Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model

AIM： TO investigate Kupffer cell dynamics and phagocytic activity, using a rat nonalcoholic steatohepatitis （NASH） model.
METHODS： Male F344 rats were fed either a control diet or a choline-deficient L-amino aciddefined （CDAA） diet, followed by contrast enhanced ultrasonography （CEUS） using Levovist. The uptake of latex beads by the Kupffer cells was determined by fluorescent microscopy. The status of the Kupffer cells was compared between the two groups, using the immunohistochemical staining technique.
RESULTS： After 4 or more wk of the CDAA diet, CEUS examination revealed a decrease in the signal intensity, 20 min after intravenous Levovist. Fluorescent microscopic examination showed that the uptake of latex beads by the Kupffer cells was reduced at week 1 and 2 in the study group, compared with the controls, with no further reduction after 3 wk. Immunohistochemical staining revealed no significant difference in the Kupffer cell counts between the control group and the CDAA group.
CONCLUSION： CEUS examination using Levovist demonstrated reduced contrast effect and phagocytic activity in the liver parenchymal phase, although the Kupffer cell numbers were unchanged, indicating reduced phagocytic function of the Kupffer cells in the rat NASH model. We believe that CEUS examination using Levovist is a useful screening modality, which can detect NASH in fatty liver patients.     

Crucial role of impaired Kupffer cell phagocytosis on the decreased Sonazoid‐enhanced echogenicity in a liver of a nonalchoholic steatohepatitis rat model

Aims: To evaluate the dynamics of Kupffer cell (KC) phagocytosis by performing both in vivo and in vitro studies using Sonazoid (GE Healthcare, Oslo) in a rat nonalcoholic steatohepatitis (NASH) model. Methods: Contrast enhanced ultrasonography (CEUS) was performed on a rat NASH model induced by a methionine choline deficient diet (MCDD) and control rats, and Sonazoid was used to measure the signal intensity in the liver parenchyma. The uptake of Sonazoid by the KCs was observed by intravital microscopy. Their phagocytic capability was evaluated in vitro using isolated and cultured KCs. The uptake of fluorescein isothiocyanate (FITC)‐labeled latex beads was observed and quantitatively analyzed by flow cytometry. Results: In the MCDD group, liver parenchymal enhancement was reduced 20 min after the Sonazoid injection. Microscopic observation of the isolated and cultured KCs revealed that the number of phagocytosed Sonazoid microbubbles was significantly decreased. Confocal laser scanning microscopic (CLSM) observation showed a decrease in the uptake of the latex beads. A decreased phagocytic capacity in the MCDD group was suggested by the quantitative analysis using flow cytometry, as well as by intravital microscopy. Conclusions: CEUS with Sonazoid is a powerful evaluation tool to diagnose NASH from an early stage of the disease.     

Phagocytosis, an unrecognized property of murine endothelial liver cells

Impairment of the phagocytic capacities of Kupffer cells, as is found in Frog Virus 3 hepatitis of mice, allows the endothelial liver cells to take up intravenously inoculated latex particles of 1.0 micron diameter. In vitro experiments with cultivated endothelial cells isolated by collagenase perfusion of the liver and purified by centrifugal elutriation demonstrate that uptake occurs via a typical mechanism of phagocytosis involving pseudopodia. Ingestion of latex is inhibited by incubation of the cells at 4 degrees C and by treatment with cytochalasin B, whereas colchicine has no effect. These results demonstrate that: the Kupffer cells are not the only cells of the hepatic sinusoid capable of phagocytosis; and under conditions where the phagocytosis in Kupffer cells is impaired, the endothelial cells may participate in the clearance of large particles from the blood.     

