Modulation of F-met-leu-phe Induced Chemotactic Activity and Superoxide Production by Neutrophils during Chronic Ethanol Intoxication

Chronic alcohol consumption has been associated with increased migration of neutrophils into liver that could contribute to the development of alcoholic liver disease. Mild endotoxemia may be at least partially responsible for this condition since endotoxemia was shown to be present in virtually all chronic alcoholics. This study examines the release of superoxide anion and chemotactic activity by Kupffer cells and sequestered hepatic as well as blood neutrophils during chronic alcohol intoxication (16 weeks) alone, and following an intravenous injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) 3 hr before cell isolation. Chronic ethanol consumption increased the total neutrophil yield per liver, but did not change the f-met-leu-phe induced chemotactic activity by both hepatic and blood neutrophils. However, the combined insults of ethanol and LPS increased the chemotactic activity and superoxide anion generation by these cells. Plasma from ethanol-fed rats was highly chemotactic to syngeneic normal rat neutrophils. This activity was increased 1.75-fold in the plasma obtained from chronic ethanol plus endotoxin-injected rats. The chemotactic activity of Kupffer cells was not significantly modulated during ethanol intoxication plus endotoxin treatment. The f-met-leu-phe-induced superoxide anion release by Kupffer cells was enhanced after LPS treatment. Chronic ethanol consumption did not induce any effect on this parameter. These observations suggest that functional alterations in neutrophils during chronic ethanol intoxication may contribute to hepatic injury.     

Chronic Alcohol Intoxication Enhances the Expression of CD18 Adhesion Molecules on Rat Neutrophils and Release of a Chemotactic Factor by Kupffer Cells

Chronic alcohol intoxication has been associated with increased migration of inflammatory leukocytes to the liver that may contribute to the development of alcoholic hepatitis in susceptible individuals. Thus, this work was performed to examine the mechanism by which neutrophils [polymorphonuclear neutrophils (PMNs)] are sequestered in the liver during prolonged consumption of alcohol. Male Sprague-Dawley rats were fed with Sustacal supplemented by 36% alcohol, or isocaloric diet for 16 weeks. Circulating blood PMNs were collected and examined for CD18 ( β ₂-integrin) adhesion molecule expression. Monoclonal antibody 1F12, an anti-CD18 antibody and potent neutropenic agent, was used to detect CD18 on PMNs. More than 97% of neutrophils obtained from pair and ethanol-fed rats were positive for the antibody. Fluorescence intensity of fluorescein iso-thiocyanate-1F12 binding to PMNs from ethanol-fed rat was significantly enhanced 2-fold compared with the pair-fed controls. The release of chemoattractant and free radical-generating activity in culture supernatants of Kupffer cells was also examined. Twenty-four hr culture supernatants of Kupffer cells from chronic alcoholic rats enhanced the migration and superoxide anion generation by normal PMNs, compared with those of the pair-fed rats. Antirat interleukin-8 antiserum inhibited chemotactic activity and superoxide generating capacity of culture supernatants. These results suggest that upregulation of adhesion molecules on PMNs and chemotactic factor release from Kupffer cells may contribute, at least in part, to enhanced migration of inflammatory leukocytes to the liver during chronic alcohol intoxication.     

Kupffer cells and alcoholic liver disease.

Liver disease is a major cause of illness and death worldwide. A central component in the complex network leading to the development of alcoholic liver disease is the activation of Kupffer cells by endotoxin and other soluble mediators. Alcohol consumption induces a state of "leaky gut increasing plasma and liver endotoxin levels. When Kupffer cells become activated, they interact with a complex of proteins located on the extracellular membrane signaling to produce a wide array of soluble factors, including cytokines, chemokines, growth factors, cyclooxygenase and lipoxygenase metabolites, and reactive oxygen species such as superoxide anion, hydrogen peroxide, and nitric oxide, all of which provide physiologically diverse and pivotal paracrine effects on all other liver cell types and, ultimately, liver injury. Kupffer cells are also central to the liver homeostatic response to injury as upon cellular degenerative changes, they immediately respond to the insult and release mediators to orchestrate inflammatory and reparative responses. Thus, the homeostatic responses are initiated by Kupffer cell-derived mediators at the cellular level and underlie the liver s defense and reparative mechanisms against injury. In order to understand better the role of Kupffer cells in the onset of liver injury, animal models in which Kupffer cells are inactivated, and cell culture settings (e.g. co-cultures) are being used with promising results that advance our understanding of alcoholic liver disease.     

