Inhibition of caspases in vivo protects the rat liver against alcohol-induced sensitization to bacterial lipopolysaccharide.

BACKGROUND: The mechanisms of liver sensitization by alcohol to Gram-negative bacterial lipopolysaccharide (LPS) remain elusive. The purpose of this study was two-fold: (1) to test the hypothesis that alcohol-enhanced liver apoptosis may be a sensitizing mechanism for LPS and (2) to further characterize the liver apoptotic response to alcohol. METHODS: Rats were fed a high-fat, alcohol-containing liquid diet for 14 weeks, treated with LPS (1.0 mg/kg of body weight, intravenously) or saline, followed by injection of a pan-caspase inhibitor IDN1965; N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid; 10 mg/kg of body weight, intraperitoneally or vehicle, and killed. The following parameters were assessed: plasma aspartate: 2-oxoglutarate aminotransferase activity (AST); liver histology and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) response; caspase-3, -8, and -9 activity; and mRNA and protein expression for two apoptosis-signaling molecules: Fas receptor and Fas ligand; and three apoptosis adaptors: Bax, Bcl-XL, and Bcl-2. RESULTS: Alcohol-feeding-induced liver steatosis, slightly increased caspases' activity, the number of TUNEL-positive nuclei, and facilitated the LPS necrotic effect without affecting mRNA expression of apoptosis signals and adaptors. LPS induced a significant increase in AST and the number of TUNEL-positive nuclei, both effects being more pronounced in alcohol-treated rats. LPS produced hepatic necrosis only in alcohol-treated rats. LPS effects were associated with up-regulation of mRNA expression for both apoptosis adaptors and signaling molecules. IDN1965 administration 3 hr after LPS injection strongly inhibited caspases' activity, particularly that of caspase-3. IDN1965 also abolished the increase in TUNEL-positive nuclei, reversed the effect of LPS on plasma AST in alcohol-treated rats, and prevented LPS-induced necrosis. CONCLUSIONS: (1) Alcohol-enhanced liver apoptosis may not involve regulatory steps at the transcriptional level. LPS-induced liver apoptosis seems to involve transcriptional regulation of several apoptosis adaptors. Therefore, alcohol and LPS may enhance liver apoptosis through different mechanisms. (2) Alcohol-enhanced liver apoptosis precedes and may facilitate the hepatic effects of LPS. LPS superimposed on alcohol further elevates the rate of apoptosis in the liver. This may exceed the phagocytosing capacity of the liver so that all the apoptotic cells are not phagocytosed, but rather die of necrosis.

How is the liver primed or sensitized for alcoholic liver disease?

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi-Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol-mediated sensitization of the liver, by Anna Colell, Carmen Garcia-Ruiz, Neil Kaplowitz, and Jose C. Fernandez-Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S-adenosylmethionine affects tumor necrosis factor-alpha gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi-Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFalpha gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut-derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman.

Chronic alcohol exposure of rats exacerbates apoptosis in hepatocytes and sinusoidal endothelial cells.

Background/aims: The liver apoptotic response to chronic alcohol consumption remains poorly characterized. The purpose of this study was to determine in rats the effects of chronic alcohol consumption on the relative magnitude of apoptosis in two major targets of alcohol-induced liver injury: the hepatocyte (Hep) and sinusoidal endothelial cell (SEC). Methods: Rats were fed a liquid diet containing either alcohol or isocaloric amounts of maltose-dextrin for 14 weeks. Hep and SEC were isolated by liver perfusion with collagenase followed by centrifugal elutriation. The state of the liver was assessed on the basis of light microscopic appearance, plasma liver enzymes (alanine and aspartate:2-oxoglutarate amino transferases), and the content of malondialdehyde in Hep. Apoptosis was assessed on the basis of DNA fragmentation in the whole organ (TUNEL), and caspase-3 and -8 activity in isolated cells. A mechanistic approach was also undertaken by measuring mRNA expression and the amount of protein for Fas/CD95, Fas ligand, caspase-3, Bax, Bcl-X(L), and Bcl-2 in the isolated Hep and SEC. Results: The livers of alcohol-fed rats displayed prominent steatosis. Oxidative stress was also present as reflected by an increase in the malondialdehyde content of Hep. Alcohol consumption increased apoptosis in the whole liver assessed on the basis of TUNEL procedure and in Hep and SEC as reflected by significant increase in caspase-3 activity. Of the multiple pro- and anti-apoptotic factors determined in this study, significant changes as assessed by both mRNA expression and the amount of proteins, were observed only in the SEC compartment. Conclusions: The data presented in this study indicate that: (1) chronic alcohol consumption in rats leads to a moderate augmentation of apoptosis in the whole liver and in two liver cell types which are targets for injury in alcoholic liver disease: Hep and SEC; (2) the mechanisms recruited/activated by these two types of liver cells to initiate and execute apoptosis in response to alcohol vary with the cell type.