A new method to monitor Kupffer cell phagocytosis continuously in perfused rat liver

A new method was developed to monitor Kupffer cell phagocytosis continuously in perfused liver using a fluorescent probe, rhodamine-gelatin, synthesized from gelatin and rhodamine isothiocyanate. In perfused rat liver, phagocytosis by Kupffer cells was assessed both by uptake of the dye and from fluorescence measured from the liver surface. Uptake of rhodamine-gelatin and surface fluorescence (520----585 nm) increased as perfusate concentrations of rhodamine-gelatin were elevated. Histological examination revealed that fluorescence caused by rhodamine-gelatin was concentrated in cells lining the sinusoid. Furthermore, when hepatic parenchymal, endothelial and Kupffer cells were isolated by centrifugal elutriation after pretreatment in vivo with rhodamine-gelatin, the fluorescent label was detected nearly exclusively in the Kupffer cell fraction. Fluorescence of rhodamine-gelatin from the liver surface was directly proportional to uptake of rhodamine-gelatin, indicating that measurement of surface fluorescence could be used as an index of phagocytic activity of Kupffer cells. Uptake of rhodamine-gelatin and fluorescent labeling of sinusoidal cells were inhibited nearly completely by dextran sulfate, which decreases phagocytic activity, and were increased by about 50% by pretreatment with endotoxin, which stimulates phagocytosis. This new method was combined with standard procedures to monitor parenchymal cell function (e.g., oxygen uptake and release of lactate dehydrogenase), Kupffer cell phagocytic activity and parenchymal cell injury simultaneously during perfusion with the hepatotoxicant allyl alcohol. Activation of phagocytosis by Kupffer cells monitored by rhodamine-gelatin fluorescence occurred within 5 min of addition of allyl alcohol. On the other hand, parenchymal cell injury, assessed from release of lactate dehydrogenase, did not begin until 40 min and reached maximal values at around 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tauroursodeoxycholic acid enhances phagocytosis of the cultured rat Kupffer cell

BACKGROUND: Ursodeoxycholic acid is used in the treatment of acute and chronic intrahepatic cholestasis because it ameliorates cholestasis and protects hepatocytes. However, few studies have examined the effect of bile acids on the function of Kupffer cells. METHODS: The effect of various bile acids on cultured rat Kupffer cells was studied in terms of phagocytic activity in response to latex particles and morphological alterations. Video-enhanced differential interference contrast microscopy was used. RESULTS: Taurochenodeoxycholic acid and taurodeoxycholic acid reduced the number of latex particles incorporated into Kupffer cells, but taurocholic and tauroursodeoxycholic acids enhanced phagocytosis of latex particles. Inhibition of phagocytosis by taurochenodeoxycholic acid or taurodeoxycholic acid was essentially dose dependent. Tauroursodeoxycholic acid also enhanced phagocytosis by Kupffer cells in which phagocytosis had been reduced by pretreatment with taurochenodeoxycholic acid or taurodeoxycholic acid. Incorporated latex particles had a distinct translocation speed of 0.084+/-0.024 microm/s (mean maximum speed+/-SD); the speed was in the same range with tauroursodeoxycholic acid treatment. Tauroursodeoxycholic acid induced a 56% expansion of cytoplasm, associated with increased ruffling and movement of intracellular organelles. CONCLUSIONS: These observations suggest that tauroursodeoxycholic acid enhances membrane trafficking without changing translocation speed.     

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells.

AIMS/BACKGROUND: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E2 (PGE2), prostacyclin (PGI), and tumour necrosis factor-alpha (TNF) production in this model. METHODS: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. RESULTS: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE2 production, phagocytosis of hepatocytes did not. CONCLUSIONS: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE2 or TNF.     

The effect of 3-palmitoyl-(+)-catechin on Kupffer cells in guinea pig liver

We report a morphometric study on the effect of 3-palmitoyl-(+)-catechin on phagocytic activity of latex particles by Kupffer cells in guinea pig liver. We found an increase in the density of latex particles in Kupffer cells of animals treated with 3-palmitoyl-(+)-catechin. The volume and density of Kupffer cells of treated animals increased (p less than 0.01); there was no significant difference in surface density of the plasma membrane. None of the parameters measured was altered in endothelial cells. These results show that 3-palmitoyl-(+)-catechin produces morphological and functional changes in Kupffer cells.     

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells

Abstract: Aims/Background: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E₂ (PGE₂), prostacyclin (PGI), and tumour necrosis factor-α (TNF) production in this model. Methods: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. Results: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE₂ production, phagocytosis of hepatocytes did not. Conclusions: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE₂ or TNF.     

