Potential conflict of interest: Nothing to report.
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Subsiding By Dividing
The outcome of toxin induced liver injury reflects a balance between killing and regeneration. Regenerating hepatocytes appear to be resistant to toxicity. Recent evidence suggests that dying hepatocytes release degradative enzymes, such as calpains, which spread the injury. Limaye et al. assessed the expression of calpastatin, an endogenous calpain inhibitor, in the resistance of dividing cells and its role in acetaminophen toxicity. CCl4, partial hepatectomy, and newborn rats with growing livers all exhibited increased calpastatin expression. To assess the importance of calpastatin, mice were treated with adenovirus containing calpastatin gene. Increased expression of calpastatin conferred resistance against acetaminophen (partial inhibition of injury and improved survival) without altering covalent binding. This work establishes an important protective role for calpastatin in inhibiting the participation of calpain in liver injury, and suggests that the resistance of dividing cells may be due to increased expression of calpastatin. However, other mechanisms of resistance may contribute in dividing cells. Also, it is not clear if overexpression of calpastatin is resisting the effects of intracellular or extracellular calpain. However, these investigators have previously shown that administration of a cell impermeant calpain inhibitor blocked the progression of acetaminophen toxicity as well as a permeant inhibitor, supporting the novel concept that the initial death of pericentral hepatocytes leads to killing of neighboring cells. Recent studies demonstrating the damaging role of Dnase-1, also released from dying cells, add support to this concept. (See HEPATOLOGY 2006;44:379-388.)
IL-15 and NKT Cells: Convergent Consequence Confounds Con A
NKT cells are known to play a pivotal role in Con A-induced liver injury. Since IL-15 is necessary for survival and homeostasis of NKT cells, Li et al. treated mice with IL-15 prior to administering Con A and observed marked protection (see Fig.). Proving that NKT cells are the target of IL-15, adoptive transfer of NKT cells from IL-15 treated mice to NKT depleted mice failed to restore Con A-injury while transfer of NKT cells from saline treated mice did restore susceptibility to Con A. In vitro treatment of NKT cells prior to transfer also inhibited the injury promoting activity in response to Con A indicating that IL-15 acts directly on these cells. This protection by IL-15 was associated with suppression of NKT-derived IL-4, IL-5, and TNF-α. Downregulation of IL-4 and IL-5 diminished downstream chemokines such as eotoxins and subsequent eosinophilic infiltration. This work provides added support for the role of NKT cells in liver injury from Con A. It remains to be seen what effect IL-15 and NKT cells will have on more clinically relevant liver diseases such as drug, viral, fatty liver and autoimmune diseases, especially when chronic inflammation is present. (See HEPATOLOGY 2006;43:1211-1219.)
Killer Hepatocytes Fight Back
An intriguing question is whether hepatocytes can express FasL and then kill inflammatory cells that infiltrate the liver. Guy et al. detected comparable FasL mRNA and protein expression in human and woodchuck primary hepatocytes, whole liver, and lymphoid cells. The hepatocytes were able to kill P815 cells which could be blocked by monoclonal antibody to Fas. FasL was upregulated by IFNγ but not exogenous TNF and this was associated with enhanced cytotoxicity of P815 cells mediated by hepatocytes. Thus, normal hepatocytes express FasL and this can be upregulated by cytokines. Although the role of hepatocyte death ligand expression in pathophysiology of liver disease remains uncertain, a number of important possibilities can be considered. Aside from possible fraticide, i.e., killing of neighboring stressed hepatocytes, this may be a mechanism for destroying invading cytotoxic Tcells which could impair eradication of viral infections. (See HEPATOLOGY 2006;43:1231-1240.)
See Why P is Bad For U
There has been considerable interest in the role of CYP2e1 in the pathogenesis of both alcoholic and nonalcoholic steatohepatitis. CYP2e1 spontaneously generates reactive oxygen species and is induced by alcohol. Lu and Cederbaum addressed the role of increased CYP2e1 and its interplay with LPS (endotoxin), another factor implicated in liver disease, in an in vivo mouse model. Pyrazole (150 mg/kg) given for 2 days markedly induced CYP2e1, but caused no injury. LPS (4 mg/kg) alone also did not injure the liver. However, the combination caused significant serum ALT elevation, histological injury, caspase-3 activation and Tunel positive apoptotic hepatocytes (see Fig.). In addition, immunohistochemical evidence of nitrotyrosine and 4-hydroxy-2-nonenal adducts was seen in the combined treatment group reflecting increased nitrosative and oxidative stress. Chlormethiazole, a CYP2e1 inhibitor, provided partial protection by attenuating ALT and necrotic foci but not apoptotic cells or caspase-3 activity. In addition, CYP2e1−/−mice were resistant to the toxicity of pyrazole+LPS. This work suggests that CYP2e1 and LPS act synergistically to induce necrosis and apoptosis. Induction of CYP2e1 appears to sensitize the liver to LPS toxicity. The question remains as to what is the mechanism for the interaction between LPS and CYP2e1. Possibilities include additional oxidative/nitrosative stress from effects of LPS on Kupffer cells or other inflammatory cells or effects of TNF production. Either could increase hepatocyte oxidative/nitrosative stress in hepatocytes already stressed by increased CYP2e1. In addition, could pyrazole have CYP2e1 inductive or other effects in nonparenchymal cells, i.e., priming them to respond to LPS? (See HEPATOLOGY 2006;44:263-274.)