Apoptosis and dysregulated ceramide metabolism in a murine model of alcohol-enhanced lipopolysaccharide hepatotoxicity.

BACKGROUND: The role of apoptosis in EtOH-induced liver injury has not been investigated much. Therefore, the question whether apoptosis is a contributory factor to alcoholic liver disease remains to be answered. The purpose of this study was to characterize the liver apoptotic response in a murine model of alcohol-enhanced lipopolysaccharide (LPS) hepatotoxicity. METHODS: Mice were fed an alcohol-containing liquid diet for 49 days followed by an acute LPS challenge. The liver state was judged on the basis of histological appearance, plasma liver enzyme activity (alanine:2-oxoglutarate and aspartate:2-oxoglutarate aminotransferases, as markers of hepatocytolysis), and plasma hyaluronan levels (as a marker of the sinusoidal endothelial cell scavenging function). The liver apoptotic response was assessed by DNA fragmentation (TUNEL procedure), and caspases-3 and -8 activity. To determine if ceramide played a role in the liver apoptotic response, the activity of acidic sphingomyelinase and tissue content of ceramide were also quantified. RESULTS: Alcohol exposure induced fat accumulation and sensitized the liver to LPS injurious effects. Plasma liver enzyme activity was elevated by alcohol and this effect was potentiated by LPS. Liver apoptosis was augmented by both alcohol and LPS treatment as reflected by high frequency of positive TUNEL staining nuclei and by an increased activity of caspase-3 and -8. Acidic sphingomyelinase activity was also increased and it was associated with an elevated tissue content of ceramide. In addition, LPS also increased plasma TNF-alpha levels. These changes were accompanied by elevated plasma hyaluronan, reflecting an impaired sinusoidal endothelial cell scavenging function. CONCLUSIONS: These results provide a more complete description of the liver apoptotic response to both alcohol and LPS and may constitute the basis for further mechanistic studies on a possible role apoptosis may play in alcoholic liver injury.

Cytokines in alcoholic liver disease.

Cytokines are low-molecular-weight mediators of cellular communication produced by multiple cell types in the liver, with the Kupffer cell critically important. Inflammatory cytokines such as tumor necrosis factor, interleukin-1, and interleukin-8, and hepatic acute-phase cytokines such as interleukin-6 play a role in modulating certain metabolic complications in alcoholic liver disease and probably play a role in the liver injury of alcoholic liver disease. Two potential inducers of cytokine production in alcoholic liver disease are endotoxin and reactive oxygen species generated after ethanol metabolism. Cytotoxic cytokines likely induce liver cell death by both necrosis and apoptosis in alcoholic liver disease. Anticytokine therapy has been highly successful in attenuating cell injury/death in a variety of toxin-induced models of liver injury, including alcohol-related liver injury. Anticytokine therapy has been used successfully in humans in disease processes such as Crohn's disease and rheumatoid arthritis. There is an emerging rationale for use of anticytokine therapy in alcoholic liver disease, with the goal of maintaining beneficial effects of cytokines and inhibition of the deleterious effects of these potentially toxic agents.

Effects of exogenous superoxide anion and nitric oxide on the scavenging function and electron microscopic appearance of the sinusoidal endothelium in the isolated, perfused rat liver.

