Correspondence: DrApBautista Department of Physiology, LSU-Medical Center, 1901 Perdido Street MEB Room 7272, New Orleans, LA 70112, USA. Email: firstname.lastname@example.org
Chemokines are implicated in the pathogenesis of alcoholic liver disease in humans and in experimental models of alcohol intoxication. The major sources of these chemokines are Kupffer cells which represent more than 80% of tissue macrophages in the body. Kupffer cells are highly responsive to the effects of ethanol, endotoxin and human immunodeficiency virus (HIV)-1 glycoprotein120. These agents, either independently or in combination, may exacerbate the production of chemokines. Chemokines are agents that are highly chemotactic to mononuclear cells and granulocytes. The levels of these chemokines in sera and tissue are elevated in patients with alcoholic hepatitis, alcoholic cirrhosis, diseased livers, viral hepatitis, and in experimental models of chronic alcohol intoxication. Alcohol-induced influx of endotoxin from the gut into the portal circulation is suggested to play an important role in the activation of Kupffer cells which leads to enhanced chemokine release. The up-regulation of chemokines during alcohol consumption is selective. During the early phase of alcoholic liver disease, C-X-C or α- chemokines predominate. This is also associated with neutrophilic infiltration of the liver. In the later stage, up-regulation of C-C or β-chemokine production and migration of mononuclear cells into the liver are observed, and this may lead to liver cirrhosis. Selective up-regulation of chemokine synthesis and release may involve differential modulation of the transcription factors required for chemokine gene expression. Increased cytokine release following alcohol consumption may also regulate chemokine secretion in Kupffer cells via paracrine and autocrine mechanisms and vice versa. In addition, infection with HIV-1 may further compromise the liver to more damage. During HIV-1 infection, a pre-existing liver disease superimposed on chronic alcohol consumption may also exacerbate HIV-1 replication and lymphocytic infiltration in the liver, because of the ability of HIV-1 gp120 to stimulate chemokine production by Kupffer cells and stimulate migration of inflammatory leucocytes in the liver.
Alcohol is recognized as an important immunomodulator because of its ability to regulate the functions of immunocompetent cells. For example, alcohol consumption has been shown to depress lymphocyte functions 1,2 and antigen presentation by Kupffer cells. 3,4 These events are likely to lead to immunosuppression. However, alcohol has also been associated with priming or activation of macrophages for enhanced release of proinflammatory cytokines and chemokines. The effect of alcohol, particularly when administered chronically is also exacerbated by endotoxins. 3,5 Alcohol consumption, either acutely 6 or chronically, 7 enhances the influx of endotoxin from the gut into the circulation. Endotoxin is then cleared by Kupffer cells and hepatic sinusoidal endothelial cells. 8 Consequently, Kupffer cells are primed or activated for enhanced production of reactive oxygen species (ROS), cytolytic proteases and proinflammatory cytokines and chemokines. 3,4,9
Excessive production of these biologically active substances during alcohol consumption is expected to contribute to the pathogenesis of alcoholic liver disease (alcoholic hepatitis and cirrhosis) in humans. Figure 1 shows a diagrammatic representation of how alcohol, its metabolites, and endotoxins contribute to the activation of hepatic macrophages or Kupffer cells for enhanced production of cytokines and chemokines. Figure 1 further shows the important role of gut-derived endotoxins in the initiation of chemokine release, and the possible interactions between cytokines and chemokines. Malondialdehyde-aldehyde protein adduct (MAA) formation has been observed following chronic ethanol consumption. 10 These adducts regulate the expression of adhesion molecules and enhance cytokine production by macrophages and hepatic sinusoidal endothelial cells. 10 These processes are likely to contribute to the development of alcoholic liver disease.
Although alcohol contributes directly or indirectly to the activation of macrophages for enhanced inflammatory reaction, it may also enhance the release of an immunosuppressant cytokine, interleukin (IL)-10. Interleukin-10 is known to depress antibody production, T helper cell-1 responses and antigen presentation. In mice, chronic alcohol feeding is associated with attenuated production of human immunodeficiency virus (HIV)-1 glycoprotein (gp)120 antibody and antigen presentation by hepatic non-parenchymal cells, as well as elevated IL-10 in the circulation. 11 However, lipopolysaccharide (LPS)-mediated production of IL-10 does not seem to depress chemokine production by human mononuclear cells. 12 In other studies, IL-10 selectively depresses chemokine production and up-regulates monocyte chemoattractant protein (MCP)-1 release. 13 This is, perhaps, one mechanism by which immunosuppression and activation of monocyte/macrophages are observed during chronic alcohol intoxication.
