Elsevier

Brain Research

Volume 889, Issues 1–2, 19 January 2001, Pages 331-338
Brain Research

Interactive report
Leflunomide inhibits activation of inducible nitric oxide synthase in rat astrocytes

https://doi.org/10.1016/S0006-8993(00)03181-4Get rights and content

Abstract

Highly reactive gaseous free radical nitric oxide (NO), generated by astrocytes 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 oxide synthase (iNOS)-dependent NO production in rat astrocytes and macrophages was investigated. Under the same cultivating conditions, leflunomide’s active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-γ+LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-γ+LPS-triggered expression of iNOS mRNA in astrocytes. In the presence 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 1726 to inhibit IFN-γ+LPS-induced expression of mRNA for an important iNOS transcription factor IRF-1. PD98059, a specific inhibitor of mitogen-activated 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 inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for A77 1726-mediated suppression of iNOS activation in astrocytes. Accordingly to results obtained with primary astrocytes, both A77 1726 and PD98059 significantly reduced IFN-γ+LPS-induced NO synthesis in the cultures of rat astrocytoma cell line C6. The ability to suppress iNOS induction in astrocytes supports potential use of leflunomide in the treatment of multiple sclerosis and other NO-dependent inflammatory brain disorders.

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.

References (36)

  • X. Xu et al.

    Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide

    J. Biol. Chem.

    (1995)
  • A.R. Amin et al.

    Nitric oxide synthase and cyclooxygenases: distribution, regulation, and intervention in arthritis

    Curr. Opin. Rheumatol.

    (1999)
  • A.S. Baldwin

    The NF-kappa B and I-kappa B proteins: new discoveries and insights

    Annu. Rev. Immunol.

    (1996)
  • R.R. Bartlett et al.

    Leflunomide (HWA 486), a novel immunomodulating compound for the treatment of autoimmune disorders and reactions leading to transplantation rejection

    Agents Actions

    (1991)
  • N.R. Bhat et al.

    Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures

    Neuroscience

    (1998)
  • L. Bo et al.

    Induction of nitric oxide synthase in demyelinating regions of multiple sclerosis brains

    Ann. Neurol.

    (1994)
  • E.D. Chan et al.

    Evaluation of the role of mitogen-activated protein kinases in the expression of inducible nitric oxide synthase by IFN-gamma and TNF-alpha in mouse macrophages

    J. Immunol.

    (1999)
  • A.S. Chong et al.

    In vivo activity of leflunomide: pharmacokinetic analyses and mechanism of immunosuppression

    Transplantation

    (1999)
  • Cited by (0)

    Published on the World Wide Web on 15 December 2000.

    View full text