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Two children, male y and female brothers, with a cholesterol ester storage disease are presented. Some pathogenic, clinical biochemical and histopathological aspects are commented. The ultrastructural hepatic finding of microcrystallized cholesterol in the Von Kupffer's cells was the determinant diagnostic parameter in both cases. The clinical expression and evolution was different, with a biggest functional impairement in the male, which was submitted to hepatic transplantation.

cDNA comprising the entire length of the rat Kupffer cell receptor (Mr = 88,000 and 77,000) for carbohydrates with an affinity for fucose and galactose was isolated, and its nucleotide sequence was determined. Receptor cDNA encoded a protein containing 550 amino acid residues with a Mr = 61,104. This Mr was consistent with the size of the deglycosylated receptor which was found to contain two polypeptides by gel electrophoresis with Mr = 58,000 and 52,000, respectively. Edman degradation of the receptor yielded a sequence which corresponded to amino acid residues 83-104 of the sequence derived from the cDNA. This confirmed that the cDNA which had been isolated corresponded to mRNA for the receptor and suggested that the smaller polypeptide in receptor preparations arises by proteolysis of the intact receptor. Amino acid composition of the receptor was nearly identical to that predicted by the cDNA. The Kupffer cell receptor was found to be homologous to other carbohydrate binding proteins including the hepatic receptors with different binding specificities. The Kupffer cell receptor also contained a series of 18 contiguous, homologous sequences with an average length of 14 residues.

Gross Cystic Disease Fluid Protein (GCDFP-15) is a 60,000 dalton glycoprotein isolated from human breast cyst fluid, composed of four 15,000 dalton monomers. Carbohydrate analysis indicates that each monomer has a single carbohydrate chain of the complex type. GCDFP-15 intravenously injected into rats showed a rapid circulatory clearance, the rate of clearance being faster in female animals [t1/2 = 12.8 (+/- 2.0) min. females, and 16.7 (+/- 2.6) min. males]. The major organs of clearance were the liver (70%) and kidneys (15%). Immunoperoxidase staining showed localization in Kupffer cells and the proximal convoluted tubules of the kidney. Removal of sialic acid from GCDFP-15 resulted in a more rapid clearance (t1/2 = 2.2 min) by the liver (85%). This clearance was inhibited by coinjection of asialo alpha 1 acid glycoprotein. About 3% of GCDFP-15 was excreted in bile with a transit time through the liver of 38 min. Examination of the uptake of GCDFP-15 by isolated rat Kupffer cells showed that yeast mannan, fucosylated BSA, and carcinoembryonic antigen (CEA) failed to inhibit uptake, though the binding of GCDFP-15 was clearly saturable. This suggests that a novel receptor system on the rat Kupffer cell may be responsible for GCDFP-15 clearance.

Turpentine injection into rats elicits enhanced secretion of acute phase proteins including alpha 2-macroglobulin (alpha 2M). Hypophysectomized rats, however, do not respond in this way unless dexamethasone is given together with turpentine. On the other hand, dexamethasone injection alone did not result in an induction of alpha 2M synthesis. When a medium of Kupffer cell cultures was added to hepatocytes, a dose-dependent stimulation of alpha 2M synthesis of up to 4-fold after 10-12 h was observed. However, the presence of low concentrations (10(-9)M) of dexamethasone was essential for the stimulatory effect. We conclude that the acute phase induction of alpha 2M in hepatocytes requires the synergistic action of glucocorticoids and a non-dialysable factor secreted by Kupffer cells.

Lipids administered by the intravenous route produced ultrastructural changes in the liver and spleen of mice. Treatment with tricaprin resulted in prominent and enlarged Kupffer cells possibly with increase numbers of dense bodies. Cholesterol oleate was found within Kupffer cells and splenic macrophages. The Kupffer cells were distended by lipid and obtruded on the sinusoidal lumen; similar changes were seen in the macrophages of the red pulp and marginal zone of the spleen. Cholesterol oleate delayed but did not prevent eventual phagocytosis or colloidal carbon; the spleen contained much more carbon than usual after treatment with this substance. Treatment with ethyl palmitate resulted in diminished retention of carbon in both liver and spleen; this lipid, unlike tricaprin or cholesterol oleate, was cytotoxic to both macrophages and lymphocytes.