Proinflammatory cytokines, and more recently chemokines, are now implicated in the pathogenesis of alcoholic liver disease. Table 1 shows a summary of the members of the chemokine superfamily, their sources, stimuli and specific targets. Several studies in humans and animal models have shown that prolonged consumption of alcohol is associated with elevated serum levels of IL-1, IL-8, tumour necrosis factor (TNF), macrophage inflammatory protein (MIP)-2, and MCP-1. 9,14 The presence of these mediators in liver homogenates from patients with alcoholic liver disease has also been documented. 15,16 The mechanism by which prolonged consumption of alcohol elevates cytokine and chemokine concentrations in the blood and tissues may involve a direct action by ethanol, or an indirect one through its metabolites, MAA-protein, or alcohol-induced endotoxaemia ( Fig. 1).
Table 1. Members of the chemokine superfamily, their sources, stimuli and target cells
Neutrophils, eosinophils, monocytes, T lymphocytes
Kupffer cells, B and T lymphocytes, monocytes
LPS, gp120 from HIV-1 and 2.
Monocytes, T lymphocytes
Kupffer cells, monocyte/macrophages
LPS, gp120 from HIV-1 and 2.
Monocytes, natural killer cells
Kupffer cells, platelets, T lymphocytes
Ethanol, LPS, PHA, gp120 from HIV-1 and 2.
Monocytes, T lymphocytes, eosinophils
Chemokines are low molecular weight, heparin-binding polypeptides (8–10 kDa) produced by a wide variety of cells in response to immunological stimuli. The latter include bacterial LPS, ethanol, gp120 from HIV-1 and HIV-2, immune complexes and phytohaemagglutinin (PHA). 13,17–20 Monocyte/macrophages exposed to endotoxin are a major source of these chemokines. 18 Chemotactic peptides are divided into two major families, i.e., the C-X-C or α-chemokines and C-C or β-chemokines. Although members of both chemokine subfamilies can attract granulocytes and lymphocytes to the sites of inflammation, C-X-C chemokines generally attract granulocytes, while the C-C types are potent chemoattractants for monocytes and lymphocytes.
Alpha-chemokines that include cytokine-induced neutrophil chemoattractant (CINC), the rodent equivalent of human IL-8, interferon-inducible protein (IP)-10, epithelial cell-derived neutrophil activating peptide (ENA)-78 and MIP-2 are potent chemoattractants for neutrophils; they are produced by LPS-treated monocyte/macrophages. 9,21 Hepatocytes are also a rich source of CINC. 22,23 Ethanol is a potent stimulus for CINC production. 22 Interferon-γ stimulates IP-10 production, 24 while TNF and IL-1 induce the release of ENA-78. 25 Macrophages treated with endotoxin produce β-chemokines; that is, MCP-1, MIP-1α, MIP-1β and regulated upon activation normal T-cell expressed and secreted (RANTES). Monocyte chemoattractant protein-1 is chemotactic for monocytes and natural killer cells, while MIP-1α, MIP-1β and RANTES are chemoattractant for monocytes and T lymphocytes.
Phytohaemagglutin stimulates RANTES production by activated T lymphocytes: 19 RANTES is chemotactic for eosinophils, 26 while MIP-1 has also been shown to attract neutrophils and eosinophils to the site of inflammation. 27 Thus, during alcohol intoxication and endotoxaemia, the liver becomes a major site of inflammation with granulocytes and mononuclear cells being attracted to this organ. The influx of inflammatory leucocytes, besides cytotoxic mediators produced (reactive oxygen species (ROS) and proteases) by activated Kupffer cells and hepatic sinusoidal endothelial cells, may further exacerbate hepatic injury.
