enhanced expression of the αM‐integrin, CD11b as reported by Seitz et al . (1998).…”

Anti-inflammatory mechanisms of methotrexate in rheumatoid arthritis

“…In addition, MTX was found to inhibit markedly the spontaneous proliferation of U937 monoblastic leukaemia cells in vitro and induce the rapid expression of the apoptosis receptor CD95 also in presence of 1,25-OH-cholecalciferol 56. Results of another recent investigation, are in agreement with these latter studies and seem to suggest that intermediate MTX concentrations (50 μg/ml), as obtained in serum after low dose treatment, can induce both a significant cell growth inhibition and apoptosis, at least in monocytic immature cells (THP-1 cell line)57 (fig 2A).…”

Monocytes/macrophages express chemokine receptor CCR9 in rheumatoid arthritis and CCL25 stimulates their differentiation

“…Monocytes migrate from the blood across the walls of synovial blood vessels and differentiate into macrophages. The clinical importance of monocytes/macrophages is revealed by the correlation between their number, disease activity and radiographic progression [2-4] and by the beneficial effect of therapies that target these cells (such as gold salts [5], methotrexate [6]) and monocyte depletion [7,8]. Furthermore, monocytes/macrophages are a major source of TNFα in the RA joint and blocking this cytokine has had a major impact on RA therapy [9,10].…”