Factors involved in the down-regulation of cytochrome P450 during Listeria monocytogenes infection

The activation of host defense mechanisms has been shown to cause a depression in hepatic cytochrome P450-mediated metabolism in rodents and humans. In a previous study, it was demonstrated that the Gram-positive bacteria Listeria monocytogenes causes a down-regulation of hepatic cytochrome P450 and related substrate metabolism as a result of a pretranslational depression of apoprotein synthesis. The objectives of this study were to determine whether the effect of listeria on hepatocyte cytochrome P450 involves hepatic nonparenchymal cells and whether the hemolysin, secreted only by hemolytic forms of the bacteria, plays any part in mediating this effect. Total cytochrome P450 levels as well as ethoxyresorufin-O-dealkylase (EROD) and benzyloxyresorufin-O-dealkylase (BROD) activities were significantly reduced in hepatic microsomes isolated from mice infected in vivo for 48 h with 15U listeria, whereas the same dose of the avirulent non-hemolytic M3D strain had no effect. Listeria (15U) significantly depressed hepatocyte EROD and BROD activities after 24 h incubations with liver cell cultures containing hepatocytes and nonparenchymal cells, as the result of both a direct effect on the hepatocyte and an interaction of listeria with hepatic nonparenchymal cells. The M3D strain of listeria had no effect on cytochrome P-450-mediated metabolism in isolated cells, confirming that hemolysin is an essential component of the mechanism responsible for the down-regulation of cytochrome P450 during listeria infections.     

刺激单核巨噬细胞对肝实质及非实质细胞脂蛋白代谢的影响

本研究结果表明酵母多糖对喂正常饲料大鼠肝非实质细胞及实质细胞的^125I-HDL及^125I-LDL代谢影响不大，但可明显增加喂高脂饲料大鼠细胞对^125I-HDL及^125I-LDL的结合、内移及降解，增加胆汁中总胆酸及胆固醇的排泄，明显降低高脂饲料导致的高胆固醇血症。     

Carbachol induces hepatocyte proliferation, but only in the presence of hepatic nonparenchymal cells

Vagal hyperactivity correlates with enhanced DNA synthesis and cell proliferation in the peripheral tissues of ventromedial hypothalamic (VMH)-lesioned rats. The infusion of an ACh receptor agonist, carbachol (Cch), induces rat duodenal and pancreatic cell proliferation to a degree comparable to the VMH lesions. Whereas the VMH lesions also induce the proliferation of hepatic cells, it is unclear whether Cch can also do this. Here we attempted to clarify the mechanism of hepatic cell proliferation induction by cholinergic stimulation. First, hepatic cell proliferation was examined in rats previously vagotomized and intraperitoneally administered with Cch via an osmotic minipump. Second, the sera from the Cch-infused rats were examined for a proliferative effect on isolated hepatic cells. And last, the effect of the presence of hepatic nonparenchymal cells (NPCs) on the proliferation of the cultured hepatocytes treated with Cch was investigated. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) showed that the 3-day Cch infusion significantly increased the number of PCNA-immunoreactive cells in the liver. Moreover, the sera from the Cch-infused rats increased the number of PCNA-immunoreactive hepatocytes in culture. However, Cch alone did not induce proliferation in monocultured hepatocytes. When compared with the monoculture of hepatocytes, the coculture of those with hepatic NPCs resulted in enhanced PCNA immunoreactivity after a 4-day treatment with 3 mM Cch. These findings suggest that ACh induces hepatocyte proliferation, which is mediated by unidentified humoral factor(s) possibly secreted from hepatic NPCs, and that it also participates in liver hypertrophy in the VMH-lesioned animals.     

