Isolated Kupffer Cells Research Articles

Febrile-range temperature modifies early systemic tumor necrosis factor alpha expression in mice challenged with bacterial endotoxin.

Fever improves survival in acute infections, but the effects of increased core temperature on host defenses are poorly understood. Tumor necrosis factor alpha (TNF-alpha) is an early activator of host defenses and a major endogenous pyrogen. TNF-alpha expression is essential for survival in bacterial infections but, if disregulated, can cause tissue injury. In this study, we show that passively increasing core temperature in mice from the basal (36.5 to 37.5 degrees C) to the febrile (39.5 to 40 degrees C) range modifies systemic TNF-alpha expression in response to bacterial endotoxin (lipopolysaccharide). The early TNF-alpha secretion rate is enhanced, but the duration of maximal TNF-alpha production is shortened. We identified Kupffer cells as the predominant source of the excess TNF-alpha production in the warmer animals. The enhanced early TNF-alpha production observed at the higher temperature in vivo could not be demonstrated in isolated Kupffer cells or in precision-cut liver slices in vitro, indicating the participation of indirect pathways. Therefore, expression of the endogenous pyrogen TNF-alpha is regulated by increments in core temperature during fever, generating an enhanced early, self-limited TNF-alpha pulse.

Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cells.

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine. Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.

Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells.

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.

The metabolic effects of estriol in female rat liver.

The effects of estriol on oxygen uptake, glucose release, lactate and pyruvate production, beta-hydroxybutyrate and acetoacetate production in perfused rat liver as well as, carbon uptake in rat liver and intracellular calcium in isolated Kupffer cells were investigated. Basal oxygen consumption of perfused liver increased significantly in estriol or ethanol-treated rats. But these increased effects were blocked by gadolinium chloride pretreatment. In a metabolic study, pretreatment with estriol resulted in a decrease in glucose production and in glycolysis while an increase in ketogenesis. A more oxidized redox state of the mitochondria was indicated by increased ratios of perfusate [lactate]/[pyruvate] and decreased ratios of perfusate [beta-hydroxybutyrate]/[acetoacetate]. Carbon uptake of Kupffer-cell increased significantly in estriol-treated rats. But these increased uptake were not shown in rats pre-treated by gadolinium chloride blocking phagocytosis. In isolated Kupffer cells from estriol-treated rats, intracellular calcium was more significantly increased after addition of lipopolysaccharide (LPS) than in controls. These findings suggest that the metabolic effects of estriol (two mg per 100 mg body wt) can be summarized to be highly toxic in rat liver, and these findings suggest that oral administration of estrogens may induce hepatic dysfunctions and play a role in the development of liver disease.

Role of Kupffer cells in the ethanol-induced oxidative stress in the liver.

These studies test the hypothesis that acute and chronic alcohol intoxication stimulate the release of oxygen-derived radicals in the liver. Male Sprague-Dawley rats received an intravenous bolus followed by continuous infusion of ethanol to maintain blood alcohol level at about 175 mg/dl for 0-18 hr. They were then allowed to recover from this "alcohol binge" and the release of free radicals during the recovery phase was monitored. In the chronic alcohol intoxication model, rats were fed with 40% ethanol in agar blocks for 16 weeks. Acute ethanol intoxication induced two phases of hepatic superoxide release. The first phase peaked during the first 3 hr of alcohol intoxication, while the second phase reached its maximum at 6 hr of recovery following a 12 hr binge. The recovery period was also associated with elevated serum transaminase activity. Kupffer cells were largely responsible for hepatic superoxide release during the first phase, while both Kupffer and hepatic sinusoidal endothelial cells contributed to the second phase of free radical formation. Acute ethanol intoxication did not induce endotoxemia. During chronic alcohol intoxication, increased levels of serum endotoxin, TNF, IL-1, and transaminase were observed and hepatic superoxide anion release was present. Superoxide release by isolated Kupffer cells, blood and hepatic PMNs of alcoholic rats was also significantly enhanced in the chronic alcoholic rats. These data indicate that acute alcohol intoxication may directly stimulate the release of reactive oxygen intermediates, whereas chronic alcohol may elicit free radical generation through enhanced endotoxin influx and cytokine release. These studies further demonstrate that free radicals produced by hepatic non-parenchymal cells are likely to play an important role in the pathogenesis of hepatic injury in susceptible individuals with alcohol-related liver disorders.

Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells

Background/ Aims: Heme oxygenase catalyzes the rate-limiting enzymatic step of heme degradation. The inducible isoform of heme oxygenase, heme oxygenase-1, is expressed at a low level in most tissues and is upregulated by its substrate heme and various stress stimuli. Kupffer cells which represent the largest population of the body's tissue macrophages serve physiological functions in the defense against various pathogens such as lipopolysaccharide. The goal of the present study was to investigate the heme oxygenase-1 gene expression in Kupffer cells of rat liver and in isolated Kupffer cell cultures during treatment with lipopolysaccharide. Methods: Cryostat sections of normal rat liver were investigated by immunofluorescence double-staining using specific antibodies for rat heme oxygenase-1 and ED2. Isolation and cell culture of Kupffer cells and primary hepatocytes from rat liver, as well as Northern and Western blot analysis, were performed with standard protocols. Results: Heme oxygenase-1 protein was highly expressed in large sinusoidal cells of normal rat liver, which were identified as Kupffer cells by staining with the macrophage surface marker ED2. By contrast, no expression of heme oxygenase-1 was detected in liver parenchymal cells. High expression of heme oxygenase-1 was also found in isolated Kupffer cells in culture by immunocytochemical staining as well as by Western and Northern blot analysis. After treatment of Kupffer cells cultures with lipopolysaccharide, heme oxygenase-1 was upregulated on the protein and mRNA level in a time- and dose-dependent manner. This increase in heme oxygenase-1 expression by lipopolysaccharide was prevented by the nitric oxide inhibitor N G-monomethyl-L-arginine which was reversed by an excess of L-arginine. Various nitric oxide donors up-regulated heme oxygenase-1 mRNA expression in Kupffer cells. Conclusions: The lipopolysaccharide-dependent upregulation of the heme oxygenase-1 gene which is highly expressed in Kupffer cells is mediated by a nitric oxide-dependent mechanism.

Role of endotoxin in the hypermetabolic state after acute ethanol exposure.

This study investigated the role of endotoxin in the hypermetabolic state or swift increase in alcohol metabolism (SIAM) due to acute ethanol exposure. Female Sprague-Dawley rats (100-120 g) were given ethanol (5 g/kg) by gavage. Endotoxin measured in plasma from portal blood was not detectable in saline-treated controls; however, 90 min after ethanol, endotoxin was increased to 85 +/- 14 pg/ml, and endotoxin clearance was diminished by approximately 50%. Oxygen uptake in perfused livers was increased 48% by ethanol, and production of PGE2 by isolated Kupffer cells was increased similarly. These effects were blunted by elimination of gram-negative bacteria and endotoxin with antibiotics before ethanol administration. To reproduce ethanol-induced endotoxemia, endotoxin was infused via the mesenteric vein at a rate of 2 ng. kg-1. h-1. Endotoxin mimicked the effect of ethanol on oxygen uptake. The specific Kupffer cell toxicant GdCl3 completely prevented increases in oxygen uptake due to endotoxin. These findings demonstrate that endotoxin plays a pivotal role in SIAM, most likely by stimulating eicosanoid release from Kupffer cells.

Upregulation of Kupffer cell β-adrenoceptors and cAMP levels during the late stage of sepsis

Although a burst of immunoresponsiveness may occur during the early stage of sepsis, late sepsis is characterized by severe immunodepression. In addition, although studies have shown that stimulation of macrophage β-adrenoceptors results in an increase in cAMP and an associated reduction in macrophage phagocytic activity, it remains unknown whether Kupffer cell β-adrenoceptor characteristics and cAMP levels are altered during polymicrobial sepsis. To study this, Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h (i.e., the early stage of sepsis) or 20 h (late sepsis) after CLP or sham operation, the liver was perfused with collagenase solution and Kupffer cells were isolated. β-Adrenoceptor characteristics of the isolated Kupffer cells were determined using [ 125I]iodopindolol, and basal levels of cAMP were measured by radioimmunoassay. The results indicate that while maximum binding capacity ( B max) of Kupffer cell β-adrenoceptors was not altered at 5 h, it increased significantly at 20 h after CLP. Similarly, basal levels of cAMP in Kupffer cells did not change at 5 h but increased markedly at 20 h after the onset of sepsis. In contrast, the dissociation constant ( K d, 1/affinity) of Kupffer cell β-adrenoceptors was not significantly affected by sepsis at both 5 h and 20 h after CLP. Thus, upregulation of β-adrenoceptors and increase in cAMP levels in Kupffer cells occur during the late stage of polymicrobial sepsis, and this may contribute to the depression of macrophage phagocytic function under such conditions.

