Activation of Kupffer cells and caspase-3 involved in rat hepatocyte apoptosis induced by endotoxin

Background/Aims: Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction, In this study Kupffer cell activation, involvement of TNF alpha and caspases downstream of the TNF alpha receptor were examined i n hepatocyte apoptosis induced by LPS, Methods: In in vivo experiments, male Sprague-Dawley rats were injected int ravenously with LPS, and small amounts of the blood and liver were sampled to evaluate apoptosis, Kupffer cells were inactivated by pretreatment with gadolinium chloride for 2 days. In in vitro experiments, hepatocytes and Ku pffer cells were separately isolated from rat livers using collagenase perf usion. Results: LPS induced time-dependent and dose-dependent increases in the num ber of TUNEL-positive cells, which coincided with the apoptotic features of hepatocytes demonstrated by electron microscopy and DNA ladder. Activation of caspase-3-like proteases was observed with an increase in the number of apoptotic hepatocytes, Immunostaining with activated caspase-3-specific an tibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-p ositive hepatocytes, Inactivation of Kupffer cells by gadolinium chloride w as concomitantly accompanied by the prevention of caspase-3 activation, hep atocyte apoptosis and liver injury induced by LPS, The coculture system of hepatocytes and Kupffer cells, but neither cell culture system, individuall y, showed LPS-induced hepatocyte apoptosis, Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNF alpha antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, a cetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decr eased the number of apoptotic hepatocytes, Conclusions: These results suggest that the activation of Kupffer cells, TN F alpha and caspases downstream of TNFR1 were involved in hepatocyte apopto sis induced by LPS.