Chronic Alcohol Intoxication Attenuates Human Immunodeficiency Virus-1 Glycoprotein 120-Induced Superoxide Anion Release by Isolated Kupffer Cells

This work tests the hypothesis that chronic alcohol intoxication suppresses the microbicidal activity of Kupffer cells by modulating the expression of cell surface receptors associated with respiratory burst and the release of potent microbicidal agents [i.e., reactive oxygen species (ROS)]. Because alcohol is also a potential risk factor in human immunodeficiency virus-1 (HIV-1) infection, this study examines the effect of HIV-1 glycoprotein 120 (gp120)-induced ROS release by isolated Kupffer cells. After 16 weeks of ethanol feeding, Kupffer cells from male Sprague-Dawley rats were isolated and assayed for HIV-1 gp120-induced superoxide release. Fluorescein iso-thiocyanate (FITC)-HIV-I gp120 binding, NADPH oxidase, and protein kinase C activity in Kupffer cells were measured. Results show that HIV-1 gp120 induced the release of superoxide anion in a dose-dependent manner in normal rats. Mannosylated-bovine serum albumin inhibited FITC-HIV-1 gp120-mediated superoxide release in normal Kupffer cells by 85%. Moreover, 83 ± 6% of Kupffer cells were FITC-HIV 1 gp120-positive, whereas <30% were CD4-positive. In alcohol-fed rats, HIV-1 gp120-induced ROS release was reduced by 70% and FITC-HIV-1 gp120 binding (in terms of fluorescence intensity per 10⁶ Kupffer cells) by 44% in Kupffer cells, without any change in percent positive cells for this ligand. Concomitantly, HIV-1 gp120-induced translocation of NADPH oxidase to the plasma membranes of Kupffer cells in alcohol-fed rats was suppressed by 60%. In contrast, alcohol consumption significantly increased total protein kinase C activity and phorbol ester-induced superoxide release by Kupffer cells. These studies demonstrate that Kupffer cells are likely targets of HIV-1 whose binding sites on macrophages could also include mannose-specific receptors. These observations further suggest that suppression of HIV-1 gp120-mediated ROS production in chronic alcoholics is due to altered cell surface receptor expression for gp120, and defective postreceptor signaling mechanisms, which in turn could lead to attenuated microbicidal activity of hepatic macrophages.     

The Production of Tumor Necrosis Factor-α by Macrophages in Rats With Acute Alcohol Loading

Background: It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease. Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol. To study the pathophysiological roles of macrophages in alcoholic liver diseases, we examined the production of TNF-α by rat Kupffer cells, splenic macrophages, and alveolar macrophages with acute alcohol loading in the presence or absence of LBP.
Methods: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from male Wistar rats given 5 mg/g body weight of ethanol intraperitoneally after an hour. The production of TNF-α by these cells incubated with endotoxin 100 ng/ml in the presence or absence of LBP (1% rat serum) was determined.
Results: Acute alcohol loading did not affect the production of TNF-α by Kupffer cells. With acute alcohol loading, splenic macrophages tended to produce more TNF-α. Alveolar macrophages produced more TNF-α than Kupffer cells, and although the production of TNF-α by alveolar macrophages tended to be suppressed by acute alcohol loading, the production of TNF-α by alveolar macrophages still remained high in the presence of rat serum.
Conclusions: Splenic macrophages and alveolar macrophages may be related to excessive production of TNF-α in acute alcoholics with endotoxemia.     

