Induction of inducible nitric oxide synthase (iNOS) in vitro and in vivo brain microgllia

To clarify the expression of inducible nitric oxide synthase (iNOS) in vitro and in vivo brain, we examined the effects of intrahippocampal microinjection with interferon-γ (IFN-γ) plus lipopolysaccharide (LPS) and treatment with IFN-γ and LPS in cultured glial cells. 130-kDa iNOS protein was induced after 12 h by intrahippocampal injection with IFN-γ plus LPS, although 160-kDa nNOS expression was not changed. On the other hand, iNOS mRNA was expressed after 6 h, but the mRNA was degraded rapidly thereafter. The injection with IFN-γ plus LPS caused numerous ameboidal microglia and induced MHC class II. In a part of these ameboidal microglia, iNOS-immunoreactivity was observed. Subsequently, immunoreactivities of iNOS and MHC class II were disappeared over 3 days after injection. These results suggest that the intrahippocampal injection with IFN-γ plus LPS induced iNOS mRNA after 6 h and iNOS protein after 12 h in a part of microglia which are activated to the ameboid type and express MHC class II in vivo brain. On the other hand, the cultivation of microglia caused ameboid type. In addition, the treatment with IFN-γ or LPS alone for 24 h induced 130-kDa iNOS protein mainly in microglia. IFN-γ induced tyrosine-phosphorylation of Jak2-tyrosine kinase and transcription factor STAT1α, but LPS did not. However, both iNOS induction by IFN-γ and LPS were inhibited by tyrosine kinase inhibitor herbimycin A. These results suggest that IFN-γ-induced iNOS induction involves activation of Jak2-STAT1α pathway in ameboidal microglia. On the other hand, LPS-induced expression was mediated by other tyrosine kinases.