BACKGROUND/AIMS: Functional and morphological alterations of the hepatic sinusoidal endothelial cell occur in several models of experimental liver injury and in clinical settings. The causes of these alterations are multiple. The aim of this study was to test the hypothesis that the early functional impairment and morphological alterations of the sinusoidal endothelial cell and hepatic sinusoid associated with liver injury are mediated by free radical species, such as superoxide anion and nitric oxide. METHODS: Isolated rat livers were perfused by recirculation with hemoglobin-free, Krebs-Henseleit bicarbonate buffer and presented with a source of superoxide anion (xanthine oxidase+hypoxanthine) or nitric oxide (S-nitroso-N-acetyl penicillamine). Hyaluronan uptake (an index of sinusoidal endothelial cell scavenging function), thiobarbituric acid-reactive substances content of the tissue (a marker of lipid peroxidation), reduced and oxidized glutathione (a marker of the thiol system oxidation/reduction state), lactate dehydrogenase and alanine aminotransferase activities (markers of cytolysis), as well as scanning and transmission electron microscopic appearance of the sinusoid were evaluated. RESULTS: At the high concentrations used, both free radical generating systems suppressed hyaluronan uptake, increased malondialdehyde content of the tissue, enhanced the release of both liver enzymes, decreased the total glutathione content of the liver, and altered the ratio of reduced/oxidized glutathione. Both free radical species induced dose-dependent morphological alterations of the sinusoid, consisting of the appearance of large gaps replacing the sieve-plated fenestration. CONCLUSIONS: The free radical species-induced functional impairment and morphological alterations of the liver sinusoid, presented in this study, closely resemble the early in vivo changes associated with liver injury under a variety of conditions, such as preservation and reperfusion, or administration of hepatotoxicants such as D-galactosamine, Gram-negative bacterial lipopolysaccharides, acetaminophen, alcohol and others. Therefore, we suggest that early liver sinusoid injury, observed under these conditions, can be attributed to the action of free radicals, such as superoxide anion and nitric oxide.

Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide.

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.

Hyperhyaluronanemia in alcoholic hepatitis is associated with increased levels of circulating soluble intercellular adhesion molecule-1.

The purpose of this study was to evaluate the role of the sinusoidal endothelial cell (SEC) during the clinical course of alcoholic hepatitis. Twenty consenting patients (mean age: 49.4 +/- 11.0 years) with moderate or severe hepatitis were studied. The patients were selected and characterized according to their history of drinking and laboratory profile, including serum aminotransferases, bilirubin, total white blood cell and neutrophil count, and prothrombin times. C-reactive protein and interleukin-6 were also measured as markers of the hepatic acute phase response. A marker of the SEC functional state, the circulating level of hyaluronan, was measured in parallel with the circulating levels of soluble intercellular adhesion molecule (sICAM)-1 over a 6-month observation period. All patients were hospitalized for the first month and encouraged to abstain from drinking for the duration of the study. The initial increased levels of both hyaluronan (542 +/- 32 ng x ml(-1) serum) and sICAM-1 (488 +/- 70 ng x ml(-1) serum), gradually fell during the 6-month observation period, eventually reaching values close to those seen in healthy subjects. A positive correlation was obtained between changes in these two markers of SEC function/activation on the one hand, and between these two tests and bilirubin, on the other hand. These data indicate that abnormalities of SEC function/activation, as reflected by serum hyaluronan and siCAM-1, are prominent in alcoholic hepatitis, and these alterations improve within relatively short periods of time after cessation of alcohol consumption.

Tumor necrosis factor and alcoholic liver disease.

