Journal of Hepatology
Volume 44, Issue 5 , Pages 984-993, May 2006

The role of apoptosis versus oncotic necrosis in liver injury: Facts or faith?

Department of Medicine, Johannes Gutenberg University, Langenbeckstrasse 1, Mainz 55101, Germany

published online 03 March 2006.

Article Outline

Abbreviations: cIAP, cellular inhibitor of apoptosis, DISC, death inducing signaling complex, ER, endoplasmic reticulum, FHF, fulminant hepatic failure, PCD, programmed cell death, ROS, reactive oxygen species, SEC, sinusoidal endothelial cells

 

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1. Introduction 

A tightly controlled balance between cell division and cell death is a basic feature for the development and maintenance of liver homeostasis. Disturbances of this balance contribute to liver diseases: too much cell death can cause liver injury, too little cell death is a prerequisite for the development of hepatocellular carcinoma. Thus, a stringent control of the equilibrium of life and death in the liver is necessary. During the last decade most research activities in hepatology dealing with liver injury focussed on the evaluation of apoptosis pathways. Therefore, our understanding of the mechanisms of apoptosis has made profound progress. Programmed cell death (PCD) in the liver enables the physiological turnover of hepatocytes and the efficient removal of unwanted cells such as aged or virus-infected cells. Fulminant hepatic failure (FHF) and hepatocellular carcinoma are prototypical settings with uncontrolled massive apoptosis on the one hand, or resistance to apoptosis on the other hand. In addition to ‘classical apoptosis’, there is accumulating evidence that liver cells can die via PCD without typical features of apoptosis such as caspase activation. Various forms of caspase-independent cell death have been described, depicted as paraptosis, autophagy, mitotic catastrophe and others (Fig. 1).

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  • Fig. 1. 

    Modes of cell death in the liver. Hepatocytes can die from different modes of cell death. Apoptosis occurs in physiologic as well as pathologic conditions and represents a highly organized and genetically controlled type of cell death leading to shrinkage of the cell and disintegration into small apoptotic bodies. Paraptosis involves cytoplasmic vacuolation independent on caspase activity and in the absence of typical nuclear changes [68]. Its role in liver diseases is not yet defined. Necrosis (or oncosis/oncotic necrosis) leads to cellular edema and disruption of the cell membrane, e.g. in reperfusion liver injury. In autophagy, the cell's own lysosomal system leads to degradation of organelles and cell death. Breakdown of autophagy has been discussed to contribute to tumorigenesis in hepatocellular carcinoma [67]. Anoikis occurs if hepatocytes loose their contact to the extracellular matrix [73]. Mitotic catastrophe occurs after mitotic failure, but has not been described to contribute to liver injury so far [71]. FHF, fulminant hepatic failure; NASH, non-alcoholic steatohepatitis; ROS, reactive oxygen species; TNF, tumor necrosis factor.

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2. Apoptosis in liver injury 

Apoptosis is a common property of multicellular organisms to eliminate unwanted and potentially harmful cells [1], [2]. This process is actively executed by specific proteases, the caspases, and occurs in a programmed fashion, thus also referred to as PCD. Apoptosis is characterized by typical morphological changes mainly caused by caspase activity, such as shrinkage of the cell, condensation of chromatin, and disintegration of the cell into small apoptotic bodies. Apoptotic bodies are removed by phagocytosis, in the liver mainly by Kupffer cells and hepatic stellate cells (HSC), which is typically not accompanied by profound inflammatory reactions in physiologic conditions [3]. However, hepatocellular apoptosis in pathologic conditions may cause inflammatory reactions such as infiltration of neutrophils resulting in activation of hepatic stellate cells and liver fibrosis (review [4]).

Hepatocellular apoptosis can be triggered by two molecular pathways, an extrinsic pathway mediated by death receptors on the cell surface, and an intrinsic pathway, which is triggered at the mitochondrial level. Death receptors belong to the tumour necrosis factor (TNF) receptor superfamily. The most widely expressed death receptors on hepatocytes are CD95 (APO-1/Fas) and TNF receptor 1 (CD120a). TNF-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1 and -R2) are also present on hepatocytes. However, TRAIL receptor expression on hepatocytes as well as sensitivity to the specific ligand TRAIL is still a matter of debate [5]. The crucial point of death receptor signaling is the formation of a multimeric complex of proteins triggered by receptor cross-linking with their natural ligands. The structure formed is called the death-inducing signaling complex (DISC) [6]. The DISC of CD95 consists of trimerized CD95, the adapter Fas-associated death domain protein (FADD/Mort1), caspase-8 and caspase-10 [7], [8].

Depending on the cell type, either of two different pathways is activated downstream of CD95 after DISC formation. In type I cells, the death signal is propagated by a cascade that is initiated by the activation of large amounts of caspase-8 at the DISC and subsequent activation of downstream caspases. In type II cells, propagation of the apoptotic signal depends on its amplification via mitochondria [9]. In vivo-studies with Bid-deficient mice indicate that hepatocytes are CD95-type II cells [10].

The intrinsic pathway is initiated at the mitochondrial level. Mitochondria are central players in apoptosis induction and act as integrating sensors of various death stimuli.