MOLECULAR MECHANISMS OF MACROPHAGE ACTIVATION
The liver is a major site of ethanol metabolism and clearance of soluble or particulate stimuli (i.e. endotoxins). Ethanol metabolism is performed largely by hepatocytes, while clearance of gut-derived endotoxin is carried out by Kupffer cells. Kupffer cells represent the majority of macrophages in the body, and are therefore considered a major, if not an important, source of inflammatory and cytotoxic mediators. Selective depletion of Kupffer cells by liposome-encapsulated dichloromethylene diphosphonate or gadolinium chloride is associated with a significant attenuation (65–95%) in LPS-induced release of cytokines and chemokines in vivo.17,28 Thus, regulation of Kupffer cell function may have a profound impact on the overall homeostasis of the host in response to alcohol, endotoxin or systemic infection.
Following prolonged consumption of alcohol, Kupffer cells become highly susceptible to the effects of ethanol, its metabolites and LPS. The biological effect of LPS is mediated through specific receptors on Kupffer cells. The LPS binds to serum lipopolysaccharide binding protein, which in turn interacts with the CD14 receptor on Kupffer cells. 29 This interaction results in the translocation and activation of nuclear transcription factor kappa B (NF-κB) to the nucleus, which is involved in gene transcription for various cytokines and chemokines. The observation that LPS activates NF-κB in the absence of CD14, implies a CD14-independent mechanism for Kupffer cell activation. 29 This is mediated through CD11c/CD18 adhesion molecule that also serves as a transmembrane signalling receptor for LPS. 30 This concept is supported by studies showing that anti-CD18 antibody stimulates Kupffer cells to enhance free radical generation. 31 The formation of free radicals in response to immunological stimuli is important because such radicals also regulate the translocation and activation of nuclear transcription factors required for chemokine and cytokine gene expression.
Ethanol, however, does not depend on specific binding sites on target cells to elicit a biological response. A number of studies have shown that ethanol alters plasma membrane fluidity. This effect of ethanol is ubiquitous (i.e. no specific cell types are targeted). It has been shown that ethanol enhances Ca2+ influx and alters membrane phosphatidyl-inositol turnover and arachidonic acid metabolism. This leads to the release of secondary messengers that consequently stimulate the translocation and activation of protein kinase C to the plasma membrane. 32,33 Protein kinase C induces the translocation of NADPH oxidase to the plasma membrane leading to the formation of oxygen-derived radicals. Reactive oxygen species, particularly H2O2, serve as secondary messengers for the translocation and activation of nuclear transcription factors leading to enhanced gene expression for a number of cytokines and chemokines. 34 For example, even in the absence of overt endotoxaemia, ethanol enhanced RANTES mRNA expression and protein release by isolated Kupffer cells. 16
Human immunodeficiency virus-1 gp120 has been shown to induce the release of cytokines and chemokines in human mononuclear cells 35,36 and rat Kupffer cells. 4,17 The mechanism by which these mediators are produced in response to HIV-1 gp120 is likely to involve ROS release, leading to the activation of transcription factors similar to those observed following ethanol or LPS treatment. Mannose specific receptors are the putative binding sites for HIV-1 gp120 in rat Kupffer cells. 11 These glycosylated binding sites on macrophages and endothelial cells are also potential targets for MAA-protein adducts. 10 As a result of ligand binding to these receptors, activation of signal transduction pathways is expected to occur ( Fig. 2). Consequently, transcription factors for specific chemokines or cytokines are up-regulated or down-regulated.
Translocation and activation of transcription factors to the nucleus, and their subsequent binding to promoter regions are important events that trigger gene expression, synthesis and secretion of chemokines. Among these factors are NF-κB. However, NF-κB binding sites are not found on all chemokine promoter regions. 13 Nuclear factor-κB binding sites are present in the promoter regions for RANTES, IL-8, and MIP-2 genes, 13 but they are not necessarily involved in LPS-induced human MIP-1α and MIP-1β gene expression. 13 The promoter region that binds MIP-1α nuclear protein-1 has been shown to play an important role in MIP-1α gene expression after LPS treatment or viral infection. 37 The basic promoter structures of β-chemokine genes are similar but not identical to those of α-chemokine. 13 These slight variations may have a role in the differential regulation of chemokine gene expression and protein release during chronic alcohol intoxication. Moreover, these minor variations may explain, in part, the differences in the kinetics of chemokine generation following an immunological stimulus.
