The effects of in vivo administration of endotoxin on the functions and interaction of hepatocytes and Kupffer cells

It was the purpose of this study to determine the effects of the in vivo administration of endotoxin on certain in vitro hepatocyte and Kupffer cell functions. An Alzet osmotic pump that contained endotoxin (LPS, 2.5 mg/100g) was implanted into the peritoneal cavity of 300g guinea pigs and delivered the endotoxin over a period of four days. In vivo administration of LPS did not cause a change in the in vitro release of albumin by isolated hepatocytes. However, when hepatocytes were co-cultured with Kupffer cells there was a significant decrease in albumin release for both control and LPS-treated animals. There was no difference between control and LPS-treated animals in the release of C3 by hepatocytes. However, there was a significant increase over the control group in C3 release by Kupffer cells from LPS-treated animals. When hepatocytes and Kupffer cells were cultured together, their release of C3 was not additive. Kupffer cells from LPS-treated animals released significantly greater amounts of PGE2 than control animals when stimulated in vitro with LPS. Thus, these Kupffer cells appeared to be primed to respond to an in vitro challenge of LPS. Kupffer cells from LPS-treated animals had significantly depressed antibody dependent cellular cytotoxicity (ADCC). This endotoxin model is useful for determining the in vivo effects of endotoxin on cellular function and gives some indirect evidence for the detrimental effects of LPS on the immune system and host defense.     

Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

Ultrastructural changes in the histohematic barrier of the liver in the early period after endotoxin administration

Investigation of the changes of the mouse liver cells revealed early reaction of endothelial sinusoid cells and Kupffer cells after endotoxin application. During first 60 min. after endotoxin injection there were certain bulge of nuclear zone, changes of nuclear shape, appearance of microfilaments in the cytoplasm of endothelial cells. An increase in the number of the pores and fenestrae grouped in sieve plates was discovered in the endothelial cells. Their luminal surface formed the bubbles. Such changes of the endothelial cells can be described as their activation. Reaction of the endothelial cells of hepatic sinusoids and activation of the Kupffer cells were revealed at the same time.     

Dietary glycine blunts lung inflammatory cell influx following acute endotoxin

Mortality associated with endotoxin shock is likely mediated by Kupffer cells, alveolar macrophages, and circulating neutrophils. Acute dietary glycine prevents mortality and blunts increases in serum tumor necrosis factor-alpha (TNF-alpha) following endotoxin in rats. Furthermore, acute glycine blunts activation of Kupffer cells, alveolar macrophages, and neutrophils by activating a glycine-gated chloride channel. However, in neuronal tissue, glycine rapidly downregulates chloride channel function. Therefore, the long-term effects of a glycine-containing diet on survival following endotoxin shock were investigated. Dietary glycine for 4 wk improved survival after endotoxin but did not improve liver pathology, decrease serum alanine transaminase, or effect TNF-alpha levels compared with animals fed control diet. Interestingly, dietary glycine largely prevented inflammation and injury in the lung following endotoxin. Surprisingly, Kupffer cells from animals fed glycine for 4 wk were no longer inactivated by glycine in vitro; however, isolated alveolar macrophages and neutrophils from the same animals were sensitive to glycine. These data are consistent with the hypothesis that glycine downregulates chloride channels on Kupffer cells but not on alveolar macrophages or neutrophils. Importantly, glycine diet for 4 wk protected against lung inflammation due to endotoxin. Chronic glycine improves survival by unknown mechanisms, but reduction of lung inflammation is likely involved.     

Endotoxin-refractory liver macrophages secrete tumor necrosis factor-alpha upon viral infection

Rat liver macrophages (Kupffer cells) secrete tumor necrosis factor-alpha (cachectin) after exposure to Newcastle disease virus or bacterial endotoxin. Macrophages treated with endotoxin become refractory and fail to release tumor necrosis factor-alpha to a secondary challenge with endotoxin. The acquisition of the refractory state is dose-dependent, requires the continuous presence of endotoxin for a minimum of 8 h, is transient, and reversible. Endotoxin, however, renders Kupffer cells unresponsive only to itself. When endotoxin-refractory macrophages are activated by Newcastle disease virus, they still secrete tumor necrosis factor-alpha in amounts expected with this stimulus. Immunoprecipitation studies show that the precursor of tumor necrosis factor-alpha is found only in lysates of endotoxin-sensitive, but not in refractory macrophages, thus arguing against a post-translational regulatory process. Whereas prostaglandin E2 inhibits the production of tumor necrosis factor-alpha in response to endotoxin and viruses, it does not appear to mediate the refractory state.     

