Complementary roles of tumor necrosis factor alpha and interferon gamma in inducible microglial nitric oxide generation

Proinflammatory cytokines and pathogen components activate microglia to release several substances such as nitric oxide (NO) produced after the induction of type II nitric oxide synthase (iNOS). The present study was designed to elucidate the interaction between the proinflammatory cytokines interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) on iNOS expression and NO production in microglial cells. In primary mouse microglial cells exposure to IFN-γ (5 and 10 ng/ml; 48 h) or TNF-α (20 ng/ml; 48 h) alone were unable to induce iNOS expression; however, when cells were exposed to both cytokines together, the expression of this enzyme and the NO production in culture media were found significantly increased. In the BV-2 microglial cell line, IFN-γ and TNF-α were shown to cooperate in nuclear factor κ B (NF-κB) activation, an essential transcription factor for iNOS gene transcription. Importantly, IFN-γ induced NF-κB binding to DNA was totally dependent on the endogenous TNF-α released via MEK/ERK signalling pathway. Thus, exposure of BV-2 cells to IFN-γ in the presence of the selective MEK inhibitor U0126 or a neutralizing anti-TNF-α antibody significantly reduced IFN-γ dependent NF-κB activation and iNOs expression. In addition, by activating the Jak/STAT pathway IFN-γ potentiated TNF-α induced NF-κB binding to DNA and activated additional transcription factors (i.e. IRF-1) known to be essential for iNOs gene expression. The present findings demonstrate that the proinflammatory cytokines IFN-γ and TNF-α have complementary roles on iNOS expression in microglial cells and this might be relevant to understand the molecular mechanisms of microglial activation associated with the pathogenesis of several neuroinflammatory disorders in which increased levels of IFN-γ and TNF-α have been reported.