In LPS-treated human mononuclear cells, MIP-1α is released earlier than IL-8. 12In vivo release of MIP-1α and MCP-1 in response to endotoxin is observed within 15 min and peaks much earlier than release of RANTES. 17 Furthermore, RANTES gene expression in lymphocytes is expressed immediately by nuclear factor for interleukin-6 that is activated within 24 h after T cell activation. 19 A second site in the RANTES promoter region binds to a novel transcription factor complex that is up-regulated between 3 and 4 days. 19 These factors may contribute to the selective up-regulation of chemokine synthesis seen in alcoholic hepatitis with or without cirrhosis. 15,16
Taken together, LPS, ethanol and HIV-1 gp120 are potent stimuli that can regulate the synthesis and secretion of proinflammatory mediators by Kupffer cells. Figure 2 shows a diagram postulating the pathway by which these immunomodulators stimulate the release of chemokines or cytokines by hepatic macrophages. Furthermore, chemokines and cytokines are known to have an autocrine function that can regulate the synthesis of proinflammatory mediators and release of free radicals. We have shown that Kupffer cells are a rich of source of MIP-2. 9 Unpublished observations in our laboratory have shown that MIP-2 induces a respiratory burst in neutrophils and Kupffer cells. Others have shown that MIP-1α and MIP-1β are capable of stimulating a respiratory burst. 38 Proinflammatory cytokines regulate ENA-78 production, 25 while MIP has been shown to enhance TNF-α production by macrophages. 18 Thus, the paracrine and autocrine actions of chemokines and cytokines in the liver could have an important role in perpetuating a persistent inflammatory response and cellular injury in alcoholic liver disease.
CHRONIC ALCOHOL INTOXICATION, HEPATIC INJURY AND ALCOHOLIC LIVER DISEASE
Prolonged alcohol consumption is associated with increased hepatic influx of inflammatory polymorphonuclear cells 9 and mononuclear cells. 39 Migration of inflammatory leucocytes is mediated by proinflammatory mediators produced by parenchymal and non-parenchymal cells in the liver. Chronic alcohol intoxication is known to induce endotoxaemia and exacerbates LPS-mediated hepatic injury. 5,21 In contrast, acute alcohol intoxication has been shown to have a protective effect against LPS-mediated injury. 40 The mechanism for this protective effect is thought to involve ethanol-induced acute endotoxaemia that leads to tolerance to the priming and stimulating action of a subsequent LPS challenge. 6 Ingestion of a single dose of alcohol also enhances the influx of LPS into the circulation, which elicits tolerance to exogenous LPS challenge within 3 h. 6 This window of opportunity is tight, because tolerance is lost after 3 h of exposure to alcohol. 6 We have also shown in a model of LPS/LPS tolerance that a low dose of LPS given to rats induces LPS tolerance when a second endotoxin challenge is given within 48 h of the initial dose. When a second LPS dose is given at 120 h, the deleterious effects of endotoxin are severely exacerbated. 41 Thus, in chronic alcohol intoxication in which rats are also endotoxaemic, increased sensitization to LPS may occur, and may have passed the stage of tolerance. Thurman’s group have shown that endotoxin is involved in the induction of tolerance and sensitization to subsequent LPS challenge via mechanisms that involve Ca2+ and protein kinase C. 6 Published data from our laboratory further show that total protein kinase C activity of Kupffer cells from chronic alcohol-fed rats is increased. 7 Increased protein kinase C activity in Kupffer cells may lead to enhanced chemokine release when these cells are challenged immunologically.
