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Alcohol-induced fatty liver and inflammation: where do Kupffer cells act?

2000; Elsevier BV; Volume: 32; Issue: 6 Linguagem: Inglês

10.1016/s0168-8278(00)80108-7

1600-0641

Alessandro Casini,

Eicosanoids and Hypertension Pharmacology

The liver is the major target organ for the toxic effects of ethanol in our body because of the extraordinary metabolic machinery contained in hepatocytes. The hepatotoxicity of alcohol results from the hepatic oxidative metabolism, which involves alcohol dehydrogenase (ADH)-mediated excessive generation of nicotinamide adenine dinucleotide, reduced form (NADH), and acetaldehyde, the main oxidative metabolite of ethanol. Acetaldehyde may also result from an accessory (but inducible) microsomal oxidising system (MEOS), which specifically contains an inducible form of cytochrome P4502E1 (CYP2E1) able to produce acetaldehyde from ethanol (1.Lieber CS DeCarli LM Hepatic microsomal ethanol oxidizing system: in vitro characteristics and adaptive properties in vivo.J Biol Chem. 1970; 245: 2505-2512Abstract Full Text PDF PubMed Google Scholar). In ADH-mediated oxidation of ethanol, acetaldehyde is produced and hydrogen is transferred from ethanol to the cofactor NAD, that is converted to its reduced form NADH. Thus, as a net result, alcohol oxidation generates an excess of reducing equivalents in the liver, primarily as NADH. Further contribution to NADH accumulation is due to acetaldehyde oxidation to acetate via the mitochondrial aldehyde-dehydrogenase (ALDH2) pathway. This metabolic process is responsible for a number of metabolic disorders including hepatic steatosis. The increased NADH/NAD ratio enhances the concentration of α-glycerophosphate, which favours hepatic triglyceride accumulation by trapping fatty acids. Moreover, excess NADH may promote fatty acid synthesis. In addition to the enhanced hepatic lipogenesis, other metabolic disturbances may contribute to the occurrence of fatty liver: decreased hepatic release of lipoproteins, increased mobilisation of peripheral fat, enhanced hepatic uptake of circulating lipids, and decreased fatty acid oxidation due to mitochondrial dysfunction (2.Lieber CS Alcohol and the liver: 1994 update.Gastroenterology. 1994; 106: 1085-1105Abstract PubMed Google Scholar). Ethanol metabolism via the NADPH-dependent CYP2E1 oxidising pathway is responsible for the generation of a great amount of reactive oxygen species (ROS), able to induce several damaging events in liver tissue, including the peroxidation of cell membrane phospholipids (lipid peroxidation) which play a pivotal role in the pathogenesis of alcoholic liver injury. Ethanol induces liver microsomal CYP2E1 up to 10-fold in humans and in rat models of chronic alcohol administration; this leads to lipid peroxidation phenomena within the liver and the formation of protein adducts with reactive aldehyde endproducts of lipid peroxidation (3.Tsutsumi M Lasker JM Shimizu M Rosman AS Lieber CS The intralobular distribution of ethanol-inducible P450IIE1 in rat and human liver.Hepatology. 1989; 10: 437-446Crossref PubMed Scopus (296) Google Scholar, 4.Clot P Bellomo G Tabone M Aricò S Albano E Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis.Gastroenterology. 1995; 108: 201-207Abstract Full Text PDF PubMed Scopus (96) Google Scholar). Thus, CYP2E1 seems to have a key role in the occurrence of ethanolinduced ROS formation in the liver and consequent lipid peroxidation. Conversely, its role in the development of hepatic steatosis appears doubtful. In fact, it has been shown in rats that CYP2E1 activity can be induced by ethanol, even in the absence of hepatic steatosis (2.Lieber CS Alcohol and the liver: 1994 update.Gastroenterology. 1994; 106: 1085-1105Abstract PubMed Google Scholar). Furthermore, the excess of hepatic reducing equivalents, coming from both ADH and ALDH activity, can also be transferred to NADPH, which is “consumed” and not accumulated by CYP2E1 activation. Against this background, the article by Järveläinen et al. (5.Järveläinen HA Fang C Ingelman-Sundberg M Lukkari TA Sippel H Lindros KO Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver.J Hepatol. 