In the case of 36 female patients who were anamnestically known to have taken laxatives, semiquantitative histological investigations with laparoscopically obtained liver needle biopsies were effected after the exposition with preparations containing phenolisatine. The time gap until exposition was 12 to 24 h (16 cases), 48 h (8 cases), 72 to 96 h (4 cases) and 7 to 14 days (4 cases). The histological result after the exposition is an acute cholangiolitis of the allergic-hyperergic type with edema and a dense eosinophile infiltration of the portal fields with destruction of the epithelium of preformed bile ducts and portally proliferated ductles. In addition, the parenchyma of the liver shows a pleomorphism of the cells in form and colour with a cellular edema and with disseminated acidophilic necroses and necrobioses of the individual cells as well as with little reactive proliferation of the Kupffer's cell. After a period of 8 days the acute process has more or less subsided. Also, in the majority of cases there are histological signs of an aggressive chronic hepatitis of type IIa, partially in the active stage with piece-meal necroses and partially stabilized or in the process of healing. A transition to the picture of hepatitic cirrhosis is possible. In serious cases the picture of a chronic non-purulent destructive cholangitis can be simulated by the hepatocellular and canalicular damage. Thirty-one bioptic pre-examinations from the same results, whereby the acute cholangiolitical exacerbation can be attributed to an exposition of the patients themselves. The clinical picture of the phenolisatine damage in its entirety is induced by medication and is described as a recurrent chronic cholangiohepatitis. Similarities exist between the liver damages caused by chlorpromazine and arsphenamine. When medication is discontinued, the morphologic substrate recedes leaving behind an inactive fibrosis or cirrhosis. The formal and known causal pathogenetic connections are discussed with regard to this clinically important liver disease. Guidelines are then given for histological diagnosis of this damage caused by medication. 14% of the female patients with a histological picture of aggressive chronic hepatitis and hepatitic cirrhosis are affected by this type of liver damage.

The effects of intravenously administered thiomolybdate on the liver and kidney of copper loaded sheep were studied using 16 ewes in three groups. Copper, iron and molybdenum concentrations were determined by spectrophotometry and the distribution of copper in the liver and kidney was studied histochemically. Following thiomolybdate administration, the concentration of copper in the liver was reduced, that of molybdenum increased and the concentration of copper and molybdenum in the kidney increased. The reduction of copper concentration in the liver was associated with reductions in the number and size of granules in hepatocytes which stained positively for copper and in the number of Kupffer cells containing positively staining granules. The decrease in the amount of copper in hepatocytes appeared to be greater than that in Kupffer cells. This effect was greatest in the centrilobular zones and least in the periportal zones. The increased concentration of copper and molybdenum in kidney was associated with an increase in the number and size of granules staining positively for copper in the epithelial cells of the proximal convoluted tubules which suggested an uptake of copper-molybdenum complexes by the lysosomes of these cells.

The cellular location and carbohydrate specificities of a glycoprotein recognition system on rat hepatic sinusoidal cells have been determined. Purified preparations of endothelial, Kupffer, and parenchymal cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. (125)I-labeled agalactoorosomucoid, an N-acetylglucosamine-terminated glycoprotein, was selectively taken up in vitro by endothelial cells. Uptake was shown to be protein dependent, calcium ion dependent, and saturable, and could be described by Michaelis-Menten kinetics (apparent K(m) 0.29 muM; apparent maximum velocity 4.8 pmol/h per 5 x 10(6) cells). Uptake was inhibited not only by N-acetylglucosamine, mannose, and mannan but also by glucose, fructose, and a glucose-albumin conjugate. Inhibition by glucose was competitive over a wide range of concentrations and was almost 100% at a glucose concentration of 56 mM. Fasting and the induction of diabetes mellitus prior to isolation of cells was associated with 60% reductions in the recovery of endothelial cells. Uptake by cells isolated from fasted rats was enhanced (apparent maximum velocity 14.3 pmol/h per 5 x 10(6) cells without change in the apparent K(m)). These observations suggest that fasting is associated with a marked increase in the mean number of glycoprotein receptors per endothelial cell isolated from normal rats. This effect of fasting could be due to upregulation of glycoprotein receptors on endothelial cells or to the selective isolation of a subpopulation of endothelial cells from fasted animals that bears more glycoprotein receptors per cell than does another subpopulation of these cells. In addition, in vivo studies of the fate of intravenously administered (125)I-agalactoorosomucoid indicated that its rate of disappearance from plasma, hepatic accumulation, and catabolism were slower in diabetic than in normal rats. The results suggest that modulation of a carbohydrate-mediated glycoprotein recognition system located on hepatic endothelial cells can be induced by glucose and glucose-conjugated proteins and by fasting and diabetes mellitus. The findings in this study suggest a mechanism for abnormal glycoprotein metabolism in diabetes mellitus.