Metabolic response of isolated liver cells to in vivo phagocytic challenge

This study elucidates the in vivo metabolic response of different liver cells after a single phagocytic challenge. In vivo glucose uptake of different tissues and isolated liver cells was determined by a sequential double labeling version of the 2-deoxyglucose technique. After latex administration, glucose uptake more than doubled in the liver, increased by about 50% in the spleen and lung and was not changed in muscle and testis. Within 10 min after intravenous injection of latex beads, neutropenia developed, with no change in the number of lymphocytes. This was accompanied by a marked influx of polymorphonuclear leukocytes into the liver. Latex was found in 52%, 35%, and 14% of the isolated Kupffer cells, polymorphonuclear leukocytes and endothelial cells, respectively. In vivo glucose uptake increased by 111%, 142%, and 43% in these cells. Glucose uptake by the latex-free hepatocytes was also elevated, presumably by way of intercellular signals between parenchymal and non-parenchymal liver cells. Indomethacin pretreatment resulted in the delay of neutrophil immigration into tissues without any change in the glucose response of different liver cells. Thus phagocytic stimulation in vivo results in marked neutropenia, migration of neutrophils into the liver, increased glucose uptake by phagocytic cells of the liver and enhanced glucose metabolism by the non-phagocytic parenchymal cells.     

Integrity of the cytoskeletal system is important for phagocytosis by Kupffer cells

The mechanism of phagocytosis by Kupffer cells is believed to involve the Ca2(+)-calmodulin system. However, the role of myosin in this system is still unknown. In this study, we found that myosin light chain kinase inhibitor (ML-9) inhibited phagocytosis by cultured Kupffer cells using polystyrene beads, a time-lapse VTR system and fluorescent staining techniques. The inhibitory effects of ML-9 suggest that myosin may be involved in this complex cellular function and that the integrity of the cytoskeletal system is essential for normal phagocytosis.     

Effects of amphotericin B on the excretory function and the colloid clearance capacity of the perfused rat liver

The effects of amphotericin B (AmB) on the hepatic excretory function and the colloid clearance capacity were investigated in the perfused rat liver. AmB at 5 or 10 microM caused dose-dependent reductions in bile and perfusate flow rates and in biliary bile acid (BA) excretion. BA concentration in bile tended to increase, due to a prominent reduction in bile water induced by the drug. At 5 microM, AmB also caused an increase in [14C]sucrose clearance by the liver and a release of hepatocytic enzymes into the perfusate. These alterations were not related to the decrease in the perfusate flow induced by AmB. In addition, the drug, at 5 microM, caused a significant decrease in the colloidal carbon clearance by the liver. In this case also, the effect was independent of the reduction in the perfusate flow induced by the drug. The toxic effects of AmB on the rat liver could be interpreted as a derangement of the cell membrane functional integrity, which causes cholestasis, enzyme leakage and an impairment of the reticuloendothelial system function. This latter effect deserves careful evaluation of its clinical implications.     

Phagocytotic activities of latex beads by FRTL cell: possible interaction of cell surface glycoprotein

We investigated the phagocytic activity of cultured rat thyroid cells (FRTL) by using latex beads. FRTL cells ingested many carboxylate-modified polystyrene latex beads (c-latex beads), which had carboxyl residues on the surface, whereas they ingested few polystyrene latex beads (p-latex beads), which had no electrostatic charge. Phagocytotic activity of c-latex beads was stimulated by TSH in a dose-related manner. Dibutyryl, 8-bromo-cAMP, and forskolin also stimulated this process. Tunicamycin significantly inhibited dibutyryl cAMP-stimulated phagocytotic activity of c-latex beads by FRTL cells. N-acetylglucosaminidase also inhibited dibutyryl cAMP-stimulated phagocytosis. These results suggest that the interaction between negative surface charge of c-latex beads and positive surface charge probably induced by N-acetylglucosamine in the cell surface glycoprotein, might play an important role in the phagocytosis of FRTL cells.     