Structural and functional characteristics of Kupffer cells at different periods of the development of a liver response in mice to injury

The number of Kupffer cells increased up to necroses and processes of liver regeneration following carbon tetrachloride-induced injury. Mouse Kupffer cells migrated to the necrotic centrolobular zones 48 h after CCl4-induced necroses. During regeneration, Kupffer cells migrated to the sinusoids from the necrotic centrolobular zones. Study of the ultrastructure of Kupffer cells indicates activation of their function during all observation periods.     

The in situ activation of cytotoxic properties in murine Kupffer cells by the systemic administration of whole Mycobacterium bovis organisms or muramyl tripeptide

Summary We determined whether the systemic administration of viable Mycobacterium bovis organisms (BCG) or a lipophilic derivative of muramyl tripeptide (MTP-PE) would lead to the activation of antitumor properties in murine Kupffer cells (KC). KC-mediated tumor cytolysis was determined by the release of radiolabeled nuclear breakdown products of target cells. KC harvested from either C57BL/6 or C3H/HEN mice treated with saline exhibited no cytotoxicity against syngeneic B16 melanoma or UV-2237 fibrosarcoma cells. In contrast, KC harvested from BCG or MTP-PE-injected mice were highly cytotoxic against the tumor targets, as measured by an in vitro radiorelease assay. The demonstration that the administration of macrophage activators can generate in situ tumoricidal activity in KC suggests that these cells can be important in the control of hepatic micrometastases.     

Structural changes produced in Kupffer cells in the rat liver by injection of lipopolysaccharide

Summary The fine structure of Kupffer cells has been studied at various times after an intravenous injection of lipopolysaccharide of Salmonella abortus equii. The most prominent effects were: an increase in the number and dimensions of phagocytic vacuoles (often containing ingested LPS and neutrophilic granulocytes); mitochondrial damage, including disintegration of the matrix and cristae; an increase in the amount of dilated, lucent rough endoplasmic reticulum; presence of fat droplets in the cytoplasm. Five days after injection of lipopolysaccharide, the Kupffer cells had resumed their normal ultrastructure.Several minutes after injection of lipopolysaccharide, platelets adhered to the Kupffer and endothelial cells. Between one and six hours, neutrophilic granulocytes accumulated in the liver sinusoids. The resulting obstruction of the hepatic microcirculation most probably affected cellular ultrastructure by ischaemia. At three days, the number of Kupffer cells was doubled, and increased further at later time intervals.     

Ultrastructural study of Kupffer cells in teleost liver under normal and experimental conditions

Summary The Kupffer cells in the liver of the teleost fish, Pimelodus maculatus, are attached by desmosomes to the endothelial cells lining the sinusoids. These provide a strong attachment allowing them to resist the passage of blood. Following perfusion with India ink, both endothelial and Kupffer cells ingest India ink particles by pinocytosis and micropinocytosis. It is suggested that both cell types may represent two different functional states of the same cell.     

A biochemical and immunocytochemical study on the targeting of alglucerase in murine liver

Summary A current hypothesis is that functional glucocerebrosidase needs to be delivered to the lysosomes of tissue macrophages to guarantee successful enzyme therapy for Gaucher's disease. In this study, biochemical and immunohistochemical techniques were applied to identify in mice the localization of intravenously administered alglucerase (human modified placental glucocerebrosidase). Only in liver and spleen was a significant increase of glucocerebrosidase activity observed, with a maximum level at 15 minutes after enzyme infusion. The uptake of enzyme by liver was sufficiently high to allow more detailed studies on the (sub)cellular distribution of human alglucerase. The enzyme in liver is localized both in the endosomallysosomal system of the Kupffer cells and the endothelial cells lining the lumen of the sinusoids. Uptake by both of these types of cell is prevented by mannan. The results suggest that the cellular mechanisms responsible for improvement of Gaucher patients receiving alglucerase treatment is probably more complicated than previously recognized.     

Acute alcohol intoxication and endotoxemia desensitize HIV-1 gp120-induced CC-chemokine production by Kupffer cells

