Up-regulation of nitric oxide production by interferon-γ in cultured peritoneal macrophages from patients with cirrhosis

Abstract

We previously described a long-lasting overproduction of nitric oxide (NO) in cirrhotic patients with spontaneous bacterial peritonitis. The aim of the present study was to investigate the presence of the inducible NO pathway in peritoneal macrophages. Ascitic fluids were collected from 29 patients with cirrhosis, aged between 35 and 82 years. Peritoneal macrophages were isolated and cultured in the presence or absence of 1 μg/ml lipopolysaccharide and/or 500 units/ml interferon-γ (IFN-γ) for 6 days. NO production was measured as nitrate+nitrite (NOx), inducible NO synthase (iNOS) protein expression was analysed by immunocytochemistry and Western blot analysis using a specific anti-(human iNOS) antibody, and the catalytic activity of NOS was revealed by cytochemical staining for NADPH-dependent diaphorase. Cultured macrophages spontaneously released small amounts of NOx [median (10–90th percentile) of 18 separate experiments: 3.3 (0–8) μmol/l]. Addition of lipopolysaccharide alone or in combination with IFN-γ to the culture medium did not change the levels of NOx, while IFN-γ alone dramatically increased NO production [13.4 (3.5–28.3) μmol/l; P< 0.001]. Macrophages were stimulated by IFN-γ to a greater extent in patients with recent spontaneous bacterial peritonitis (n = 13) than in those in a stable clinical condition (n = 18) [19.8 (10.5–30.1) and 10.0 (3.2–14.5) μmol/l respectively; P< 0.001]. Macrophages freshly isolated or stimulated with IFN-γ expressed iNOS protein, as shown by Western blot and immunocytochemical analysis, and stained for NADPH diaphorase. Our findings demonstrate the presence of iNOS protein in peritoneal macrophages from cirrhotic patients. The role of IFN-γ appears to be a determinant for the up-regulation of NO production, particularly under conditions of infection. Therefore peritoneal macrophages producing large amounts of NO at the site of infection may contribute to maintaining splanchnic vasodilation in these patients.