Leflunomide inhibits activation of inducible nitric oxide synthase in rat astrocytes

Highly reactive gaseous free radical nitric oxide (NO), generated by astroc ytes and infiltrating macrophages is implicated in inflammatory destruction of brain tissue, including that occurring in multiple sclerosis. Therefore , the influence of immunosuppressive drug leflunomide on inducible nitric o xide synthase (iNOS)-dependent NO production in rat astrocytes and macropha ges was investigated. Under the same cultivating conditions, leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO productio n in IFN-gamma +LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppresse d IFN-gamma +LPS-triggered expression of iNOS mRNA in astrocytes. In the pr esence of transcription inhibitor actinomycin D, A77 1726 failed to inhibit astrocyte NO production, suggesting transcriptional regulation of iNOS by leflunomide. This assumption was further supported by the ability of A77 17 26 to inhibit IFN-gamma +LPS-induced expression of mRNA for an important iN OS transcription factor IRF-1. PD98059, a specific inhibitor of mitogen-act ivated protein kinase kinase (MAPKK/MEK), but not genistein, an unselective protein tyrosine kinase inhibitor, completely mimicked cell type-specific inhibition of NO synthesis by A77 1726. Therefore, previously described inh ibition of MEK/MAP pathway by leflunomide could present a possible mechanis m for A77 1726-mediated suppression of iNOS activation in astrocytes. Accor dingly to results obtained with primary astrocytes, both A77 1726 and PD980 59 significantly reduced IFN-gamma +LPS-induced NO synthesis in the culture s of rat astrocytoma cell line C6. The ability to suppress iNOS induction i n astrocytes supports potential use of leflunomide in the treatment of mult iple sclerosis and other NO-dependent inflammatory brain disorders. (C) 200 1 Elsevier Science B.V. All rights reserved.