2000; 32: 900-910Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) in this issue of Journal of Hepatology reports an interesting finding. When the authors treated rats with an ethanol-containing liquid diet and “inactivated” Kupffer cells with gadolinium chloride (GdC13), they obtained a significant improvement in liver pathology by reduction of fatty liver but not of inflammation. Moreover, GdC13 treatment significantly diminished the liver induction of CYP2E1 due to ethanol consumption. Decreased hepatic steatosis was associated with a parallel reduction of CYP2E1 expression with a remarkable linear correlation, thus suggesting a steatopathogenetic role for this enzyme. These findings render the role of CYP2E1 in ethanol hepatotoxicity even more intriguing. Although a moderate expression of CYP2E1 has been found in Kupffer cells (6.Koop DR Chernosky A Brass EP Identification and induction of cytochrome P450 2E1 in rat Kupffer cells.J Pharmacol Exp Ther. 1991; 258: 1072-1076PubMed Google Scholar), as underlined by the authors, the contribution of these cells to total liverCYP2E1 activity is obviously negligible, because of the hepatocyte content of this enzyme. Therefore, a direct inhibitory effect of GdC13 on hepatocyte CYP2E has to be postulated (7.Badger DA Kuester RK Sauer JM Sipes IG Gadolinium chloride reduces cytochrome P450: relevance to chemical-induced hepatotoxicity.Toxicology. 1997; 121: 143-153Crossref PubMed Scopus (55) Google Scholar) in order to explain the findings mentioned above. We must, however, point out that other factors, namely the high fat content of the liquid diet used in the present work (44% of calories), might have influenced the “steatopathogenetic” role of CYP2E1. The recent manipulation of different dietary compositions has brought attention to factors in the classical pair-feeding models that could enhance the extent of liver damage. Rats fed ethanol with fish oil develop more severe liver injury than those fed ethanol with corn oil. Fish oil contains a larger percentage of fatty acids with more than two double bonds and thus a greater number of single methylenes flanked by double bonds that increase susceptibility to lipid peroxidation (8.Ekstrom G Ingelman-Sundberg M Rat microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P450 IIE1).Biochem Pharmacol. 1989; 38: 1313-1319Crossref PubMed Scopus (505) Google Scholar). Interestingly, themore severe liver injury in animals fed fish oil is associated with greater induction of CYP2E1 and increased lipid peroxidation, shown by enhanced levels of microsomal-conjugated dienes (9.Nanji AA Zhao S Sadrzadech SMH Dannenberg AJ Tahan SR Waxman DJ Markedly enhanced cytochrome P450 2E1 induction and lipid peroxidation is associated with severe liver injury in fish oil-ethanol-fed rats.Alcohol Clin Exp Res. 1994; 18: 1280-1285Crossref PubMed Scopus (237) Google Scholar). In ethanol-fed rats on a fish oil diet in which hepatic steatosis, inflammation and necrosis developed, discontinuing ethanol and switching to a diet enriched in saturated fatty acids reversed the liver injury; in contrast, continuing the fish oil diet without ethanol did not improve the degree of hepatic injury (10.Nanji AA Yang EK Fogt F Sadrzadech SMH Dannenberg AJ Medium chain triglycerides and vitamin E reduce the severity of established experimental alcoholic liver disease.J Pharmacol Exp Ther. 1996; 277: 1694-1700PubMed Google Scholar). The classical animal model of chronic alcohol intoxication by Lieber & DeCarli (11.Lieber CS DeCarli LM The feeding of alcohol in liquid diets: two decades of applications and 1982 update.Alcohol Clin Exp Res. 1982; 6: 523-531Crossref PubMed Scopus (610) Google Scholar) relied on voluntary ethanol intake in liquid-diet form with pair-fed control animals receiving an isocaloric diet where ethanol was replaced by carbohydrates. This dietary treatment could induce a certain amount of hepatic steatosis, but no progression to more severe forms of liver damage was evident. The more “aggressive” model of Tsukamoto et al. (12.Tsukamoto H Towner SJ Ciofalo LM French SW Ethanolinduced liver fibrosis in rats fed high fat diets.Hepatology. 