The Bcl-2 family is a well-established family of proteins playing a pivotal role for mitochondrial integrity in hepatocytes. Anti-apoptotic members of the Bcl-2 family such as Bcl-2, Bcl-xL and myeloid cell leukemia-1 (Mcl-1) stabilize mitochondria, whereas pro-apoptotic members, such as Bid, Bax or Bak, actively initiate permeabilization of the mitochondrial outer membrane and trigger release of effector molecules such as cytochrome c. Once cytochrome c is released into the cytosol, the apoptosome is assembled, a multi-protein complex in which Apaf-1 serves as an oligomerization platform for assembly and autoproteolytic activation of caspase-9 [11].

Caspases are cysteinyl-aspartate-specific proteases, synthesized as inactive precursors, and play an important role in liver injury [12]. Upon specific cleavage at defined aspartate residues, caspases get activated. The so called ‘initiator caspases’ including caspase-8, -10, and -9, are recruited to large protein complexes such as the DISC or the apoptosome upon activation of the extrinsic or intrinsic death pathways. These caspases cleave and activate ‘executioner’ caspases, mainly caspase-3, -6, and -7. The extent of liver injury is mirrored by caspase activity. For example, increased caspase activation can be found not only in liver biopsies, but also in sera from patients with hepatitis C [13], [14]. In addition, serum caspase activity correlates with the extent of steatosis and the progression of fibrosis [15].

Apart from caspases, other protease families such as cathepsins have been implicated in hepatocellular apoptosis. Cathepsins belong to the most abundant lysosomal enzymes and are involved in degradation processes. Activation of death receptors as well as different stress stimuli can trigger permeabilization of lysosomes resulting in a translocation of cathepsins into the cytoplasm. Cathepsin activity can result in apoptotic features, dependent and independent of caspase activity [16]. Cathepsins can directly cleave and activate caspases contributing to apoptotic cascades [17]. Cathepsin B actively participates in hepatocellular apoptosis, e.g. after exposure of hepatocytes to TNF [18], [19], [20]. In cathepsin B knockout mice, hepatocytes are resistant to TNF-mediated apoptosis [21]. However, cathepsin B activation may also contribute to liver injury independent of apoptosis, since inhibition of cathepsin B reduces liver injury in models, in which oncotic necrosis is involved (e.g. ischemia-reperfusion injury and cholestasis) [22], [23], [24], [25].

Deregulation of the apoptosis program is pathophysiologically involved in acute as well as chronic liver diseases, including cholestasis [26], hepatitis C [27] as well as alcoholic and non-alcoholic steatohepatitis [28], [29] (recent review [30]). The most dramatic liver disease, FHF, is characterized by an uncontrolled massive death of hepatocytes [31]. Several studies in humans and rodents have shown that apoptosis of hepatocytes in acute and chronic liver diseases is generally mediated by death receptors, in particular CD95 [31], [32], [33], [34]. Hepatocytes constitutively express CD95 on their surface rendering the liver highly sensitive towards CD95-mediated apoptosis. Mice injected with agonistic CD95 antibodies rapidly die of liver failure [32], [35]. Correspondingly, primary human hepatocytes are highly sensitive to CD95-mediated apoptosis in vitro [31], [36]. Increased CD95 expression has been observed in various liver diseases including viral hepatitis and alcohol induced liver disease [31]. Recent studies have demonstrated that the phosphorylation pattern of CD95 is important for the survival of hepatocytes: bile salts trigger epidermal growth factor receptor (EGF-R)-mediated tyrosine phosphorylation of CD95 and thereby enable formation of the DISC at the cell membrane [37]. High CD95 expression makes hepatocytes quite sensitive to cytotoxic T cells expressing CD95L. In addition, the specific ligand of CD95, CD95L, can be detected in sera of patients with liver diseases. CD95L derived from the placenta can enter the liver via the feto-maternal circulation causing liver damage in patients with HELLP syndrome [38]. In models of chronic liver injury, CD95-mediated apoptosis has been associated with liver fibrosis [39]. However, inflammatory reactions caused by liver resident and recruited inflammatory cells rather than apoptosis of liver parenchymal cells are supposed to play a major role in liver fibrosis [40].

During liver injury, hepatocytes, endothelial cells and cholangiocytes are endangered to die by apoptosis as a result of the activity of cytokines such as TNF, IL-1β and IFN-γ, as well as toxic metabolites, ROS, bile acids and other substances. Several anti-apoptotic mechanisms antagonize apoptosis induction in hepatocytes, including activation of NF-κB [41], [42] and survival pathways such as the MEK/Erk and PI3K signaling pathways [43]. TNF is present abundantly during acute and chronic liver failure and activates TNFR1. However, hepatocytes are well protected against apoptosis induction by TNF due to an activation of NF-κB as a feedback loop [44]. NF-κB translocates to the nucleus and induces expression of several anti-apoptotic genes [45]. Inhibition of NF-κB induces apoptosis in hepatocytes indicating its prominent role in the induction of transcription of anti-apoptotic genes [46], [47].

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3. Oncotic necrosis in liver injury 

Contrary to the controlled cellular death program in apoptosis, necrosis (or recently renamed oncosis or oncotic necrosis [48]) is a more chaotic way of dying. It results from metabolic disruption with energy depletion and loss of adenosine triphosphate (ATP). Loss of ATP leads to cellular edema, rounding and swelling of mitochondria, dilations of the endoplasmic reticulum (ER), lysosomal disruption and formation of plasma membrane protrusions called blebs [49]. Bleb formation which is caused by disrupted cellular volume control and cytoskeletal disturbances, is reversible, e.g. after reoxygenation. Necrotic cell death occurs by failure of the plasma membrane permeability barrier leading to the disruption of the plasma membrane and the release of cellular components. The release of cellular contents triggers an inflammatory response in the surrounding tissue [3]. In addition, plasma membrane permeability enables vital dyes such as trypan blue to enter the cell.

