Quantitative ultrastructure of isolated Kupffer cells of rat liver

The average volume of isolated Kupffer cells of rat liver is 821 +/- 64 microns 3, the average surface being 423 +/- 24 microns 2 (599 microns 2, with cell processes included). The surface structure (pseudopodia, lamellipodia, filopodia, microvilli) of isolated cells is much less developed than that of Kupffer cells in situ. By morphometric characterization volume densities are 0.1264 +/- 0.0077 (SE) for mitochondria and 0.3591 +/- 0.0169 for lysosomal structures. The volume of mitochondria amount to 0.79 +/- 0.04 microns 3.     

Identification of G6PDH-active sinusoidal cells as Kupffer cells in the rat liver

Summary The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded. Fat-storing cells were specifically marked by vital staining with vitamin A and identified by fluorescence microscopy. Kupffer cells could be detected after vital staining with carmine. Both staining methods allowed a subsequent incubation for the demonstration of G6PDH activity in the same unfixed cryostat section. Whereas more than 80% of the fluorescent particles were found outside the enzyme-positive cells, all G6PDH-active cells contained carmine particles. After counting the G6PDH-active cells, an estimation of 0.217 × 10⁸ cells/g liver tissue was obtained. The results indicate that high G6PDH activity is common to all Kupffer cells, and is therefore a highly specific marker enzyme for this class of sinusoidal liver cells.The essential parts of this study will be presented as an Inaugural-Dissertation to the Medical Faculty of the University of Freiburg by W. HosemannSupported by a grant from the SFB 46 (Molgrudent)     

Enhanced tumour growth in the rat liver after selective elimination of Kupffer cells

The evidence that Kupffer cells are capable of controlling metastatic growth in the liver in vivo is largely circumstantial. The best approach when studying natural cytotoxicity activities of Kupffer cells is to investigate the effect of Kupffer cell elimination on tumour growth. Until now it has not been possible to eliminate Kupffer cells without affecting other cell populations. We have recently developed a new method to eliminate Kupffer cells selectively: intravenous injection of liposome-encapsulated (dichloromethylene)bisphosphonate (Cl₂MDP-liposomes) leads to effective elimination of all Kypffer cells, without affecting non-phagocytic cells. Wag/Rij rats were injected with Cl₂MDP-liposomes. After 48 h, rats were inoculated with syngeneic CC531 colon carcinoma cells by injection in the portal system. The results show a strongly enhanced tumour growth in the liver of the Cl₂MDP-liposometreated rats. In these animals, livers were almost completely replaced by tumour and had increased in weight, whereas in the control groups only a few (four to eight) small (1-mm) tumour nodules were found. These data show that selective elimination of Kupffer cells results in enhanced tumour growth in the liver, implying that Kupffer cells play a crucial role in controlling tumour growth in the liver.     

Isolation and characterization of Kupffer and endothelial cells from the rat liver

Non-parenchymal cell suspensions were prepared from rat livers by three different methods based on a collagenase, a pronase and a combined collagenase-pronase treatment. The highest yield of Kupffer and endothelial cells was obtained with the pronase treatment. Attempts were made for a further purification of these cells by Metrizamide density gradient centrifugation after preferentially loading lysosomal structures in Kupffer cells with Triton WR 1339, Jectofer^®, Neosilvol^®, Zymosan or colloidal carbon. After loading with Triton WR 1339 or Jectofer^®, highly purified endothelial cell suspensions were obtained, but the final Kupffer cell preparations were contaminated with about 20% of endothelial cells. Kupffer and endothelial cells purified in this way showed an altered ultrastructure and contained increased activities of the lysosomal enzymes acid phosphatase, arylsulphatase B and cathepsin D. As an alternative procedure for the purification of Kupffer and endothelial cells, a method based on centrifugal elutriation was employed. With this procedure, highly purified preparations of Kupffer or endothelial cells with a well preserved ultrastructure were obtained. Compared with endothelial cells, purified Kupffer cells had a three times higher cathepsin D activity, whereas the arylsulphatase B activity was three times higher in endothelial cells. The high cathepsin D activity in Kupffer cells could be nearly completely inhibited by the specific cathepsin D inhibitor pepstatin, which excludes a possible contribution to this activity by proteases endocytosed during the isolation of the cells.     

Cadmium metabolism by rat liver endothelial and Kupffer cells.