1986; 6: 814-822Crossref PubMed Scopus (249) Google Scholar) was based on a continuous intragastric supply of high concentrations of ethanol. This treatment gave rise to more severe pictures of liver damage, with inflammation and progression to liver fibrosis, particularly when combined with dietary inclusion of unsaturated fats. Järveläinen et al. (5.Järveläinen HA Fang C Ingelman-Sundberg M Lukkari TA Sippel H Lindros KO Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver.J Hepatol. 2000; 32: 900-910Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) used a liquid lowcarbohydrate diet pair-feeding model, which results in a much more severe liver injury than the standard liquid diet of Lieber-DeCarli, as evident by the induction of macro- and microvescicular steatosis and the incidence of inflammation (13.Lindros KO Järveläinen HA A new oral low-carbohydrate alcohol liquid diet producing liver lesions: a preliminary account.Alcohol Alcohol. 1998; 33: 347-353Crossref PubMed Scopus (62) Google Scholar). Taken together, all these data indicate that there may be a significant metabolic component that contributes to the onset and extension of liver damage associated with chronic alcohol administration, and confirm the concept that the amount of dietaryfat intake is a main determinant of the degree of hepatic fatty infiltration, both in animals (14.Lieber CS DeCarli LM Quantitative relationship between amount of dietary fat and severity of alcoholic fatty liver.Am J Clin Nutr. 1970; 23: 474-478Crossref PubMed Scopus (246) Google Scholar) and in humans (15.Lieber CS Spritz N Effects of prolonged ethanol intake in man: role of dietary, adipose, and endogenously synthesized fatty acids in the pathogenesis of alcoholic fatty liver.J Clin Invest. 1966; 45: 1400-1411Crossref PubMed Scopus (101) Google Scholar) ingesting alcohol. The article by Järveläinen et al. (5.Järveläinen HA Fang C Ingelman-Sundberg M Lukkari TA Sippel H Lindros KO Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver.J Hepatol. 2000; 32: 900-910Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) in this issue of Journal of Hepatology deals with an additional, and not fully understood, important issue in the field of alcoholic liver disease: the role of endotoxins and endotoxin-induced cytokines in the hepatotoxicity of ethanol. Endotoxins have become relevant candidates as important contributors to the development of the alcohol-induced liver injury. Endotoxins have been found to be elevated in the blood of alcoholics and in animal models of alcoholic liver disease; they may induce the release of a number of cytokines from macrophages and other cells, and their blood levels correlate with high levels of circulating proinflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) (16.Bode C Kugler V Bode JC Endotoxemia in patients with alcoholic and non-alcoholic cirrhosis and subjects with no evidence of chronic liver disease following acute alcohol excess.J Hepatol. 1987; 4: 8-14Abstract Full Text PDF PubMed Scopus (478) Google Scholar, 17.Nanji AA Khettry U Sadrzadech SMH Yamanaka T Severity of liver injury in experimental alcoholic liver disease.Am J Pathol. 1993; 142: 367-373PubMed Google Scholar). Endotoxin lipopolysac-charide (LPS) derives from the cell wall material of Gram-negative bacteria, including the bacterial flora of the gut. Once it has penetrated through the intestinal wall, LPS is transported to the liver bound to an LPS-binding protein (LBP), where it is cleared mainly by Kupffer cells. Several classes of cell surface receptors for endotoxins are present, among which the CD14 receptors are functionally most prominent. Chronic ethanol administration may induce such receptors in Kupffer cells, as reported by the paper of Järveläinen et al. (5.Järveläinen HA Fang C Ingelman-Sundberg M Lukkari TA Sippel H Lindros KO Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver.J Hepatol. 