Effect of aging on cytoskeleton system of Kupffer cell and its phagocytic capacity

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Kupffer cell function during the erythocytic stage of malaria

Background and Aim: Previous studies using isolated perfused rat liver in vivo have suggested that during the erythrocytic phase of malaria infection, overall phagocytosis by Kupffer cells is enhanced. The aim of the present study was to further investigate the individual phagocytic capacity and prostaglandin E₂ (PGE₂) secretion of isolated Kupffer cells in vitro, and the immunohistochemical characteristics of Kupffer cells in vivo. Methods: Malaria was induced in male Sprague–Dawley rats (n = 12) by inoculation with parasitized red cells containing Plasmodium berghei. Kupffer cells were isolated by centrifugal elutriation. Results: A significantly increased yield of Kupffer cells was obtained from malaria‐infected livers compared to controls (36.7 ± 4.5 vs 11.8 ± 1.1 ×10⁶ cells, P < 0.0001, n = 12). There was an increased internalization by phagocytosis of [³H]‐BSA latex microspheres after 60 min in malaria‐infected Kupffer cells compared to controls (65.05 ± 1.5 vs 48.6 ± 0.7, P < 0.001, n = 12). PGE₂ secretion into the cell culture medium was significantly suppressed in malaria‐infected Kupffer cells compared to controls (1167 ± 88 vs 4537 ± 383 pg per 10⁶ cells, P < 0.001, n = 5). Staining of ED1, ED2 and PCNA was greater in malaria‐infected livers compared to control. Conclusion: The results indicate that the number of Kupffer cells is significantly increased and their phagocytic activity on a cell‐by‐cell basis is enhanced during the erythrocytic stage of malaria.     

Phagocytic function in the isolated perfused rat liver. An experimental model

An experimental model for measuring the phagocytic function of the isolated perfused rat liver is described. A progressive rise in phagocytosis was observed with increasing liver blood flow. This is due to an increase in total particle uptake by the liver with no alteration in the rate constant for phagocytosis except at the highest flow rate. Phagocytosis is substantially greater in the livers of 100-day-old rats than in 21-day-old rats, but the number of particles ingested per unit weight by the older rats is significantly less. Liver phagocytosis is shown to be both temperature- and oxygen-dependent, but independent of nutritional status and animal gender. This model may be useful for assessing the effects of drugs and toxins on hepatic phagocytosis.     

Hepatic Kupffer cell phagocytotic function in rats with erythrocytic-stage malaria

BACKGROUND: In the erythrocytic phase of malaria, Kupffer cells show marked hypertrophy and hyperplasia and are filled with malarial pigment. However, phagocytic function in this state has not been well characterized. The aim of the present study was to use mouse Plasmodium berghei to infect rats with malaria and study the phagocytic function and morphology of Kupffer cells. METHODS: We used a recirculating isolated perfused rat liver (IPRL) to quantitate Kupffer cell phagocytic clearance of radiolabeled albumin-latex over 120 min in high parasitemia (53 +/- 6%; n = 7) and low parasitemia (approximately 1%; n = 4) malaria-infected rats and littermate controls (n = 7 and n = 4, respectively). In a further group of high-parasitemic rats, perfusion was ceased after 7 min and liver radioactivity also measured. Electron microscopy was performed after perfusions. RESULTS: In high-parasitemia malaria rats, clearance of radiolabeled latex from IPRL perfusate over 120 min was significantly (P < 0.01) faster than in controls, with a lower area under the curve (0.19 +/- 0.02 vs 0.43 +/- 0.07 /mL per min, respectively) and shorter half-life (t1/2k; 2.4 +/- 0.6 vs 10.0 +/- 2.3 min, respectively). Low-parasitemia rats were identical to controls. After 7 min perfusion in high-parasitemic rats (n = 4), total radioactivity in liver homogenates was higher than in controls (n = 4; 33.1 +/- 6.2 vs 18.4 +/- 1.9% of injected radiolabel; P < 0.05). Electron microscopy showed latex in Kupffer cells, more abundantly seen in high-parasitemic animals. CONCLUSIONS: Total Kupffer cell phagocytic activity of the liver is markedly increased in rats with a high parasitemic load of malarial P. berghei infection. This is presumed to reflect an upregulation of scavenger activity phagocytosing erythrocytes and their breakdown products.     