Homozygous beta-thalassaemia is a disease in which there is a progressive iron overload from infancy to death in early adulthood. Liver biopsies from 10 patients in various stages of this disease were examined by electron microscopy. A number of round or oval lysosomal structures, containing lamellae different from myelin figures, were seen in all patients, including those with minimal iron overload. Ferritin molecules were seen either in relationship with the lamellae forming arrays, or in paracrystalline arrangement, or with no organized form. There were practically no ferritin molecules in sub-cellular compartments other than cell sap and lysosomes. The density of cell sap ferritin was constant beyond infancy, but the number of iron-laden lysosomes increased with age. The stages in the process of iron seclusion, seen even in advanced phases of iron overload, are described. Ferritin is thought to accumulate in lysosomes by a transmembraneous movement, but other explanations are considered.

A small-scale affinity chromatographic procedure was developed to screen for the presence of fucose and mannose/N-acetylglucosamine-binding lectins in small amounts of rat tissues. Of all tissues examined, only the liver contained the fucose-binding lectin, whereas both liver and blood serum contained the mannose/N-acetylglucosamine lectin. By means of immunocytological methods using antibodies to hepatic lectins, the fucose lectin was shown to be uniquely present in Kupffer cells and absent in all other types of rat macrophages examined. The binding and uptake of different neoglycoproteins by nonparenchymal cell fractions of liver indicated that the fucose-binding lectin was either not responsible for the uptake or that more than one lectin was acting. With the identification of another lectin (Mr = 180,000) by the above screening procedure for hepatic lectins and the results of studies in the following paper (Haltiwanger, R.S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7440-7444) two lectins appear to be involved. A small amount of the hepatic mannose/N-acetylglucosamine lectin was found by the above screening procedure to have a higher affinity for L-fucosyl-bovine serum albumin-Sepharose than the majority of the lectin in hepatocytes. This lectin, called the high affinity form, was purified and its properties examined. On a weight basis the high affinity form bound 7-12 times more ligand than the normal form. Its Ka for L-fucosyl-bovine serum albumin was 2.3 X 10(9) M-1 compared to 3.5 X 10(8) M-1 for the normal form. Moreover, the concentrations of monosaccharides required to inhibit the high affinity form were about 3 times less than those required to inhibit binding of the normal form. The two forms, however, have identical molecular weights (32,000) under reducing and nonreducing conditions, bind anti-lectin antibodies in the same way, and give identical peptide maps after V-8 protease digestion. The structural basis for the different binding affinities of the two forms remains unknown.

Fc receptors for immunoglobulin G in the liver sinusoidal wall were studied in the normal rat and in humans by applying peroxidase-antiperoxidase immunoglobulin G complexes to the frozen sections. Fc receptors were found to exist continuously along the sinusoidal lining. The receptors showed no zonal distribution in the rat, and they were generally scarce near the central veins and portal areas in humans. To characterize the sinusoidal cells, carbon or latex was given intravenously and endogenous peroxidase was demonstrated for the rat, whereas factor VIII-related antigen and endogenous peroxidase were demonstrated for the humans. In the rat, Fc receptors were detected on Kupffer cells, which were characterized by an intense endogenous peroxidase activity and ingestion of latex or quantities of carbon. They were also detected on sinusoidal endothelial cells, which were characterized by undetectable peroxidase activity and no ingestion of latex nor of a small quantity of carbon. In humans, Fc receptors were also present on Kupffer cells as well as sinusoidal endothelial cells, as identified by endogenous peroxidase and factor VIII-related antigen, respectively.

Hepatocellular adenomas are usually visualized as defects on technetium-99m-sulfur colloid liver scans, a fact which has been attributed to the absence of phagocytic Kupffer cells in the tumors. To determine whether this is true, seven hepatocellular adenomas were subjected to immunoperoxidase staining for lysozyme, a marker of mononuclear phagocytes. The Kupffer cells were counted in the tumors and surrounding non-neoplastic liver. All hepatocellular adenomas studied were found to contain Kupffer cells. Three tumors had fewer Kupffer cells than the surrounding liver. Three had about the same number as the surrounding liver, and one had more Kupffer cells than the non-neoplastic liver. Thus, the lack of phagocytosis of colloid in liver scans is probably due to something other than a deficiency of Kupffer cells in the hepatocellular adenomas.

