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 immunosu ppressive activities. We describe here a molecular mechanism contributing t o this effect. Materials and Methods: Murine T cell lymphoma EL4-6.1 cells were activated with the physiological stimulus interleukin (IL)-1 beta to express IL-2 mRN A in the presence or absence of subtoxic concentrations of the physiologica l spontaneous NO donor S-nitrosocysteine (SNOC). subsequently, semiquantita tive RT-PCR and gel shift assays with nuclear extracts were performed to an alyze 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-1 beta-induced IL-2 mRNA expression in EL4-6.1 cell s. The suppressive activity of NO was concentration dependent and found to be completely reversible. Importantly, NO at the concentrations used induce d neither apoptosis nor necrosis. Dominant regulation of IL-2 gene expressi on 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 acti vities of recombinant Sp1 and EGR-1. More importantly, gel shift assays als o showed a lack of DNA binding of native Sp1 derived from NO-treated nuclea r extracts and that from NO-treated viable lymphocytes. This effect is sele ctive, 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 mam mals, thus contributing to NO-mediated inhibition of IL-2 gene expression a fter physiological stimuli. The exact understanding of the molecular mechan ism leading to NO-mediated, fully reversible suppression of immune reaction s may lead to use of this naturally occuring tool as an aid in inflammatory diseases.