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 isothiocyanate (FITC)-HIV-1 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 10[6] 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.

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

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

Relationship between cellular shape and receptor-mediated endocytosis: an ultrastructural and morphometric study in rat Kupffer cells.

The relationship between cellular shape (i.e., size, volume, presence of microvilli, pseudopodia, flat or round shape) and receptor-mediated endocytotic activities (i.e., binding and internalization) was investigated using intact liver as well as freshly isolated Kupffer cells and Kupffer cells in culture. The morphological features of Kupffer cells were reconstructed by three-dimensional analysis from in situ experiments and by densitometric analysis of cells in suspension and in culture. By morphometry at the ultrastructural level, different cellular shapes were compared with the respective capacities for binding and internalization of glycoproteins with terminal galactosyl residues. The number of asialoglycoprotein-gold particles bound to the cell surface or internalized into endosomes was calculated. Our data show that differences in cellular shape, mainly related to the reduction of projection and microvilli and to the roundness of cell surface, accompany modulation of galactose-specific receptors in rat Kupffer cells, thus supporting the hypothesis that cell morphology is affected by endocytic activities. In fact, the progressive reduction in microvilli projections and cellular roundness is paralleled by the progressive decrement of both binding and uptake capacity from in situ, freshly isolated and cultured Kupffer cells.

Pharmacokinetic analysis of scavenger receptor-mediated uptake of mucopolysaccharides in various cells

Although the scavenger receptor-mediated uptake has been qualitatively investigated in the research fields of biochemistry and pathology, pharmacokinetic characteristics of the scavenger receptors are poorly understood. In this review, we summarized basic findings on scavenger receptors reported in available literature, and introduced our recent studies on the quantitative characteristics of the scavenger receptor-mediated uptake. High molecular weight fractionated [3H]heparin (HMWFH, 16,000-24,000 Da), one of the model mucopolysaccharides, was investigated to elucidate its uptake mechanism into isolated rat Kupffer cells, isolated peritoneal macrophages and liver parenchymal cells in primary culture. The equilibrium bindings of HMWFH to isolated Kupffer cells and peritoneal macrophages were concentration-dependent with the respective dissociation constants (Kd) of 5.7 and 6.0 nM and with the respective maximum binding capacities (Bmax) of 1.5 and 1.9 pmol/10(6) cells. Several ligands of scavenger receptors inhibited the binding of HMWFH to macrophages, suggesting the involvement of scavenger receptors in the uptake of HMWFH by these macrophages. It was also suggested that the scavenger receptor-mediated uptake is different from the receptor-mediated endocytosis of polypeptides and phagocytosis, based on the evidence of the now inhibitory effects of an inhibitor of receptor-mediated endocytosis of polypeptides(phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) on the internalization. The involvement of scavenger-like receptors was also suggested in the uptake of HMWFH by liver parenchymal cells in primary culture by demonstrating inhibitory effects of ligands for scavenger receptors. The internalization into liver parenchymal cells by scavenger-like receptors was not affected by an inhibitor of receptor-mediated endocytosis of polypeptides and phagocytosis inhibitors, similarly to the results in the macrophage scavenger receptors. The Kd of 53.5 nM and Bmax of 32.8 pmol/10(6) cells in parenchymal cells were both in the order of magnitude larger than those in isolated Kupffer cells, suggesting the binding of HMWFH to scavenger-like receptors in parenchymal cells with lower affinity and higher capacity. On the other hand, an apparent internalization rate constant (kint, app) of 0.0056 min-1 was comparable with that in Kupffer cells (0.0118 min-1). Thus, we demonstrated the involvement of scavenger receptors in the uptake of HMWFH by rat Kupffer cells, peritoneal macrophages and liver parenchymal cells, and succeeded in characterizing the uptake kinetically. These findings should provide useful information for not only establishing the rational clinical use of mucopolysaccharides but also developing new drugs such as antiatherosclerotic agents and peptides delivered to cells with scavenger receptors.

Endothelin-Stimulated Nitric Oxide Production in the Isolated Kupffer Cell

Endothelin (ET) is a potent peptide mediator exhibiting a wide variety of effects in both the parenchymal and nonparenchymal hepatic cells. In the Kupffer cell, ET activates several transmembrane signaling pathways to generate numerous second messengers including the phospholipase C-generated products inositol-1,4,5-trisphosphate and diacylglycerol and the cyclooxygenase product prostaglandin E2via specific ETB-type receptors. In addition to these findings, we have now demonstrated that endothelin stimulates the production of nitric oxide (NO) in the Kupffer cell in a time- and concentration-dependent manner. Western blot analysis indicates that ET-stimulated NO production occurs though activation of the inducible form of the nitric oxide synthase enzyme. These findings have important implications as the stimulation of NO production by ET may be part of the physiological response to inflammation or infection. Elevated levels of ET and NO have been found to be associated with numerous hepatic pathophysiological conditions that may contribute to derangements in the vascular system seen in these conditions.

