DUAL MECHANISM FOR THE CONTROL OF INDUCIBLE-TYPE NO SYNTHASE GENE-EXPRESSION IN MACROPHAGES DURING ACTIVATION BY INTERFERON-GAMMA AND BACTERIAL LIPOPOLYSACCHARIDE - TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION

Production of nitric oxide (NO) by macrophages is enhanced upon activa tion by bacterial endotoxins and cytokines mainly via an increase of t he intracellular content of the inducible isoform of nitric oxide synt hase (i-NOS). We have studied in detail the effect of several modulato rs of macrophage activity on steady state levels of i-NOS mRNA in the mouse macrophage-like cell line RAW 264.7. Bacterial lipopolysaccharid e (LPS) and interferon-gamma (IFN-gamma) were found to be effective in ducers of i-NOS mRNA, in accordance with their known ability to stimul ate both i-NOS activity and NO production in macrophages from differen t sources, while TNF-alpha, IL-1, or IL-6 was ineffective in this rega rd. Accumulation of i-NOS mRNA in response to either LPS or IFN-gamma stimulation was accompanied by increased i-NOS gene transcription, as detected both by using a nuclear ''run-on'' transcription assay and by transient transfection of the cloned gene promoter in RAW 264.7 cells . Co-stimulation of the cells with both inducers resulted in higher st eady state levels of i-NOS mRNA in the absence, however, of a correspo nding potentiation of the rate of gene transcription. This was due pri marily to a considerable effect of LPS on i-NOS mRNA stability, with p rolongation of its half-life from 1-1.5 h, in the presence of LFN-gamm a alone, to 4-6 h in the presence of both LPS and IFN-gamma.