Shunhei Yamashina

NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease

In North America, liver disease due to alcohol consumption is an important cause of death in adults, although its pathogenesis remains obscure. Despite the fact that resident hepatic macrophages are known to contribute to early alcohol-induced liver injury via oxidative stress, the exact source of free radicals has remained a mystery. To test the hypothesis that NADPH oxidase is the major source of oxidants due to ethanol, we used p47(phox) knockout mice, which lack a critical subunit of this major source of reactive oxygen species in activated phagocytes. Mice were treated with ethanol chronically, using a Tsukamoto-French protocol, for 4 weeks. In wild-type mice, ethanol caused severe liver injury via a mechanism involving gut-derived endotoxin, CD14 receptor, production of electron spin resonance-detectable free radicals, activation of the transcription factor NF-kappaB, and release of cytotoxic TNF-alpha from activated Kupffer cells. In NADPH oxidase-deficient mice, neither an increase in free radical production, activation of NF-kappaB, an increase in TNF-alpha mRNA, nor liver pathology was observed. These data strongly support the hypothesis that free radicals from NADPH oxidase in hepatic Kupffer cells play a predominant role in the pathogenesis of early alcohol-induced hepatitis by activating NF-kappaB, which activates production of cytotoxic TNF-alpha.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production

Recently, it was demonstrated that liver injury and TNF‐a production as a result of endotoxin (lipopolysaccharide, LPS) were attenuated by feeding animals a diet enriched with glycine. This phenomenon was shown to be a result of, at least in part, activation of a chloride channel in Kupffer cells by glycine, which hyperpolarizes the cell membrane and blunts increases in intracellular calcium concentrations ([Ca2+]i) similar to its action in the neuron. It is well known that hepatotoxicity due to LPS has a neutrophil‐mediated component and that activation of neutrophils is dependent on increases in [Ca2+]i. Therefore, the purpose of this study was to determine if glycine affected agonist‐induced increases in [Ca2+]i in rat neutrophils. The effect of glycine on increases in [Ca2+]i elicited either by the bacterial‐derived peptide formyl‐methi‐onine‐leucine‐phenylalanine (FMLP) or LPS was studied in individual neutrophils using Fura‐2 and fluorescence microscopy. Both FMLP and LPS caused dose‐dependent increases in [Ca2+]i, which were maximal at 1 μM FMLP and 100 μg/ml LPS, respectively. LPS increased intracellular calcium in the presence and absence of extracellular calcium. Glycine blunted increases in [Ca2+]i in a dose‐dependent manner with an IC50 of ~0.3 mM, values only slightly higher than plasma levels. Glycine was unable to prevent agonist‐induced increases in [Ca2+]i in chloride‐free buffer. Moreover, strychnine (1 μM), an antagonist of the glycine‐gated chloride channel in the central nervous system, reversed the effects of glycine (1 mM) on FMLP‐ or LPS‐stimulated increases in [Ca2+]i. To provide hard evidence for a glycine‐gated chloride channel in the neutrophil, the effect of glycine on radioactive chloride uptake was determined. Glycine caused a dose‐dependent increase in chloride uptake into neutro‐phils with an ED50 of ~0.4 mM, an effect also prevented by 1 μM strychnine. Glycine also significantly reduced the production of superoxide anion from FMLP‐stimulated neutrophils. Taken together, these data provide clear evidence that neutrophils contain a glycine‐gated chloride channel that can attenuate increases in [Ca2+]i and diminish oxidant production by this important leukocyte.—Wheeler, M., Stachlewitz, R. F., Yamashina, S., Ikejima, K., Morrow, A. L., and Thurman, R. G. Glycine‐gated chloride channels in neutrophils attenuate calcium influx and superoxide production. FASEB J. 14, 476–484 (2000)

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

Adenoviral gene delivery can inactivate Kupffer cells: role of oxidants in NF‐κB activation and cytokine production