A typical stimulus for oncotic necrosis in the liver is cold hepatic ischemia followed by reperfusion, which leads to necrosis within minutes [25].

In cholestasis, CD95-mediated apoptosis has been discussed to contribute to liver injury [26]. Recent studies report a major role of necrosis in cholestasis. In the model of experimental bile duct ligation in rats, liver injury has been shown to be almost exclusively caused by oncotic necrosis correlating with the severity of the inflammatory response [24], [50], [51]. Interestingly, necrosis can also be regulated and may involve in some cases caspase activation [52].

In addition, oncotic necrosis is involved in drug-induced hepatotoxicity, which is a frequent cause of acute liver failure and a major reason for drug withdrawal from pharmaceutical development and clinical use. Mitochondria are prominent targets for the hepatotoxicity of many drugs. Dysfunction of mitochondria results in impairment of energy metabolism and an intracellular oxidative stress with excessive formation of reactive oxygen species and peroxynitrite triggering necrosis [53]. Drug-mediated induction of cytochrome P450 isoenzymes also promotes oxidative stress and cell injury. In acetaminophen-induced hepatotoxicity, production of reactive nitrogen and oxygen species cause protein nitrosylation, lipid peroxidation and, finally, hepatic necrosis [54], [55]. Other studies also demonstrate a contribution of death receptor-mediated apoptosis in drug-induced liver cell death, e.g. after exposure to chemotherapeutic agents [56].

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4. Other forms of cell death in liver injury 

PCD is an active cellular process, which can be intercepted by inhibition of intracellular signaling. On the contrary, necrosis is an accidental process independent of energy [57]. The terms ‘programmed cell death’ and ‘apoptosis’ have been frequently used as synonyms in the past. In recent years, various forms of PCD have been described, which cannot be readily classified as apoptosis or necrosis, since they do not show a typical morphology. These forms of cell death are often characterized by an absence of caspase activity, which is a prerequisite for the typical apoptotic morphology. Already a decade ago, it was shown in leukemia cells that inhibition of caspase activity did not inhibit Bax-induced cell death but changed the apoptotic morphology of the dying cells [58]. Caspase-independent cell death is an important protective mechanism for an organism to eliminate harmful cells in case of a failure of caspase activation. Different organelles, such as mitochondria, lysosomes and the ER can be involved in the induction of caspase-independent (as well as caspase-dependent) PCD by the release of proteases including cathepsins and calpains (calcium-activated neutral proteases) [59], [60]. While cathepsin B has been identified to trigger caspase activation, cathepsin D can induce cell death independent of caspase activity via triggering of apoptosis inducing factor (AIF) release from mitochondria [20], [61].

An entirely different form of PCD is called autophagy [62], [63]. In this form of active cell death cells die by chewing themselves up. Unlike apoptosis, cells are degraded with little or no help from phagocytes and use their own lysosomal system for degradation. It is characterized by the appearance of autophagic vacuoles originating from lysosomes, followed by mitochondrial dilatation and enlargement of the ER and Golgi [64]. Autophagy helps to eliminate long-lived proteins and plays an important role in cellular remodeling due to damage, stress or differentiation. Hepatocytes of liver grafts show autophagic vacuoles/autolysosomes after reperfusion suggesting that warm reperfusion acted as a stress stimulus to hepatocytes triggering autophagic degeneration [65]. The role of autophagy in the pathogenesis of other liver diseases continues to be explored. In carcinogenesis of hepatocellular carcinoma breakdown of autophagy might play an important role [66], [67].

The differentiation between autophagy and another form of cell death, called paraptosis, is not clear so far. Paraptosis is characterized by swelling of mitochondria and the ER leading to cytoplasmatic vacuolation [68]. Paraptosis can be induced, e.g. by insulin-like growth factor I receptors via activation of mitogen-activated protein kinases [69]. The role of paraptosis in liver injury has not been defined yet. The same applies for another type of cell death depicted as mitotic catastrophe. It results from abnormal mitosis due to defective cell cycle checkpoints and leads to the formation of interphase cells with multiple micronuclei (review [70], [71]). It can be accompanied by caspase activation, but caspase inhibition has been reported not to prevent catastrophic mitosis [72].

Epithelial cells such as hepatocytes require contact with extracellular matrix to survive. In the absence of integrin-mediated signals, normal epithelial cells detach and subsequently undergo a form of apoptosis termed anoikis. In anoikis, indicators of apoptosis do not appear until the cell has become non-adherent, because the loss of adhesion is the cause and not the result of cell death. For hepatocyte survival, β1-integrin-mediated attachment to hepatic extracellular matrix is a prerequisite to avoid anoikis [73].

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5. Conflicting patterns of cell death in liver injury 

Deregulation of apoptosis contributes to liver injury in pathologic conditions. However, other types of PCD are observed in liver injury. The contribution of the different death modes including oncotic necrosis often remains elusive (Fig. 2). First of all, differentiation between death patterns, even between apoptosis and necrosis, can be difficult, since morphological and molecular overlaps occur. Thus, different modes of cell death can neither be classified as apoptosis nor necrosis.

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  • Fig. 2. 

