INDUCTION OF HEPATIC ITO CELL NITRIC-OXIDE PRODUCTION AFTER ACUTE ENDOTOXEMIA

Nitric oxide is a highly reactive mediator released in the liver by hepatocytes, Kupffer cells and endothelial cells during endotoxin-induced inflammation. In this study we determined whether Ito cells also produce nitric oxide after exposure to endotoxin. For induction of endotoxemia, rats were injected intravenously with Escherichia coli lipopolysaccharide (2.5 mg/kg). Ito cells were isolated from the animals 48 hr later by means of in situ perfusion of the liver with protease and collagenase followed by purification on an arabinogalactan gradient. Ito cells from untreated and endotoxemic rats were found to produce low levels of nitric oxide in response to interferon-gamma. In both cell types, this response depended on L-arginine and was blocked by N-G-monomethyl-L-arginine, a specific nitric oxide synthase inhibitor. Cells from rats treated with endotoxin produced significantly more nitric oxide than did cells from untreated animals; this was due, at least in part, to increased expression of protein for an inducible form of nitric oxide synthase. These cells also responded to stimulation with lipopolysaccharide in vitro, as well as the combination of interferon-gamma and lipopolysaccharide, which was synergistic in stimulating nitric oxide production. Tumor necrosis factor-alpha and macrophage colony-stimulating factor were also found to stimulate nitric oxide production by Ito cells from endotoxemic rats. In addition, in these cells, tumor necrosis factor-alpha synergized with interferon-gamma in inducing nitric oxide production. The combination of interferon-gamma and lipopolysaccharide was also found to inhibit Ito cell DNA synthesis, as measured on the basis of [H-3]-thymidine uptake. This inhibitory effect was readily blocked by N-G-monomethyl-L-arginine. In contrast, macrophage colony-stimulating factor, as well as granulocyte/macrophage colony-stimulating factor, stimulated Ito cell DNA synthesis, which was not significantly affected by N-G-monomethyl-L-arginine. These observations suggest that the effects of inflammatory mediators on Ito cell DNA synthesis depend in part on their ability to stimulate nitric oxide production. Our finding that Ito cells produce nitric oxide indicates that these cells have the capacity to participate in hepatic inflammatory responses during acute endotoxemia.