Identification of factors from rat neutrophils responsible for cytotoxicity to isolated hepatocytes

Abstract

Activated polymorphonuclear neutrophils (PMNs) have been shown to be cytotoxic to rat hepatic parenchymal cells in vitro. This cytotoxicity could be observed without direct cell-cell contact, since the conditioned medium from PMNs activated with formyl-Met-Leu-Phe (fMLP) was effective in hepatocyte killing. To identify the toxic factor(s) released by PMNs, degranulation was induced by fMLP in PMNs pretreated with cytochalasin B. The contents released from the phagocytes were subjected to gel filtration on a Sephadex G-100 column. Resulting fractions were tested for cytotoxicity to isolated hepatocytes by using release of alanine aminotransferase as a marker for hepatocyte injury. Cytotoxicity was associated with fractions containing cathepsin G and elastase and not with other fractions, including those containing myeloperoxidase. The former two enzymes were purified to homogeneity with a carboxymethyl cellulose column. Each of these enzymes demonstrated concentration-dependent cytotoxicity to hepatocytes at concentrations > 2 microgram/mL. Moreover, they exhibited an additive cytotoxic effect. Effective concentrations for the combined cathepsin G and elastase in the incubation mixture were similar to the concentrations of these enzymes in PMN-conditioned medium that produced cytotoxicity to hepatocytes. Cytotoxicity of either purified enzyme or of conditioned medium could be prevented by plasma alpha-1-antitrypsin or soybean trypsin-chymotrypsin inhibitor, which were also potent inhibitors of enzymic activity of both cathepsin G and elastase. By contrast, the serine protease inhibitors, aprotinin and 4-(2-aminoethyl)-benzene-sulfonyl fluoride, were less effective in inhibiting cathepsin G and elastase activities as well as cytotoxicity caused by the purified proteases or PMN-conditioned medium. These results support the hypothesis that cathepsin G and elastase are important mediators of hepatic parenchymal cell killing produced by activated PMNs in vitro.

Coagulation-Mediated Hypoxia and Neutrophil-Dependent Hepatic Injury in Rats Given Lipopolysaccharide and Ranitidine

“…4) almost abolished hepatocellular injury suggested an interdependence of hemostasismediated hypoxia and PMNs in LPS/RAN-induced liver injury. Because toxic PMN proteases are important mediators of hepatocellular killing both in vitro and in vivo in other models of PMN-dependent liver injury (Ho et al, 1996;Ishii et al, 2002), we explored the potential for interaction between hypoxia and PMN elastase in HPC killing. Using isolated HPCs, preliminary experiments were conducted to identify a reduction in chamber O 2 concentration to which HPCs could be exposed without evidence of significant cytotoxicity, as evaluated by the release of ALT into culture medium.…”

“…Activated PMNs release several cytotoxic factors, including the proteases such as PMN elastase, which can kill rat HPCs in vitro (Ho et al, 1996). Few studies have examined the relationship between hypoxia and cellular sensitivity to toxic proteases.…”

“…Hepatocellular injury in rats given a large, hepatotoxic dose of LPS and in several models of LPS-augmented xenobiotic hepatotoxicity occurs by a neutrophil (PMN)-dependent mechanism (Hewett et al, 1992;Barton et al, 2000;Yee et al, 2003a). Activated PMNs release proteases (i.e., elastase and cathepsin G) that kill hepatocytes (HPCs) in vitro (Ho et al, 1996), and inhibition of PMN elastase reduces LPS-induced liver injury in vivo (Ishii et al, 2002). Histopathological evaluation of livers from LPS/RAN-treated rats revealed midzonal hepatocellular oncotic necrosis accompanied by a marked infiltration of PMNs (Luyendyk et al, 2003b).…”

Chemokine involvement in hepatic ischemia/reperfusion injury in mice: Roles for macrophage inflammatory protein-2 and kupffer cells

“…18 It has been shown that the neutrophil proteases elastase and cathepsin G are highly cytotoxic to hepatocytes in vitro. 19 Still another mechanism by which neutrophils may contribute to hepatic injury is through impedance and occlusion of hepatic sinusoids. Neutrophils must deform to pass through tissue capillaries, including hepatic sinusoids.…”

Potential applications of N-terminal recombinant fragments of bactericidal/permeability-increasing protein in liver surgery

“…[81][82][83] Here, neutrophils can generate tissue injury through the liberation of toxic oxygen radicals and proteolytic enzymes, such as elastase and cathepsin G, which are highly cytotoxic to hepatocytes. 84 Also, the adherence of neutrophils to the endothelium leads to an impaired microcirculation, which results in the plugging of capillaries, the occlusion of hepatic sinusoids, and an aggravation of the hepatic injury and dysfunction. 82,83,85 The important role of neutrophils in the development of postresection hepatic injury was clearly shown by Pagetti et al, 86 who showed that neutrophil depletion before injury greatly attenuated liver dysfunction.…”