Chronic alcohol intoxication induces hepatic injury through enhanced macrophage inflammatory protein-2 production and intercellular adhesion molecule-1 expression in the liver

This study tested the hypothesis that prolonged consumption of alcohol directly or indirectly, through endotoxin influx in the circulation, stimulates the Kupffer cells to produce macrophage inflammatory protein-2 (MIP₂) and up-regulates the expression of adhesion molecules, i.e., CD18 on PMNs and its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on hepatic cells. As a result, enhanced sequestration and cell-cell interaction among these cell types may occur in the liver, which in turn could result in altered hepatic function and hepatotoxicity. This hypothesis was tested in alcohol-fed, specific pathogen-free, male Sprague-Dawley rats. After 16 weeks of feeding, endotoxin (0.2 +/- 0.043 EU/mL) and MIP₂ (625 +/- 100 pg/mL) were detected in the sera of alcoholic rats but not in the pair-fed rats. Concomitantly, serum aspartate transaminase (AST) activity was significantly increased. Small lipid deposition and inflammatory-like changes in the liver were also observed. Isolated Kupffer cells from alcohol-fed rats released large amount of MIP₂ (> 600 pg/10⁶ Kupffer cells/24 hr) in vitro compared with Kupffer cells from pair-fed rats (< 150 pg/10⁶ Kupffer cells/24 hr). At the same time, the expression of CD18 and ICAM-1 on polymorphonuclear neutrophils (PMNs) and hepatic cells was increased more than twofold. Monoclonal antibody 1F12, an anti-CD18 antibody, attenuated hepatic injury in vivo, and in PMN-hepatocyte coculture in vitro in the alcohol-fed group. Another factor that could contribute to hepatic injury was MIP₂, which was cytotoxic to alcoholic hepatocytes in vitro. This was reversed by cycloheximide, thus suggesting the indirect hepatotoxic effect of MIP₂. In addition, isolated PMNs and Kupffer cells from alcohol-fed rats released large amounts of superoxide, which may also play a role in hepatic injury. These results demonstrate that MIP₂ and adhesion molecules may contribute, at least in part, in the initiation of hepatic injury during alcohol intoxication.(Hepatology 1997 Feb;25(2):335-42)     

Chemokines and the pathophysiology of neuropathic pain

Chemokines and chemokine receptors are widely expressed by cells of the immune and nervous systems. This review focuses on our current knowledge concerning the role of chemokines in the pathophysiology of chronic pain syndromes. Injury- or disease-induced changes in the expression of diverse chemokines and their receptors have been demonstrated in the neural and nonneural elements of pain pathways. Under these circumstances, chemokines have been shown to modulate the electrical activity of neurons by multiple regulatory pathways including increases in neurotransmitter release through Ca-dependent mechanisms and transactivation of transient receptor channels. Either of these mechanisms alone, or in combination, may contribute to sustained excitability of primary afferent and secondary neurons within spinal pain pathways. Another manner in which chemokines may influence sustained neuronal excitability may be their ability to function as excitatory neurotransmitters within the peripheral and central nervous system. As is the case for traditional neurotransmitters, injury-induced up-regulated chemokines are found within synaptic vesicles. Chemokines released after depolarization of the cell membrane can then act on other chemokine receptor-bearing neurons, glia, or immune cells. Because up-regulation of chemokines and their receptors may be one of the mechanisms that directly or indirectly contribute to the development and maintenance of chronic pain, these molecules may then represent novel targets for therapeutic intervention in chronic pain states.     