    Conflicting patterns of cell death in the liver. Inappropriate death of hepatocytes causes liver injury in different pathological conditions. However, the classic dichotomy between apoptosis and necrosis does not reflect the complexity of cell death patterns in liver injury. Apoptosis and necrosis can share features and mechanisms, which often makes discrimination difficult. ROS, reactive oxygen species.

As a consequence of the increasing understanding of apoptosis pathways many biochemical and immunologic assays have been developed to characterize apoptosis apart from the classical morphological criteria, e.g. enzyme assays for caspase activity, annexin-V labeling for phosphatidyl serine externalization, measurement of mitochondrial membrane permeabilization and cytochrome c release (review [74], [75]). Other methods analyze the typical pattern of DNA cleavage in apoptosis leading to characteristic DNA fragments with multiples of 190 base pairs due to DNA cleavage between nucleosomes. To detect these DNA fragments, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay is frequently used. However, none of the changes monitored in these assays, is unique to apoptosis. Cytochrome c release also occurs in oncotic necrosis. Annexin-V labeling occurs in necrotic cells as well due to membrane permeability. In necrotic cells DNA cleavage is usually not internucleosomal. However, a typical DNA ladder pattern after gel electrophoresis has also been described for necrotic cells [76]. In addition, TUNEL assay may not distinguish internucleosomal DNA cleavage of apoptosis from the pattern of DNA cleavage in necrosis [77]. In the model of bile duct ligation-associated liver injury TUNEL staining is frequently positive. However, an absence of any relevant morphological, biochemical, and immunohistochemical evidence of apoptosis has been described [50]. Thus, controversial conclusions in models of liver injury may have been due to the use of TUNEL assay [39]. In conclusion, no specific assay or parameter, except the morphological changes such as chromatin condensation and nuclear fragmentation in vivo, allows a clear distinction between apoptosis and oncotic necrosis. Furthermore, it is important to mention that the specific morphological changes of apoptosis can disappear in the course of time, so that apoptotic cell death might be underestimated. One of the most exclusive characteristics of apoptosis is activation of caspase-3, albeit apoptosis induction does not require active caspase-3. However, caspase activity does not automatically reflect apoptosis cascades. Caspases can trigger secondary necrosis when they cleave and inactivate plasma membrane Ca2+ transport systems causing secondary Ca2+ overload and membrane lysis [52].

In various liver diseases, critical death stimuli have been identified. However, death stimuli such as cytokines, bile acids or ROS can trigger different modes of cell death in parallel. ROS, including superoxide anions, hydroxyl radicals and H2O2, are generated by mitochondria and cytochrome P450 enzymes in hepatocytes, but also derive from Kupffer cells and inflammatory cells includinig neutrophils [78]. Normally, ROS are scavenged by enzymes like the superoxide dismutase and antioxidants like glutathione, which is present in high amounts in hepatocytes [79]. If detoxification mechanisms are impaired, oxidative stress occurs which can induce cell death, e.g. in obstructive cholestasis [80] and hepatic ischemia-reperfusion [25]. A decision between necrotic and apoptotic cell death upon oxidative stress may depend on the intracellular ATP level. A marked depletion of ATP by mitochondrial permeability transition facilitates necrotic cell death, whereas high levels of ATP favor the development of apoptosis. Manipulating the intracellular ATP level can cause necrosis although typical apoptosis stimuli occur [81]. In addition, molecular switches between apoptosis and necrosis have been described [82]. For example, apoptosis induction may be followed by secondary necrosis, when ATP is eventually depleted during apoptosis [83]. On the contrary, if the necrosis pathway is inhibited, death stimuli may eventually force the cell into apoptosis [25]. Changes in energy levels and the switches between cell death programs lead to a situation in vivo, in which different cell death patterns may coexist [84]. In addition, ROS generation is often accompanied by activation of lysosomes, which can lead to different death patterns. Lysosomal proteases can directly cleave and activate caspases confirming that lysosomal permeabilization also contributes to apoptosis cascades [17]. However, the amount of released lysosomal proteases is important for the type of cell death: low release triggers apoptosis-like cell death, whereas massive release leads to necrosis [85]. Ineffective elimination of apoptotic bodies may also result in an inflammatory response leading to a switch from apoptosis to necrosis [30].

Several mediators, such as NO, have been described to function as potent modulators of death programs [86]. NO can move from one cell to the other as a versatile messenger with an extreme short half-life and has been implicated in death of hepatocytes [84]. NO may cause a switch from apoptosis to necrosis by depletion of ATP or inactivation of caspases, e.g. in ROS-stimulated rat hepatocytes. On the contrary, NO can also increase caspase activity in hepatocytes by a cGMP-dependent mechanism [86]. Signaling pathways leading to NO-induced cell death are still poorly understood. The intensity of the NO stimulus and the time to NO exposure may influence the type of cell death and can also result in an inhibition of death [87].

The complex situation of death programs in liver injury is further complicated by complex cross talks between different proteolytic systems, such as caspases, calpains and cathepsins. Calpains, a family of cytosolic proteases, reside within the cytoplasm as inactive zymogens and can be activated by intracellular calcium reflux. Calpains can synergize or in contrast hinder caspases in the execution of cell death [88]. The cross talk between calpains and cathepsins may also influence the mode of cell death in liver injury as it has been shown in neuronal cells [89].

