BACKGROUND/AIMS: Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction. In this study, Kupffer cell activation, involvement of TNFalpha and caspases downstream of the TNFalpha receptor were examined in hepatocyte apoptosis induced by LPS. METHODS: In in vivo experiments, male Sprague-Dawley rats were injected intravenously 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 Kupffer cells were separately isolated from rat livers using collagenase perfusion. RESULTS: LPS induced time-dependent and dose-dependent increases in the number 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 antibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-positive hepatocytes. Inactivation of Kupffer cells by gadolinium chloride was concomitantly accompanied by the prevention of caspase-3 activation, hepatocyte apoptosis and liver injury induced by LPS. The co-culture system of hepatocytes and Kupffer cells, but neither cell culture system, individually, showed LPS-induced hepatocyte apoptosis. Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNFalpha antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, acetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decreased the number of apoptotic hepatocytes. CONCLUSIONS: These results suggest that the activation of Kupffer cells, TNFalpha and caspases downstream of TNFR1 were involved in hepatocyte apoptosis induced by LPS.
BACKGROUND/AIMS: Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction. In this study, Kupffer cell activation, involvement of TNFalpha and caspases downstream of the TNFalpha receptor were examined in hepatocyte apoptosis induced by LPS. METHODS: In in vivo experiments, male Sprague-Dawley rats were injected intravenously 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 Kupffer cells were separately isolated from rat livers using collagenase perfusion. RESULTS: LPS induced time-dependent and dose-dependent increases in the number 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 antibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-positive hepatocytes. Inactivation of Kupffer cells by gadolinium chloride was concomitantly accompanied by the prevention of caspase-3 activation, hepatocyte apoptosis and liver injury induced by LPS. The co-culture system of hepatocytes and Kupffer cells, but neither cell culture system, individually, showed LPS-induced hepatocyte apoptosis. Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNFalpha antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, acetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decreased the number of apoptotic hepatocytes. CONCLUSIONS: These results suggest that the activation of Kupffer cells, TNFalpha and caspases downstream of TNFR1 were involved in hepatocyte apoptosis induced by LPS.
Authors: Patricio Godoy; Nicola J Hewitt; Ute Albrecht; Melvin E Andersen; Nariman Ansari; Sudin Bhattacharya; Johannes Georg Bode; Jennifer Bolleyn; Christoph Borner; Jan Böttger; Albert Braeuning; Robert A Budinsky; Britta Burkhardt; Neil R Cameron; Giovanni Camussi; Chong-Su Cho; Yun-Jaie Choi; J Craig Rowlands; Uta Dahmen; Georg Damm; Olaf Dirsch; María Teresa Donato; Jian Dong; Steven Dooley; Dirk Drasdo; Rowena Eakins; Karine Sá Ferreira; Valentina Fonsato; Joanna Fraczek; Rolf Gebhardt; Andrew Gibson; Matthias Glanemann; Chris E P Goldring; María José Gómez-Lechón; Geny M M Groothuis; Lena Gustavsson; Christelle Guyot; David Hallifax; Seddik Hammad; Adam Hayward; Dieter Häussinger; Claus Hellerbrand; Philip Hewitt; Stefan Hoehme; Hermann-Georg Holzhütter; J Brian Houston; Jens Hrach; Kiyomi Ito; Hartmut Jaeschke; Verena Keitel; Jens M Kelm; B Kevin Park; Claus Kordes; Gerd A Kullak-Ublick; Edward L LeCluyse; Peng Lu; Jennifer Luebke-Wheeler; Anna Lutz; Daniel J Maltman; Madlen Matz-Soja; Patrick McMullen; Irmgard Merfort; Simon Messner; Christoph Meyer; Jessica Mwinyi; Dean J Naisbitt; Andreas K Nussler; Peter Olinga; Francesco Pampaloni; Jingbo Pi; Linda Pluta; Stefan A Przyborski; Anup Ramachandran; Vera Rogiers; Cliff Rowe; Celine Schelcher; Kathrin Schmich; Michael Schwarz; Bijay Singh; Ernst H K Stelzer; Bruno Stieger; Regina Stöber; Yuichi Sugiyama; Ciro Tetta; Wolfgang E Thasler; Tamara Vanhaecke; Mathieu Vinken; Thomas S Weiss; Agata Widera; Courtney G Woods; Jinghai James Xu; Kathy M Yarborough; Jan G Hengstler Journal: Arch Toxicol Date: 2013-08-23 Impact factor: 5.153