Functional studies of purified rat hepatic macrophages

The release of reactive oxygen intermediates by Kupffer cells or inflammatory hepatic macrophages and their role in the pathogenesis of a rat model of liver injury have been investigated. When macrophages were recruited into the liver by administration of intravenous Corynebacterium parvum there was a mild granulomatous hepatitis. The subsequent administration of intravenous Salmonella typhosa endotoxin resulted in massive and often fatal hepatic necrosis, whereas the same dose of endotoxin produced no abnormality in normal rats. Pretreatment of C parvum exposed rats with allopurinol or superoxide dismutase reduced the severity of hepatic injury. Furthermore superoxide dismutase decreased mortality if given before endotoxin. These results suggest that reactive oxygen intermediates released by hepatic macrophages were involved in the pathogenesis of this rat model of liver injury. In vitro studies of the production of reactive oxygen intermediates during the respiratory burst of normal Kupffer cells and C parvum elicited hepatic macrophages (± endotoxin) were therefore performed. A technique for isolation and purification of Kupffer cells and C parvum elicited hepatic macrophages using counterflow centrifugal elutriation was established and validated. Highly purified viable cells that were capable of performing complex receptor-mediated functions were obtained. Initial studies demonstrated that it was not possible to measure superoxide production by hepatic macrophages using the standard assay of superoxide dismutase-inhibitable reduction of ferricytochrome C, owing to the oxidative effects of other generated reactive oxygen intermediates and/or cytochrome oxidase activity. Catalase (1000u/ml) and cyanide (0.1mM) were introduced to prevent or inhibit these reactions and their use in this assay was validated. The respiratory burst activity of isolated cells was subsequently assessed both by this improved technique for measuring superoxide production and by the completely independent method of measuring ^14 C glucose oxidations via the hexose monophosphate shunt. Four important observations were made: i) When triggered by phorbol myristate acetate, normal Kupffer cells exhibited a low level of respiratory burst activity. ii) C parvum elicited-hepatic macrophages demonstrated significantly increased respiratory burst activity compared with normal Kupffer cells. iii) Hepatic macrophages isolated from C parvum rats that had been treated with S typhosa endotoxin demonstrated significantly increased respiratory burst activity compared with those isolated from C parvum and saline treated control rats. iv) Endotoxin did not act as a triggering stimulus for the respiratory burst activity of C parvum-elicited hepatic macrophages in vitro. These results indicate that there is an in vivo stepwise sequence of hepatic macrophage activation after exposure of rats to C parvum followed by endotoxin and suggest that these effects may be mediated by two separate mechanisms.