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.

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.

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-beta1 (TGF-beta1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-beta1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand on NK Cells Protects From Hepatic Ischemia-Reperfusion Injury

BACKGROUND: Ischemia-reperfusion injury (IRI) significantly contributes to graft dysfunction after liver transplantation. Natural killer (NK) cells are crucial innate effector cells in the liver and express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent inducer of hepatocyte cell death. Here, we investigated if TRAIL expression on NK cells contributes to hepatic IRI. METHODS: The outcome after partial hepatic IRI was assessed in TRAIL-null mice and contrasted to C57BL/6J wild-type mice and after NK cell adoptive transfer in RAG2/common gamma-null mice that lack T, B, and NK cells. Liver IRI was assessed by histological analysis, alanine aminotransferase, hepatic neutrophil activation by myeloperoxidase activity, and cytokine secretion at specific time points. NK cell cytotoxicity and differentiation were assessed in vivo and in vitro. RESULTS: Twenty-four hours after reperfusion, TRAIL-null mice exhibited significantly higher serum transaminases, histological signs of necrosis, neutrophil infiltration, and serum levels of interleukin-6 compared to wild-type animals. Adoptive transfer of TRAIL-null NK cells into immunodeficient RAG2/common gamma-null mice was associated with significantly elevated liver damage compared to transfer of wild-type NK cells. In TRAIL-null mice, NK cells exhibit higher cytotoxicity and decreased differentiation compared to wild-type mice. In vitro, cytotoxicity against YAC-1 and secretion of interferon gamma by TRAIL-null NK cells were significantly increased compared to wild-type controls. CONCLUSIONS: These experiments reveal that expression of TRAIL on NK cells is protective in a murine model of hepatic IRI through modulation of NK cell cytotoxicity and NK cell differentiation.

Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells.

Immunization with Plasmodium sporozoites that have been attenuated by gamma-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain long-lived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p(-)), a member of the P48/45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p(-) sporozoites abort during development within the hepatocyte. Immunization with p36p(-) sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p(-) sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p(-) sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.

In the human urothelium and suburothelium, intradetrusor botulinum neurotoxin type A does not induce apoptosis: preliminary results

Intradetrusor injections of botulinum neurotoxin type A (BoNTA) are emerging as the preferred second-line treatment for neurogenic and idiopathic overactive bladder (OAB). In animal experiments, intradetrusor BoNTA injections have been shown to cause apoptosis in the bladder urothelium and suburothelium but not the detrusor.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Inhibition of the kynurenine-NAD+ pathway leads to energy failure and exacerbates apoptosis in pneumococcal meningitis

Pneumococcal meningitis causes neurological sequelae, including learning and memory deficits in up to half of the survivors. In both humans and in animal models of the disease, there is apoptotic cell death in the hippocampus, a brain region involved in learning and memory function. We previously demonstrated that in an infant rat model of pneumococcal meningitis, there is activation of the kynurenine (KYN) pathway in the hippocampus, and that there was a positive correlation between the concentration of 3-hydroxykynurenine and the extent of hippocampal apoptosis. To clarify the role of the KYN pathway in the pathogenesis of hippocampal apoptosis in pneumococcal meningitis, we specifically inhibited 2 key enzymes of the KYN pathway and assessed hippocampal apoptosis, KYN pathway metabolites, and nicotinamide adenine dinucleotide (NAD) concentrations by high-performance liquid chromatography. Pharmacological inhibition of kynurenine 3-hydroxylase and kynureninase led to decreased cellular NAD levels and increased apoptosis in the hippocampus. The cerebrospinal fluid levels of tumor necrosis factor and interleukin-1? and -? were not affected. Our data suggest that activation of the KYN pathway in pneumococcal meningitis is neuroprotective by compensating for an increased NAD demand caused by infection and inflammation;this mechanism may prevent energy failure and apoptosis in the hippocampus.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

N-myc downstream regulated gene-1 expression correlates with reduced pancreatic cancer growth and increased apoptosis in vitro and in vivo

The role of N-myc downstream regulated gene-1 (NDRG1) in cancer has recently gained interest, as potential regulator of cell death and tumor suppressor. Although its normal function in the pancreas is largely unknown, loss of NDRG1 expression is associated with a more aggressive tumor phenotype and poor outcome in pancreatic cancer patients.