The metabolism of cadmium was investigated in Wistar-rat liver non-parenchymal cells. Kupffer and endothelial cells, the major cell populations lining the sinusoidal tracts, were isolated by collagenase dispersion and purified by centrifugal elutriation. At 20 h after subcutaneous injection of the metal salt (1.5 mg of Cd/kg body weight), endothelial cells accumulated 2-fold higher concentrations of Cd than did Kupffer or parenchymal cells. Most of the Cd in non-parenchymal cells was associated with cytosolic metallothionein (MT), the low-Mr heavy-metal-binding protein(s). When MT was quantified in cytosols from cells isolated from control rats by a 203Hg competitive-binding assay, low levels were found to be present in Kupffer, endothelial and parenchymal cells. Cd injection significantly increased MT levels in all three cell types. The induction of MT synthesis was investigated in vitro by using primary monolayer cultures. The incorporation of [35S]cysteine into MT increased 47% over constitutive levels in endothelial-cell cultures after the addition of 0.8 microM-Cd2+ to the medium for 10 h. MT synthesis in Kupffer cells was not observed. The lack of MT synthesis by monolayer cultures of Kupffer cells in vitro was associated with a decreased capacity of these cells to accumulate heavy metals from the extracellular medium. This apparent decreased ability to transport metals did not reflect a general defect in either cellular function or metabolic activity, since isolated Kupffer cells incorporated [3H]leucine into protein at rates comparable with those shown by liver parenchymal cells and readily phagocytosed particles.     

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.     

The fate of lipopolysaccharide in cultured rat Kupffer cells

Cultured rat Kupffer cells were incubated in presence of biologically tritiated Salmonella abortus equi lipopolysaccharide. Uptake of lipopolysaccharide increased rapidly during the first 2 h of incubation and then levelled off. Within the first h of incubation 10(6) Kupffer cells were able to ingest up to 18 micrograms lipopolysaccharide. Kupffer cells metabolised lipopolysaccharide and released lipopolysaccharide-related substances, but neither the cell-associated lipopolysaccharide nor the released lipopolysaccharide products were detoxified, as measured by the mouse lethality test.     

Separation of Kupffer and endothelial cells of the rat liver by centrifugal elutriation

Isolated non-parenchymal cells from rat liver were separated by centrifugal elutriation into two fractions consisting of structurally intact Kupffer and endothelial cells with purities of 91 and 95%, respectively. Purified Kupffer and endothelial cells showed nearly equal specific activities for the lysosomal enzyme acid phosphatase, whereas the specific activity of cathepsin D was about 3 times higher in Kupffer cells. It was calculated that a significant amount of the cathepsin D activity in the liver is present in the Kupffer cells.     

Ethanol-dependent induction of bilirubin UDP-glucuronosyl-transferase in rat liver is mediated by Kupffer cells

Recently we have reported that bilirubin UDP-glucuronosyltransferase (UGT1A1) is induced in rat liver by chronic ethanol treatment. Several studies have shown that Kupffer cells play a central role in the mediation of various hepatic effects of chronic alcohol consumption. In the present work, the participation of Kupffer cells in the ethanol dependent induction of UGT1A1 was investigated. A group of rats was pretreated with gadolinium chloride, a known Kupffer-cell-depleting agent. We compared the effect of chronic ethanol ingestion on UGT1A1 expression in the liver of normal and gadolinium chloride treated rats. The effect of ethanol on bilirubin glucuronidation was completely prevented in Kupffer cell deficient rats. The western and northern blot analyses showed that the increase of both the protein and mRNA of UGT1A1 was prevented in these animals. These results suggest that Kupffer cells play a major role in the mediation of ethanol-stimulated induction of UGT1A1 in liver parenchymal cells.     

Demonstration of collagenase activity in rat liver homogenate

Collagenase activity became detectable in rat liver homogenate by washing liver tissue repeatedly with buffered saline before homogenization. This enzyme activity was inhibited by adding minute quantities of serum. These data suggest that collagenase is active $\text{in situ}$ in the liver, but is made inactive during the homogenization by forming a complex with contaminating serum factors.     

