GASTROENTEROLOGY 1997;112:2056 – 2064 Nitric Oxide Production by Peritoneal Macrophages of Cirrhotic Rats: A Host Response Against Bacterial Peritonitis MANUEL MORALES – RUIZ,* WLADIMIRO JIME ´ NEZ,* JOSEFA ROS,* MANUEL SOLE ´ , ‡ ALBERT LEIVAS,* MARTA BOSCH – MARCE ´ ,* FRANCISCA RIVERA,* VICENTE ARROYO, § and JUAN RODE ´ S § *Hormonal Laboratory, ‡ Pathology Department, and § Liver Unit, Hospital Clı B nic i Provincial, University of Barcelona, Barcelona, Spain Background & Aims: Patients and rats with cirrhosis son for claiming the possible role of NO in the pathogen- and ascites are prone to develop peritonitis. The aim esis of arterial vasodilation in cirrhosis. 4 of this study was to assess whether peritoneal macro- L-arginine and molecular oxygen are the cosubstrates of phages of cirrhotic rats without peritoneal infection pro- the reaction leading to NO formation. In most cases, how- duce nitric oxide and express inducible NO synthase ever, NO production is regulated by NO synthase (NOS) (iNOS). Methods: NO 2 0 accumulation produced by mac- activity rather than by substrate availability. NOS is a fam- rophages from control rats and cirrhotic rats with asci- ily of isozymes with important differences in their biological tes was determined. iNOS messenger RNA and protein actions and regulatory properties. In the vasculature, a low- expression were analyzed by Northern and Western blot output Ca 2/ /calmodulin-dependent constitutive pathway and immunocytochemical analysis. The in vivo effects has been localized in the endothelial cells (endothelial of inhibiting iNOS were investigated by giving the spe- NOS). 5 Another Ca 2/ /calmodulin-independent isozyme, cific iNOS inhibitor L-N-(1-iminoethyl)-lysine (L-NIL) or sterile saline to 9 and 7 cirrhotic rats with ascites, producing large amounts of NO and only present when its respectively. Results: Cirrhotic macrophages produced expression has been induced by proinflammatory stimuli, NO 2 0 that was around fourfold greater than that of con- has been identified in fibroblast and vascular smooth muscle trol macrophages after 30 hours in culture. Northern and endothelial cells (inducible NOS [iNOS]). 6 Attempts and Western blot and immunocytochemical analysis to identify what is/are the enzyme(s) involved in the in- showed the presence of iNOS messenger RNA and pro- creased NO production in advanced liver disease suggest tein in macrophages of cirrhotic rats. Ascites cultures that both, endothelial NOS and iNOS are overexpressed in were positive in all rats administered L-NIL and negative arterial vessels of rats with cirrhosis and ascites. 7,8 However, in those administered saline. Conclusions: Macro- these investigations do not allow for elucidation of whether phages of cirrhotic rats produce NO and express iNOS these isozymes are also involved in pathophysiological func- messenger RNA and protein, and these changes are tions other than vasodilation. not a consequence of overt bacterial infection. Be- cause iNOS inhibition results in peritoneal infection, The hypothesis of this study was that if iNOS is present these results suggest that iNOS induction in macro- in the vasculature of cirrhotic rats, then the agents promot- phages of cirrhotic rats is a host defense response to ing iNOS expression should also induce the enzyme in other prevent bacterial peritonitis. nonvascular cell types in which the biological functions of this gas are more related with host defense mechanisms than with arterial vasodilation. Therefore, the investigation A was aimed to assess whether peritoneal macrophages from rterial vasodilation is a major disturbance in patients with cirrhosis and ascites that is defined by arterial cirrhotic rats with ascites, which are nonvascular cells highly hypotension, high cardiac output, and low peripheral sensitive to proinflammatory stimuli, produce NO and ex- resistance. 1 During the last decade, our comprehension press iNOS messenger RNA (mRNA) and protein. More- of the mechanisms accounting for this syndrome has grown in parallel with the identification of several endog- Abbreviations used in this paper: GPDH, glyceraldehyde-3-phos- enous vasoactive factors able to regulate, in an endocrine phate dehydrogenase; IBMX, 3-isobutyl-1-methylxanthine; iNOS, in- ducible NO synthase; L-NIL, L-N-(iminoethyl)-lysine; LPS, lipopoly- and/or paracrine fashion, blood volume and vascular saccharide; NNA, N w -nitro-L-arginine; NOS, NO synthase; SOD, tone. 2,3 Among these substances, nitric oxide has occu- superoxide dismutase. pied a paramount position. The powerful vasodilator  1997 by the American Gastroenterological Association 0016-5085/97/$3.00 properties of this gaseous compound were the main rea- / 5e1d$$0027 05-12-97 08:10:02 gasas WBS-Gastro