Regulation of nitric oxide synthase II in primary human fetal astrocytes: A critical role for interleukin-1 and extracellular ATP

Abstract

The inducible isoform of nitric oxide synthase (NOS), NOS II or iNOS, is believed to play an important role in pathogenesis of inflammatory reactions in the CNS. NOS II regulated by transcription and induced by inflammatory stimuli. Differences in the expression of NOS II by different cell types in different species have important consequences for disease pathogenesis. The overall objective of this thesis was to determine which cell types express NOS II in the human CNS, and to define how expression is regulated in these cells.;Using both in situ hybridization and the histochemical marker, NADPH diaphorase, NOS II activity was localized primarily to astrocytes in MS lesions. The level of NOS II mRNA and NOS activity correlated well with the extent of inflammation observed in the lesions. Since NOS II has been shown to be expressed in MS lesions, we hypothesized that cytokines associated with MS are critical in regulating NOS II.;In purified cultures of human fetal astrocytes and microglia, NOS II was expressed only by astrocytes following stimulation with interleukin (IL)-1 while lipopolysaccharide (LPS) had no effect. The availability of components of the IL-1 system was found to be critical for regulation of NOS II expression in astrocytes. In particular, addition of the IL-1 receptor antagonist (IL-1ra) to the cytokine cocktail resulted in the complete inhibition of the IL-1-induced expression of NOS II. We hypothesized that the relative amounts of IL-1 and IL-1ra in the CNS could also be regulated by other cytokines. We showed in our cell culture system that only microglia could be activated to express IL-1 and IL-1ra, and anti-inflammatory cytokines, IL-4 and IFN beta, enhanced the production IL-1ra preferentially over IL-1.;In addition to cytokine effects, input from other systems make affect gene expression in astrocytes. In particular, blockade of purinergic system, receptors that respond to extracellular nucleotides, resulted in down-regulation of NOS II expression in astrocytes. It was determined that these inhibitors inhibited nuclear factor-kappaB (NF-kappaB) translocation and activity, which is known to activated by IL-1. The activation of purinergic receptors was critical for maximal astrocyte induction of NOS II.