Phagocytosis in rat Kupffer cells in vitro

Phagocytosis was studied in rat Kupffer cells in vitro by using opsonized sheep red cells as objects and inducing attachment and ingestion through the Fc and C₃ receptors. The Fc receptors functioned by and large in the same manner as in the peritoneal macrophages. When the red cells were opsonized with IgM and complement, there was attachment but little ingestion in a serum-free medium. Newborn calf serum was found to trigger ingestion. Our experiments provided no conclusive evidence as to the nature of this triggering mechanism. The limiting factor in phagocytosis was the cytoplasmic volume of the phagocyte rather than the availability of surface receptors. The expression of surface receptors on cells in culture depended on length of culture and degree of spreading. We confirmed the available information on the energy requirements of phagocytosis as studied in peritoneal macrophages. As judged by isotope release, digestion of the red cells was in process shortly after ingestion. However, morphological examination failed to detect any changes in appearance prior to 4 h. After a blocking dose of sheep red cells, a rather long period (40 h) was required before cells fully recovered their phagocytic capacity.     

The presence of collagenase in collagen preparations

The frequently observed instability of neutral salt solutions of native collagen extracted from various sources and partially purified by standard procedures has been studied by disc electrophoresis in polyacrylamide gel and by electron microscopic examination of segment long spacing crystallites. The phenomenon has revealed time and temperature dependency, pH optima near neutrality, and inhibition by sodium EDTA and serum. In addition, collagen breakdown has been found to be quantitatively related to the state of aggregation of the substrate, being more marked in reconstituted collagen gels than in collagen in solution. A typical pattern of animal collagenase degradation of native collagen into two fragments designated as TC^A and TC^B has been observed under certain conditions. It is concluded that the degradation of native collagen in neutral salt solution is due to a specific collagenase, and that this enzyme probably remains bound to collagen throughout the process of extraction and partial purification. Experiments with gelatin suggest that, in addition to collagenase, a nonspecific proteolytic activity may also be present in collagen preparations.     

The development of the sinusoids of fetal rat liver: localization of endogenous peroxidase in fetal Kupffer cells.

Endogenous peroxidase is the cytochemical marker used to identify Kupffer cells in the adult liver. In this study, we show by ultrastructural cytochemistry that Kupffer cells of the fetal rat liver are endogenous peroxidase positive. The reaction product is localized in the endoplasmic reticulum including the perinuclear cisternae and in a few lysosome-like dense bodies. Serial sections of Golgi regions suggest that GERL and not the Golgi stacks, is peroxidase positive. As in the adult liver, peroxidase is not localized in endothelial cells. Kupffer cells do not appear to transform from endothelial or extravascular developing monocytic cells and are present prior to bone marrow formation. The relevance of these observations with respect to the possible origin of the Kupffer cell is discussed.     

High activity of glucose-6-phosphate dehydrogenase in Kupffer cells isolated from rat liver

Summary Sinusoidal cells in the rat liver react intensively for G6DPH activity after appropriate incubation (Rieder et al. 1978). After isolation and purification of the sinusoidal Kupffer and endothelial cells, it was demonstrated that Kupffer cells exhibit a 5–8 times higher G6PDH activity on a per cell basis by comparison with endothelial cells, while the specific G6PDH activity was 3–4 times higher in Kupffer cells. The Kupffer cells can be divided into two groups which differ significantly in G6PDH activity calculated on a per cell basis. In histochemical studies, G6PDH can be used as a marker for Kupffer cell identification.     

BETA-glucuronidase and chloroacetate-esterase staining discriminates rat liver sinusoidal endothelial cells from Kupffer cells in primary culture

Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.     

枯否细胞对大鼠再生肝细胞转录活性和LDH的影响

目的与方法 SD大鼠随机分成3组,即C组(切除2/3肝叶)、L组(切除2/3肝叶后注射LPS)和G组(GdCl3预处理后切除2/3肝叶),研究大鼠肝再生期间(0～144 h)再生肝重量比、AgNORs数量、乳酸脱氢酶的变化.结果 L组肝重量比在16～48 h低于C组(P<0.05),AgNORs的数量在48 h达到最大值(P<0.01,与正常对照组相比).G组再生肝重量比在16～36 h低于C组(P<0.05),AgNORs的数量在36 h即达到高峰,乳酸脱氢酶新增一条LDH4谱带,其平均密度在36 h达到最高值.结论 肝切除后注射LPS,抑制了早期的肝再生进程,注射GdCl3灭活枯否细胞后利于肝脏的早期再生.