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6. Targeting cell death in liver diseases 

Liver injury is caused by a complex picture of cell death patterns. Since the apoptosis program is deranged in a variety of liver diseases, many therapeutic approaches target the apoptosis cascade (Fig. 3). Inhibition of apoptosis has been proved to be effective, e.g. in FHF. A series of publications describe successful rescue of animals from FHF by blocking caspase activity [90], expression of anti-apoptotic Bcl-2 family members such as Bcl-2 [91] and expression of dominant negative FADD/MORT1 [92], [93]. The discovery of RNA interference opens up new opportunities to specifically target apoptosis pathways, e.g. through downregulating genes encoding CD95 or caspase-8 [94], [95].

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  • Fig. 3. 

    Pro-survival strategies for the treatment of liver diseases. Over the past years, much effort has been devoted to the search of strategies to inhibit liver injury. Most of these efforts target apoptosis pathways, such as inhibition of caspases and death receptor signaling. KC, Kupffer cells; HSC, hepatic stellate cells; cIAP, cellular inhibitors of apoptosis; DISC, death inducing signaling complex.

Caspases are an attractive target not only for anti-apoptotic interventions in acute, but also in chronic hepatitis [96]. Caspase inhibitors have already entered phase II clinical trials in patients with hepatitis C unresponsive to approved anti-viral agents and in patients after liver transplantation [97]. Interestingly, caspase inhibition by pharmacological inhibitors as well as by siRNA approaches has been shown to reduce liver injury in models, which have been discussed to be mainly mediated by oncotic necrosis, such as the model of cold ischemia-warm reperfusion injury [98], [99].

Another approach to interrupt the caspase cascade is overexpression of cIAP family members, which have been proved to protect hepatocytes from apoptosis in vitro [41].

Other approaches to reduce cell death in liver injury include inhibition of calpains and cathepsins. Calpain inhibition reduces the number of apoptotic sinusoidal endothelial cells (SEC). This may be of special interest for the prevention of reperfusion injuries in clinical transplantation [100]. Inhibition of cathepsin B is of therapeutic interest, e.g. for the attenuation of hepatic injury in cholestasis [101] or reperfusion injury [22].

However, interference with cell death pathways in liver diseases, e.g. inhibition of caspases, may not be effective in complex chronic pathophysiologic conditions (Fig. 4: overview on putative pitfalls of pro-survival therapy strategies in liver diseases). Interference with caspase activity may not alter the extent of death, but rather the shape of demise [81], since caspase inhibitors cannot prevent the appearance of all features of apoptosis [102]. Several forms of demise including apoptosis-like death can occur in a caspase independent manner or are even accelerated by caspase inhibitors [103]. Furthermore, deletion of single caspases may have only localized and partial effects on cell death, whereas broad-range caspase inhibitors may inhibit unrelated proteases, whose activity may be necessary for survival. In addition, caspase inhibition may increase oxidative stress, because caspases contribute to the removal of damaged and ROS producing mitochondria, e.g. after stimulation with TNF [104].

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  • Fig. 4. 

    Pitfalls of anti-apoptotic therapy strategies liver diseases. Targeting death pathways in the liver represents a powerful tool for the treatment of liver diseases. However, some potential side effects should be considered. Senescence: a signal transduction program leading to irreversible cell cycle arrest. HSC, hepatic stellate cells.

The design of anti-apoptotic therapeutic strategies should consider the overlapping death programs in hepatocytes. Inhibition of apoptosis may have little effect on the survival of liver cells, if a decrease in apoptosis is compensated by an increase in the fractions of cells that undergo permanent growth arrest with features of cell senescence, or die through the process of mitotic catastrophe. Considering the fact that switches between different cell death modes can occur, dependent on the mediators and tissue conditions, targeting of individual death pathways may not be sufficient to ameliorate liver injury effectively. In addition, blocking of cell death in the liver may not only promote survival of hepatocytes, but also that of potentially harmful cells, including hepatic NKT cells. NKT cells are abundant in the normal liver and are discussed to contribute to the pathophysiology of FHF and other forms of liver injury such as viral hepatitis [105], [106]. An anti-apoptotic therapy regimen may also trigger liver fibrosis by preventing cell death of hepatic stellate cells [107]. Finally, promotion of cell survival in liver injury may favor carcinogenesis of hepatocellular carcinoma, since death of abnormal cells in the liver is an important process to prevent clonal expansion and tumor formation.

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7. Conclusion 

Death programs in the liver enable the elimination of damaged and unwanted cells in vivo. In acute and chronic liver injury, the balance between death and survival is disturbed. Enhanced liver cell death through apoptosis and necrosis is a key pathogenic feature of liver diseases. However, under pathological conditions, various cell death patterns in hepatocytes can neither be classified as necrosis nor as apoptosis, e.g. various forms of programmed cell death independent on caspase activity. In addition, apoptosis and necrosis pathways are often interwined and apoptotic and necrotic death markers concomitantly occur at the same time. Hepatocytes may even switch between different death pathways. Thus, overlaps between the signaling pathways of different death programs make exclusive definitions artificial. It still remains elusive which factors determine the type of death. Research being currently conducted in this exciting field is likely to be beneficial for the future treatment of liver diseases.

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References 

  1. Krammer PH. CD95's deadly mission in the immune system. Nature. 2000;407:789–795
  2. Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004;116:205–219
  3. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–257
  4. Canbay A, Friedman S, Gores GJ. Apoptosis: the nexus of liver injury and fibrosis. Hepatology. 2004;39:273–278
  5. Mori E, Thomas M, Motoki K, Nakazawa K, Tahara T, Tomizuka K, et al. Human normal hepatocytes are susceptible to apoptosis signal mediated by both TRAIL-R1 and TRAIL-R2. Cell Death Differ. 2004;11:203–207
  6. Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH, et al. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. Eur. Mol. Biol. Org. J. 1995;14:5579–5588
  7. Walczak H, Sprick MR. Biochemistry and function of the DISC. Trends Biochem Sci. 2001;26:452–453
  8. Lavrik I, Golks A, Krammer PH. Death receptor signaling. J Cell Sci. 2005;118:265–267
  9. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et al. Two CD95 (APO-1/Fas) signaling pathways. Eur. Mol. Biol. Org. J. 1998;17:1675–1687
  10. Li S, Zhao Y, He X, Kim TH, Kuharsky DK, Rabinowich H, et al. Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis. J Biol Chem. 2002;277:26912–26920
  11. Hengartner MO. The biochemistry of apoptosis. Nature. 2000;407:770–776
  12. Jones RA, Johnson VL, Buck NR, Dobrota M, Hinton RH, Chow SC, et al. Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases. Hepatology. 1998;27:1632–1642
  13. Kronenberger B, Wagner M, Herrmann E, Mihm U, Piiper A, Sarrazin C, et al. Apoptotic cytokeratin 18 neoepitopes in serum of patients with chronic hepatitis C. J Viral Hepat. 2005;12:307–314
  14. Bantel H, Lugering A, Poremba C, Lugering N, Held J, Domschke W, et al. Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infection. Hepatology. 2001;34:758–767
  15. Seidel N, Volkmann X, Langer F, Flemming P, Manns MP, Schulze-Osthoff K, et al. The extent of liver steatosis in chronic hepatitis C virus infection is mirrored by caspase activity in serum. Hepatology. 2005;42:113–120
  16. Jaattela M. Multiple cell death pathways as regulators of tumour initiation and progression. Oncogene. 2004;23:2746–2756
  17. Schotte P, Van Criekinge W, Van de Craen M, Van Loo G, Desmedt M, Grooten J, et al. Cathepsin B-mediated activation of the proinflammatory caspase-11. Biochem Biophys Res Commun. 1998;251:379–387
  18. Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, et al. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest. 2000;106:1127–1137
  19. Werneburg NW, Guicciardi ME, Bronk SF, Gores GJ. Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent. Am J Physiol Gastrointest Liver Physiol. 2002;283:G947–G956
  20. Roberts LR, Adjei PN, Gores GJ. Cathepsins as effector proteases in hepatocyte apoptosis. Cell Biochem Biophys. 1999;30:71–88
  21. Guicciardi ME, Miyoshi H, Bronk SF, Gores GJ. Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications. Am J Pathol. 2001;159:2045–2054
  22. Baskin-Bey ES, Canbay A, Bronk SF, Werneburg N, Guicciardi ME, Nyberg SL, et al. Cathepsin B inactivation attenuates hepatocyte apoptosis and liver damage in steatotic livers after cold ischemia-warm reperfusion injury. Am J Physiol Gastrointest Liver Physiol. 2005;288:G396–G402
  23. Canbay A, Guicciardi ME, Higuchi H, Feldstein A, Bronk SF, Rydzewski R, et al. Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis. J Clin Invest. 2003;112:152–159
  24. Gujral JS, Liu J, Farhood A, Jaeschke H. Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation. Hepatology. 2004;40:998–1007
  25. Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology. 2003;125:1246–1257
  26. Miyoshi H, Rust C, Roberts PJ, Burgart LJ, Gores GJ. Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas. Gastroenterology. 1999;117:669–677
  27. Patel T, Roberts LR, Jones BA, Gores GJ. Dysregulation of apoptosis as a mechanism of liver disease: an overview. Semin Liver Dis. 1998;18:105–114
  28. Ribeiro PS, Cortez-Pinto H, Sola S, Castro RE, Ramalho RM, Baptista A, et al. Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB in the liver of nonalcoholic and alcoholic steatohepatitis patients. Am J Gastroenterol. 2004;99:1708–1717
  29. Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD, et al. Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology. 2003;125:437–443
  30. Guicciardi ME, Gores GJ. Apoptosis: a mechanism of acute and chronic liver injury. Gut. 2005;54:1024–1033
  31. Galle PR, Hofmann WJ, Walczak H, Schaller H, Otto G, Stremmel W, et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med. 1995;182:1223–1230
  32. Galle PR, Krammer PH. CD95-induced apoptosis in human liver disease. Semin Liver Dis. 1998;18:141–151
  33. Faubion WA, Gores GJ. Death receptors in liver biology and pathobiology. Hepatology. 1999;29:1–4
  34. Strand S, Hofmann WJ, Grambihler A, Hug H, Volkmann M, Otto G, et al. Hepatic failure and liver cell damage in acute Wilson's disease involve CD95 (APO-1/Fas) mediated apoptosis. Nat Med. 1998;4:588–593
  35. Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, et al. Lethal effect of the anti-Fas antibody in mice. Nature. 1993;364:806–809
  36. Schulze-Bergkamen H, Untergasser A, Dax A, Vogel H, Buchler P, Klar E, et al. Primary human hepatocytes–a valuable tool for investigation of apoptosis and hepatitis B virus infection. J Hepatol. 2003;38:736–744
