Mannose 6-Phosphate/Insulin-like Growth Factor–II Receptor Targets the Urokinase Receptor to Lysosomes via a Novel Binding Interaction

The urokinase-type plasminogen activator receptor (uPAR) plays an important role on the cell surface in mediating extracellular degradative processes and formation of active TGF-β, and in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. We have searched for mechanisms that determine the cellular location of uPAR and may participate in its disposal. When using purified receptor preparations, we find that uPAR binds to the cation-independent, mannose 6-phosphate/insulin-like growth factor–II (IGF-II) receptor (CIMPR) with an affinity in the low micromolar range, but not to the 46-kD, cation-dependent, mannose 6-phosphate receptor (CDMPR). The binding is not perturbed by uPA and appears to involve domains DII + DIII of the uPAR protein moiety, but not the glycosylphosphatidylinositol anchor. The binding occurs at site(s) on the CIMPR different from those engaged in binding of mannose 6-phosphate epitopes or IGF-II. To evaluate the significance of the binding, immunofluorescence and immunoelectron microscopy studies were performed in transfected cells, and the results show that wild-type CIMPR, but not CIMPR lacking an intact sorting signal, modulates the subcellular distribution of uPAR and is capable of directing it to lysosomes. We conclude that a site within CIMPR, distinct from its previously known ligand binding sites, binds uPAR and modulates its subcellular distribution.     

Endothelial binding of transferrin in fractionated liver cell suspensions

Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that hepatocytes have transferrin receptors. When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions. Neither hepatocytes nor Kupffer cells contained transferrin receptors. In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity. Incubation of these fractions with 125I- or 59Fe-labeled transferrin led to exclusive binding to endothelial cells but not hepatocytes nor Kupffer cells. Kinetic analysis demonstrated Kd of 1.9 X 10(-7) M, Bmax of 3.1 pmol/10(6) cells per min, corresponding to 2.1 X 10(5) molecules/cell per min. At 4 degrees C, the binding reached a steady-state plateau within 5 min. Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells. Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells.     

Distribution of insulin receptors in liver cell suspensions using a minibead probe : Highest density is on endothelial cell

The distribution of insulin receptors was studied in rat liver cell suspensions using a latex minibead covalently bound to insulin. This probe can be visualized by electron microscopy (EM). Using this visual probe, the highest density of the receptor was found on endothelial cells in the cell suspension, with hepatocytes having only few receptors and Kupffer cells having none. Fractionation of liver cell suspensions on metrizamide gradients yielded two populations of cells; large cells (hepatocytes) and small cells which consisted mostly of Kupffer cells and endothelial cells, distinguishable by their surface and cytoplasmic features. Again, by the use of an insulin-minibead probe, the highest density of receptors was found on endothelial cells. It is suggested that the endothelium has a crucial role in the uptake and transport of the hormone across the tissue-blood barrier.     

Cell surface-expressed cation-independent mannose 6-phosphate receptor (CD222) binds enzymatically active heparanase independently of mannose 6-phosphate to promote extracellular matrix degradation

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate, an important structural component of the extracellular matrix (ECM) and vascular basement membrane (BM). The cleavage of heparan sulfate by heparanase-expressing cells, such as activated leukocytes, metastatic tumor cells, and proliferating endothelial cells, facilitates degradation of the ECM/BM to promote cell invasion associated with inflammation, tumor metastasis, and angiogenesis. In addition to its enzymatic function, heparanase has also recently been shown to act as a cell adhesion and/or signaling molecule upon interaction with cell surfaces. Despite the obvious importance of the mechanisms for the binding of heparanase to cell surfaces, the receptor(s) for heparanase remain poorly defined. In this study, we identify the 300-kDa cation-independent mannose 6-phosphate receptor (CIMPR) as a cell surface receptor for heparanase. Purified platelet heparanase was shown to bind the human CIMPR expressed on the surface of a transfected mouse L cell line. Optimal binding was determined to be at a slightly acidic pH (6.5-7.0) with heparanase remaining on the cell surface for up to 10 min at 37 degrees C. In contrast, mouse L cells or Chinese hamster ovary cells expressing the cation-dependent mannose 6-phosphate receptor (CDMPR) showed no binding of heparanase. Interestingly, the binding of heparanase to CIMPR was independent of Man-6-P moieties. Significantly, primary human T cells upon activation were shown to dramatically up-regulate levels of cell surface-expressed CIMPR, which showed a concomitant increase in their capacity to bind heparanase. Furthermore, the tethering of heparanase to the surface of cells via CIMPR was found to increase their capacity to degrade an ECM or a reconstituted BM. These data suggest an important role for CIMPR in the cell surface presentation of enzymatically active heparanase for the efficient passage of T cells into an inflammatory site and have implications for the use of this mechanism by other cell types to enhance cell invasion.     

