TRANSCELLULAR BIOSYNTHESIS OF CYSTEINYL LEUKOTRIENES BY KUPFFER CELL—HEPATOCYTE COOPERATION IN RAT LIVER

We previously proposed that an enzymatic cooperation between Kupffer cells and hepatocytes may play an important role in cysteinyl leukotriene (LT) production in rat liver. Anin vitrotranscellular synthesis cysteinyl LTs by a Kupffer cell—hepatocyte coculture system was characterized here. Kupffer cells alone, with A23187 stimulation, did not generate cysteinyl LTs until supplemented either with isolated hepatocytes or with LTC₄synthase and glutathione, indicating that Kupffer cells can synthesize LTA₄but not convert it into LTC₄. In contrast, hepatocytes converted the LTA₄into cysteinyl LTs and further degraded the cysteinyl LTs. Cysteinyl LT production by the Kupffer cell—hapatocyte coculture system was optimized by addition of 1–3% serum albumin to the culture and by bringing the cell—cell distance closer to less than 3μ. Tumour necrosis factor also stimulated cysteinyl LT production by the coculture system. From these results, it is expected that the Kupffer cell—hepatocyte transcellular system for cysteinyl LT production actually functionsin vivo.     

Opposite Cross-Talk by Oleate and Palmitate on Insulin Signaling in Hepatocytes through Macrophage Activation

Chronic low grade inflammation in adipose tissue during obesity is associated with an impairment of the insulin signaling cascade. In this study, we have evaluated the impact of palmitate or oleate overload of macrophage/Kupffer cells in triggering stress-mediated signaling pathways, in lipoapoptosis, and in the cross-talk with insulin signaling in hepatocytes. RAW 264.7 macrophages or Kupffer cells were stimulated with oleate or palmitate, and levels of M1/M2 polarization markers and the lipidomic profile of eicosanoids were analyzed. Whereas proinflammatory cytokines and total eicosanoids were elevated in macrophages/Kupffer cells stimulated with palmitate, enhanced arginase 1 and lower leukotriene B₄ (LTB₄) levels were detected in macrophages stimulated with oleate. When hepatocytes were pretreated with conditioned medium (CM) from RAW 264.7 or Kupffer cells loaded with palmitate (CM-P), phosphorylation of stress kinases and endoplasmic reticulum stress signaling was increased, insulin signaling was impaired, and lipoapoptosis was detected. Conversely, enhanced insulin receptor-mediated signaling and reduced levels of the phosphatases protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homolog (PTEN) were found in hepatocytes treated with CM from macrophages stimulated with oleate (CM-O). Supplementation of CM-O with LTB₄ suppressed insulin sensitization and increased PTP1B and PTEN. Furthermore, LTB₄ decreased insulin receptor tyrosine phosphorylation in hepatocytes, activated the NFκB pathway, and up-regulated PTP1B and PTEN, these effects being mediated by LTB₄ receptor BTL1. In conclusion, oleate and palmitate elicit an opposite cross-talk between macrophages/Kupffer cells and hepatocytes. Whereas CM-P interferes at the early steps of insulin signaling, CM-O increases insulin sensitization, possibly by reducing LTB₄.     

Different immunolocalizations of cathepsins B, H, and L in the liver

Different localizations of cathepsin B, H, and L in normal rat liver were revealed immunohistochemically with anticathepsin Fab'-horseradish peroxidase conjugates. Staining of cathepsin B was strong in the periportal sinusoids, possibly in Kupffer cells; and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of the cytoplasm, possibly representing the peribiliary dense bodies; and weaker in periportal sinusoidal cells, possibly Kupffer cells. Staining of cathepsin L was strongest in centrilobular hepatocytes and weaker in periportal sinusoidal cells, possibly Kupffer cells. These findings, revealed for the first time in the present study, show that the histologic and intracellular localizations of the three cathepsins are different, suggesting that they have different roles in degradation of exogenous and endogenous proteins.     

Distribution of hepatic phosphofructokinase isozymes in parenchymal and sinusoidal cells.

