Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

TRAIL-induced apoptosis: between tumor therapy and immunopathology

The death ligand members of the tumor necrosis factor (TNF) family are potent inducers of apoptosis in a variety of cell types. In particular, TNF-related apoptosis-inducing ligand (TRAIL) has recently received much scientific and commercial attention because of its potent tumor cell-killing activity while leaving normal untransformed cells mostly unaffected. Furthermore, TRAIL strongly synergizes with conventional chemotherapeutic drugs in inducing tumor cell apoptosis, making it a most promising candidate for future cancer therapy. Increasing evidence indicates, however, that TRAIL may also induce or modulate apoptosis in primary cells. A particular concern is the potential side effect of TRAIL-based tumor therapies in the liver. In this review we summarize some of the recent findings on the role of TRAIL in tumor cell and hepatocyte apoptosis.

Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

Tauroursodeoxycholic Acid Affects PPAR gamma and TLR4 in Steatotic Liver Transplantation

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia-reperfusion (I/R). We examined whether tauroursodeoxycholic acid (TUDCA), a known inhibitor of endoplasmic reticulum (ER) stress, protects steatotic and nonsteatotic liver grafts preserved during 6 h in University of Wisconsin (UW) solution and transplanted. The protective mechanisms of TUDCA were also examined. Neither unfolded protein response (UPR) induction nor ER stress was evidenced in steatotic and nonsteatotic liver grafts after 6 h in UW preservation solution. TUDCA only protected steatotic livers grafts and did so through a mechanism independent of ER stress. It reduced proliferator-activated receptor-gamma(PPAR gamma) and damage. When PPAR gamma was activated, TUDCA did not reduce damage. TUDCA, which inhibited PPAR gamma, and the PPAR gamma antagonist treatment up-regulated toll-like receptor 4 (TLR4), specifically the TIR domain-containing adaptor inducing IFN beta (TRIF) pathway. TLR4 agonist treatment reduced damage in steatotic liver grafts. When TLR4 action was inhibited, PPAR gamma antagonists did not protect steatotic liver grafts. In conclusion, TUDCA reduced PPAR gamma and this in turn up-regulated the TLR4 pathway, thus protecting steatotic liver grafts. TLR4 activating-based strategies could reduce the inherent risk of steatotic liver failure after transplantation.

Connexin and pannexin (hemi) channels in the liver

The liver was among the first organs in which connexin proteins have been identified. Hepatocytes harbor connexin32 and connexin26, while non-parenchymal liver cells typically express connexin43. Connexins give rise to hemichannels, which dock with counterparts on adjacent cells to form gap junctions. Both hemichannels and gap junctions provide pathways for communication, via paracrine signaling or direct intercellular coupling, respectively. Over the years, hepatocellular gap junctions have been shown to regulate a number of liver-specific functions and to drive liver cell growth. In the last few years, it has become clear that connexin hemichannels are involved in liver cell death, particularly in hepatocyte apoptosis. This also holds true for hemichannels composed of pannexin1, a connexin-like protein recently identified in the liver. Moreover, pannexin1 hemichannels are key players in the regulation of hepatic inflammatory processes. The current paper provides a concise overview of the features of connexins, pannexins and their channels in the liver.

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL-Jun kinase-Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.

Macrophage activating syndrome is associated with lobular hepatitis and severe bile duct injury with cholestasis

Macrophage activating syndrome (MAS) is a rare hematological disorder associated with uncontrolled systemic T-cell activation. Persistent fever, fatigue and hepatosplenomegaly are frequent clinical manifestations, whereas hyperferritinemia, elevated serum lactate dehydrogenase levels and cytopenia are key criteria for the diagnosis of MAS. The nature of liver pathology in MAS has been partially elucidated but destructive biliary lesions have been rarely described. This report illustrates four cases of MAS developing marked cholestasis, leading to one case of biliary cirrhosis necessitating liver transplantation. Histologically, liver involvement was characterized in all cases by acute lobular hepatitis, marked hepatocyte apoptosis and small bile duct injury similar to the vanishing bile duct syndrome. Immuno-histological studies showed that the inflammatory changes and bile duct lesions were dominated by the presence of activated macrophages and T-cells, in particular CD8+ lymphocytes, and in part NK-cells. These findings suggest that in MAS, various T-cell triggers such as infection, autoimmune disease and malignancy might result in the release of cytokines, which in turn activate macrophages to trigger a systemic acute phase response and local tissue damage. This communication suggests that a macrophage, T- and NK-cell network is operational in the pathogenesis of the cholangiocyte, hepatocyte and sinus endothelial cell damage in MAS.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis

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5-HT1A AND 5-HT2C RECEPTOR GENE EXPRESSION AND FUNCTIONAL REGULATION DURING RAT HEPATOCYTE PROLIFERATION AND APOPTOSIS

The work is an attempt to understand the role of 5-HT, 5-HT1A and 5-HT2C receptors in the regulation of liver cell proliferation using in vivo and in vitro models. The work also focuses on the brain serotonergic changes associated with hapatocyte proliferation and apoptosis to delineate its regulatory function. The investigation of mechanisms involving different models of hepatocyte proliferation contributes to our knowledge about serotonergic regulation of cell growth, apoptosis and carcinogenesis of liver. The study reveals that the alteration of the 5-HT1A and 5-HT2C receptor function and gene expression in the brain stem, cerebral cortex and hypothalamus play an important role in the sympathetic regulation of cell proliferation, neoplastic transformation and apoptosis. The functional balance between 5-HT1A and 5-HT2C receptor plays an important role in regulating hepatocyte proliferation, neoplastic transformation and hepatic apoptosis. The regulatory role of 5-HT1A and 5-HT2C receptor during neoplastic transformation and apoptosis could lead to possible therapeutic intervention in the treatment of cancers and have immense clinical importance.

Endogenous Hepatocyte Growth Factor Attenuates Inflammatory Response in Glycerol-Induced Acute Kidney Injury

Background: Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats. Methods: Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated. Results: We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-alpha, IL-1 beta) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment. Conclusion: Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI. Copyright (C) 2008 S. Karger AG, Basel

Hepatocyte lesions and cellular immune response in yellow fever infection

The study of the in-situ cellular immune response is very important for the understanding of different liver infections. In the present study, 53 liver samples obtained by viscerotomy from patients who died during the course of jungle yellow fever were analyzed. The diagnosis was confirmed by serology, viral isolation and virus-specific immunohistochemistry. The specimens were analyzed by immunohistochemistry using specific antibodies for apoptosis, CD45RO, CD4, CD8, CD20, S100, CD57 and CD68. Quantitative analysis of the labeling pattern showed a clear predominance of the different phenotypes in the portal tract and midzone region of the acini. There was a predominance of T CD4+ lymphocytes, accompanied by the presence of T CD8+ lymphocytes, natural killer cells (CD57), macrophages and antigen-presenting cells (S100). The disproportion between the intensity of inflammation and the degree of hepatic injury was probably due to the intense apoptotic component, which classically does not induce an inflammatory response. The present study demonstrates that, despite the disproportion between injury and inflammation, the cellular immune response plays an important role in the pathogenesis of the hepatocytic injury observed in yellow fever, probably as a result of cytolytic actions through mechanisms involving MHC II and the activation of Fas receptors and granzymes/perforins. (C) 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Long-term ethanol consumption promotes hepatic tumorigenesis but impairs normal hepatocyte proliferation in rats

Chronic and excessive alcohol consumption has been related to an increased risk of several cancers, including that of the liver; however, studies in animal models have yet to conclusively determine whether ethanol acts as a tumor promoter in hepatic tumorigenesis. We examined whether prolonged alcohol consumption could act as a hepatic tumor promoter after initiation by diethylnitrosamine (DEN) in a rat model. Male Sprague-Dawley rats were injected with 20 mg DEN/kg body weight 1 wk before introduction of either an ethanol liquid diet or an isoenergic control liquid diet. Hepatic pathological lesions, hepatocyte proliferation, apoptosis, PPARα and PPARγ, and plasma insulin-like growth factor 1 (IGF-1) levels were assessed after 6 and 10 mo. Mean body and liver weights, plasma IGF-1 concentration, hepatic expressions of proliferating cellular nuclear antigen and Ki-67, and cyclin D1 in ethanol-fed rats were all significantly lower after 10 mo of treatment compared with control rats. In addition, levels of hepatic PPARγ protein, not PPARα, were significantly higher in the ethanol-fed rats after prolonged treatment. Although ethanol feeding also resulted in significantly fewer altered hepatic foci, hepatocellular adenoma was detected in ethanol-fed rats at 10 mo, but not in control rats given the same dose of DEN. Together, these results indicate that chronic, excessive ethanol consumption impairs normal hepatocyte proliferation, which is associated with reduced IGF-1 levels, but promotes hepatic carcinogenesis. © 2011 American Society for Nutrition.