  37. Reinehr R, Haussinger D. Inhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95. Gastroenterology. 2004;126:249–262
  38. Strand S, Strand D, Seufert R, Mann A, Lotz J, Blessing M, et al. Placenta-derived CD95 ligand causes liver damage in hemolysis, elevated liver enzymes, and low platelet count syndrome. Gastroenterology. 2004;126:849–858
  39. Canbay A, Higuchi H, Bronk SF, Taniai M, Sebo TJ, Gores GJ. Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis. Gastroenterology. 2002;123:1323–1330
  40. Ramadori G, Saile B. Inflammation, damage repair, immune cells, and liver fibrosis: specific or nonspecific, this is the question. Gastroenterology. 2004;127:997–1000
  41. Schoemaker MH, Ros JE, Homan M, Trautwein C, Liston P, Poelstra K, et al. Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis. J Hepatol. 2002;36:742–750
  42. Miyoshi H, Rust C, Guicciardi ME, Gores GJ. NF-kappaB is activated in cholestasis and functions to reduce liver injury. Am J Pathol. 2001;158:967–975
  43. Schulze-Bergkamen H, Brenner D, Krueger A, Suess D, Fas SC, Frey CR, et al. Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95-mediated apoptosis via Akt. Hepatology. 2004;39:645–654
  44. Bradham CA, Qian T, Streetz K, Trautwein C, Brenner DA, Lemasters JJ. The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release. Mol Cell Biol. 1998;18:6353–6364
  45. Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science. 2002;296:1634–1635
  46. Bellas RE, FitzGerald MJ, Fausto N, Sonenshein GE. Inhibition of NF-kappa B activity induces apoptosis in murine hepatocytes. Am J Pathol. 1997;151:891–896
  47. Schuchmann M, Galle PR. Dead or alive—NF-kappaB, the guardian which tips the balance. J Hepatol. 2002;36:827–828
  48. Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol. 1995;146:3–15
  49. Lemasters JJ, DiGuiseppi J, Nieminen AL, Herman B. Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes. Nature. 1987;325:78–81
  50. Fickert P, Trauner M, Fuchsbichler A, Zollner G, Wagner M, Marschall HU, et al. Oncosis represents the main type of cell death in mouse models of cholestasis. J Hepatol. 2005;42:378–385
  51. Schoemaker MH, Gommans WM, Conde de la Rosa L, Homan M, Klok P, Trautwein C, et al. Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation. J Hepatol. 2003;39:153–161
  52. Schwab BL, Guerini D, Didszun C, Bano D, Ferrando-May E, Fava E, et al. Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis. Cell Death Differ. 2002;9:818–831
  53. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci. 2002;65:166–176
  54. Michael SL, Pumford NR, Mayeux PR, Niesman MR, Hinson JA. Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species. Hepatology. 1999;30:186–195
  55. Gujral JS, Knight TR, Farhood A, Bajt ML, Jaeschke H. Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis?. Toxicol Sci. 2002;67:322–328
  56. Muller M, Strand S, Hug H, Heinemann EM, Walczak H, Hofmann WJ, et al. Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. J Clin Invest. 1997;99:403–413
  57. Leist M, Jaattela M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol. 2001;2:589–598
  58. Xiang J, Chao DT, Korsmeyer SJ. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci USA. 1996;93:14559–14563
  59. Broker LE, Kruyt FA, Giaccone G. Cell death independent of caspases: a review. Clin Cancer Res. 2005;11:3155–3162
  60. Ferri KF, Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol. 2001;3:E255–E263
  61. Bidere N, Lorenzo HK, Carmona S, Laforge M, Harper F, Dumont C, et al. Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J Biol Chem. 2003;278:31401–31411
  62. Baehrecke EH. Autophagic programmed cell death in Drosophila. Cell Death Differ. 2003;10:940–945
  63. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 2004;23:2891–2906
  64. Schweichel JU, Merker HJ. The morphology of various types of cell death in prenatal tissues. Teratology. 1973;7:253–266
  65. Lu Z, Dono K, Gotoh K, Shibata M, Koike M, Marubashi S, et al. Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation. Arch Histol Cytol. 2005;68:71–80
  66. Schwarze PE, Seglen PO. Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats. Exp Cell Res. 1985;157:15–28
  67. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science. 2004;306:990–995
  68. Sperandio S, de Belle I, Bredesen DE. An alternative, nonapoptotic form of programmed cell death. Proc Natl Acad Sci USA. 2000;97:14376–14381
  69. Sperandio S, Poksay K, de Belle I, Lafuente MJ, Liu B, Nasir J, et al. Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix. Cell Death Differ. 2004;11:1066–1075
  70. King KL, Cidlowski JA. Cell cycle and apoptosis: common pathways to life and death. J Cell Biochem. 1995;58:175–180
  71. Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G. Cell death by mitotic catastrophe: a molecular definition. Oncogene. 2004;23:2825–2837
  72. Roninson IB, Broude EV, Chang BD. If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Drug Resist Updat. 2001;4:303–313
  73. Pinkse GG, Voorhoeve MP, Noteborn M, Terpstra OT, Bruijn JA, De Heer E. Hepatocyte survival depends on beta1-integrin-mediated attachment of hepatocytes to hepatic extracellular matrix. Liver Int. 2004;24:218–226
  74. Jaeschke H, Gujral JS, Bajt ML. Apoptosis and necrosis in liver disease. Liver Int. 2004;24:85–89