Basal levels of serum chemokines (CINC, MIP-2, MCP-1, RANTES, MIP-1α, MIP-1 β) and cytokines (TNF- α, IL-1β) were elevated in alcohol-fed rats compared with pair-fed controls. 4 The serum chemokine and cytokine levels were further increased when alcohol-fed rats were challenged with exogenous endotoxin. 3–5 This was accompanied by an exacerbation of hepatic injury and inflammation. 3–5 The precise mechanism for these observations has not been well defined. However, it is likely that previous exposure to low levels of endotoxin during chronic alcohol intoxication primes and sensitizes the Kupffer cells for enhanced protein kinase C activity, ROS release and NF-κB activation. We have previously shown that Kupffer cells from alcohol-fed rats release significant amounts of ROS spontaneously. 3,4,21 The release of ROS is further enhanced when alcohol-fed rats are treated with LPS. 3,4,8 Others have shown that NF-κB is activated in Kupffer cells from rats fed with alcohol for 10–17 weeks. 42 Expression of chemokine and cytokine mRNA is also up-regulated under these conditions. 4,42,43
During chronic alcohol intoxication, chemokine production by Kupffer cells is an important event, because migration of inflammatory leucocytes (induced by these peptides) to the liver is likely to provoke more damage to the hepatocytes. Chemokines are not directly hepatotoxic. However, a previous study has shown that MIP-2 is directly cytotoxic to isolated hepatocytes from alcohol-fed and iron-treated rats. 9,21 Another chemokine, CINC, is hepatotoxic during adenovirus infection. 44 The cytotoxic potential of other chemokines is more likely to be indirect. Chemokines (MIP-2, MIP-1, RANTES) can stimulate the release of ROS by neutrophils and Kupffer cells, and the degranulation of eosinophils. These processes are likely to damage hepatocytes. The migration of cytotoxic T lymphocytes to the liver may also exacerbate tissue injury.
During chronic alcohol intoxication, up-regulation of chemokine release is not universal at one particular time. Thus, Fig. 3 shows that serum RANTES, MIP-1α, and keratinocyte were up-regulated in BALB/c mice fed agar blocks containing ethanol for 12 weeks, but serum MIP-2 was not altered. Alcohol feeding for 16 weeks up-regulated α-chemokines in serum and release by rat Kupffer cells, with small or undetectable levels of β-chemokines. 4 Increased cytokine (TNF-α, transforming growth factor-β, IL-6) gene expression and protein release by isolated Kupffer cells are observed at 10 weeks of alcohol feeding with a high-fat diet (the Tsukamoto–French Diet). 42 Concomitantly, liver injury and lipid peroxidation are increased. 42 At 17 weeks of alcohol feeding, further increases in cytokine gene expression, protein release, tissue injury and lipid peroxidation are observed. 42
During the early phase of alcohol consumption, the predominant leucocyte types are the neutrophils. 9 After 25–30 weeks of alcohol feeding, there is a switch to higher serum levels of β-chemokines, 4 increased mononuclear cell infiltration and pericentral fibrosis in the liver. 39 This could lead to cirrhosis. Protein and mRNA expression for MIP-1α, MIP-1β, MCP-1 and IL-8 in the livers of patients with alcoholic hepatitis and alcoholic cirrhosis are greater than in normal liver. 16 In another study, GRO was more predominant in human liver homogenates from patients with alcoholic hepatitis, while RANTES was elevated in diseased livers and in those from patients with viral hepatitis. 15 These observations reinforce the idea that further prolongation of alcohol consumption is likely to result in a more severe inflammatory reaction in the liver involving mononuclear cells, particularly lymphocytes. This process is likely to occur because members of the C-C-chemokine subfamily are the predominant chemokines up-regulated during the latter stages of alcoholic liver disease, with or without infection. As stated above, members of this group of chemokines are chemoattractants for lymphocytes. Thus, infiltrated lymphocytes are likely to play a role in the pathogenesis of alcoholic hepatitis. The precise mechanism by which lymphocytes contribute to hepatic dysfunction is not clearly defined. However, it has been shown that in the alcohol-fed rat, CD4+ cells contribute to concanavalin A-mediated acute liver injury by mobilizing TNF-α, interferon-γ and IL-6. 45
ALCOHOLIC LIVER DISEASE AND HIV-1