Natural antitumor defense system of the murine liver

Murine nonparenchymal liver cells from various genetic strains isolated by collagenase digestion and differential sedimentation contain both lymphocytes and macrophages. Nonparenchymal liver cells as well as spleen cells, mononuclear blood cells, and peritoneal exudate cells from C3HeB/FeJ mice were tested for natural cytotoxicity against YAC-1 (sensitive to NK cells) and P815 (resistant to NK cells) tumor cell lines. Resident peritoneal exudate cells exerted no cytotoxicity against either tumor cell, whereas spleen and mononuclear blood cells lysed only YAC-1. In contrast, nonparenchymal liver cells lysed both YAC-1(4 h) and P815 (18 h) tumor cells. Treatment of nonparenchymal liver cells with anti-asialo GM1 and complement abolished the antitumor activity against both tumor cell lines but not the phagocytic activity. Nonadherent nonparenchymal liver cells exerted greater cytotoxicity against YAC-1 tumor cells but little cytotoxicity against P815 tumor cells when compared with unfractionated cells. Adherent nonparenchymal liver cells (macrophages) from untreated mice exerted no antitumor activity against either tumor cell. In contrast, adherent nonparenchymal liver cells from Corynebacerium parvum treated mice were directly cytotoxic to P815 tumor cells. Spleen cells that are normally not cytotoxic to P815 tumor cells (18 h) became cytotoxic when mixed with adherent nonparenchymal liver cells from untreated mice. These results indicate that the tumoricidal effector cell in nonparenchymal liver cells from untreated mice appears to be the NK cell. Apparently, murine liver macrophages from untreated mice do not have tumoricidal activity per se but can "activate" NK cells to kill tumor cells normally resistant to NK cells.     

Ethanol effects on protein synthesis in nonparenchymal liver cells, hepatocytes, and density populations of hepatocytes

Rats were given ethanol chronically (20-30% of the energy) in a nutritionally sufficient diet regimen. Controls received lipid as an isoenergetic substitute for ethanol. Protein synthesis in hepatocytes isolated from ethanol-fed rats was decreased compared with controls, but not in isolated nonparenchymal liver cells. Ethanol added in vitro inhibited protein synthesis in hepatocytes by 30%, but not in nonparenchymal cells for both ethanol-fed and control rats. Protein export and protein degradation in isolated hepatocytes were not affected by long-term ethanol treatment. Isolated hepatocytes were separated according to their buoyant density in linear metrizamide gradients. They were distributed in a bell-shaped manner regardless of donor rat treatment. Cells of low density contained three times as much lipid as high density cells. They were probably enriched in periportal cells, since histologic examination indicated a predominantly periportal localization of cells containing lipid droplets. Distribution of the intra-acinar marker alanine aminotransferase supported this conclusion. Protein synthesis was similar in the low-density hepatocyte populations of the respective groups of rats, whereas it was inhibited in a high-density population of ethanol-treated rats compared to the controls. Inhibition of protein synthesis by 80 mM ethanol was lower in the low-density hepatocytes of ethanol-fed rats.     

Role of transforming growth factor-[beta]1 in inhibiting endothelial cell proliferation in experimental alcoholic liver disease.

We used the intragastric feeding rat model for alcoholic liver disease to investigate the relationship between transforming growth factor (TGF)-beta 1 and inhibition of endothelial cell proliferation. Twelve groups of male Wistar rats (four to five rats per group) were fed ethanol or dextrose with either corn oil or saturated fat for 1-, 2-, and 4-week periods. All control animals were pair fed the same diets as ethanol-fed rats except that ethanol was isocalorically replaced by dextrose. In the ethanol-fed groups, nonparenchymal cells were isolated and TGF-beta 1 was measured in the nonparenchymal cell supernatant. Liver pathology and endothelial cell proliferation with an antibody to proliferating cell nuclear antigen were studied in all groups. Plasma TGF-beta 1 was measured in all rats. Pathological changes (fatty liver, necrosis, and inflammation) were observed only in the corn oil/ethanol-fed rats at 4 weeks. Significantly higher levels of TGF-beta 1 were seen in both plasma and nonparenchymal cell supernatant in rats fed corn oil and ethanol; plasma levels of TGF-beta 1 were not significantly different between the dextrose-fed controls and saturated fat/ethanol-fed rats. A significant inverse correlation (r = -0.89, P < 0.01) was seen between plasma TGF-beta 1 and the number of endothelial cells arrested at G1/S. Immunohistochemistry revealed the presence of TGF-beta 1 staining in interstitial macrophages only in rats fed corn oil and ethanol. The present study provides evidence for a role for TGF-beta 1 in inhibiting endothelial cell proliferation in experimental alcoholic liver disease. Arrest of endothelial cells may lead to their differentiation and/or to produce mediators that could stimulate other cells such as Ito cells. Sustained TGF-beta 1 may also lead to Ito cell production of extracellular matrix.     

