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

We previously described a long-lasting overproduction of nitric oxide (NO) in cirrhotic patients with spontaneous bacterial peritonitis. The aim of th e 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 i solated and cultured in the presence or absence of 1 mu g/ml lipopolysaccha ride and/or 500 units/ml interferon-gamma (IFN-gamma) for 6 days. NO produc tion was measured as nitrate + nitrite (NOx), inducible NO synthase (iNOS) protein expression was analysed by immunocytochemistry and Western blot ana lysis using a specific anti-(human iNOS) antibody, and the catalytic activi ty of NOS was revealed by cytochemical staining for NADPH-dependent diaphor ase. Cultured macrophages spontaneously released small amounts of NOx [medi an (10-90th percentile) of 18 separate experiments: 3.3 (0-8) mu mol/l]. Ad dition of lipopolysaccharide alone or in combination with IFN-gamma to the culture medium did not change the levels of NOx, while IFN-gamma alone dram atically increased NO production [13.4 (3.5-28.3) mu mol/l; P < 0.001]. Mac rophages were stimulated by IFN-gamma 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) mu mol/l respectively; P < 0.001]. Macrophages freshly isolated or stimulated with IFN-gamma expressed iNOS protein, as shown by Western blot and immunocytoch emical analysis, and stained for NADPH diaphorase. Our findings demonstrate the presence of iNOS protein in peritoneal macrophages from cirrhotic pati ents. The role of IFN-gamma appears to be a determinant for the up-regulati on of NO production, particularly under conditions of infection. Therefore peritoneal macrophages producing large amounts of NO at the site of infecti on may contribute to maintaining splanchnic vasodilation in these patients.