Differential expression of inducible NO synthase in two murine macrophage cell lines

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Abstract
Although primary macrophages and most murine macrophage cell lines such as RAW 264.7 cells respond to interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS) by producing large amounts of nitrite, i.e. the oxidation product of nitric oxide (NO) produced by inducible NO synthase (iNOS), other cell lines like P388.D1 cells do not produce significant amounts. To gain insight into the signalling pathway that leads to the induction of iNOS activity, we compared iNOS expression in RAW 264.7 and P388.D1 cells. We showed that IFN-gamma binds to each cell line with a similar affinity. Furthermore, no differences in iNOS gene structure were detectable by Southern blot analysis. Even though no significant nitrite secretion was found in the supernatant of P388:D1 cells stimulated with IFN-gamma and/or LPS, iNOS mRNA expression was induced. In addition, IFN-gamma induced the interferon regulatory factor-1 (IRF-1) gene and activated the binding of this factor to its target sequence in the iNOS gene. This binding was recently shown to be necessary for iNOS expression. However, in P388.D1 cells, we were unable to detect the corresponding iNOS protein. These results indicate a deficiency in P388.D1 cells which appears to be restricted to the signalling pathway controlling iNOS protein synthesis. This deficiency does not affect the overall IFN-gamma biological response, but rather a convergent post-transcriptional step common to IFN-gamma and LPS.

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