2000; 32: 900-910Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), thus “sensitizing” these cells to the effects of LPS. Binding of LPS to its specific receptors initiates a chain of intracellular signalling events that lead to the activation of Kupffer cells, resulting in the synthesis and secretion of cytokines and other substances, including the proinflammatory cytokines TNF-α and IL-1β, and prostaglandins, such as PGE2 and PGD. These soluble factors generated by Kupffer cells may promote an inflammatory response and thus contribute to liver failure. The major cause of the alcohol-induced elevation of circulating endotoxin is thought to be an ethanol-induced increase of the intestinal wall permeability to endotoxin; however, an increased bacterial flora associated with alcohol abuse and a reduced capacity for endotoxin scavenging in alcoholics have been hypothesised (18.Schenker S Bay MK Alcohol and endotoxin: another path to alcoholic liver injury.Alcohol Clin Exp Res. 1995; 19: 1364-1366Crossref PubMed Scopus (40) Google Scholar). Taken together, these data indicate that Kupffer cells may have a pivotal role in the development of alcohol-induced liver injury. Destruction of these cells with GdC13 or reduction of bacterial endotoxin by sterilisation of the gut with antibiotics is able to block the inflammatory response and to reduce hepatic necrosis due to ethanol (19.Thurman RG Alcoholic liver injury involves activation of Kupffer cells by endotoxin.Am J Physiol. 1998; 275: G605-G611PubMed Google Scholar). Similar results have been obtained with anti-TNF-α antibodies (20.Iimuro Y Gallucci RM Luster MI Kono H Thurman RG Antibodies to tumor necrosis alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat.Hepatology. 1997; 26: 1530-1537Crossref PubMed Scopus (454) Google Scholar). In their experimental model of alcoholic liver disease Järveläinen et al. (5.Järveläinen HA Fang C Ingelman-Sundberg M Lukkari TA Sippel H Lindros KO Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver.J Hepatol. 2000; 32: 900-910Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) reported an increased expression of CD14 receptor on Kupffer cells, associated with enhanced levels of hepatic LBP mRNA, but inactivation of Kupffer cells by GdC13 did not affect either of these ethanol-induced effects. Conversely, GdC13 treatment eliminated the periportal large ED2 positive Kupffer cells that produce other mediators, such as eicosanoids, able to affect the hepatic metabolism of fat (21.Armbrust T Ramadori G Functional characterization of two different Kupffer cell populations of normal rat liver.J Hepatol. 1996; 25: 518-528Abstract Full Text PDF PubMed Scopus (70) Google Scholar) and thus capable of influencing the development of fatty liver. Alcohol feeding was devoid of any significant effect on both TNF-α and IL-1β liver mRNA expression. In a previous paper (22.Järveläinen HA Fang C Ingelman-Sundberg M Lindros KO Effect of chronic coadministration of endotoxin and ethanol on rat liver pathology and proinflammatory and anti-inflammatory cytokines.Hepatology. 1999; 29: 1503-1510Crossref PubMed Scopus (105) Google Scholar) the same group of investigators reported similar results, not only for proinflammatory cytokines but also for anti-inflammatory IL-4 and IL-10. In contrast, LPS treatment of rats induced all such cytokines in the liver. Although the ethanol diet used by the authors caused a 3-fold increase in plasma LPS, there was no evidence of significant potentiation of alcoholic liver damage by endotoxin. The authors concluded that sustained elevation of LPS levels could induce a tolerance able to attenuate the potential damage associated with acute endotoxin exposure and suggested that a similar adaptation might occur in alcoholics with continuous endotoxemia. If this happened in humans, sustained circulating levels of endotoxin should be protective against some adverse effects of alcohol on the liver, including the ethanol-induced pro-fibrogenic action since Järveläinen and co-workers, in their previous paper (22.Järveläinen HA Fang C Ingelman-Sundberg M Lindros KO Effect of chronic coadministration of endotoxin and ethanol on rat liver pathology and proinflammatory and anti-inflammatory cytokines.Hepatology. 