Prolonged consumption of alcohol has also been considered a risk factor in HIV-1/aquired immune deficiency syndrome (AIDS). However, there are no studies available to suggest that alcoholic liver disease exacerbates HIV-1 infection or vice versa. Current evidence suggests that chemokines are involved in the pathogenesis of both disorders. The ability of ethanol and endogenous endotoxins to stimulate the release of chemokines that can contribute to the development of alcoholic liver disease, may also have an important role in the regulation of HIV-1 replication in target cells. As stated above, chronic alcohol intoxication is associated with activation of Kupffer cells and increased production of ROS, cytokines and chemokines. It has been shown that these mediators, particularly TNF-α and IL-1 are important factors for HIV-1 replication. Recently, the role of chemokines in viral replication has gained considerable attention, because receptors for these factors are the coreceptors for HIV-1 gp120. 46 Alpha chemokine receptors (C-X-CR4) are present on monocyte/macrophages. Lymphocytes express both C-X-CR4 and C-CR5, which are the binding sites of α- and β-chemokines, respectively. 46 In lymphocytes, HIV-1 replication may be exacerbated when a β-chemokine cocktail of MIP-1α, MIP-1β and RANTES interact with the C-CR5 receptors on target cells. Beta-chemokines have been shown to induce lymphocyte proliferation. 47
Activation of lymphocytes by PHA or chemokines exacerbates viral replication in CD4+ cells, and β-chemokines also enhance simian immunodeficiency virus-1 replication in simian lymphocytes. 48 The role of chemokines in alcoholic liver disease and HIV-1 infection is extremely relevant, because the Kupffer cells in the liver are the major sources of these chemotactic peptides. The presence of lymphocytes, which are also a rich source of MIP-1α, MIP-1β and RANTES, in the livers of individuals with alcoholic hepatitis or cirrhosis, may further exacerbate local activation and sequestration of inflammatory leucocytes into the liver. Thus, pre-existing liver disorders (i.e. alcoholic liver disease, hepatitis C) may be exacerbated by HIV-1 because of the ability of viral gp-120 to induce the synthesis of chemokines by Kupffer cells. The resultant proliferation of lymphocytes could further exacerbate hepatic dysfunction.
This paper shows the potential of Kupffer cells to be an important factor in the pathogenesis of alcoholic liver disease, because of their ability to produce a wide spectrum of biologically active substances, including ROS, cytokines, and chemokines in response to ethanol, LPS and HIV-1 gp120. These agents share similar signal transduction pathways that lead to the regulation of chemokine and cytokine gene expression and protein secretion. During chronic alcohol intoxication, gut-derived LPS seems to play an important role in the priming and activation of Kupffer cells. The release and protein expression of various chemokines is also selective. During the early phase of chronic alcohol intoxication, C-X-C chemokines are significantly elevated and result in neutrophilic accumulation in the liver. During the latter part of chronic alcohol consumption, at the least in the rat, C-C-chemokines are involved in the migration of mononuclear cells to the liver. This is potentially a more serious pathology because lymphocytes can proliferate in this organ in the presence of stimuli that include C-C chemokines. The exacerbated production of these chemokines during chronic alcohol intoxication may perpetuate persistent lymphocytic inflammation in the liver, a factor that would lead to cirrhosis. The liver may also become a potential target for HIV-1 because of the preponderance of lymphocytes in this organ in individuals with alcoholic hepatitis. It has been shown that the liver is one of the last organs to be infected by HIV-1 during the terminal stages of HIV-1/AIDS. Such a condition may be further exacerbated by pre-existing liver diseases, such as alcoholic hepatitis or cirrhosis.
Kupffer cells may play an important role in the development of alcoholic liver disease. However, other hepatic non-parenchymal cells (i.e. large granular lymphocytes (pit cells), hepatic sinusoidal endothelial cells and stellate cells) are likely to be involved because they are potential sources of cytotoxic and proinflammatory mediators. For example, hepatic sinusoidal endothelial cells produce ROS, cytokines and chemokines. Stellate cells, which are primarily involved in the development of fibrosis, secrete MCP-1 in response to LPS only when they are transformed into myofibroblast-like cells. 49 The precise role of pit cells or natural killer cell receptor protein-1 positive lymphocytes in alcoholic liver disease is not well defined, although lymphocytes have been shown to augment cytokine production by Kupffer cells. 50 Thus, Kupffer cells and other hepatic non-parenchymal cells are likely to participate in the evolution of alcoholic liver disease, perhaps at different stages of the disease. Kupffer cells may initiate a number of pathophysiological changes within minutes following exposure to immunological stimuli, viral products, alcohol and/or endotoxin. Biologically active substances produced by Kupffer cells may, in turn, regulate the functions of other hepatic non-parenchymal cells and vice versa that could have an important impact in the pathogenesis of alcoholic liver disease.
This work was supported by the National Institutes of Health (USA) grants RO1 AA 08846, RO1 AA 10466, RO1 AA 10746 and P50 AA 09803 to Dr Abraham P Bautista.