Interferon (IFN)-gamma and Herpes simplex virus/tumor necrosis factor-alpha synergistically induce nitric oxide synthase 2 in macrophages through cooperative action of nuclear factor-kappa B and IFN regulatory factor-1.

The radical nitric oxide (NO) constitutes an important part of the innate immune response to many viruses, and among these notably Herpes simplex virus (HSV). We have previously shown that HSV/tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma synergistically induce NO production in macrophages, and here we have investigated the molecular mechanism underlying this phenomenon. The enhancement of NO production was regulated at the level of NO synthase 2 (NOS2, iNOS) transcription. The ISRE element of the NOS2 promoter, which binds IFN regulatory factor (IRF)-1, was essential both for full responsiveness to IFN-gamma and the synergistic response. The GAS motif, binding signal transducer and activator of transcription 1 (STAT1), did not contribute to the cross-talk with virus/TNF-induced signals, but was necessary for full responsiveness to IFN-gamma. The distal binding site for nuclear factor (NF)-kappa B was important for the cooperative response, while the proximal kappa B site was not involved in the cooperative promoter activation but played a role in full promoter inducibility. By ectopic expression of IRF-1 and NF-kappa B (p65), we found that these factors synergistically induce NO accumulation. Together, our results show that binding of IRF-1 and NF-kappa B to their respective sites in the distal domain of the NOS2 promoter, creates a potent trans-activating complex with the ability to induce NOS2 transcription synergistically in response to simultaneous HSV-2/TNF-alpha and IFN-gamma treatment.