Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes.

BACKGROUND Nitric oxide (NO) has frequently been shown to display immunosuppressive activities. We describe here a molecular mechanism contributing to this effect. MATERIALS AND METHODS Murine T cell lymphoma EL4-6.1 cells were activated with the physiological stimulus interleukin (IL)-1beta to express IL-2 mRNA in the presence or absence of subtoxic concentrations of the physiological spontaneous NO donor S-nitrosocysteine (SNOC). Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyze the effects of NO on IL-2 mRNA expression and on the activity of the dominant regulating transcription factors Sp1, EGR-1, and NFATc. RESULTS NO inhibits IL-1beta-induced IL-2 mRNA expression in EL4-6.1 cells. The suppressive activity of NO was concentration dependent and found to be completely reversible. Importantly, NO at the concentrations used induced neither apoptosis nor necrosis. Dominant regulation of IL-2 gene expression is known to reside in the zinc finger transcription factors Sp1 or EGR-1 and in the non-zinc finger protein NFAT. NO abrogates the DNA binding activities of recombinant Sp1 and EGR-1. More importantly, gel shift assays also showed a lack of DNA binding of native Sp1 derived from NO-treated nuclear extracts and that from NO-treated viable lymphocytes. This effect is selective, as the DNA binding activity of recombinant NFATc was not affected by NO. CONCLUSION Inactivation of zinc finger transcription factors by NO appears to be a molecular mechanism in the immunosuppressive activity of NO in mammals, thus contributing to NO-mediated inhibition of IL-2 gene expression after physiological stimuli. The exact understanding of the molecular mechanism leading to NO-mediated, fully reversible suppression of immune reactions may lead to use of this naturally occurring tool as an aid in inflammatory diseases.