Early-phase redistribution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

It has been shown that the treatment with 3-[2-(diethylamino)ethoxy] androst-5-en-17-one (U18666A) causes the accumulation of cholesterol and the cation-independent mannose 6-phosphate receptor (CIMPR) in late endosomal/lysosomal compartments in BHK cells. The present study reports on a study of the effect of U18666A on CIMPR distribution in more detail in HeLa cells. When cells were treated with U18666A for 20 h, the intense perinuclear signal for CIMPR corresponding to the trans-Golgi network (TGN) disappeared and lamp1-negative punctate signals, scattered in the perinuclear region were detected. CIMPR then began to accumulate in lamp1-positive compartments 48 h after addition of the drug. Double immunofluorescence microscopy showed that U18666A-induced mannose 6-phosphate receptor-containing compartments (U-MPRCs), which were formed in the early phase of the redistribution, contained no marker for the TGN, late endosomes or lysosomes. Approximately half of the structures contained transferrin that had been internalized for 20 min, and cathepsin D, the majority of which appeared to be its precursor form. Immunoelectron-microscopic analysis revealed that U-MPRCs are composed of multivesicular bodies, irregularly shaped structures, and vesicular structures adjacent to the multivesicular bodies. These results suggest that U18666A treatment primarily suppresses the CIMPR transport pathways to late endosomes and from transferrin-containing endosomes, both of which may be dependent on cholesterol function.This work was supported by grants from Japan Ministry of Education, Culture, Sports, Science, and Technology.     

Carcinoma autoantigens T and Tn and their cleavage products interact with Gal/GalNAc-specific receptors on rat Kupffer cells and hepatocytes

We studied interactions of isolated Thomsen-Friedenreich (T)- and Tn-specific glycoproteins with the Gal/GalNAc-specific receptors on rat Kupffer cells and compared them to those with rat hepatocytes. Immunoreactive T and Tn are specific pancarcinoma epitopes. Electron microscopy of gold-labelled T and Tn antigens revealed their specific binding to Kupffer cells, followed by their uptake via the coated pit/vesicle pathway of receptor-mediated endocytosis. Preincubation of Kupffer cells with GalNAc and GalNAc-BSA, but not GlcNAc or GlcNAc-BSA specifically inhibited binding of the T and Tn glycoproteins. Desialylated, isologous erythrocytes (T RBC) are known to bind to the Gal/GalNAc receptors of rat Kupffer cells and hepatocytes. This attachment was specifically inhibited by T and Tn in a concentration-dependent manner: 50% T RBC-Kupffer cell contacts were inhibited at 8.5.10(-6) mM T and 8.5.10(-5) mM Tn antigen concentrations, respectively. The corresponding figures for hepatocytes were 6.10(-6) mM T and 1.2.10(-6) mM Tn antigen. Amino-terminal cleavage products of the T glycoprotein, possessing clusters terminating in non-reducing Gal/GalNAc, inhibited T RBC binding to Kupffer cells and hepatocytes usually at 10(-2) to 10(-5) mM concentrations, whereas GalNAc, galactose and galactose glycosides inhibited at millimolar concentrations. Galactose-unrelated carbohydrates were inactive at concentrations greater than or equal to 50 mM.     

The effect of a conditioned medium from neonatal rat hepatocytes on a mixed culture of Kupffer cells and fibroblast-like liver cells]

The study was carried out on primary coculture Kupffer cells and liver fibroblasts of newborn and mature rats. At first Kupffer cells and liver fibroblasts were taken on the equal quantity. There were observed significant decrease of Kupffer cells quantity and fibroblasts death after 4-5 days in coculture. Mitotic and functional activity liver fibroblasts, adhesion of Kupffer cells were increased by the using of conditioned medium of newborn rat hepatocytes. The rise of mitotic activity of liver fibroblasts and quantity of nucleolus in their nuclei forestalls of the increase Kupffer cells size, appearing of Kupffer cells with some nuclei.     

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.     

Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

大鼠再生肝8种细胞的丝氨酸族氨基酸代谢相关基因的转录谱

为了解大鼠肝再生中8种肝脏细胞的丝氨酸族氨基酸代谢相关基因转录谱, 文章用Percoll密度梯度离心结合免疫磁珠分选分离大鼠的8种再生肝细胞, 用Rat Genome 230 2.0芯片等检测它们中丝氨酸族氨基酸代谢相关基因的表达变化, 用Cluster和Treeview等软件分析上述基因在肝再生中表达模式, 用生物信息学和系统生物学等方法分析上述细胞中丝氨酸族氨基酸代谢活动。结果表明, 在27个发生有意义表达变化的基因中, 肝细胞、胆管上皮细胞、卵圆细胞、肝星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞的基因数分别为13、16、11、14、13、11、12、14, 相应细胞的上调、下调和上/下调的基因数分别为7、6和0, 2、10和4, 2、8和1, 8、3和3, 6、5和2, 4、6和1, 2、10和0, 6、6和2。总的来看, 肝再生中各细胞的表达下调基因占优势, 但在肝再生启动阶段, 肝星形细胞和窦内皮细胞的表达上调基因占优势。上述丝氨酸族氨基酸代谢相关基因转录谱预示丝氨酸族氨基酸的合成主要在肝再生启动阶段的肝细胞、肝星形细胞、窦内皮细胞和库普弗细胞中增强, 它们的降解主要在肝再生进展阶段的肝细胞、胆管上皮细胞、陷窝细胞和树突状细胞中进行。     

