Endotoxin-inducible granulocyte-mediated hepatocytotoxicity requires adhesion and serine protease release

In primary cultures of Kupffer cells and hepatocytes, human granulocytes potentiated toxicity of endotoxin about 1000-fold. Granulocyte elastase activity was found to correlate with toxicity. The serine protease inhibitors alpha1-antitrypsin, eglin C, and aprotinin protected against toxicity. Tumor necrosis factor-alpha (TNF-alpha) induced cytotoxicity and elastase release, whereas neutralization of TNF-alpha blocked both events. We conclude that TNF-alpha formed by Kupffer cells activates granulocytes. Experiments in cultures where cells were separated by membranes permeable to mediators indicated that cell contact is needed for toxicity. Scanning electron microscopy showed granulocytes adhering to and interdigitating with hepatocytes. Using liver cells from ICAM-1-deficient mice had no effect on toxicity. However, neutralizing CD31 inhibited toxicity and elastase release but not granulocyte adhesion. Our findings demonstrate that adhesion of granulocytes is a necessary but not sufficient condition for the synergistic interaction of endotoxin-stimulated liver macrophages and granulocytes in the proteolytic killing of hepatocytes.