The neutral lipid and phospholipid compositions of purified sinusoidal (fat-storing, endothelial and Kupffer) cells, parenchymal cells and liver homogenates were determined by thin layer chromatography. In addition, the retinoid content of the same purified cell populations was determined by high performance liquid chromatography. From each cell type, both a lipid droplet fraction and a pellet fraction (containing the majority of the remaining cell organelles) were prepared by differential centrifugation. Electron microscopic analysis showed that lipid droplets isolated from fat-storing cells were larger (up to 8 microns) than those isolated from parenchymal cells (up to 2.5 microns). Moreover, the parenchymal lipid droplets seemed to be surrounded by a membranous structure, while the fat-storing lipid droplets seemed not to be. Both fat-storing and parenchymal cells contained high concentrations of neutral lipids, 57.9 micrograms and 71.0 micrograms/10(6) cells, respectively, while endothelial and Kupffer cells contained only 8.6 micrograms and 13.8 micrograms/10(6) cells of neutral lipids, respectively. Sixty-five percent of fat-storing cell lipid droplet fractions comprised esters of retinol and cholesterol. This combined ester fraction contained mainly retinyl esters. In addition, considerable quantities (20%) of triglycerides were present. Parenchymal cell lipid droplet fractions comprised triglycerides (62%) and cholesteryl esters (up to 30%). The pellet fractions prepared from all four cell types consisted mainly of cholesterol (41-67%) and free fatty acids (20-28%). The phospholipid content was much higher in parenchymal cells than in the sinusoidal liver cell types. The relative proportions of the four major phospholipid classes were comparable in all liver cell types analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

The main hepatic change in erythropoietic protoporphyria is the deposition of protoporphyrin. Brown deposits of this pigment occur in bile canaliculi and ductules, discretely in hepatocytes, and secondarily in macrophages and Kupffer cells. The pigment is deposited in a crystalline form. Under the fluorescence microscope with a mercury maximum pressure burner (HO 50) at a wave length of 380--500 nm, it shows a typical red fluorescence even after paraffin embedding. Its crystalline structure results in a characteristic double refraction under the polarising microscope. Light-microscopically, hepatocellular reactions are characterised mainly by discrete alterations in the ergastoplasm. However, cell damage is indicated by diffusely distributed, hyaline single cell necrosis and by cytolytic piecemeal necrosis at the peripheries of hepatic lobules. Numerous, often disturbed mitoses produce binuclear and multinuclear hepatocytes. The obligatory secretion of protoporphyrin into the bile ducts leads to an alteration in the canalicular and ductular excretion apparatus which involves distinct ductular proliferation and accompanying fibrosis. Piecemeal necrosis is a further consequence of this process. The resulting histological picture is similar to sclerosing cholangitis with which it also has in common the slowly progressive development of hepatic cirrhosis.

Fetal mouse liver explants were cultured and the culture media shown to possess an erythropoietically active substance neutralizable by antiserum to sheep plasma erythropoietin, suggesting that the media contain erythropoietin. Immunofluorescent and carbon particle ingestion techniques suggest that the erythropoietin was elaborated by macrophages or Kupffer cells of the hepatic reticuloendothelial system.

Hepatic stellate cells store the majority of the liver's retinoid (vitamin A) reserves as retinyl esters in stellate cell lipid droplets. A study was conducted to explore the effects of differences in dietary retinoid and triglyceride intake on the composition of the stellate cell lipid droplets. Weanling rats were placed on one of five diets that differed in retinoid or triglyceride contents. The dietary groups were: 1) control (2.4 mg retinol (as retinyl acetate)/kg diet and 20.5% of the calories supplied by triglyceride (as peanut oil]; 2) low retinol (0.6 mg retinol/kg diet and control triglyceride levels); 3) high retinol (24 mg retinol/kg diet and control triglyceride levels); 4) low triglyceride (2.4 mg retinol/kg diet and 5% of the calories supplied by triglyceride); and 5) high triglyceride (2.4 mg retinol/kg diet and 45% of the calories supplied by triglyceride). Stellate cells were isolated using the pronase-collagenase method and stellate cell lipid droplets were isolated by differential centrifugation. The levels of retinoids and other lipids were measured by high performance liquid chromatography. The stellate cells from control rats contained 113 micrograms total lipid/10(6) cells. Control stellate cell lipid droplets had the following mean percent lipid composition: 39.5% retinyl ester; 31.7% triglyceride; 15.4% cholesteryl ester; 4.7% cholesterol; 6.3% phospholipids; and 2.4% free fatty acids. Both the concentration of stellate cell lipids and the composition of stellate cell lipid droplets were markedly altered by changes in dietary retinoid. The low and high retinol groups contained, respectively, 82 and 566 micrograms total lipid/10(6) cells, with retinyl ester representing, respectively, 13.6% and 65.4% of the lipid present in the stellate cell lipid droplets. Low and high triglyceride groups were similar to controls in both stellate cell lipid content and the composition of the stellate cell lipid droplets. These findings indicate that the composition of stellate cell lipid droplets is strongly regulated by dietary retinoid status but not by dietary triglyceride intake.