Promoter polymorphism of the CD14 endotoxin receptor gene as a risk factor for alcoholic liver disease

Twin concordance studies indicate that genetic factors influence the individual susceptibility for alcoholic liver disease (ALD). Both clinical and experimental data suggest that Kupffer cell activation by gut-derived endotoxins and other bacterial products is an important pathogenic factor. Activated Kupffer cells release proinflammatory cytokines, a process that is regulated by the CD14 endotoxin receptor (CD14). Recently, a C-->T (-159) polymorphism in the promoter region of the CD14 gene was detected and found to confer increased CD14 expression. In the present study, the association of CD14 promoter polymorphism with different forms of ALD was examined in 3 separate autopsy series. Among 442 men with valid alcohol-consumption data, 381 men had been moderate or heavy alcohol consumers. The allele frequency of the CD14 promoter genotype, determined by a modified cycle minisequencing technique, was 0.34 (CC), 0.51 (CT), and 0.16 (TT). The T allele was found to be associated with advanced ALD, i.e., with alcoholic hepatitis (odds ratio [OR]: 2.48; P = .018), and especially with cirrhosis (OR: 3.45; P = .004), but not with fatty liver, periportal fibrosis, or bridging fibrosis. The overall age-adjusted risk for cirrhosis was 3.08 (P = .01) for the carriers of the CT genotype, and 4.17 (P = .005) for the homozygous TT genotype. These results suggest that in the relatively isolated Finnish population, the T allele confers increased risk of alcoholic liver damage. In particular, TT homozygotes are at a high risk to develop cirrhosis.     

An essential role for monocyte chemoattractant protein-1 in alcoholic liver injury: regulation of proinflammatory cytokines and hepatic steatosis in mice

The importance of chemokines in alcoholic liver injury has been implicated. The role of the chemokine, monocyte chemoattractant protein-1 (MCP-1), elevated in patients with alcoholic liver disease is not yet understood. Here, we evaluated the pathophysiological significance of MCP-1 and its receptor, chemokine (C-C motif) receptor 2 (CCR2), in alcoholic liver injury. The Leiber-DeCarli diet containing alcohol or isocaloric control diets were fed to wild-type (WT) and MCP-1-deficient knockout (KO) mice for 6 weeks. In vivo and in vitro assays were performed to study the role of MCP-1 in alcoholic liver injury. MCP-1 was increased in Kupffer cells (KCs) as well as hepatocytes of alcohol-fed mice. Alcohol feeding increased serum alanine aminotransferase in WT and CCR2KO, but not MCP-1KO, mice. Alcohol-induced liver steatosis and triglyceride were attenuated in alcohol-fed MCP-1KO, but high in CCR2KO mice, compared to WT, whereas serum endotoxin was high in alcohol-fed WT and MCP-1KO mice. Expression of liver proinflammatory cytokines tumor necrosis factor alpha, interleukin (IL)-1β, IL-6, KC/IL-8, intercellular adhesion molecule 1, and cluster of differentiation 68 was induced in alcohol-fed WT, but inhibited in MCP-1KO, mice independent of nuclear factor kappa light-chain enhancer of activated B cell activation in KCs. Oxidative stress, but not cytochrome P450 2E1, was prevented in chronic alcohol-fed MCP-1KO mice, compared to WT. Increased expression of peroxisome proliferator-activated receptor (PPAR)α and PPARγ was accompanied by nuclear translocation, DNA binding, and induction of fatty acid metabolism genes acyl coenzyme A oxidase and carnitine palmitoyltransferase 1A in livers of alcohol-fed MCP-1KO mice, compared to WT controls. In vitro assays uncovered an inhibitory effect of recombinant MCP-1 on PPARα messenger RNA and peroxisome proliferator response element binding in hepatocytes independent of CCR2. Conclusion: Deficiency of MCP-1 protects mice against alcoholic liver injury, independent of CCR2, by inhibition of proinflammatory cytokines and induction of genes related to fatty acid oxidation, linking chemokines to hepatic lipid metabolism.     