Kupffer cell function in chronic ethanol-fed rats

In an attempt to clarify the Kupffer cell function in alcoholism, chronic ethanol-fed rats were investigated. The clearance of latex particles in the rat was analysed to estimate the function of the reticuloendothelial system in the liver, and the phagocytic function of Kupffer cells was measured by counting particles in the cell after isolation of non-parenchymal cells by collagenase digestion of the liver following an injection of latex particles and subsequently by staining of endogenous peroxidase activities. In addition, the number of Kupffer cells and their phagocytic function were examined histologically in fresh frozen sections of liver after an injection of particles. Serum ethanol concentration in the ethanol-fed rats was 10-60 mumol/l. The clearance of latex particles was markedly reduced in the ethanol-fed rats as compared with the paired controls (P less than 0.01). Markedly decreased-phagocytic function was found in 20% of Kupffer cells in the chronic ethanol-fed rats. The number of Kupffer cells in the ethanol-fed rats was increased as compared with the paired control rats. Chemotaxis analysis revealed that hepatocytes when incubated with ethanol, produced chemotactic factor for Kupffer cells and polymorphonuclear cells. These abnormal Kupffer cell functions may contribute to the pathogenesis of alcoholic liver disease.     

The effects of splenectomy and glucocorticoids on survival and hepatic uptake of damaged red cells in the mouse

We have studied the effects of splenectomy and glucocorticoids on the survival and sequestration of Heinz body-containing red blood cells (RBC-HZB). Mice were injected with phenylhydrazine damaged 51Cr labeled isologous red blood cells (RBCs). The spleen removed 36% and the liver 19% of the injected dose after 120 hrs. Red cell survival (T 1/2) fell from 180 hrs for undamaged red cells to 16 hrs for RBC-HZB. Splenectomy resulted in an increase in hepatic uptake of damaged RBCs (36% of the injected dose) and a modest improvement in red cell survival (T 1/2 54 hrs). Treatment of non-splenectomized mice with glucocorticoids reduced the splenic uptake to 16% and the hepatic uptake to 14% of the injected dose. The reduction of splenic upatke was associated with a decrease in splenic mass rather than a decrease in uptake per unit weight of splenic tissue, while reduction in hepatic uptake was associated with both a decrease in hepatic mass and uptake per unit weight. A marked decrease was observed in hepatic uptake and in phagocytosis by Kupffer cells in glucocorticoid-treated splenectomized mice. These data suggest that increased hepatic uptake may decrease the effectiveness of splenectomy in RBC-HZB hemolytic anemia and that glucocorticoids may decrease the hepatic uptake by reducing phagocytosis by Kupffer cells.     

Isolation and primary culture of rat Kupffer cells

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48–110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 μm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80–110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80–120 times 10⁶ Kupffer cells per liver.     

Alteration of xanthine oxidase activity in sinusoidal endothelial cells and morphological changes of kupffer cells in hypoxic and reoxygenated rat liver

In the model of the perfused rat liver, we investigated the alterations of sinusoidal cells in the pathogenesis of liver injury caused by hypoxia and reperfusion. In sinusoidal endothelial cells, the activity of xanthine oxidase (XOX), a cytoplasmic marker enzyme, was located cytochemically and determined biochemically. Kupffer cells, identified by their endogenous peroxidase staining, were studied with regard to changes in their ultrastructure. In our experiments, parenchymal cells were shown to be severely damaged in contrast to sinusoidal lining cells, which showed minor signs of injury. In comparison with the control group, XOX activity increased significantly in the sinusoidal endothelial cells after low-flow hypoxia; however, after reoxygenation of only 5 minutes, that activity was lower after hypoxia but higher after control perfusion. In Kupffer cells, hypoxia resulted in a strong suppression of phagocytic and endocytotic activity and in a disappearance of the lamellopodia. Kupffer cells were flattened, resembling sinusoidal endothelial cells. After reoxygenation phagocytic vesicles, lamellopodia, and cell volume of Kupffer cells increased markedly in comparison with the control group. In the hypoxia/reperfusion injury model, our observations revealed significant alterations of sinusoidal lining cells. It appears that sinusoidal endothelial cells respond to the hypoxic phase by producing oxygen-derived free radicals and that Kupffer cells respond to the subsequent reperfusion phase by activation followed by the release of toxic mediators.