Demonstration of glucose-6-phosphatase and peroxisomal catalase activity by ultrastructural cytochemistry in oval cells from livers of carcinogen-treated rats.

Oval cells isolated from livers of carcinogen-treated rats have morphologic and biochemical features of immature hepatocytes but seem to lack glucose-6-phosphatase (G6Pase) activity. The authors reinvestigated this question using histochemical methods for visualization of G6Pase activity by light and electron microscopy and the polyene antibiotic filipin to facilitate the penetration of the substrate. Oval cells that proliferate in the liver of animals receiving a carcinogenic diet (choline-deficient, containing 0.05% ethionine) contained G6Pase activity; 50-60% of nonparenchymal epithelial cells isolated from these livers by centrifugal elutriation contained G6Pase activity; and oval cell cultures displayed intense G6Pase activity at confluence but did not have detectable enzyme activity during exponential growth. These results and the demonstration that clofibrate induces peroxisomal proliferation in cultured oval cells strengthen the view that oval cells (or some subpopulation of cells in this compartment) are part of the hepatocyte lineage.     

Inhibition of growth of early passage normal rat liver epithelial cell lines by epidermal growth factor

The proliferation of newly derived or early passage nonparenchymal rat liver epithelial cell lines in serum-containing medium was inhibited consistently by epidermal growth factor (EGF). Inhibition of proliferation by EGF was a dominant effect which could not be overcome by insulin, phorbol ester or substances which raised the intracytoplasmic concentration of cyclic-AMP (cholera toxin and dibutyryl cyclic-AMP). EGF has a heterogeneous effect on colony formation by individual cells of early passage cell lines. Whereas the colony-forming ability of a majority of these cells was inhibited by EGF, a small fraction of the cells resisted this effect and formed colonies in the presence of EGF. Clonal strains of EGF-inhibited and EGF-stimulated cells were isolated. Measurement of 125I-EGF binding capacity of these cell strains revealed no significant differences between the EGF-inhibited and EGF-stimulated cells. In general, EGF-stimulated cell strains demonstrated faster growth rates and higher colony-forming efficiencies than EGF-inhibited strains. EGF-induced growth inhibition however, was an unstable phenotype that might be diminished or lost after multiple passages, especially when cells were allowed to remain confluent for many days between passages. These observations may explain the consistent findings that in late passage, rat liver epithelial cell lines in which the conditions for culture and times of subcultures have not been controlled rigorously, the inhibitory effect of EGF usually is not observed.     

Transforming Growth Factor α Levels in Liver and Blood Correlate Better than Hepatocyte Growth Factor with Hepatocyte Proliferation during Liver Regeneration

Transforming growth factor α (TGFα) and hepatocyte growth factor (HGF) are mitogens for hepatocytes in vitro and in vivo, produced by hepatocytes or nonparenchymal cells such as stellate cells in the liver. It is still uncertain whether TGFα and HGF are essential for liver regeneration. To assess the role of these growth factors in liver regeneration, their circulating and hepatic levels were studied in various rat models of liver regeneration. Hepatic and plasma HGF levels were increased with increased number of mitotic hepatocytes in rats after partial hepatectomy or carbon tetrachloride intoxication. However, hepatic HGF levels were decreased despite an increased number of mitotic hepatocytes and increased or unchanged plasma HGF levels in rats given phenobarbital and in rats after dimethylnitrosamine intoxication, which can induce hepatic necrosis after apoptosis of hepatic stellate cells. In contrast, hepatic and serum TGFα levels were increased in all of the models. In sham-operated rats with no increased number of mitotic hepatocytes, hepatic and circulating levels of HGF were increased, whereas those levels of TGFα were unchanged. The results indicate that TGFα levels in liver and blood more closely correlate with hepatocyte mitogenesis than HGF levels.     

Extramedullary eosinophilopoiesis in the liver ofSchistosoma japonicum-infected mice,with reference to hemopoietic stem cells

Extramedullary hemopoiesis recognized as hemopoietic foci increased in the liver ofSchistosoma japonicum-infected mice in parallel with kinetic changes in periovular granuloma formation. At the peak of the response, about 65% of the hepatic hemopoietic foci were of eosinophil lineage. WhenS. japonicum-infected mice were irradiated, hepatic hemopoietic foci rapidly disappeared within 3 days, whereas inflammatory cells in the periovular granulomas slowly reduced in number. When the number of hemopoietic stem cells in the liver were examined by spleen-colony assay, kinetic changes in the number of hemopoietic stem cells in hepatic nonparenchymal cells paralleled those of hepatic hemopoietic foci. Hemopoietic stem cells were rare in the granuloma cells. These results indicate that in response to the increased demand for eosinophils and other inflammatory cells, the liver acts as an extramedullary hemopoietic organ in which inflammatory cells are generated from hemopoietic stem cells.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - NPC nonparenchymal cell - CFU-S spleen colony forming unit - ECF eosinophil chemotactic factor     

In vitro differentiation of liver progenitor cells derived from healthy dog livers

Naturally occurring liver disease in dogs resemble human liver disease in great detail; including the activation of liver progenitor cells (LPC) in acute and chronic liver disease. The aim of the present study was to isolate, culture, and characterize progenitor cells derived from healthy mature dog livers. A nonparenchymal cell fraction enriched with small hepatocytes was isolated and cultured in Hepatozyme-serum-free media (SFM) to stimulate the growth of colony-forming small epithelial cells. After 2 weeks of culturing, clonal expansion of keratin 7 (K7) immunopositive small cells with a large nucleus/cytoplasm ratio emerged in the hepatocyte monolayer. These colonies expressed genes of several hepatocyte (CYP1A1, ALB, and KRT18), cholangiocyte/LPC (KRT7 and KRT19), and progenitor cell markers (alpha-fetoprotein, CD44, prominin1, KIT, THY1, and neural cell adhesion molecule 1), indicating their immature and bipotential nature. Gene-expression profiles indicated a more pronounced hepatic differentiation in Hepatozyme-SFM compared to William's Medium E (WME). Furthermore, colony-forming cells differentiated toward intermediate hepatocyte-like cells with a more pronounced membranous K7 immunostaining. In conclusion, colony-forming small epithelial cells in long-term canine liver cell cultures express LPC markers and have differentiating capacities. These cells may therefore be considered as progenitor cells of the liver.     

Developmental Changes of Cell Adhesion Molecule Expression in the Fetal Mouse Liver

Developmental changes of cell adhesion molecule expression, especially in nonparenchymal cells, have hardly ever been analyzed in the murine liver. The present study was undertaken to immunohistochemically examine the expression of NCAM, ICAM, VCAM, and N‐cadherin during mouse liver development and in fetal liver cell cultures. NCAM was transiently expressed in mesenchymal cells of the septum transversum and sinusoidal cells in liver development. In vitro studies demonstrated that desmin‐positive stellate cells expressed this cell adhesion molecule. NCAM expression in periportal biliary epithelial cells and connective tissue cells also coincided well with bile duct remodeling processes in the perinatal periods. Expression of ICAM and VCAM was transiently restricted to hepatoblasts, hepatocytes and hemopoietic cells in fetal stages. N‐cadherin was expressed not only in hepatoblasts and hepatocytes, but also in nonparenchymal cells such as endothelial cells, stellate cells and connective tissue cells, however the expression was weak. These results suggest that each cell adhesion molecule may play an important role during development in hepatic histogenesis, including hepatoblast/hepatocyte‐stellate cell interactions, hemopoiesis, and bile duct morphogenesis. Anat Rec 293:1698–1710, 2010. © 2010 Wiley‐Liss, Inc.     

Morphometric Evaluation of Pulmonary Lesions in Rats Exposed to Ozone

Four hours exposure to 3 ppm ozone produces morphometrically quantifiable alterations in the lungs of rats. Lungs of exposed rats were fixed either immediately after exposure or after a 12-hour recovery period, and the tissue was evaluated using stereologic technics with both the light and electron microscopes. Immediately after exposure, the volume fraction of nonparenchymal tissue was larger (0.057) than for controls (0.045); that of the lumina of large vessels decreased (0.039) from the control value (0.059). The nonparenchymal tissue fraction was 0.053 12 hours later, while the luminal fraction (0.054) resembled the control value. The volume fractions of parenchyma, total tissue and air space showed only slight changes. Ozone also affects all three components of the air-blood barrier, primarily by the production of intracellular edema. The arithmetic mean thickness of the pulmonary blood-air barrier increased from 1.385 ± 0.095 μ in unexposed rats to 1.700 ± 0.189 μ in rats immediately after exposure. The primary increase was in thickness of membranous epithelial and capillary endothelial cells. Partial recovery of total barrier thickness (1.582 ± 0.292 μ) occurred 12 hours after exposure.     

Hepatic organoid formation in collagen sponge of cells isolated from human liver tissues

We examined whether small hepatocytes (SHs), which are hepatic progenitor cells, could be isolated from a normal human liver and whether human hepatic cells could form hepatic organoids in a collagen sponge. Normal liver tissues were obtained from resected specimens from nine patients who underwent hepatic resection. Isolated hepatic cells were plated on dishes and a collagen sponge. More than 1 month later, SH-like cells appeared and proliferated on the dishes, whereas cell aggregates were formed in the sponge and showed characteristic tissue architecture: columnar and/or cuboidal epithelial cells lined the surface of the sponge. Clusters of epithelial cells with a large cytoplasm and ductular structures were observed under the lining cells. The lining and ductular cells were positive for cytokeratins 7 and 19, which indicated they were biliary epithelial cells (BECs), and the epithelial cells forming clusters were positive for the anti-human hepatocyte antibody, identifying them as hepatocytes. Some lining cells were positive for both the hepatic marker and the BEC markers. The cells in the collagen sponge actively proliferated and the hepatocytes excreted albumin into the medium. Thus, hepatic organoids could be reconstructed in a collagen sponge by normal human liver cells.     

Interaction between monensin and CCl4 after chronic treatment: dolichol and retinol content of rat liver sinusoidal cells

The aim of this investigation was to study retinol, which is known to decrease in hepatic stellate cells during fibrogenesis, and dolichol, which influences membrane fluidity and decreases in liver injury, in freshly isolated liver parenchymal and nonparenchymal cells after intoxication of rats with CCl4 combined with the ionophore monensin for 3, 5 and 7 weeks. To study the interrelationship between dolichol and vitamin A transport, a load of vitamin A was given to batches of rats 3 days before sacrifice. Monensin did not modify the action of CCl4 in hepatocytes. On administration of CCl4 and CCl4-monensin, dolichol decreased independently of vitamin A load, while retinol increased, especially when a load of vitamin A was given to rats 3 days before sacrifice. Hepatocytes appeared to no longer be able to export or metabolize vitamin A. In a subfraction of hepatic stellate cells (Ito-1 cells) dolichol always decreased, while retinol was no longer stored after each treatment; dolichol and retinol showed the same behavior but the decrease was more pronounced in monensin after vitamin A load and after 3 weeks. These data support the hypothesis that by modulating membrane characteristics, dolichol might be involved in intracellular or intercellular retinol transport and that altered transport between hepatocytes and Ito-1 cells might accompany liver injury. The data regarding another subfraction, Ito-2 cells, partly resemble those for the Ito-1 fraction and are in agreement with the heterogeneity of hepatic stellate cells. In Kupffer and sinusoidal endothelial cells, dolichol and retinol content was not homogeneous and was only slightly altered after the treatments. Monensin and CCl4 are not interactive. Although both drugs alter membrane lipids, their association allows some sinusoidal cell responses to be differentiated.     

A novel monoclonal antibody identified hepatic stem-like cells in rats

Both liver epithelial and oval cells are believed to be liver stem cells. We investigated the identification by producing monoclonal antibodies against liver epithelial cells. Monoclonal antibodies against hepatic stem-like cells (HSL cells) have been selected to follow the hepatic stem cells during hepatic regeneration and developmental changes in the liver. Monoclonal antibodies were induced by immunization of BALB/c mice with HSL cells established from the epithelial cells of the adult rat liver. The hybridomas were screened by indirect immunofluorescence staining of HSL cells. We produced a unique monoclonal antibody against HSL cells, MabH, which specifically recognizes liver epithelial cells. MabH did not react with liver parenchymal cells but did react with bile ductule cells under normal conditions in the adult liver. This antibody also reacted with oval cell lines and with the oval cells that appeared during liver regeneration. In addition, fetal liver cells showed immunoreactivity with MabH. Although the level of staining decreased after birth, some cells in the portal area remained highly reactive. These results suggested that liver epithelial cells, oval cells, and fetal liver cells possess a common cell marker of liver stem cells.