Interactive reportLeflunomide inhibits activation of inducible nitric oxide synthase in rat astrocytes☆
Introduction
The isoxazol derivative leflunomide is a recently developed immunosuppressive agent that is shown effective in a broad array of animal models of autoimmune diseases and transplant rejections, as well as in rheumatoid arthritis [3], [6]. Leflunomide acts through its active metabolite A77 1726, inhibiting protein tyrosine kinase (PTK) activity, and de novo pyrimidine biosynthesis [6], [11]. These actions may account for its suppressive effects on T and B cell growth and antibody production [6], [11]. Highly reactive free radical gas nitric oxide (NO), generated by microbial products and/or cytokine-activated inducible NO synthase (iNOS) has a wide spectrum of immunomodulatory and tissue destructive actions. NO has been implicated in pathogenesis of several autoimmune diseases, such as multiple sclerosis, autoimmune diabetes, rheumatoid arthritis, and their experimental animal models [1], [14], [29]. It is also involved in tissue destruction observed during allograft rejection [10]. We have recently reported the ability of A77 1726 to suppress iNOS activation in fibroblasts [18], which might partly account for the anti-inflammatory effects of leflunomide in rheumatoid arthritis and transplant rejection. Although leflunomide has been shown beneficial in the experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS) [31], there are no reports concerning mechanisms responsible for such action of leflunomide. Since both astrocytes and macrophages are supposed to be an important source of NO in MS and EAE [14], we investigated effects of leflunomide on NO production in these cells.
The results presented in this report indicate cell type-specific action of leflunomide on iNOS-mediated NO synthesis, probably through interaction with IFN-γ+LPS-triggered iNOS gene expression in astrocytes.
Section snippets
Reagents
Fetal calf serum (FCS), RPMI-1640, and phosphate-buffered saline (PBS) were from Flow Laboratories (Irvine, UK). Leflunomide’s active metabolite A77 1726 was kindly provided by Dr. Robert Bartlett (Hoechst Kalle-Albert, Wiesbaden, Germany). Recombinant rat IFN-γ was obtained from Holland Biotechnology (Leiden, Netherlands). Cycloheximide (CHX) was obtained from U.S. Biochemical Corporation (Cleveland, OH). Genistein (GEN), l-leucine-methyl-ester (LLME), actinomycin D (Act D), naphthylenediamine
The effects of A77 1726 on NO production in astrocytes and macrophages
Combination of IFN-γ and LPS has been reportedly effective in activation of iNOS in various cell types, including rodent astrocytes and macrophages [4], [20], [22]. Accordingly, a significant increase of nitrite accumulation, as an indicator of NO production, was observed in 48 h culture supernatants of both rat primary astrocytes and macrophages stimulated with IFN-γ+LPS, compared to the low basal levels in unstimulated cultures (1.3±0.1 μM and 4.3±0.3 μM for astrocytes and macrophages
Discussion
The presented results clearly demonstrate that novel immunosuppressive drug leflunomide inhibits iNOS-mediated NO synthesis in rat primary astrocytes and the C6 astrocytoma cell line, but not in peritoneal macrophages. While leflunomide’s active metabolite A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-γ+LPS-triggered expression of mRNA for iNOS in astrocytes, probably through inhibition of iNOS gene transcription. The reduced expression of IRF-1 message in A77
Acknowledgements
This work was supported by grants from Ministry of Science and Technology, Republic of Serbia, Yugoslavia.
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Published on the World Wide Web on 15 December 2000.