Stimulation of adenylate cyclase from isolated hepatocytes and Kupffer cells.

Hepatocytes and Kupffer cells were separated from rat liver after prelabeling the Kupffer cells with colloidal iron and perfusion of the liver with digestive enzymes. The activity of several enzymes from Kupffer cells and hepatocytes was compared to validate this method of cell separation. The ratios of hepatocyte to Kupffer cell specific activities of glucose-6-phosphatase, 5'-nucleotidase, adenylate cyclase, and acid phosphatase were 20, 0.39, 0.18, and 0.078, respectively. Adenylate cyclases from hepatocytes and Kupffer cells were stimulated by fluoride ion, GTP, and catecholamines. Hepatocyte adenylate cyclase was also stimulated by glucagon, secretin, vasoactive intestinal polypeptide, and by prostaglandin E1, whereas, the Kupffer cell enzyme was completely insensitive to these hormones. The stimulation of hepatocyte adenylate cyclase by combinations of glucagon plus secretin, or glucagon plus vasoactive intestinal polypeptide, were equivalent to the sum of the individual stimulations. This suggests that the hepatocyte has specific receptors for glucagon and for vasoactive intestinal polypeptide and secretin. Prostaglandin E1 stimulation of hepatocyte adenylate cyclase was not additive to the stimulation caused by polypeptide hormones or catecholamines, nor did prostaglandin E1 decrease stimulation caused by these hormones. Although prostaglandin-sensitive adenylate cyclase was recovered with hepatocytes, 40 to 50% of the total liver prostaglandin-sensitive activity was recovered in a fraction of cell debris mixed with small cells which did not phagocytize colloidal iron.     

Lectin binding sites in normal and phenobarbitale/halothane treated rat liver. A histochemical study

The content of carbohydrate residues of both normal and phenobarbitale-halothane-hypoxia exposed rat liver has been examined by means of lectin histochemistry. Eight biotinylated lectins specific to galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose and mannose were applied to paraffin sections of rat liver at light microscopic level. The most distinct binding was observed at the structures of the "perisinusoidal functional unit": Kupffer cells are bound by S-WGA, SBA and PNA. Bile canaliculi display binding sites for RCA I and WGA. Cytoplasm of hepatocytes appears lectin-negative, except for PSA. The enhanced reaction of S-WGA, PNA and SBA after the preincubation of the sections with neuraminidase indicates the occurrence of sialic acid in Kupffer cells. The phenobarbitale-halothane-hypoxia exposed rat liver shows centrolobular degeneration of hepatocytes with a diminished amount of hepatocyte and Kupffer cells as well. The lectin binding pattern of sinusoidal walls, membranes of hepatocytes and bile canaliculi remains the same compared to that of normal rat liver. This finding suggests that at least the carbohydrate content of membranes in the liver resists severe destruction under phenobarbitale-halothane-hypoxia. It is assumed that there exists a connection between intact carbohydrate residues and the regeneration of liver parenchyma.     

Quantification and regulation of apolipoprotein E expression in rat Kupffer cells

Apolipoprotein E (apoE) is synthesized by a wide variety of cells including cells of the monocyte-macrophage lineage. In order to assess the quantitative significance of apoE synthesis in a mature tissue macrophage, apoE synthesis was compared in Kupffer cells and hepatocytes isolated from rat liver. Immunoreactive apoE synthesized by both cell types exhibited identical isoform patterns when examined by high-resolution two-dimensional gel analysis. ApoE synthesis was not detected in hepatic endothelial cells. Northern blot analysis using a rat apoE cDNA probe demonstrated a single mRNA species of approximately 1200 nucleotides in freshly isolated hepatocytes and Kupffer cells. The absolute content of apoE mRNA in each cell type was determined with a DNA-excess solution hybridization assay. The apoE mRNA content (pg/microgram RNA) for Kupffer cells and hepatocytes was 35.7 and 98.8, respectively. Accounting for cellular RNA content and the population size of each cell type in the liver, Kupffer cells were calculated to contain about 0.7% of liver apoE mRNA; hepatocytes account almost quantitatively for the remainder. These results suggest that Kupffer cells are not major contributors to the plasma apoE pool. After intravenous injection of bacterial endotoxin, apoE mRNA was decreased in freshly isolated Kupffer cells whereas whole liver showed no change in apoE mRNA. Endotoxin treatment had no effect on the apoE mRNA content in several peripheral tissues. These results indicate that apoE expression in vivo is differentially regulated by endotoxin in Kupffer cells as compared to hepatocytes or apoE-producing cells in peripheral tissues.     