1999; 29: 1503-1510Crossref PubMed Scopus (105) Google Scholar), reported a remarkable finding: the striking LPS-induced increase in transforming growth factor β1 (TGF-β1) in the liver was significantly reduced by combined LPS-ethanol administration. These studies indicate that ethanol-induced elevation of endotoxin may be critical in the development of alcoholic liver disease, since the LPS-induced release of cytotoxic factors, namely TNF-α and prostaglandin E2, by Kupffer cells appears to be a key step in initiating inflammatory reactions and generating tissue damage. This type of damage may be further enhanced by free radicals, notably ROS, produced by Kupffer cells in addition to ROS generated by ethanol-induced CYP2E1. Several studies have shown that stimulation of Kupffer cells elicits the production of superoxide anion. This has been reported both in perfused isolated liver and in isolated Kupffer cells after in vivo administration of either LPS or ethanol (23.Bautista AP Mészaros K Bojta J Spitzer JJ Superoxide anion generation in the liver during the early stage of endotoxemia in rats.J Leukoc Biol. 1990; 48: 123-128PubMed Google Scholar, 24.Bautista AP Spitzer JJ Acute ethanol intoxication stimulates superoxide anion production by in situ perfused rat liver.Hepatology. 1992; 15: 892-898Crossref PubMed Scopus (72) Google Scholar). Through this mechanism, Kupffer cells may contribute to the progress of alcoholic liver disease toward the development of fibrosis. There is now overwhelming evidence that hepatic stellate cells (HSC) are the principal cell type involved in hepatic fibrosis. In different forms of liver injury it has been shown that they proliferate and undergo phenotypic transformation into “active” matrix-producing, myofibroblast-like cells in response to necroinflammatory or toxic agents (25.Friedman SL The cellular basis of hepatic fibrosis.N Engl J Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar). Several reports have indicated that ROS-induced lipid peroxidation may contribute to the development of hepatic fibrosis. Specifically, it has been reported that liver tissues from alcoholics show abundant amounts of the reactive aldehydes end-product of lipid peroxidation, as well as of acetaldehyde, inside groups of hepatocytes close to sites of active fibrogenesis (26.Niemela O Juvonen T Parkkila S Immunohistochemical demonstration of acetaldehyde-modified epitopes in human liver after alcohol consumption.J Clin Invest. 1991; 87: 1367-1374Crossref PubMed Scopus (123) Google Scholar, 27.Bedossa P Houglum K Trautwein C Holstege A Chojkier M Stimulation of collagen α1(I) gene expression is associated with lipid peroxidation in hepatocellular injury: a link to tissue fibrosis?.Hepatology. 1994; 19: 1262-1271PubMed Google Scholar). Moreover, several in vitro data have confirmed that different pro-oxidant stimuli may induce the synthesis of collagen and other extracellular matrix components in HSC. Exposure of human HSC to ascorbate/iron results in early induction of lipid peroxidation and in a significant increase of the constitutive expression of procollagen type I mRNA, paralleled by accumulation of the protein in the cell culture medium; this stimulatory effect is strongly reduced by pretreating HSC with antioxidants such as vitamin E or diphenyl-phenylendiamine (28.Parola M Pinzani M Casini A Albano E Poli G Gentilini A et al.Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen α1 (I) gene expression in human liver fat-storing cells.Biochem Biophys Res Comm. 1993; 194: 1044-1050Crossref PubMed Scopus (303) Google Scholar). More recently, it has been shown that human neutrophils, activated in vitro to produce significant amounts of ROS, induce a strong increase in collagen synthesis by cultured human HSC. This effect is hampered by antioxidants (vitamin E), by inhibitors of aldehydeprotein adducts, and by the presence of nitric oxide acting as a scavenger of superoxide anion (29.Casini A Ceni E Salzano R Biondi P Parola M Galli A et al.Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells. Role of nitric oxide.Hepatology. 