Frog virus 3 inoculated into mice induces an acute degenerative hepatitis. This hepatitis is of toxic origin since the virus is unable to multiply at 37 degrees C. The Kupffer cells, which are the target cells for FV3, reveal the presence of viral particles, viral DNA and proteins. Although the hepatocytes present early and drastic nuclear lesions, viral particles were never observed in these cells. Viral proteins however but not DNA, could be found inside parenchymal cells.

Orcein-positive material in Kupffer cells obtained from 1,377 needle biopsies of the liver was studied for hepatitis B surface antigen (HBsAg). Fifty-five (57%) out of 96 cases with orcein-positive Kupffer cells had acute hepatitis, and 46 (84%) of these 55 were in the late stage of their illness. Orcein-positive material in Kupffer cells was not associated with HBsAg as evaluated by the immune peroxidase method. Orcein-positive material in Kupffer cells appears to consist mainly of lipofuscins developing in the course of acute hepatitis.

An immunoenzyme histochemical study was conducted to localize fibrin degradation products (FDPs) in rat tissues during disseminated intravascular coagulation (DIC). Serial measurements of FDP levels in serum after thrombin-induced DIC showed peak levels to be found at 30 minutes; the FDPs were rapidly cleared from the circulation (half-life about one hour). Rat tissues obtained from 10 minutes to 3 hours after the induction of DIC were studied by means of immunohistochemistry. A method was developed to differentiate FDPs from fibrin in tissue sections. This method is based on the observation that, in paraplast-embedded tissues, FDPs can be demonstrated following ethanol fixation only, and that fibrin is demonstrable after both paraformaldehyde fixation and ethanol fixation. Moreover, FDPs will react to some of the antiserums employed only, while fibrin will react to all antiserum used (antiserums against fibrin monomer, against the constituent chains of fibrinogen, and against FDP-D and -E). At 10-20 minutes after the induction of DIC, FDPs were found in kidney proximal tubule epithelial cells. These FDPs could be demonstrated using antiserum against the constituent chains of fibrinogen, but not by antiserums against FDP-D or -E. At 30-90 minutes, FDPs were found inside liver macrophages. The FDPs in liver did not react to anti-chain antiserums, though they did react to antiserums against FDP-D and -E. Since no FDPs were found in other tissues, rat FDPs are apparently cleared by kidney (earlier phase) and liver (later phase) only. In human cases of DIC, FDPs, could be demonstrated in kidney proximal tubules cells and in liver macrophages as well.

The dendritic processes and perinuclear cytoplasm of stellate-shaped perisinusoidal cells in frozen sections of rat liver were specifically labeled with antisera raised independently to glial fibrillary acidic protein (GFAP), the major component of intermediate filaments in astrocytes. A liver protein co-migrating with authentic GFAP and immunoreactive with GFAP antisera was demonstrated with immunoblots of brain and liver extracts enriched in intermediate filament proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study presents yet another example of immunoreactivity to GFAP, or a highly similar protein localized outside the CNS, in cells of mesenchymal origin exhibiting some morphological features common to astroglia.