The distribution profile of the isozymes of phosphofructokinase (PFK) in different cell types of rat liver is established using the techniques of electrophoresis and immunodiffusion. Agarose gel electrophoresis of the extracts of parenchymal cells, Kupffer or sinusoidal cells, and whole liver indicated that two PFK isozymes are present in whole liver and that the faster moving hepatic PFK isozyme is present only in parenchymal cells; whereas, the slower moving hepatic PFK isozyme is only in sinusoidal cells. Immunodiffusion studies using antiserum specific for the major hepatic PFK isozyme (PFK-L₂) revealed that PFK-L₂ is present only in whole liver or parenchymal cell extracts and is absent from sinusoidal cells. It is apparent that the other hepatic PFK isozyme (PFK-L₁) is normally found only in sinusoidal cells.     

Cholera toxin‐sensitive GTP‐binding protein‐coupled activation of augmenter of liver regeneration (ALR) receptor and its function in rat kupffer cells

Mitogenic effect of augmenter of liver regeneration (ALR), a protein produced and released by hepatocytes, on hepatocytes in vivo but not in vitro suggests that the effect is mediated by nonparenchymal cells. Since mediators produced by Kupffer cells are implicated in hepatic regeneration, we investigated receptor for ALR and its functions in rat Kupffer cells. Kupffer cells were isolated from rat liver by enzymatic digestion and centrifugal elutriation. Radioligand ([¹²⁵I] ALR) receptor binding, ALR‐induced GTP/G‐protein association, and nitric oxide (NO), tumor necrosis factor (TNF)‐α, and interleukin‐6 (IL‐6) synthesis were determined. High‐affinity receptor for ALR, belonging to the G‐protein family, with K_d of 1.25 ± 0.18 nM and B_max of 0.26 ± 0.02 fmol/µg DNA was identified. ALR stimulated NO, TNF‐α, and IL‐6 synthesis via cholera toxin‐sensitive G‐protein, as well as p38‐MAPK activity and nuclear translocation of NFκB. While inhibitor of NFκB (MG132) inhibited ALR‐induced NO synthesis, MG132 and p38‐MAPK inhibitor (SB203580) abrogated ALR‐induced TNF‐α and IL‐6 synthesis. ALR also prevented the release of mediator(s) from Kupffer cells that cause inhibition of DNA synthesis in hepatocytes. Administration of ALR to 40% partially hepatectomized rats increased expression of TNF‐α, IL‐6, and inducible nitric oxide synthase (iNOS) and caused augmentation of hepatic regeneration. These results demonstrate specific G‐protein coupled binding of ALR and its function in Kupffer cells and suggest that mediators produced by ALR‐stimulated Kupffer cells may elicit physiologically important effects on hepatocytes. J. Cell. Physiol. 222: 365–373, 2010. © 2009 Wiley‐Liss, Inc.     

Effect of d-galactosamine administration on nucleotide and protein metabolism in isolated rat Kupffer cells.

The nucleoti-e contents of isolated rat Kupffer cells were found to be smaller than those of hepatocytes. The rate of UDPGal formation from D-galactose was much lower in Kupffer cells than in hepatocytes. The viability of the former was checked by measuring the leakage of enzymes and the formation of UTP from uridine. Addition of GalN to isolated Kupffer cells did not decrease their UTP and UDPG contents as much as those of hepatocytes. The same results were obtained when cells were isolated from GalN-pretreated animals. The incorporation of labeled amino acids into protein after GalN addition was much less reduced in Kupffer cells than in hepatocytes. The data suggest that Kupffer cells do not contribute to GalN-induced liver injury as a result of uridylate trapping.     

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.     

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.     

Electron histochemical detection of intracellular and extracellular cathepsin D in the liver

Cathepsin D localization was studied in the liver of white rats by ultrastructural cytochemistry. It was shown that the product of reaction was present in lysosomes of hepatocytes, Kupffer's and endothelial cells and in fibroblasts from portal tracts am small granules or their conglomerate of different electron density. The lowest activity of cathepsin D was observed in hepatocytes, the most intensive reaction--in Kupffer cells. The extracellular activity of cathepsin D in vivo was revealed. It means that besides participation in intracellular degradation of different proteins, cathepsin D is secreted to extracellular space by liver cells (hepatocytes, Kupffer cells, fibroblasts) and it may participate in catabolism of intercellular matrix.     