Chemokines are involved in the inhibition of HIV-1 infection and in the pathogenesis of tissue injury in a number of conditions, including endotoxemia and alcoholic liver disease. CC chemotactic peptides (MIP-1alpha, MCP-1 and RANTES) are produced by a wide variety of cell types in response to immunological stimuli, bacterial endotoxin and gp120 from HIV-1 and HIV-2. This work tests the hypothesis that prior exposure to endotoxin and/or ethanol in vivo inhibits the production of CC-chemokines following a secondary challenge with HIV-1 gp120 in vitro. Male Sprague-Dawley rats received in intravenous infusion of ethanol to maintain blood ethanol level at 170 mg/dl for 3 hr. Escherichia coli LPS (1 mg/Kg) was given intravenously 5 min after the ethanol bolus was injected. Control groups received similar volumes of saline. Three hr after LPS treatment, Kupffer cells were obtained and treated with HIV-1 gp120 (5 microg/10(6) cells/24 hr). At the end of the incubation period, cells were obtained for RT-PCR analysis of CC-chemokine mRNA expression. Chemokine release in culture supernatants was measured by ELISA. Results show that in vivo ethanol was associated with downregulation of MIP-1alpha and MCP-1 mRNA expression and protein release in primary cultures of Kupffer cells. However, ethanol alone primed isolated Kupffer cells for enhanced RANTES mRNA and protein release in the presence or absence of HIV-1 gp120. These results demonstrate that acute ethanol intoxication and endotoxemia may selectively act as a desensitizing agent in response to a secondary challenge with bacterial or viral products.     

Derepression of hepatocyte proliferation with changes in the functional state of the reticuloendothelial system

Experiments on rats and mice were performed to study the effects of different substances modifying RES functions on hepatocyte proliferation. It was shown that as early as 24 hours after Kupffer cell (KC) overloading with colloidal iron particles the number of hepatocytes in mitosis increased. The mitotic rate increased by 32 h and decreased between 48 and 72 h following iron injection. Forty-eight h after injection of latex particles the hepatocyte mitotic peak could be identified. Twenty-four and 48 h after zymozan injection DNA synthesis in sinusoidal liver cells correspondingly increased. Hepatocytes in mitosis appeared 5 days later, reaching the peak value after 9 days followed by a decrease 12 days after zymozan injection. The depression of the hepatocyte mitotic rate was also observed 9 days after BCG and 15 days after prodigiozan injection. The data are suggestive of the importance of KC as potential inducers of hepatocyte proliferation.     

The role of Kupffer cell alpha(2)-adrenoceptors in norepinephrine-induced TNF-alpha production

Although previous studies have demonstrated that plasma levels of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) increase during early sepsis, the precise mechanism responsible for its upregulation remains to be elucidated. Since recent studies have shown that the gut is an important source of norepinephrine (NE) release during early sepsis and enterectomy prior to the onset of sepsis attenuates TNF-alpha production, we hypothesized that gut-derived NE plays a major role in upregulating TNF-alpha via the activation of alpha(2)-adrenoceptors on Kupffer cells. To confirm that NE increases TNF-alpha synthesis and release, Kupffer cells were isolated from normal rats and incubated with NE (20 or 50 nM) or another alpha(2)-adrenergic agonist clonidine (50 nM) without addition of Escherichia coli endotoxin. Supernatant levels of TNF-alpha were then measured. In additional animals, intraportal infusion of NE (20 microM) with or without the specific alpha(2)-adrenergic antagonist yohimbine (1 mM) at a rate of 13 microl/min was carried out for 2 h. Plasma and Kupffer cell levels of TNF-alpha were assayed thereafter. Moreover, the effects of NE and yohimbine on TNF-alpha production was further examined using an isolated perfused liver preparation. The results indicate that both NE and clonidine increased TNF-alpha release by approximately 4-7-fold in the isolated cultured Kupffer cells. Similarly, intraportal infusion of NE in vivo or in isolated livers increased TNF-alpha synthesis and release which was inhibited by co-infusion of yohimbine. Furthermore, the increased cellular levels of TNF-alpha in Kupffer cells after in vivo administration of NE was also blocked by yohimbine. These results, taken together, suggest that gut-derived NE upregulates TNF-alpha production in Kupffer cells through an alpha(2)-adrenergic pathway, which appears to be responsible at least in part for the increased levels of circulating TNF-alpha observed during early sepsis as well as other pathophysiologic conditions such as trauma, hemorrhagic shock, or gut ischemia/reperfusion.     

Free radicals, chemokines, and cell injury in HIV-1 and SIV infections and alcoholic hepatitis

Oxidative stress has been observed in HIV-1 infection and alcoholic liver disease. The formation of reactive oxygen species (ROS) contributes to cell injury through apoptosis and/or necrosis and secretion of proinflammatory cytokines and chemokines. The major sources of ROS and chemokines are the Kupffer cells. During chronic ethanol consumption they are primed and activated for enhanced formation of pro-inflammatory factors, probably as a result of ethanol-induced translocation of gut-derived endotoxin into the circulation. Pro-inflammatory factors produced in the liver stimulate neutrophilic and/or lymphocytic infiltration to this organ. The presence of inflammatory cells in the liver may compromise the hepatocytes to injury by releasing cytotoxic factors, i.e., ROS, cytolytic proteases. Kupffer cells also interact with the glycoprotein 120 of SIV and HIV-1, which can induce oxidative stress and chemokine release. CD4+ lymphocytes that are infected with the virus generate intracellular ROS, which in turn leads to apoptosis and cell death. Downregulation of CD4+ cells contributes to immunodeficiency, while enhanced sequestration of inflammatory cells in the liver during chronic ethanol use with or without HIV-1/SIV may result in hepatocyte injury and exacerbation of alcoholic liver disease.     