1997; 25: 361-367Crossref PubMed Scopus (272) Google Scholar). In this context of free radical generation and induction of lipid peroxidation, Kupffer cells may further provide an additional adverse event by secreting prostaglandin E2 that stimulates O2 consumption in hepatocytes from the perivenular zones of the liver acinus (30.Qu W Zhong Z Goto M Thurman RG Kupffer cell prostaglandin E2 stimulates parenchymal cell O2 consumption: alcohol and cell-cell communication.Am J Physiol. 1996; 270: G574-G580PubMed Google Scholar). This would promote a transient hypoxic condition, exposing regions of liver tissue to anoxia-reperfusion episodes, which in turn would contribute further to free radical formation. In this complicated network of events Kupffer cells might be, at the same time, targets and effectors of different, self-perpetuating events that involve endotoxin, proinflammatory cytokines, fat metabolism, and free radical generation. Chronic alcohol consumption could alter the gut membrane structure, making it more permeable to endotoxin. Circulating endotoxin would then enter the liver, where it would be taken up by Kupffer cells, which could become activated, releasing TNF-α, ROS and prostaglandin E2. Elevation of hepatocyte oxygen consumption on one hand and increased ethanol metabolism, due to CYP2E1 induction, on the other, generate a large amount of ROS, induce metabolic disturbances of hepatic fat metabolism, and provide maintenance for lipid peroxidation phenomena. As mentioned earlier, HSC undergo a process of activation in vivo when they are exposed to different damaging stimuli. This phenotypic transformation is associated with loss of the intracellular vitamin A deposits as fat droplets by these cells (25.Friedman SL The cellular basis of hepatic fibrosis.N Engl J Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar). Although several studies have tried to elucidate this phenomenon, no convincing data have been produced to explain the mechanisms that link activation of HSC to loss of fat droplets. A promising contribution is coming from very recent studies on a group of nuclear receptors, named peroxisome proliferator-activated receptors (PPAR), that regulate the “fat-phenotype” of several cell types (31.Braissant O Foufelle F Scotto M Bauca M Wahli W Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR alpha, beta and gamma in the adult rats.Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1737) Google Scholar). The γ isoform of these receptors (PPARγ) acts as obligate heterodimer with another nuclear receptor, 9-cis retinoic acid receptor (RXR), and has been shown to have a key role in adipogenesis (32.Tontonoz P Hu E Spigelman BM Regulation of adipocyte gene expression and differentiation by peroxisome proliferator-activated receptor gamma.Curr Opin Gene Dev. 1995; 5: 571-576Crossref PubMed Scopus (407) Google Scholar). In a very recent work (33.Galli A Crabb D Price D Ceni E Salzano R Surrenti C Casini A Peroxisome proliferator-activated receptor γ transcriptional regulation is involved in platelet-derived growth factor-induced proliferation of human hepatic stellate cells.Hepatology. 2000; 31: 101-108Crossref PubMed Scopus (205) Google Scholar), the presence of PPARγ receptors has been demonstrated in human HSC, where they play a regulatory role in the activation process of such cells. In “resting” (fat-storing) HSC the abundance of PPARγ may contribute to specific expression of genes that help to maintain the fat-storing pheno-type. During HSC activation PPARγ activity declines due to both reduction in expression of the receptor and possible loss of putative endogenous ligands. This study clearly indicates that different stimuli able to down-regulate PPARγ expression may initiate and maintain HSC transdifferentiation. The ethanol-induced disturbance of fat metabolism, in which Kupffer cells play an important role as outlined above, might be the crucial event that links toxic injury to the progression of alcoholic liver disease to end-stage fibrosis. Whether Kupffer cells are more involved in alcoholinduced inflammatory response than in hepatic metabolic alterations remains so far controversial.