Kupffer cells play a significant role in the pathogenesis of several liver diseases; therefore, a potential therapeutic strategy would be to inactivate the Kupffer cell with a gene‐delivery system. Although recombinant adenovirus provides robust, transgene expression in parenchymal cells, whether adenovirus transduces Kupffer cells is unclear. Thus, the purpose of this study was to evaluate this possibility. In animals infected with adenovirus, Kupffer cells were identified positively to express adenoviral transgenes by immunohistochemical techniques and Western blot analysis, indicating that Kupffer cells are transduced in vivo. Indeed, isolated Kupffer cells were transduced in vitro with recombinant adenovirus in a dose‐dependent manner. Moreover, adenoviral transduction of Kupffer cells was blocked by inhibitors of αVβ5 integrin, the co‐receptor for adenovirus binding, supporting the hypothesis that adenovirus transduces Kupffer cells via an αVβ5 integrin‐dependent mechanism. Indeed, it is shown here that Kupffer cells express αVβ5 integrins. In a functional assay, infection of isolated Kupffer cells with adenovirus containing superoxide dismutase or IκBα super‐repressor blunted LPS‐induced nuclear transcription factor kappa B (NF‐κB) activation and tumor necrosis factor α (TNF‐α) production but not IL‐10 production. Moreover, superoxide production was blocked by expression of superoxide dismutase. These data support the hypothesis that LPS‐induced NF‐κB activation and TNF‐α production in Kupffer cells are oxidant‐dependent. These findings suggest that Kupffer cell‐targeted approaches may be a potential therapeutic strategy against many inflammatory diseases including early alcohol‐induced liver injury.

Role of Kupffer cells and gut-derived endotoxins in alcoholic liver injury1

The hepatotoxic effects of alcohol have been described in detail, but factors responsible for its hepatotoxicity have only partially been characterized. For example, it is known that chronic ethanol ingestion increases hepatotoxicity and produces fatty liver, hepatitis and cirrhosis. However, acute ethanol consumption reduces endotoxin hepatotoxicity. It now appears that Kupffer cells participate in several aspects of these phenomena. Previously, most studies on the effects of alcohol on liver function have focused chiefly on the hepatocyte. Recently, attention has been directed towards the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut‐derived endotoxins (lipopolysaccharides) via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol‐induced liver injury. Here we will review new evidence for the proposal that Kupffer cells and endotoxins play a pivotal role in hepatotoxicity following alcohol exposure, based on studies using the continuous intragastric enteral feeding model developed by Tsukamoto and French and an acute model developed by us.

Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy.

BackgroundAutophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine.MethodsInduction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-3 to replicon cells. The antiviral effect of chloroquine and/or interferon-α (IFNα) was evaluated.ResultsThe transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNα enhanced the antiviral effect of IFNα and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNα but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine.ConclusionThe results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.

Abnormality of autophagic function and cathepsin expression in the liver from patients with non‐alcoholic fatty liver disease

Recent evidences indicate that hepatic steatosis suppresses autophagic proteolysis. The present study evaluated the correlation between autophagic function and cathepsin expression in the liver from patients with non‐alcoholic fatty liver disease (NAFLD).

Interleukin-11 Links Oxidative Stress and Compensatory Proliferation

In dying cells, reactive oxygen species stimulate the production of a cytokine that triggers the healthy neighboring cells to proliferate. Dying Cells Protect the Neighborhood In addition to releasing a number of factors that induce the production of proinflammatory cytokines, dying cells can promote wound healing and tissue homeostasis by inducing the proliferation of surrounding cells in a process known as compensatory proliferation. Nishina et al. found that in dying hepatocytes reactive oxygen species triggered the production of the cytokine interleukin-11 (IL-11), which induced the proliferation of surrounding cells by activating the transcription factor STAT3. Consistent with these in vitro findings, IL-11 signaling protected hepatocytes in a mouse model of acetaminophen-induced liver injury, and mice deficient in a component of the IL-11 receptor had exacerbated disease. Together, these findings suggest that IL-11 stimulates compensatory proliferation in response to oxidative stress. Apoptotic cells can stimulate the compensatory proliferation of surrounding cells to maintain tissue homeostasis. Although oxidative stress is associated with apoptosis and necrosis, whether it contributes to compensatory proliferation is unknown. Here, we showed that interleukin-11 (IL-11), a member of the IL-6 family of proinflammatory cytokines, was produced by cells in an oxidative stress–dependent manner. IL-11 production depended on the activation in dying cells of extracellular signal–regulated kinase 2, which in turn caused the phosphorylation and accumulation of the transcription factor Fra-1 by preventing its proteasome-dependent degradation. Fra-1 was subsequently recruited to the Il11 promoter and activated gene transcription. Upon acute liver injury in mice, IL-11 was mainly produced by hepatocytes in response to reactive oxygen species that were presumably released from dying hepatocytes. IL-11 that was secreted by the dying cells then induced the phosphorylation of the transcription factor STAT3 in adjacent healthy hepatocytes, which resulted in their compensatory proliferation. Furthermore, an IL-11 receptor (IL-11R) agonist enhanced the proliferation of hepatocytes and ameliorated oxidative stress upon acetaminophen-induced liver injury. Conversely, the effects of acetaminophen were exacerbated in mice deficient in the IL-11R α subunit. Together, these results suggest that IL-11 provides a functional link between oxidative stress and compensatory proliferation.

Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.