Alcohol consumption and liver cirrhosis mortality with and without mention of alcohol--the case of Canada

Aims To analyse post‐war variations in per capita alcohol consumption in relation to gender‐specific liver cirrhosis mortality in Canadian provinces and to assess the extent to which alcohol bears a different relation to cirrhosis deaths with mention of alcohol (alcoholic cirrhosis) compared to cirrhosis deaths without mention of alcohol (non‐alcoholic cirrhosis). Data and method Annual liver cirrhosis mortality rates by 5‐year age groups were converted into gender‐specific and age‐adjusted mortality rates. Outcome measures included total cirrhosis—the conventional measure of liver cirrhosis—alcoholic cirrhosis and non‐alcoholic cirrhosis. Per capita alcohol consumption was measured by alcohol sales and weighted with a 10‐year distributed lag model. A graphical analysis was used to examine the regional relationship and the Box–Jenkins technique for time‐series analysis was used to estimate the temporal relationship. Findings Geographical variations in alcohol consumption corresponded to variations in total liver cirrhosis and particularly alcoholic cirrhosis, whereas non‐alcoholic cirrhosis rates were not associated geographically with alcohol consumption. In general, for all provinces, time‐series analyses revealed positive and statistically significant effects of changes in alcohol consumption on cirrhosis mortality. In Canada at large, a 1‐litre increase in per capita consumption was associated with a 17% increase in male total cirrhosis rates and a 13% increase in female total cirrhosis rates. Alcohol consumption had a stronger impact on alcoholic cirrhosis, which increased by fully 30% per litre increase in alcohol per capita for men and women. Although the effect on the non‐alcoholic cirrhosis rate was weaker (12% for men and 7% for women) it was nevertheless statistically significant and suggests that a large proportion of these deaths may actually be alcohol‐related. Conclusions Some well‐established findings in alcohol research were confirmed by the Canadian experience: per capita alcohol consumption is related closely to death rates from liver cirrhosis and alcohol‐related deaths tend to be under‐reported in mortality statistics.     

Endotoxin clearance and its relation to hepatic and renal disturbances in rats with liver cirrhosis

BACKGROUND/AIMS: Little is known about endotoxin clearance and secretion of cytokines from macrophages in liver cirrhosis. The aims of this study were to investigate the relationship of endotoxin clearance and release of tumor necrosis factor alpha by various macrophages to hepatic and renal disturbances in liver cirrhosis. Methods: Male Sprague-Dawley rats were given 0.04% thioacetamide orally for 6 or 12 months. The organ distribution of infused [3H]-endotoxin (10 microg/kg b.w.) was analyzed at 30 min or at 24 h. Uptake of [3H]-endotoxin and secretion of tumor necrosis factor alpha by Kupffer cells, splenic macrophages and peripheral blood monocytes (1 x 10(4) cells/ml) from cirrhotic and control rats were determined. RESULTS: In cirrhotic rats, more endotoxin was left in the body and more endotoxin accumulated in the spleen and kidney, and thus was related to elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine and tumor necrosis factor alpha. Endotoxin uptake and tumor necrosis factor alpha release by the Kupffer cells were decreased and those by the splenic macrophages and peripheral blood monocytes were increased in cirrhotic rats. CONCLUSIONS: In liver cirrhosis, impaired clearance of endotoxin together with increased secretion of tumor necrosis factor alpha by extrahepatic macrophages may play an important role in the progression of hepatic and renal disturbances.     

Hospital contacts with alcohol problems prior to liver cirrhosis or pancreatitis diagnosis

AIM To evaluate prior hospital contacts with alcohol problems in patients with alcoholic liver cirrhosis and pancreatitis. METHODS This was a register-based study of all patients diagnosed with alcoholic liver cirrhosis or pancreatitis during 2008-2012 in Denmark. Hospital contacts with alcohol problems(intoxication, harmful use, or dependence) in the 10-year period preceding the diagnosis of alcoholic liver cirrhosis and pancreatitis were identified.RESULTS In the 10 years prior to diagnosis, 40% of the 7719 alcoholic liver cirrhosis patients and 40% of the 1811 alcoholic pancreatitis patients had at least one prior hospital contact with alcohol problems. Every sixth patient(15%-16%) had more than five contacts. A similar pattern of prior hospital contacts was observed for alcoholic liver cirrhosis and pancreatitis. Around 30% were diagnosed with alcohol dependence and 10% with less severe alcohol diagnoses. For the majority, admission to somatic wards was the most common type of hospital care with alcohol problems. Most had their first contact with alcohol problems more than five years prior to diagnosis.CONCLUSION There may be opportunities to reach some of the patients who later develop alcoholic liver cirrhosis or pancreatitis with preventive interventions in the hospital setting.     