The plasma clearance of human α2-macroglobulin-trypsin complex in the rat is mainly accounted for by uptake into hepatocytes

Rats were given intravenous injections of ¹²⁵I-labelled human α₂-macroglobulin·trypsin. The half-time of disappearance of radioactivity from arterial blood was 2 min. External counting showed that radioactivity in the liver was maximal by 10 min and then decreased slowly. 87% of the injected dose was recovered in the liver by 10 min. Light- and electron microscopic autoradiography carried out on samples of liver fixed with glutaraldehyde 3 min or 30 min after the injection showed that 85–90% of the grains were over the hepatocytes and 4–9% were over the Kupffer cells. Thus, uptake into hepatocytes, and not into Kupffer cells as believed previously, appears to account for the major part of the uptake of α₂-macroglobulin·trypsin by the liver and thereby for its rapid removal from the blood.     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.     

Distribution of lysosomal enzymes in different types of rat liver cells.

Eight lysosomal enzymes were measured in different types of rat liver cells. Hepatocytes were purified by low speed centrifugation of a cell suspension obtained by treating the perfused liver with collagenase. Nonparenchymal cells (NPC) were purified by centrifugation after treating the initial cell suspension with pronase, which selectively destroys the parenchymal cells (PC). Kupffer cells were found to attach selectively to tissue culture dishes after overnight culture of an NPC suspension. The specific activity of lysosomal enzymes was generally higher in NPC than in hepatocytes, but the different enzymes were concentrated to different degrees in the NPC. Specific activity of acid phosphatase was 1.7 times higher in NPC than in hepatocytes. Specific activity of acid DNAase, on the other hand, was 8 times higher in NPC than in hepatocytes. Other enzymes showed intermediate values. Assuming that 30% of the liver cells are nonparenchymal it may be calculated that from 7% (acid phosphatase) to 25% (acid DNAase) of the hepatic lysosomal enzymes are present in the NPC. The pattern of lysosomal enzymes in cultured Kupffer cells was similar to that of the NPC from which the Kupffer cells were derived. Cathepsin D and β-glucuronidase were, however, elevated in Kupffer cells as compared with NPC. The enzyme pattern in Kupffer cells was almost identical with that of rat peritoneal macrophages.     

中国大鲵肝脏的超微结构

应用透射电镜对中国大鲵的肝脏进行了超微结构研究.观察表明,大鲵肝不具肝小叶,与其他脊椎动物有所不同.肝细胞含有单个卵圆形的核;细胞质内含有粗面内质网、高尔基囊泡、线粒体、糖原颗粒和脂滴等细胞器和内含物.胆小管由两个相邻肝细胞质膜凹陷围成,而肝血窦则由内皮细胞的胞质构成.胆小管腔和窦周隙内浸润许多由肝细胞发出的微绒毛结构.还发现了枯否氏细胞和贮脂细胞.还讨论了中国大鲵肝脏的一般形态结构特点.     

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.     

Isolation of Kupffer cell lysosomes, with observations on their chemical and enzymic composition

The purpose of the present investigation was twofold: The isolation of Kupffer cell lysosomes by changing their density in vivo through uptake of colloidal silver iodide (Neosilvol^R), and the characterization of the isolated fraction. No significant changes in the activities or distribution of acid phosphatase, aryl sulphatase, and cathepsin D were found after the injection of Neosilvol^R. A method is presented for the isolation of silver-loaded lysosomes from rat liver Kupffer cells by means of ultracentrifugation in sucrose gradients. Morphological and biochemical data indicate that the lysosomal fraction was contaminated with other subcellular organelles only to a minor degree. The lysosomal fraction showed non-parallel enrichment of various acid hydrolases, with the highest degree of purification found for aryl sulphatase and the lowest for acid phosphatase. The lysosomal enzyme activity pattern was similar to that found in Kupffer cell preparations.     

Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes.When expressed as rates of hydrolysis per 10⁶ cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2–4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.