To determine the frequency of talc microcrystals in liver tissue of intravenous (IV) drug abusers and the significance of this finding, the authors reviewed, with light and polarizing microscopy, sections of liver tissue from 70 patients with chronic hepatitis and a history of active (45) or past (25) IV drug abuse. Birefringent crystalline particles consistent with talc were found in 44 cases (63%), 31 associated with active and 13 with past drug abuse. The microcrystals were situated predominantly in hypertrophied portal macrophages; there were no well-formed granulomas. Scanning electron microscopic and energy-dispersive spectrophotometry performed on eight of the positive cases showed the characteristic "flake-pastry" appearance and chemical composition (silicon and magnesium) of talc. For comparison, the authors similarly examined 70 cases of posttransfusion chronic hepatitis, all of which had negative findings for talc, and 70 cases of chronic hepatitis with no documented risk factors for viral hepatitis, of which two had positive findings for talc, even though IV drug abuse was denied by the two patients. The authors conclude that talc is frequently present in the liver of IV drug abusers and whenever encountered it strongly suggests IV drug abuse. Only two patients (1.4%) with a negative history also had talc.

The hepatic granulomas in experimental cryptococcosis were analyzed by peroxidase (PO) cytochemistry. Cryptococcus neoformans was inoculated intravenously into rats (group A), and some rats were administrated with dextran sulphate to suppress Kupffer cell functions before inoculation (group B). All rats were sacrificed 7 days after inoculation. The livers were examined PO cytochemically. In addition, the liver, spleen, lungs, kidneys and brain were also examined histopathologically. The hepatic granulomas consisted of the following four type cells; exudate macrophages (type I), PO-negative macrophages (type II), Kupffer cells (type III), and other inflammatory cells (type IV) such as neutrophils and lymphocytes. The percentages of the granuloma-composing cells in group A were 10.3% (type I), 27.3% (type II), 52.9% (type III) and 9.5% (type IV), respectively. In contrast in group B, type II cells outnumbered type III cells by a ratio of 5:3. In group B, necrosis and hemorrhage were observed in the granuloma. The lesions in the lungs changed from granulomatous to cystic ones after suppression of the Kupffer cell functions. These results suggest that resident macrophages such as Kupffer cells may play an important role in the formation of cryptococcal lesions.

Localization of ferritin using a pre-embedding diffusion technique and an indirect localization sequence has been made in 34 cases of human liver under normal and several pathological conditions. With light microscopic observation, positive immuno-staining for ferritin was demonstrated as diffuse deposits in the hepatocytes and Kupffer cells. Intensity of the positive immuno-staining for ferritin in these cells appeared to roughly coincide with serum ferritin levels of each patient, but showed no disease specificity, although hepatoma cells contained weak deposits or were negative from immuno-staining for ferritin. With electron microscopic studies, intracellular antigen was well preserved in the hepatocytes and Kupffer cells in most cases with the positive immuno-staining for ferritin being observed in cytosol and a few cisternae of rough endoplasmic reticulum. Content of the positive immuno-staining for ferritin differed considerably from one case to another and one cell to another even in the same case. There was no immuno-staining for ferritin in hemosiderin pigment, lysosome, most of rough endoplasmic reticulum, Golgi complexes, and nucleus in both cells.

It was investigated how to extract both morphometrical and X-ray elemental information from scanning electron microscopical (SEM) or scanning transmission electron microscopical (STEM)-images and how to integrate these two information streams either on line or off-line after storage. Cytochemical reaction products in cell organelles in ultrathin sections are the biological structures of interest. In such organelles four different situations can be met: morphologically the structures are homomorph or heteromorph; chemically the elements are distributed either homogeneously or heterogeneously. A new program has been proposed and described, which permits determination of both the area and the mean net-intensity value of chemical elements, inhomogeneously distributed over heteromorph organelles. The value of this integration method is demonstrated by three examples of increasing complexity, starting with two elements which are more or less homogeneously distributed over one lysosome, the establishing of a platinum discontinuity in an acidophilic granule and finally the localization of two chemical elements inhomogeneously distributed over a rather heteromorph phagolysosome. In two examples Chelex ion exchange beads, maximally loaded with the element also present in the structure of interest, are co-embedded with the tissue as internal standards. In such cases the absolute elemental concentration in the structures analysed can be established. The presence of such cross-sectioned beads in the ultrathin sections is also used: to demonstrate their function as models to select the proper conditions for the digital-controlled raster analysis of the unknown cell- or tissue structures, to prove the value of this method.