  75. Lovborg H, Gullbo J, Larsson R. Screening for apoptosis—classical and emerging techniques. Anticancer Drugs. 2005;16:593–599
  76. Dong Z, Saikumar P, Weinberg JM, Venkatachalam MA. Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases. Am J Pathol. 1997;151:1205–1213
  77. Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology. 1995;21:1465–1468
  78. Jaeschke H, Ho YS, Fisher MA, Lawson JA, Farhood A. Glutathione peroxidase-deficient mice are more susceptible to neutrophil-mediated hepatic parenchymal cell injury during endotoxemia: importance of an intracellular oxidant stress. Hepatology. 1999;29:443–450
  79. Xu Y, Jones BE, Neufeld DS, Czaja MJ. Glutathione modulates rat and mouse hepatocyte sensitivity to tumor necrosis factor toxicity. Gastroenterology. 1998;115:1229–1237
  80. Gujral JS, Farhood A, Bajt ML, Jaeschke H. Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology. 2003;38:355–363
  81. Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med. 1997;185:1481–1486
  82. Nicotera P, Melino G. Regulation of the apoptosis–necrosis switch. Oncogene. 2004;23:2757–2765
  83. Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, et al. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta. 1998;1366:177–196
  84. Leist M, Gantner F, Jilg S, Wendel A. Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. J Immunol. 1995;154:1307–1316
  85. Bursch W. The autophagosomal–lysosomal compartment in programmed cell death. Cell Death Differ. 2001;8:569–581
  86. Li J, Bombeck CA, Yang S, Kim YM, Billiar TR. Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. J Biol Chem. 1999;274:17325–17333
  87. Vodovotz Y, Kim PK, Bagci EZ, Ermentrout GB, Chow CC, Bahar I, et al. Inflammatory modulation of hepatocyte apoptosis by nitric oxide: in vivo, in vitro, and in silico studies. Curr Mol Med. 2004;4:753–762
  88. Neumar RW, Xu YA, Gada H, Guttmann RP, Siman R. Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis. J Biol Chem. 2003;278:14162–14167
  89. Yamashima T. Ca2+-dependent proteases in ischemic neuronal death: a conserved ‘calpain-cathepsin cascade' from nematodes to primates. Cell Calcium. 2004;36:285–293
  90. Rodriguez I, Matsuura K, Ody C, Nagata S, Vassalli P. Systemic injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death. J Exp Med. 1996;184:2067–2072
  91. Lacronique V, Mignon A, Fabre M, Viollet B, Rouquet N, Molina T, et al. Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat Med. 1996;2:80–86
  92. Streetz K, Leifeld L, Grundmann D, Ramakers J, Eckert K, Spengler U, et al. Tumor necrosis factor alpha in the pathogenesis of human and murine fulminant hepatic failure. Gastroenterology. 2000;119:446–460
  93. Schuchmann M, Varfolomeev EE, Hermann F, Rueckert F, Strand D, Koehler H, et al. Dominant negative MORT1/FADD rescues mice from CD95 and TNF-induced liver failure. Hepatology. 2003;37:129–135
  94. Zender L, Hutker S, Liedtke C, Tillmann HL, Zender S, Mundt B, et al. Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc Natl Acad Sci USA. 2003;100:7797–7802
  95. Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 2003;9:347–351
  96. Bajt ML, Vonderfecht SL, Jaeschke H. Differential protection with inhibitors of caspase-8 and caspase-3 in murine models of tumor necrosis factor and Fas receptor-mediated hepatocellular apoptosis. Toxicol Appl Pharmacol. 2001;175:243–252
  97. Valentino KL, Gutierrez M, Sanchez R, Winship MJ, Shapiro DA. First clinical trial of a novel caspase inhibitor: anti-apoptotic caspase inhibitor, IDN-6556, improves liver enzymes. Int J Clin Pharmacol Ther. 2003;41:441–449
  98. Contreras JL, Vilatoba M, Eckstein C, Bilbao G, Anthony Thompson J, Eckhoff DE. Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice. Surgery. 2004;136:390–400
  99. Natori S, Higuchi H, Contreras P, Gores GJ. The caspase inhibitor IDN-6556 prevents caspase activation and apoptosis in sinusoidal endothelial cells during liver preservation injury. Liver Transpl. 2003;9:278–284
  100. Sindram D, Kohli V, Madden JF, Clavien PA. Calpain inhibition prevents sinusoidal endothelial cell apoptosis in the cold ischemic rat liver. Transplantation. 1999;68:136–140
  101. Guicciardi ME, Gores GJ. Cheating death in the liver. Nat Med. 2004;10:587–588
  102. McCarthy NJ, Whyte MK, Gilbert CS, Evan GI. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J Cell Biol. 1997;136:215–227
  103. Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med. 1998;187:1477–1485
  104. Cauwels A, Janssen B, Waeytens A, Cuvelier C, Brouckaert P. Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2. Nat Immunol. 2003;4:387–393
  105. Ajuebor MN, Aspinall AI, Zhou F, Le T, Yang Y, Urbanski SJ, et al. Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells. J Immunol. 2005;174:8027–8037
  106. Chen Y, Wei H, Gao B, Hu Z, Zheng S, Tian Z. Activation and function of hepatic NK cells in hepatitis B infection: an underinvestigated innate immune response. J Viral Hepat. 2005;12:38–45
  107. Wright MC, Issa R, Smart DE, Trim N, Murray GI, Primrose JN, et al. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats. Gastroenterology. 2001;121:685–698

PII: S0168-8278(06)00086-9

doi:10.1016/j.jhep.2006.02.004

Journal of Hepatology
Volume 44, Issue 5 , Pages 984-993, May 2006

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