Cell-specific Regulation of Expression of Plasma-type Platelet-activating Factor Acetylhydrolase in the Liver

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that causes hypotension, increases vascular permeability, and has been implicated in anaphylaxis, septic shock and several other inflammatory responses. PAF is hydrolyzed and inactivated by the enzyme PAF-acetylhydrolase. In the intact rat, a mesenteric vein infusion of lipopolysaccharide (LPS) served as an acute, liver-focused model of endotoxemia. Plasma PAF-acetylhydrolase activity increased 2-fold by 24 h following LPS administration. Ribonuclease protection experiments demonstrated very low levels of plasma-type PAF-acetylhydrolase mRNA transcripts in the livers of saline-infused rats; however, 24 h following LPS exposure, a 20-fold induction of PAF-acetylhydrolase mRNA was detected. In cells isolated from endotoxin-exposed rat livers, Northern blot analyses demonstrated that Kupffer cells but not hepatocytes or endothelial cells were responsible for the increased PAF-acetylhydrolase mRNA levels. In Kupffer cells, plasma-type PAF-acetylhydrolase mRNA was induced by 12 h, peaked at 24 h, and remained substantially elevated at 48 h. Induction of neutropenia prior to LPS administration had no effect on the increase in PAF-acetylhydrolase mRNA seen at 24 h. Although freshly isolated Kupffer cells contain barely detectable levels of plasma-type PAF-acetylhydrolase mRNA, when Kupffer cells were established in culture, PAF-acetylhydrolase expression became constitutively activated concomitant with cell adherence to the culture plates. Alterations in plasma-type PAF-acetylhydrolase expression may constitute an important mechanism for elevating plasma PAF-acetylhydrolase levels and an important component in minimizing PAF-mediated pathophysiology in livers exposed to endotoxemia.

Involvement of nitric oxide synthesis in hepatic perturbations induced in rats by a necrogenic dose of thioacetamide.

1. The biological actions of nitric oxide (NO), a highly diffusible and short-lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg-1 body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration. 2. Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P < 0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P < 0.001), 13 (P < 0.001) and 13 (P < 0.001) times the initial values, respectively. 3. In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%; P < 0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h. 4. As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P < 0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide-treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P < 0.001) that of the control. 5. These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post-necrotic hepatocellular regeneration following thioacetamide administration.

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)

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/10(6) Kupffer cells/24 hr) in vitro compared with Kupffer cells from pair-fed rats (< 150 pg/10(6) 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.

The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91% and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= delta 125-rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-delta 125-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Liposome-encapsulated dichloromethylene diphosphonate induces macrophage apoptosis in vivo and in vitro.

Dichloromethylene diphosphonate (MDPCl2) encapsulated in multilamellar liposomes was selectively incorporated by macrophages, immediately transferred to lysosomes, then released from liposomes into lysosomes by enzymatic digestion of the liposomal lipid layers. From 4 h after ingesting liposome-encapsulated MDPCL2 murine macrophages in vivo and in vitro acquired the ultrastructural features of apoptosis, such as condensed nuclear chromatic, nuclear fragmentation, cell shrinkage, and blebbing of the plasma membrane. Murine peritoneal macrophages and isolated rat Kupffer cells incubated in the medium containing liposome-encapsulated MDPCl2 increased DNA fragmentation in a dose-dependent manner. Electrophoretic analysis of extracted DNA from the isolated Kupffer cells showed DNA fragmentation. Another diphosphonate, Alendronate (4-amino-1-hydroxy-butylidene-1,1-diphosphonate) had less potent macrophage cytotoxicity. However, MDPCl2, Alendronate, and gadolinium chloride in solution were not cytotoxic to macrophages. These results implied that the intralysosomal accumulation of MDPCl2 generates signals to induce macrophage apoptosis.

Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells.

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae. Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed. The liver uptakes ranged between 65.9% for B. burgdorferi IRS and 40.5% for B. turicatae. Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium. The in vitro uptake of B. burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B. burgdorferi antibody. The reculturing of B. burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative. The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors.