Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor beta and interleukin 6.

In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H-TdR uptake and the number of 3H-TdR-labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24-48 h. This action of TNF was abrogated by DNA polymerase alpha inhibitor, aphidicolin and blocked specifically by anti-TNF antibody. The actions of rmTNF and rhTNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2-fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL-6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor beta, which is known as a potent inhibitor of hepatocyte growth. Interferon gamma also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration.     

Galactose-specific receptors on liver cells. II. Characterization of the purified receptor from macrophages reveals no structural relationship to the hepatocyte receptor

We have isolated a galactose-specific receptor protein from rat liver macrophages by three techniques, all using EDTA extraction and subsequent affinity chromatography. The purified receptor has an apparent molecular mass of 30 kDa and exhibits hemagglutinating activity. Monospecific receptor-antisera produce one precipitation line with the macrophage receptor in Ouchterlony double diffusion but show no cross-reaction with the hepatocyte receptor. Sinusoidal cells, but not hepatocytes, are stained with monoclonal antibodies to the macrophage receptor, whereas anti-hepatocyte receptor antibodies stain hepatocyte surfaces but not sinusoidal cells. We conclude that the galactose-specific receptor from liver macrophages is structurally different from the hepatocyte receptor, although the two lectins share a similar binding specificity.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (Mr) 55,000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of Mr 52,000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55,000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types.(ABSTRACT TRUNCATED AT 400 WORDS)     

Study of insulin-receptor interactions in plasma membrane of rat liver using antibodies against insulin

Preincubation of rat liver cell plasma membranes with antiinsulin serum (AIS) results in a 3-fold increase of specific binding of [125I]insulin added to AIS-free membranes. The degree of such increase depends on preincubation time and AIS concentration; at a rise of both parameters the dependence curve shown a plateau. The Scatchard plots suggest that preincubation with AIS increases the high and low affinity of binding sites; their number remains thereby unchanged. Using several types of immune and non-immune sera for preincubation the specificity of this effect was established. It was assumed that the antibodies initiate the immunological reaction of the membrane. Study of AIS effect on dissociation of membrane-bound labelled insulins differing in their affinities for the given AIS revealed that the antibodies can interact with the newly formed hormone-receptor complex but cannot affect the receptor within insulin. It is concluded that the intact hepatocyte membrane contains a receptor-bound insulin which is a target of AIS action. The antigen-antibody interaction changes the functional state of occupied and free receptors which bind [125I]insulin. The latter becomes repeatedly involved in this process as a result of destabilization of site-site interactions provoked by antibodies against the hormones.     

Alteration in the regulation of plasma membrane glycoproteins of the hepatocyte during ontogeny

The expression of four integral membrane glycoproteins was examined in detail utilizing monospecific antibodies during liver development. These included asialoglycoprotein receptor, a hepatocyte glycoprotein residing in the sinusoidal domain, and three bile canalicular glycoproteins, leucine aminopeptidase, dipeptidyl peptidase IV, and a Mr 110,000 glycoprotein denoted GP 110. It was observed that asialoglycoprotein receptor, GP 110, and dipeptidyl peptidase IV were present in low amounts in fetal liver and reached adult levels between 1 to 3 weeks. In contrast, leucine aminopeptidase was present in nearly adult amounts in 18-day-old fetal livers. These observations were qualitatively confirmed by indirect immunofluorescent staining of frozen thin liver sections obtained from fetal and adult rats. Further, in fetal livers it was found that leucine aminopeptidase was not localized to typical bile canalicular areas. Immunoprecipitation studies performed in the presence of proteolytic inhibitors using detergent-solubilized extracts of metabolically labeled liver minces revealed that GP 110 was present in low amounts as Mr 110,000 and Mr 105,000 polypeptides in 17-day fetal livers but by 21 days of gestation the larger polypeptide was the major synthesis product. Conversely, the apparent molecular weights of leucine aminopeptidase and dipeptidyl peptidase IV were not altered during development. Experiments determining relative rates of synthesis using excess amounts of antibodies showed that the concentrations of the three bile canalicular glycoproteins in liver during ontogeny reflect their rates of synthesis. These results underscore that plasma membrane constituents of the hepatocyte undergo dramatic changes in expression and localization as the liver changes its physiological role at birth.     

Ontogeny of the mouse liver studied with hepatocyte membrane antigens and a comparison with alpha-fetoprotein synthesis

The mouse liver was studied during embryogenesis and postnatal development using indirect immunoperoxidase method with rat monoclonal antibodies to two membrane antigens of the hepatocytes. B10 antigen was first detected in the liver of 16 day old embryos on the hepatocyte membrane in the area of biliary capillary in the center of an acinar structure, thus reflecting the onset of formation of acinar structure in the liver. Formation of the liver beams after birth is accompanied by changes in the pattern of the antigen distribution--it is present on the lateral side of the hepatocyte membrane. D2 antigen is equally distributed over the hepatocyte membrane. When the acinar organization of the liver is replaced by the beam-like structure after birth, the antigen is redistributed--the part of the hepatocyte membrane facing circulatory sinus contains this antigen in a much greater quantity.     

Regulation of hepatocyte fate by interferon-γ

Interferon (IFN)-γ is a cytokine known for its immunomodulatory and anti-proliferative action. In the liver, IFN-γ can induce hepatocyte apoptosis or inhibit hepatocyte cell cycle progression. This article reviews recent mechanistic reports that describe how IFN-γ may direct the fate of hepatocytes either towards apoptosis or a cell cycle arrest. This review also describes a probable role for IFN-γ in modulating hepatocyte fate during liver regeneration, transplantation, hepatitis, fibrosis and hepatocellular carcinoma, and highlights promising areas of research that may lead to the development of IFN-γ as a therapy to enhance recovery from liver disease.     

A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice

The ability of hepatocytes to enter the cell cycle and regenerate the liver after tissue loss provides an in vivo model to study the regulation of proliferation and organ regeneration. The extent of hepatocyte proliferation is directly proportional to the amount of resected liver tissue, and 2/3 partial hepatectomy (2/3 PH) leads to highly synchronized hepatocyte cell-cycle entry and progression. This surgical technique was first described in rats and requires modification for application in mice. Lack of standardization of 2/3 PH in mice has caused discrepancies in the results obtained in different laboratories. Here, we provide a protocol and a movie describing a straightforward surgical technique, which takes 15-20 min, to consistently remove two-thirds of the liver in mice. As this protocol is not associated with mortality and gives highly reproducible results, we hope that it will be widely used and serve to standardize 2/3 PH in mice.     

Studies in vivo of the tissue uptake, cellular distribution and catabolic turnover of exogenous glucocerebrosidase in rat.

The kinetics of plasma clearance of highly purified human placental glucocerebrosidase in rats were biphasic with 75% of the infused dose showing a rapid clearance (t1/2 = 11 min) and the remaining 25% a considerably lower rate (t1/2 = 60 min). The majority of the enzyme (60%) was taken up by the liver. Although saturation kinetics for the clearance or uptake were not observed, the very high hepatic endocytic index (217 microliter/min) of glucocerebrosidase uptake indicated that liver uptake was mediated by an adsorptive endocytic process. Analysis of the cellular distribution of recovered glucocerebrosidase revealed predominantly parenchymal cell uptake with 38% of the exogenous enzyme in hepatocytes and only 2% in sinusoidal cells. High-mannose glycoproteins blocked hepatocyte and sinusoidal cell uptake of glucocerebrosidase equally. Kinetic experiments failed to demonstrate a transfer or shuttle of exogenous glucocerebrosidase from sinusoidal cells to hepatocytes. The possibility was raised that uptake of enzyme by the liver may be mediated by a common receptor that functions in both hepatocytes and sinusoidal cells. The catabolic turnover of exogenous glucocerebrosidase in rat liver was biphasic and the rate of decline was similar in hepatocytes and sinusoidal cells.     

Ultrastructural observations on the infection of rat liver by Plasmodium berghei sporozoites in vivo

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.     

Ultrastructural Observations on the Infection of Rat Liver by Plasmodium berghei Sporozoites In Vivo1

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.     

Cholangiocyte marker-positive and -negative fetal liver cells differ significantly in their ability to regenerate the livers of adult rats exposed to retrorsine

We have used monoclonal antibodies against cell-surface developmental epitopes in combination with micromagnetic beads to isolate phenotypically defined subpopulations of cholangiocyte marker-positive fetal liver epithelial cells (CMP-FLEC). Differentiation potential was evaluated by injecting cell isolates from dipeptidyl peptidase IV (DPPIV) positive (DPPIV+) Fischer donor rats into the spleen of partially hepatectomized, DPPIV negative (DPPIV-) Fischer host rats exposed to retrorsine. At various time points, liver tissue was harvested and cells in DPPIV+ colonies were phenotyped by immunofluorescence and histochemical protocols. Functional differentiation and liver replacement were determined by comparing donor and host hepatocyte protein expression patterns and DPPIV enzyme activity in extracts from livers of host rats receiving CMP-FLEC. Our results showed that bipotentiality was retained during differentiation and maturation of CMP-FLEC, indicating that the acquisition of ductal morphology and phenotype were not indicative of lineage commitment. CMP-FLEC transplanted into the adult rat liver lost ductal and gained hepatocyte markers, and acquired protein expression patterns in 2D gels with a close similarity (>75% spot match) to host hepatocytes but differing significantly from the transplanted CMP-FLEC cell isolate (<25% spot match). The average size of donor hepatocyte colonies increased with time so that by 1 year, up to 70% of the host rat liver was replaced by CMP-FLEC derived DPPIV+ hepatocytes. Depletion of CMP-FLEC from fetal liver isolates resulted in a marked decrease in adult liver colonization, suggesting that a high percentage of the hepatocyte colonies in animals receiving total fetal liver isolates are derived from CMP-FLEC.     

Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

We have used pulse-chase metabolic radiolabeling with L-[35S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates. Approximate half-times for arrival are in the range of 90-120 min for aminopeptidase N and dipeptidylpeptidase IV whereas only 15-20% of the mature radiolabeled HA 4 associated with the hepatocyte plasma membrane fraction has become apical even after 150 min of chase. Our results suggest a mechanism for hepatocyte plasma membrane biogenesis in vivo in which all integral plasma membrane proteins are shipped first to the basolateral domain, followed by the specific retrieval and transport of apical proteins to the apical domain at distinct rates.     

Antibodies to hepatocyte membrane antigens in chronic liver disease: detection by immunofluorescence after Bouin's fixation

Using a modified indirect immunofluorescent (IF) technique in which cryostat tissue sections were fixed in Bouin's solution for ten minutes prior to reaction with sera under test, we have looked for antibodies to the hepatocyte membrane (HMA) in the sera of patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). Samples were tested initially in parallel on unfixed and Bouin's-fixed rat composite blocks (kidney, stomach, liver) at a titer of 1:100 and those found to be positive were diluted further and reexamined. Conventional unfixed sections of rat composite block showed no liver membrane immunofluorescence although antinuclear (ANA), mitochondrial (AMA), and smooth muscle antibodies (SMA) were detected as anticipated. By contrast, prior Bouin's fixation abolished most of the fluorescence due to ANA, AMA and SMA but resulted in brilliant fluorescence of rat hepatocyte membranes in eleven of twelve patients with CAH (93%) and all ten patients with PBC. Only one of 22 normals (5%), one of 20 with collagen-vascular diseases (5%), and one of seven with nonimmunologic liver disease (14%) were positive for this hepatocyte membrane antibody. Bouin's fixation prior to IF is a simple technique which appears to alter the hepatocyte membrane so that HMA become detectable. The strong association of HMA with CAH and PBC suggests that this test might be of value and may contribute towards a further understanding of the pathogenesis of these conditions.     

Immunolocalization of hepatocyte growth factor and its receptor (c-Met) during mouse liver development

Although hepatocyte growth factor (HGF) was discovered as a potent hepatotrophic factor responsible for liver regeneration and may involve some organ development in embryogenesis, it remains to be revealed what roles HGF plays in liver development. The present study was undertaken to determine which cells express HGF and its receptor c-Met and when c-Met is activated in mouse liver development by using immunoblotting and immunohistochemical techniques. HGF was detected in hepatocytes and non-parenchymal cells, including biliary epithelial cells, periportal connective tissue cells, megakaryocytes, endothelial cells, and sinusoidal cells, throughout liver development. Positive HGF immunostaining in hepatocytes increased during postnatal development, and reached the maximal level in the adult stage. c-Met protein was also expressed in hepatocytes throughout liver development, but maximal staining was obtained in 1- or 2-week-old livers. Phosphorylation of tyrosine residues in the c-Met beta chain also occurred in these stages. These results suggest that HGF signaling is implicated in hepatocyte growth during postnatal liver development, and its action could be in a paracrine mode; HGF produced by non-parenchymal cells such as sinusoidal cells acts on hepatocytes expressing c-Met receptors. Positive immunostaining in adult and postnatal hepatocytes may be derived from their blood clearance of HGF.     

Partial purification and characterisation of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy.

We have investigated the influences that nonparenchymal cells from regenerating rat liver exert on hepatocyte proliferation. When primary adult rat hepatocytes isolated from resting liver were co-cultured with nonparenchymal cells (NPCs) from resting liver of a different syngeneic animal, the proliferative response of hepatocytes to epidermal growth factor (EGF) was unaffected by the presence of NPCs. In the presence of NPCs taken from livers that had undergone partial hepatectomy 24 hours before (regen-NPCs), the response of hepatocytes from resting liver to EGF, TGF-alpha, and hepatocyte growth factor (HGF) was markedly inhibited. Inhibitory activity was not dependent on cell-to-cell contact, and conditioned-medium from regen-NPCs, but not normal NPCs, inhibited EGF-induced hepatocyte DNA synthesis by approximately 50%. After concentration by gel chromatography and lyophilisation, inhibition was 98%. The inhibitory activity migrated on SDS-PAGE gel electrophoresis with an apparent molecular weight of 14 to 17 kDa and was trypsin-sensitive but relatively heat-stable. The effects of blocking antibodies established that it was not TGF-beta 1, IL1-beta, or IL6. Investigations of regen-NPCs taken at different time points demonstrated that inhibitory activity was released into conditioned medium of cells harvested at 24 and 48 hours after partial hepatectomy, but not 10 or 72 hours. This powerful inhibitor of hepatocyte response to proliferogens is released by cultures of NPCs with a time course suggesting that it may be involved in terminating the surge of hepatocyte replication induced by partial hepatectomy.     

Monoclonal antibodies against tryptophanyl-tRNA-synthetase

Monoclonal antibodies designated as Am1 and Am2 were prepared against purified beef pancreas tryptophanyl-tRNA synthetase (EC 6.1.1.2). Both antibodies were able to bind the native enzyme in a solid-phase assay and to precipitate enzyme activity in immune complexes. Am2 inhibited the tryptophanyl-tRNA synthetase activity in ATP-[32P]pyrophosphate exchange and in tRNATrp aminoacylation reactions; Am1 had no influence on both the enzyme activity and the inhibiting action of Am2. Only Am2, not Am1, bound elastase-modified form of the enzyme which consists of two subunits shortened by 20 000 daltons from the N-end of the molecule. These results were interpreted as an evidence for non-overlapping of Am1 and Am2 antigenic determinants along the polypeptide chains of the enzyme.