Effects of inhibitors of ADP-ribosylation on macrophage activation.

Nitric oxide (NO) is a potent mediator involved in many biological functions including macrophage cytotoxicity and non-specific immunity against parasites, bacteria and viruses. Murine macrophages possess the capacity to express the inducible NO synthase (iNOS) which is not constitutively expressed but induced at the transcriptional level by interferon gamma (IFN-gamma) alone or synergistically with LPS. We have investigated the possible role of ADP-ribosylation reactions in the signaling pathway involved in NO synthase induction, since ADP-ribosylation has been reported to be involved in the expression of certain IFN-gamma and LPS-inducible genes. We found that inhibitors of ADP-ribosylation inhibited nitrite synthesis in RAW 264.7 macrophages after stimulation by IFN-gamma and LPS. These ADP-ribosylation inhibitors acted by preventing NO synthase mRNA induction, without inhibiting NO synthase enzyme activity. IRF-1, a transcription factor induced and activated by IFN-gamma was recently shown to be involved in iNOS induction. We showed that inhibitors of ADP-ribosylation had no effect on IFN-gamma-mediated mRNA induction of IRF-1 nor on its activation and binding to its target sequence in the iNOS gene. In addition, the inhibitors failed to impair the IFN-gamma-mediated antiviral activity against VSV virus. Since induction by IFN-gamma of IRF-1 and induction of the antiviral state proceed through the JAK/STAT signalling pathway, our results imply that ADP-ribosylation reactions are not involved in triggering this pathway. Although the precise mechanism requires further investigation, our results indicate that ADP-ribosylation is a crucial step restricted to the signalling pathway which leads to iNOS mRNA induction, as well as TNF and MHC class II induction during macrophage activation.