CAR is a transcription factor which regulates detoxification and has been shown to protect against bile acid-induced injury. Since bile acid injury depends on the Fas death receptor, Baskin-Bey et al. addressed the possibility that CAR regulates genes of the apoptosis pathway. When a potent CAR agonist, TCPOBOP, was administered to wild type mice, marked protection against Jo2 (agonistic Fas monoclonal antibody)-induced apoptosis caspase activity, histological injury and lethality was seen but TCPOBOP did not protect CAR knockout mice (see Fig.). TCPOBOP, although known to be a mitogen, did not further enhance hepatocyte proliferation in response to Jo2. Similar results were seen in the acute Con A model. Furthermore, the fibrosis seen in response to repeated dosing with Con A was attenuated in the TCPOBOP treated mice. Starting with the most upstream components of the Fas death pathway, TCPOBOP did not alter caspase-8 activation or Bid cleavage and tBid translocation to mitochondria. However, TCPOBOP markedly decreased the expression of pro-apoptotic Bax and Bak and increased expression of anti-apoptotic Mcl-1 in wild type but not CAR knockout mice. A functional CAR binding site in the Mcl-1 gene promoter was verified and Mcl-1 overexpressing transgenic mice were resistant to Jo2. Thus, this very elegant work demonstrates that CAR, a nuclear hormone receptor family member, can regulate apoptosis due to decreased expression of pro-apoptotic and increased expression of anti-apoptotic Bcl-2 family members. It will be of interest to see if phenobarbital, another CAR activator, exerts similar effects and if CAR protects against TNF induced apoptosis. Also the mechanism for decreased Bax and Bak levels will require more detailed study. However, this work demonstrates the potential for CAR activation as a strategy to treat liver diseases where Fas plays an important role in injury. (See HEPATOLOGY 2006;44:252-262.)
A Flipping Game: Aces Win
ATP8B1 is a P-type ATPase that functions as a flippase which translocates phospholipids from the outer to the inner leaflet of membranes. ATP8B1 is located in the apical membrane of hepatocytes and cholangiocytes. Mutations in this gene cause progressive familial intrahepatic cholestasis type1 (PFIC1). Paulusma et al. addressed the heretofore elusive mechanism for impaired bile acid excretion and cholestasis in this disorder. Using mice harboring a homozygous mutation in Atp8b1 (G308V), they demonstrated that infusion of bile salt in mutant mice caused increased biliary output of phospholipids including phosphatidylserine, as well as cholesterol and canalicular membrane enzymes, alkaline phosphatase, and aminopeptidase N. Phosphatidylserine is normally found in the inner leaflet and is not found in normal bile. Immunohistochemistry of PFIC1 patients' livers showed absence of canalicular ectoenzymes such as γ-glutamyltranspeptidase. However, integral membrane transporters were not altered. Mutant mice accumulated vesicular structures within canaliculi similar to what has been described in PFIC1 patients. Despite normal expression and localization of the bile salt export pump in mutant livers, hydrophobic bile acid excretion was impaired in the isolated perfused mutant livers. This work demonstrates that the canalicular membrane of Atp8b1 mutant mice is more susceptible to the detergent action of hydrophobic bile acids exemplified by increased bile cholesterol and ectoenzymes. Phosphatidylserine, normally not present in the outer leaflet, is extracted by the lumenal bile acids because the absence of functional Atp8b1 results in failure to retain phosphatidylserine in the inner leaflet. The findings support the hypothesis that phospholipids, such as phosphatidylserine, present in the outer leaflet due to failure of their flipping to the inner leaflet, allowing bile acids to remove membrane lipids including cholesterol. Removal of cholesterol then impairs the function of the bile salt pump. It is of interest to contrast PFIC1 and PFIC3. Both are flippase defects which lead to detergent effects on apical membranes. In the former case, the outer leaflet becomes vulnerable to bile acid dissolution because of the failure to retain the phospholipids, phosphatidylserine, in the inner leaflet, whereas in the latter, bile acid dissolution of membranes occurs because of failure to flip phospholipids such as phosphatidylcholine from the inner to the outer leaflet so that it can be removed or secreted to “neutralize” the detergent bile acids. (See HEPATOLOGY 2006;44:195-204.)