Apoptotic death of hepatocytes a feature and contributing factor of many

Apoptotic death of hepatocytes a feature and contributing factor of many chronic and acute liver diseases can be a consequence of over-activation of the immune system. as potential therapeutic targets for treatment of inflammatory liver diseases. and in hepatocytes of mice injected with LPS+GalN (Chen et al. 2007 Zhao et al. 2001 Notably most studies on TNFα-induced apoptosis have been performed with cells in culture and very little is known about the mechanisms by which over-activation of the immune system causes TNFα-mediated immuno-pathological tissue destruction. Our experiments with gene-targeted mice demonstrated that TNFα-mediated hepatocyte apoptosis requires caspase-8 and involves the pro-apoptotic BH3-only proteins Bid activated by caspase-8 and Bim activated by JNK respectively. These cell death inducers and effectors can therefore be considered potential therapeutic targets for immuno-pathological liver disorders. RESULTS Caspase-8 Is Essential for LPS plus GalN-Induced Hepatocyte Destruction Experiments with gene-targeted mice demonstrated that expression of caspase-8 within hepatocytes is essential for anti-Fas-antibody induced hepatocyte killing and fatal hepatitis (Kang et al. 2004 It is however not clear whether caspase-8 is also essential for pathological killing of hepatocytes by TNFα. In fact several studies with cultured cells have indicated that TNFα kills cells by caspase-independent perhaps even non-apoptotic mechanisms (reviewed in (Ding and Yin 2004 When mice lacking caspase-8 selectively in hepatocytes (homozygotes Proscillaridin A expressing Proscillaridin A the Cre recombinase under control of the hepatocyte-specific albumin promoter) were challenged with LPS plus GalN they Proscillaridin A showed only minor elevation of serum ALT and AST levels (Figure 1A) retained normal liver structure (Figure 1B) and all mice survived long-term (Figure 1C). In contrast all littermate controls succumbed to this treatment within 8-10 h (Figure 1C) presenting at autopsy with abnormally elevated serum levels of ALT and AST (Figure 1A; Alb-Cre/vs control mice: p<0.015 for ALT p<0.0015 for AST) and extensive disruption of liver architecture (Figure 1B). Consistent with these observations Western blot analysis of liver extracts from LPS+GalN ITGA9 treated control animals revealed processing of Bid (p22) into its active p15 form tBid as well as extensive processing of caspase-3 and -7 whereas no Bid-cleavage and no activation of effector caspases could be detected in Alb-Cre/mice (Figure S2). Figure 1 LPS plus GalN-Induced Hepatitis Requires the Initiator Caspase Caspase-8 Is Inhibited by a Pan-Caspase Inhibitor and Involves Cleavage of the Pro-Apoptotic BH3-Only Bcl-2 Family Member Bid Consistent with the experiments using mice lacking caspase-8 in their hepatocytes treatment of C57BL/6 (wt) mice with the pan-caspase inhibitor Q-VD-oph resulted in a highly significant protection from LPS+GalN induced hepatitis as Proscillaridin A assessed by serum levels of ALT/AST and histological examination (Figures 1D 1 and S3A). However administration of Q-VD-oph even at multiple dosages afforded less protection than loss of caspase-8 (compare Figures S3B and 1C) presumably because this treatment did not achieve complete blockade of this enzyme. Similar to Fas-activation (Li et al. 1998 Luo et al. 1998 injection of wt mice with LPS+GalN caused rapid processing of pro-caspase-8 to produce the active p18 fragment cleavage of Bid (p22) into its active truncated p15 form (tBid) as well as processing and activation of effector caspases such as caspase-7 (p17) (Figure 1F). No processing of caspase-8 Bid or effector caspases was seen in liver extracts from LPS+GalN injected mice lacking TNFα (Figure S4). Collectively these results demonstrate that upon LPS+GalN injection activation of caspase-8 within hepatocytes is required for TNFα-mediated liver destruction and fatal hepatitis. Bid Is a Minor Contributor to LPS plus GalN-Induced Hepatocyte Apoptosis Caspase-8-mediated activation of Bid is essential for anti-Fas antibody induced liver destruction (Yin et al. 1999 We confirmed this observation (Kaufmann et al. 2007 and found that killing of hepatocytes by TNFα wt and Models of Hepatitis For Fas-mediated hepatitis mice were injected intravenously (i.v.) with 0.25 μg/g body weight recombinant soluble Fas ligand (FLAG? tagged Apotech) that had been crosslinked with 2 μg anti-FLAG? antibody (M2 SIGMA) per μg of FasL. For the LPS+GalN model mice were.