Endotoxin stimulates glycogenolysis in the liver by means of intercellular communication

Escherichia coli endotoxin (lipopolysaccharide) was shown to increase glycogenolysis in the perfused liver 2-3-fold. In isolated parenchymal liver cells, however, endotoxin did not influence glycogenolysis, whereas stimulation by endotoxin of glycogenolysis in the perfused liver could be blocked by aspirin. This suggests that the effect of endotoxin on liver glycogenolysis is mediated by eicosanoids. The amount of prostaglandin D2 (which is the major prostanoid formed by Kupffer cells) in the liver perfusates was increased 5-fold upon endotoxin addition, with a time course which preceded the increase in glucose output. It is concluded that endotoxin stimulates glycogenolysis in the liver by stimulating prostaglandin D2 release from Kupffer cells, with a subsequent activation of glycogenolysis in parenchymal liver cells. This mechanism of intercellular communication may be designed to provide the carbohydrate source of energy necessary for the effective destruction of invaded microorganisms, by phagocytic cells, including the Kupffer cells.     

Plasmodium yoelii: Influence of immune modulators on the development of the liver stage

Plasmodium sporozoites suppress the respiratory burst and antigen presentation of Kupffer cells, which are regarded as the portal of invasion into hepatocytes. It is not known whether immune modulation of Kupffer cells can affect the liver stage. In the present study, we found that sporozoites inoculated into Wistar rats could be detected in the liver, spleen, and lung; however, most sporozoites were arrested in the liver. Sporozoites were captured by Kupffer cells lined with endothelial cells in the liver sinusoid before hepatocyte invasion. Pretreatment with TLR3 agonist poly(I:C) and TLR2 agonist BCG primarily activated Kupffer cells, inhibiting the sporozoite development into the exoerythrocytic form, whereas Kupffer cell antagonists dexamethasone and cyclophosphamide promoted development of the liver stage. Our data suggests that sporozoite development into its exoerythrocytic form may be associated with Kupffer cell functional status. Immune modulation of Kupffer cells could be a promising strategy to prevent malaria parasite infection.     

Use of the avidin-biotin-peroxidase complex (ABC) method for the localization of rabbit cathepsin B in cells and tissues

Visualization of rabbit cathepsin B was achieved utilizing monospecific sheep antibodies and the avidin-biotin-peroxidase complex (ABC) method. This technique was applied to stain 1) paraffin sections of the liver, 2) fixed fibroblasts from tissue culture, and 3) fixed mesenteries. Cathepsin B was found to be localized within cells of the lining of the liver sinusoids (most probably Kupffer cells), in perinuclear granules of cultured fibroblasts, and within histiocytes of the mesentery. The results demonstrate that the method permits precise and highly sensitive localization of cathepsin B within cells and tissues. Compared to fluorescent staining of cathepsin B, the ABC method has the advantage that routine paraffin sections can be stained, and that all the orthodox histological staining procedures can still be carried out.     

Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by flow cytometry.

BACKGROUND: The triggering of cellular responses during endotoxic shock is initiated for the binding of endotoxin (lipopolysaccharide; LPS) to the cell surface. Kupffer and endothelial liver cells, involved in the removal of endotoxin from blood circulation, show in vitro a rapid response to LPS in the absence of serum. METHODS: A double-labeling fluorescent assay was designed to evaluate the binding properties of Escherichia coli O111:B4 LPS to individual endothelial and Kupffer cells in suspension, where both populations occurred in the same relative proportion as in liver. After immunolabeling of the Kupffer cell population with the monoclonal antibody ED1 conjugated to R. phycoerythrin, the binding characteristics of LPS labeled with fluorescein to both endothelial and Kupffer cells were simultaneously studied by flow cytometry in serum-free conditions. RESULTS: Specific and saturable binding of endotoxin was observed with both populations, showing properties of a receptor-mediated process. The Kupffer cell population showed a faster capacity and a higher affinity for LPS binding. The Hill coefficients indicated positive cooperativity in the LPS interaction with both populations. CONCLUSIONS: Specific endotoxin binding to liver sinusoidal cells occurs in a serum-independent manner, particularly at high LPS concentrations. Flow cytometry is a fast, precise, and efficient technique to evaluate the simultaneous interaction of a ligand with two different cell types.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED₁₀₀; ED = effective dose) and a toxic (LD₂₅; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.