Increased levels of hepatic and serum tumor necrosis factor (TNF) have been documented in animal models of alcoholic liver disease and in human alcoholic liver disease. This dysregulated TNF metabolism has been postulated to play a role in many of the metabolic complications and the liver injury of alcoholic liver disease. One potential therapy for alcoholic liver disease may be agents that downregulate TNF production or block TNF activity. Indeed, agents such as prostaglandins and glucocorticoids (both inhibit TNF production) have been used in both human liver disease and experimental models of liver injury, and anti-TNF antibody has recently been shown to attenuate the hepatotoxicity in an animal model of alcoholic-related liver disease. In this study, we demonstrate that a simple ex vivo system can be used to initially assess potential efficacy of anticytokine agents when administered to humans. Both prednisone and a prostaglandin analog were effective in downregulating TNF and interleukin-8 production. The liver is normally resistant to TNF cytotoxicity. Sensitivity to TNF cytotoxicity is thought to occur when there is inadequate production of hepatic protective factors. In this study, we showed that, when patients with acute alcoholic hepatitis were matched with trauma patients for serum levels of interleukin-6, they had similar depressions in the negative acute phase protein, albumin, but markedly different increases in the major acute phase protein, C reactive protein. Patients with alcoholic hepatitis had a very blunted response. We also showed that inhibiting activation of the redox sensitive transcription factor NFkappaB sensitizes to TNF-induced hepatocyte death in vitro. This transcription factor is important for the production of both cytokines and many acute phase protective factors. Several hepatic protective factors are induced by TNF. One possible mechanism for liver injury in alcoholic hepatitis may be inadequate generation of hepatic protective factors. Our future understanding of mechanisms of alcoholic liver disease will involve understanding the balance between noxious and protective factors in the liver, and this should lead to rational therapy for this disease process.

Tumor necrosis factor-alpha internalization and degradation by isolated hepatocytes of rats exposed to ethanol.

Internalization and degradation of human recombinant [125I]TNF-alpha was studied in hepatocytes isolated from rats exposed to ethanol (EtOH) either acutely (i.p. injection, 2.2 g kg(-1) b.wt.) or chronically (14-16 weeks of EtOH feeding in liquid diet). Both acute and chronic EtOH exposure diminished cytokine binding to the cell-surface receptors. In the acute group, EtOH increased internalization of the cytokine, accelerated its disappearance from the cell surface, and markedly reduced its conversion into acid-soluble 125I-containing compounds. In the chronic group, EtOH did not markedly affect these parameters. Internalization and degradation of the cytokine in the chronic group was much lower than in the acute group. It is concluded that EtOH interferes not only with the cytokine binding to the cell-surface receptors, as demonstrated in previous studies, but also with postbinding events, such as internalization and intracellular degradation of TNF-alpha. Possible mechanisms of action of EtOH are discussed.

Dose- and time-dependent effects of ethanol on functional and structural aspects of the liver sinusoid in the mouse.

Increasing evidence implicates injury of hepatic sinusoidal endothelial cells as an important component in the development of several forms of liver injury. The purpose of this study was to test the hypothesis that alcohol [ethanol (EtOH)]-induced pathological changes of the sinusoidal endothelial cell of the liver precede, and may lead to, hepatocyte injury. BALB/c mice were treated with EtOH either acutely (1.5 or 3.0 g.kg-1 body weight, i.p.) or chronically [by feeding an EtOH-containing (4%, w/v) liquid diet]. Acutely treated animals were killed 3, 6, and 12 hr after EtOH administration, whereas chronically treated animals were killed 12, 28, and 56 days after the initiation of EtOH feeding. The levels of plasma EtOH, hyaluronan (a functional marker for sinusoidal endothelial cell), and alanine-2: oxoglutarate aminotransferase (ALT) activity (a marker for hepatocyte damage) were measured in all groups. The livers were examined by electron and light microscopy. Between 3 and 6 hr after intraperitoneal injection of EtOH, the plasma EtOH levels were relatively stationary (5 and 11 mM for the low- and high-dose groups, respectively). At 12 hr, EtOH was almost completely cleared from the plasma. Hyaluronan levels were increased 3 hr after EtOH exposure at both doses and reached a peak at 6 hr after EtOH administration. In the low EtOH dose animals, the hyaluronan level declined toward normal values at 12 hr. In the high EtOH dose group, hyaluronan levels were still above normal values 12 hr after EtOH administration. No changes in the plasma ALT level were observed in either acutely EtOH-treated groups. In animals treated chronically, plasma hyaluronan levels were markedly increased at 12, 28, and 56 days of EtOH feeding. Plasma ALT levels were elevated at 28 and 56 days, but not at 12 days, of EtOH feeding. Scanning electron microscopy of the liver sinusoid in the acutely treated animals showed the presence of large gaps co-existing with normal sieve-plate fenestrations in sinusoidal endothelial cells. Such changes were seen 3 hr after the high dose and 6 hr after the low dose of EtOH. They disappeared 12 hr after low dose, but lasted well beyond this time point after the high dose of EtOH. Twelve days after the start of EtOH feeding, no changes in the electron microscopic appearance of the sinusoid could be observed. However, 26 days after the initiation of EtOH feeding, the sinusoidal endothelial cells displayed a reduced number of fenestrae. Moreover, the remaining fenestrae were distributed uniformily rather than in organized sieve plates. In addition, at these latter time points, transmission electron microscopy demonstrated the presence of fibrous material in the space of Disse. Both light and transmission electron microscopy demonstrated the presence of lipids within the hepatocyte. The picture observed 56 days after the start of EtOH feeding was essentially the same as at 28 days, except that the reduction in the number of fenestrae was more accentuated. These data document EtOH-induced pathological changes in sinusoidal endothelial cell before either biochemical or histological hepatocyte damage.

Alcohol and cytokine networks.

Experimental and clinical data pertaining to alcohol (ethanol, EtOH) interference with cytokine networks are analyzed from the viewpoint of their importance for understanding the deleterious effects of EtOH on various functions. The data are grouped and analysed according to several major directions that have emerged in the last decade since the research on EtOH interference with cytokine networks started. These are: cytokine secretion, including the effects of the drug on in vivo cytokine levels and on in vitro cytokine secretion by various cells; cytokine-receptor interaction; intracellular fate of cytokines; and cellular responses to cytokines. Correspondingly, the data are presented in four tables. Attention is paid to the fact that the effects of EtOH on various aspects of cytokine biology (e.g., secretion by various cells) are diversified. Contradictory data have been reported but the cause for discrepancy is poorly understood. EtOH effects vary with the cytokine studied, the method of EtOH administration (i.e., acute or chronic), the species used, or with other experimental conditions. It is important to note, however, that the vast majority of experimental and clinical data show that EtOH interferes with cytokine networks and that research on such interference may lead to important steps in understanding the mechanisms of action of EtOH. An attempt is made herein to select aspects of cytokine biology, as affected by EtOH, that have been studied to a lesser extent, thus calling for more research efforts in those areas.

Effect of chronic alcohol consumption by rats on tumor necrosis factor-alpha and interleukin-6 clearance in vivo and by the isolated, perfused liver.

The effects of chronic (16-week) alcohol consumption by rats on [125I] tumor necrosis factor (TNF)-alpha and [125I]interleukin (IL)-6 plasma clearance and organ distribution in vivo and uptake and metabolism by the isolated, perfused liver were studied. Alcohol was administered to rats in a liquid diet for 16 weeks, and caused a decreased (48%) plasma clearance rate of IL-6 and converted the plasma clearance kinetics of the cytokine from a biphasic exponential in normal rats to a monophasic exponential decay. Alcohol feeding significantly increased (101%) plasma clearance of TNF-alpha, which followed a biphasic exponential decay and decreased the T1/2 for both the alpha (67%) and beta (76%) elimination components. The distribution of both cytokines in trichloroacetic acid precipitable and non-precipitable fractions of liver, spleen, stomach, small intestine (ileum), lung, kidney, and blood was also studied. The only effect of alcohol treatment was a significant decrease in IL-6 uptake and metabolism by the small intestine. Perfused livers, isolated from alcohol-fed rats, took up and metabolized larger amounts of IL-6 than did livers isolated from pair-fed rats. TNF-alpha uptake and metabolism by the isolated, perfused liver were not affected by chronic alcohol consumption. Regardless of the animal treatment, the isolated perfused liver took up and metabolized significantly larger (17-fold) amounts of TNF-alpha than IL-6, in spite of identical concentrations of cytokines (6 nM) in the perfusion medium. The data presented in this study along with our previous results demonstrating the effects of alcohol consumption on TNF-alpha and IL-6 receptors on various liver cells suggest that the effects of chronic alcohol treatment on cytokine clearance cannot be ascribed to changes in the receptors for the two cytokines. Also, no correlation was found between the effects of alcohol treatment on plasma cytokine clearance and uptake and metabolism of cytokines by the isolated, perfused liver. Experimental data and theoretical considerations suggest that cytokine receptor recycling may play an important role in mediating alcohol effects on cytokine clearance.

Chronic alcohol feeding in liquid diet or in drinking water has similar effects on electron microscopic appearance of the hepatic sinusoid in the rat.

Electron microscopic appearance of the liver sinusoid was examined in rats fed alcohol chronically in a complete liquid diet or in sucrose-containing drinking water. The animals were kept on liquid diet (+/- alcohol) for 14 weeks or on sucrose-containing drinking water (+/- alcohol) for 12.5 weeks and sacrificed thereafter. To rule out possible artifact induced by fixation procedure, livers were fixed by immersion (no perfusion), immersion preceded by perfusion, and by perfusion with glutaraldehyde and examined with both scanning and transmission electron microscopy. Regardless of the mode of its administration, and of the fixation procedure used, alcohol induced similar changes in liver sinusoid ultrastructure. Such changes included disruption of the sieve-plate pattern of the sinusoidal endothelial cell fenestrations with the appearance of large gaps and resulting in a meshwork lining, wherein large areas of the sinusoid communicated freely with the underlying hepatocytes. Transmission electron microscopy complemented these findings. The results reported in this study demonstrate that alcohol-induced structural changes of the liver sinusoid in the rat are similar whether alcohol is fed via a liquid diet or in drinking water. Therefore, alcohol administration in drinking water may provide a simple, inexpensive, and convenient method of inducing structural changes in the rat liver sinusoid.

Hepatic sinusoidal endothelial cell in alcoholemia and endotoxemia.

The experimental data reviewed in this study tend to indicate that the hepatic sinusoidal endothelial cell (SEC) is, chronologically, the first hepatic cell that undergoes pathologic changes in alcoholemia. Due to its strategic position in the liver sinusoid, SEC dysfunction and structural alterations have far-reaching repercussions for the whole liver. The authors gather experimental evidence suggesting that alcohol-induced SEC alterations are mostly due to Kupffer cell activation induced by alcohol rather than to a direct action of alcohol on SEC. Once activated, the Kupffer cell secretes a spectrum of mediators that affect both function and structure of SEC. Kupffer cell activation is regarded as a result of both direct and indirect actions of alcohol on the cell. The indirect action of alcohol is ascribed to alcohol-induced elevated plasma levels of Gram-negative bacterial lipopolysaccharide (LPS), a strong activator of Kupffer cell. However, a comparison of alcohol and LPS effects on SEC functions and structure reveals that these two agents may have, under many circumstances, different actions on the SEC, at least in laboratory animals. However, this issue continues to be a matter of debate. Also the review presents justification for the necessity to extend research on mechanisms underlying alcoholic liver disease to the effects of alcohol on the SEC. Finally, several future research directions are suggested in this review to better understand the mechanisms underlying alcohol-induced liver dysfunction.

Interleukin-6 tumor necrosis factor-alpha clearance and metabolism in vivo and by the isolated, perfused liver in the rat: effect of acute alcohol administration.

Plasma clearance and organ distribution of intravenously injected human recombinant [125I]interleukin (IL)-6 and [125I]tumor necrosis factor (TNF)-alpha were studied in male rats, 2 hr after intravenous alcohol (ethanol) administration (single dose, 2.2 g.kg-1 body weight). Also, the rate of uptake and degradation of the two cytokines by the isolated, perfused rat liver was studied in the absence or in the presence of ethanol (35 mM) in the perfusate. Acute ethanol administration significantly increased plasma clearance rate for both cytokines (36% and 72%, for IL-6 and TNF-alpha, respectively), decreased the t1/2 alpha (30% and 11%, for IL-6 and TNF-alpha, respectively), abolished the slow (beta)-phase component for TNF-alpha, and increased t1/2 beta for IL-6 (31%). Although alcohol did not affect organ distribution of TNF-alpha, it increased the IL-6 content in the liver, kidney, and blood. IL-6 uptake rate by the isolated, perfused rat liver was 2-fold higher than TNF-alpha uptake, whereas the rate of degradation was larger for TNF-alpha than for IL-6, despite the fact that both cytokines were presented to the liver at the same concentration (6 nM). Ethanol addition to the perfusate (35 mM, final concentration) significantly increased TNF-alpha uptake (24%), without affecting IL-6 uptake or the degradation rate of either cytokine. Also, the kinetics of degradation by the isolated, perfused rat liver was linear for TNF-alpha, but exponential for IL-6. Data presented in this study demonstrate that: (1) acute alcohol consumption can alter the kinetic behavior of IL-6 and TNF-alpha in the bloodstream, mainly by accelerating their clearance which, in turn, may counteract the outcome of cytokine secretion and delivery to the blood; and (2) short exposure of liver to ethanol levels commonly seen in humans after binge drinking may alter its capacity to take up cytokines.

Kupffer cell inactivation prevents lipopolysaccharide-induced structural changes in the rat liver sinusoid: an electron-microscopic study.

Scanning and transmission electron-microscopic examination of the rat liver sinusoid was performed in this study after in vivo treatment of rats with gram-negative bacterial lipopolysaccharide (LPS, 1 mg/Kg(-1) body weight), with or without pretreatment with gadolinium chloride (GdCl3 10 mg(Kg(-1) body weight). Twenty-seven and 48 hours after GdCl3 administration, to inactivate/eliminate part of the Kupffer cell population, a decrease in the number of visualized Kupffer cells was observed, without evident effects on the sinusoidal endothelial cell or on the hepatocyte. Three and 24 hours after its administration, LPS produced ultrastructural changes in the sinusoid characterized by morphological evidence of Kupffer cell activation (i.e., swelling and expanded philopodia anchoring the Kupffer cell to the luminal surface of the sinusoidal wall), and a marked decrease in the population of endothelial cell fenestration. The reduction in the number of fenestrae was associated with a change in the diameter of fenestrae and can be interpreted as a component of the "capillarization" process of the hepatic sinusoid. Such ultrastructural changes were prevented by the administration of GdCl3 24 hours before LPS injection. Hence, these findings suggest that LPS-induced structural changes in the liver sinusoid are mediated by an LPS-induced Kupffer cell activation. Coupled with previous experimental data, showing similar effects of GdCl3 on one of the hepatic sinusoidal endothelial cell (SEC) functions, i.e., hyaluronan scavenging, the data presented in this study strongly support the view that Kupffer cells modulate both the hepatic SEC's functional as well as ultrastructural properties.

Alcohol modulates alveolar macrophage tumor necrosis factor-alpha, superoxide anion, and nitric oxide secretion in the rat.

We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to produce tumor necrosis factor-alpha (TNF-alpha), superoxide anion (O2-), and nitric oxide (NO)--three critical components of pulmonary host defense. Male rats were treated with alcohol either acutely (priming dose 175 mg/100 g of body weight, followed by a 7-hr continuous intravenous infusion of 30 mg/100 g of body weight/hr) or chronically (12-14 weeks of feeding ethanol in a liquid diet). Three hours before sacrifice, the rats received an intravenous injection of saline or lipopolysaccharide (LPS; Escherichia coli, 026:B6, 100 micrograms/100 g of body weight). Alveolar macrophages (AMs) were then isolated by bronchoalveolar lavage and assessed for their in vitro capacity to produce TNF-alpha, O2-, and NO spontaneously and in response to different stimuli. Acute alcohol administration suppressed in vitro LPS-stimulated AM TNF-alpha secretion by 52%. AMs from both pair-and alcohol-fed rats secreted TNF-alpha spontaneously in culture. However, the AMs from chronic alcohol-fed group secreted 42-53% less TNF-alpha spontaneously and in response to LPS, interferon-gamma (IFN-gamma) or IFN-gamma + LPS compared with the AMs from pair-fed group. Systemic LPS treatment inhibited in vitro LPS-stimulated AM TNF-alpha secretion (50%) only in the control rats. Acute alcohol administration enhanced significantly in vitro phorbol 12-myristate 13-acetate (PMA)- and opsonized zymosan (OPZ)-induced AM O2- secretion (4-and 1.8-fold, respectively). Systemic LPS treatment primed the AMs from control rats to secrete 83% more O2- in response to PMA but not OPZ; however, in the acute alcohol-treated group, it suppressed both PMA (54%)- and OPZ (66%)-induced AM O2- release (loss of priming effect of LPS). Chronic alcohol feeding suppressed PMA-induced AM O2- secretion (40%) without affecting the OPZ-induced release. Although systemic LPS treatment had no significant effect on PMA or OPZ-induced AM O2- secretion in the pair-fed group, it enhanced the PMA-stimulated AM O2- release (88%) in the alcohol-fed group. AMs recovered from control or acute alcohol-treated rats did not secrete NO spontaneously in vitro. However, AMs from both pair and chronic alcohol-fed rats secreted NO spontaneously with AMs from chronic alcohol-fed group secreting 34% less. Both acute and chronic alcohol treatment inhibited AM NO secretion in response to IFN-gamma, LPS, and IFN-gamma + LPS significantly. Systemic LPS had no effect on AM NO production in response to different in vitro stimuli in any of the treatment groups. These data suggest that (1) both acute and chronic alcohol administration to rats inhibit AM TNF-alpha and NO secretion; (2) acute and chronic alcohol treatment have differential effects on AM O2- secretion; and (3) alcohol-induced alteration in AM TNF-alpha, O2-, and NO secretion may in part explain the increased susceptibility of alcohol-consuming individuals to pulmonary infections.

Liver sinusoid during chronic alcohol consumption in the rat: an electron microscopic study.

Transmission and scanning electron microscopic studies were performed on the liver sinusoid, with emphasis on sinusoidal endothelial cells, in rats fed a liquid diet containing either alcohol or dextrin (control) for 14 weeks. Animals were also treated with either Gram-negative bacterial lipopolysaccharide (LPS; 100 micrograms/100 g body weight, intravenously) or sterile saline (control). All specimens were prepared after perfusion fixation of the liver. Livers of rats fed dextrin-containing liquid diet displayed the ultrastructural features typical of the sinusoid and its endothelial cells. Livers from alcohol-fed animals, however, were characterized by massive loss of sieve-plate architecture of the sinusoidal endothelium, which was virtually replaced with a meshwork of enlarged openings with diameters frequently exceeding 1 micron. Morphological evidence of Kupffer cell activation could also be seen along with significant fatty infiltration of the hepatocyte. Conversely, LPS administration to dextrin-fed animals induced an apparent decrease in fenestration of the sinusoidal endothelial cell, accompanied by morphological evidence of enhanced endocytotic activity and cytoplasmic swelling. The changes seen 3 hr after LPS administration were markedly advanced at 24 hr. LPS administration to alcohol-fed rats accentuated the alterations observed after alcohol treatment alone. Additionally, the presence of platelets in the sinusoid as well as adhering to the hepatocyte microvilli in the space of Disse, along with the presence of Ito and Kupffer cell activation, greater than that observed in the alcohol-treated rats, is morphological evidence consistent with the disruption of vascular integrity in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor-alpha cell-surface receptors of liver parenchymal and nonparenchymal cells during acute and chronic alcohol administration to rats.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-alpha could be due, at least in part, to alterations in TNF-alpha cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchymal (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 micrograms/100 g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (Kd) and capacity (Bmax) of binding sites, using recombinant human-[125I]TNF-alpha as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (Kd1, in the range of 150-200 pM), low-capacity (Bmax, in the range of 2-3 fmol/10(6) cells) binding site and a low-affinity (Kd2, in the range of 2-9 nM), high-capacity (Bmax2, in the range of 3-15 fmol/10(6) cells) binding site.(ABSTRACT TRUNCATED AT 250 WORDS)