Modulation of cell surface expression of liver carbohydrate receptors during in vivo induction of apoptosis with lead nitrate

Cell surface expression of carbohydrate receptors (i.e. mannose and galactose receptors) and phagocytosis of apoptotic cells by sinusoidal liver cells was studied. Binding sites and phagocytic activity were quantified at different time intervals (1, 3, 5, 7, 9, 11, 13, 15, 20, 30, 40 and 60 days) after the in vivo administration to rats of a potent liver mitogen, lead nitrate, that also induces apoptosis. The number and distribution of binding sites was receptor and cell-type dependent during the days following the metal injection. The use of competing saccharides in inhibition uptake experiments suggests that sinusoidal liver cells actively phagocytose apoptotic hepatocytes and circulating apoptotic cells by using both receptors. In particular, Kupffer cells at 5 and 15 days after the lead nitrate injection are very active in internalizing apoptotic cells (two- to threefold control). However, phagosomes containing apoptotic hepatocytes are often seen inside the cytoplasm of parenchymal and endothelial cells.     

Cytokeratin 14 expression in rat liver cells in culture and localization in vivo

Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES6+ cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.     

利用NASH大鼠原代肝细胞与原代Kupffer细胞共培养建立NASH原代细胞模型*

目的: 通过分离并提纯非酒精性脂肪性肝炎(NASH)大鼠原代肝细胞以及原代Kupffer细胞建立体外NASH原代细胞模型,为研究NASH提供可靠的细胞实验技术支持。方法: 选择SD大鼠40只,随机分为2组(n=20):对照组和NASH组,对照组大鼠利用普通饲料喂养,NASH组大鼠利用高脂饲料(88%基础饲料+10%猪油+ 2%胆固醇)喂养,6～8周后,利用NASH评分表,病理观察下肝组织切片脂肪变+小叶内炎症+气球样变评分≥4 分,表明大鼠NASH模型的成功建立,利用胶原酶原位灌注法分离并提纯NASH模型大鼠原代肝细胞以及原代Kupffer细胞,利用CK-18及CD68免疫荧光以及墨汁吞墨实验进行细胞鉴定,利用油红O染色、试剂盒测定谷丙转氨酶(ALT)、谷草转氨酶(AST)含量观察NASH大鼠原代肝细胞脂质累积和肝功情况,Western blot检测原代Kupffer细胞炎症因子表达情况,最后采用原代肝细胞:原代Kupffer细胞=6∶1比例共培养,显微镜下观察细胞状态。结果: 实验成功分离并提纯NASH原代肝细胞以及原代Kupffer细胞,通过油红O染色,NASH组大鼠原代肝细胞存在明显的脂肪沉积,且NASH组大鼠原代肝细胞中AST、ALT明显高于对照组,存在明显肝损伤(P＜0.05),Western blot测定原代Kupffer细胞TNF-α、IL-1β以及MCP-1,NASH组大鼠明显高于对照组(P＜0.05)。结论: 通过胶原酶原位灌注法可以成功分离NASH大鼠原代肝细胞以及原代Kupffer细胞,同时成功建立比例共培养大鼠体外原代细胞NASH模型。     

Hepatocyte innervation in primates

The efferent innervation and some characteristics of nerve fibers of the liver lobule in the tree shrew, a primate, are described. Nerve endings on hepatocytes were encountered regularly and were determined to be efferent adrenergic nerves. Transmission electron microscopy revealed nerve endings and varicosities in close apposition to the hepatocytes adjacent to the connective tissue of the triads as well as within the liver lobule in the space of Disse. Fluorescence microscopy indicated the existence of adrenergic nerves with a similar distribution. Autoradiography of the avid uptake of exogenous [3H]norepinephrine indicated that all intralobular nerves are potentially norepinephrinergic (adrenergic). Chemical sympathectomy with 6-OH-dopamine resulted in the degeneration of all intralobular liver nerve fibers as revealed by fluorescence microscopy and electron microscopy. Substantial regeneration occurred after 60-90 days but was not completed by that time. Some nerves were also observed in close association with von Kupffer cells and endothelial cells. The functional significance of the efferent liver innervation is discussed.     

Cellular organization of normal mouse liver: a histological,quantitative immunocytochemical,and fine structural analysis

The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 μm) and small (0.03 μm) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately, 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function.