The influence of leflunomide on cell cycle, IL-2-receptor (Il-2-R) and its gene expression

Abstract

Leflunomide is a novel immunomodulatory drug shown to be very effective in animal models of autoimmune diseases and transplantation rejection, as well as in human rheumatoid arthritis. Leflunomide's main metabolite, A77 1726, has been shown to be reversibly antiproliferativein vitro. Pursuing this, we performed cell cycle analysis by flow cytometry of a B-cell lymphoma line and found that at concentrations >2.5 μM cells accumulated in the early S-phase. In order to determine A77 1726's effects on cell activation, human peripheral blood lymphocytes (PBL) were cultured in the presence of PHA or OKT 3 antibody. Flow cytometric evaluation of IL-2 and transferrin receptor expression exhibited a dose-dependent inhibition of these activation markers (10–100 μM). Further, using the polymerase chain reaction, we investigated the ability of leflunomide to impair the transcription of the IL-2-R gene. We found that A77 1726 did not significantly decrease IL-2-R α-chain mRNA expression regardless of stimulation. It seems that leflunomide's main metabolite did not affect IL-2-R at the level of gene transcription, and thus its effects could be due to impairment of post-translational events. Taken together, these studies demonstrate that leflunomide: (1) impairs activation of quiescent lymphocytes (reduction of IL-2-R expression), without inhibiting IL-2-R mRNA formation; (2) reversibly inhibits proliferation by holding resting lymphocytes in the G0-phase and activated cells in the early S-phase of the cell cycle. Thus, this compound may exert its effects through influencing two important aspects of an immune response, i.e. activation and proliferation of lymphocytes.