Plasma endotoxin concentrations in patients with alcoholic and non-alcoholic liver disease: reevaluation with an improved chromogenic assay

Plasma endotoxin concentration was measured in 85 patients with alcoholic liver disease (alcoholic cirrhosis (n = 64), alcoholic hepatitis without cirrhosis (n = 11), fatty liver (n = 10), and in patients with non-alcoholic cirrhosis (n = 15]. Endotoxin concentration was determined with an improved chromogenic substrate assay, using individual standard curves for each plasma sample. In patients with alcoholic cirrhosis the mean endotoxin concentration was significantly higher than in patients with non-alcoholic cirrhosis (p less than 0.05). In addition, distinctly higher endotoxin concentrations (greater than 20 pg/ml) were more frequently observed in patients with alcoholic cirrhosis than in non-alcoholic cirrhosis (34.4 vs. 14.3%, p less than 0.05). Mean endotoxin concentration was not significantly higher in cirrhotics with ascites or esophageal varices as compared with the subgroup without ascites or esophageal varices. The endotoxin concentration did not correlate with serum bilirubin, prothrombin concentration or serum enzyme activities. In patients with alcoholic liver disease, however, endotoxin concentration revealed a negative correlation (p less than 0.05) with the concentration of high density lipoprotein cholesterol. On admission endotoxin concentrations in alcoholics with fatty liver were similarly elevated as observed in alcoholic cirrhosis. In six out of 12 patients with fatty liver or alcoholic hepatitis, in whom a second sample of plasma was investigated after 6 to 8 days, endotoxemia was no longer detectable; in the remaining patients, the endotoxin concentration decreased markedly. The results indicate that, irrespective of the stage of liver disease, alcohol abuse favours the development of endotoxemia. They support the hypothesis that gut-derived endotoxins might play a role in the initiation and aggravation of alcohol-induced liver disease.     

Endotoxemia and its compensatory mechanisms in experimental liver cirrhosis

This study was performed in order to elucidate the mechanisms of systemic endotoxemia in liver cirrhosis. For this purpose, the method of measuring biliary endotoxin was established. Endotoxin levels between in the bile and in the plasma in cirrhosis were compared using a modified method of chromogenic quantitative endotoxin assay in an attempt to clarify liver function of clearing portal endotoxin originated from the gut. And functional activities of the reticuloendothelial system were also examined by radioassay with 59Fe-labelled iron-chondroitin sulfate colloid and by enzymohistochemistry of acid phosphatase. Both the plasma and biliary endotoxin levels in liver cirrhosis were significantly higher, compared to those in control. The functional activities of the reticuloendothelial system, particularly of the Kupffer cells, were decreased in liver cirrhosis. These data provide evidence that in liver cirrhosis systemic endotoxemia is mainly due to a decrease of functional activities of Kupffer cells. On the other hand, the excretion of endotoxin into the bile is increased, compensating the decreased functional activities of Kupffer cells. This implies that the uptake of endotoxin and its excretion into the bile by hepatocytes might be one of the mechanisms of clearing excess endotoxin in the plasma in liver cirrhosis. This study also draws the importance of measurements of endotoxin levels in the bile in liver diseases.     

内毒素受体、内毒素血症与肝硬化

内毒素在肝脏内可激活Kupffer细胞,合成和释放多种细胞因子和炎症介质,使肝细胞受损,其作用机制主要是通过Kupffer细胞上的内毒素受体启动宿主免疫反应和效应功能。肝硬化患者因多种原因引起肠道菌群生长过度和菌群易位,可导致内毒素血症;反之,内毒素本身又可加重肝脏损伤。因此,通过改变肝硬化患者的肠道微生态,调节肠道菌群,可减少肠道内毒素的产生,防止内毒素血症的发生,减轻肝脏损伤,延缓肝硬化的进程。