Cultures of Kupffer cells and of hepatocytes, prepared from single rat livers, synthesized ferritin protein equally efficiently. In culture but not in suspension, both sorts of cells responded significantly to stimulation with iron by increased ferritin synthesis. As determined by isoelectric focusing, the isoferritin profiles of newly synthesized 14C-labeled Kupffer cell and hepatocyte ferritin were identical, each having three bands. However, unlabeled ferritin, extracted from nonparenchymal liver cells (mainly Kupffer and endothelial cells) of iron-loaded rats, contained an acidic isoferritin that was not present in hepatocyte ferritin. Investigation of ferritin synthesis in cultured peritoneal and alveolar macrophages yielded similar results. The isofocusing profile of newly synthesized peritoneal macrophage ferritin was indistinguishable from the profile of fresh Kupffer cell or hepatocyte ferritin. Thus, the three isoferritins common to Kupffer cells, hepatocytes, and extrahepatic macrophages are neither cell- nor tissue-specific. However, modifications on intracellular storage may affect the isofocusing properties. The findings, although consistent with the LnH24-n subunit model of ferritin protein, indicate identical restrictive genomic control of the H:L ratios in these sorts of cells. Further, they make it probable that Kupffer cell ferritin iron, originating by endogenous synthesis, is the principal source of Kupffer cell hemosiderin iron.

The mechanism of phagocytosis by Kupffer cells is still unknown. In this study we found that trifluoperazine, chlorpromazine, and W-7, drugs which bind to Ca2+-calmodulin and inhibit its interaction with other proteins, inhibit phagocytosis by cultured Kupffer cells using polystyrene beads, time-lapse VTR systems, and fluorescent staining techniques. Inhibitory effects of these drugs on phagocytosis suggests that the Ca2+-calmodulin system may be involved in this complex cellular function and the integrity of the cytoskeletal system of Kupffer cells is essential to this phenomenon.

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED = effective dose) and a toxic (LD25; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.

Analytical microscopy was used to study the distribution and chemical composition of thorium deposits in bone marrow and liver after injection of thorium dioxide and thorium nitrate. Thorotrast (thorium dioxide) was identified as being localized in bone marrow macrophages of a patient who had undergone cerebral arteriography forty two years ago. Large thorotrast deposits were also present in liver cells. We show that non-colloidal thorium (thorium nitrate) injected in rats concentrates in a non soluble form in bone marrow macrophages, hepatocytes and Kupffer cells. These deposits of thorium associated with phosphorus can be explained by the formation of thorium phosphate in lysosomes and we demonstrate that they remain in tissue for a long time. Microanalysis was performed with ion microscopy, and electron probe microanalysis by X ray spectrometry, which can identify and localize thorium and associated elements at cellular or intracellular level.

The role of leukotrienes was investigated in frog virus 3-induced hepatitis in rats. Frog virus 3 elicited an enhanced generation of cysteinyl leukotrienes in vivo as monitored by measurement of N-acetyl-leukotriene E4 as the major endogenous metabolite of cysteinyl leukotrienes secreted into rat bile. N-Acetyl-leukotriene E4 concentrations were elevated for more than 4 hr after frog virus 3 injection. In vitro experiments using cultured rat liver Kupffer cells of high purity indicated that these cells can produce and metabolize leukotrienes and are thus a possible source of leukotrienes elicited in vivo by frog virus 3. The selective 5-lipoxygenase inhibitor AA 861 and the dual inhibitor of arachidonate lipoxygenase and cyclooxygenase, BW 755C, reduced the hepatocellular injury after a high dose of frog virus 3 by about 50 and 80%, respectively, as judged from plasma activities of ALT and sorbitol dehydrogenase at 24 hr after frog virus 3 administration. Our in vivo and in vitro studies argue in favor of an important role of leukotrienes as mediators in frog virus 3 hepatitis in rats.

The localization of alpha 2macroglobulin (alpha 2M) has been examined by an indirect immunofluorescent technique in frozen sections of various human tissues. The results indicate that alpha 2M is present only in connective tissues and blood. The outer medulla of the kidney and the submucosa of the gut showed the strongest reaction. Epithelia or endothelial cells were unreactive. In liver, only the Kupffer cells were stained. These results were confirmed withe the immunoperoxidase technique and by the study of tissue extracts in crossed immunoelectrophoresis (CIE). As a positive control a polyspecific antiserum prepared against whole human fibroblasts as well as anti-albumin were used. Our findings are interpreted in the light of the observations that alpha 2M is synthesized and selectively ingested by fibroblasts.

Sensitive immunofluorescence and immunoperoxidase techniques were used to test an extensive range of monoclonal antibodies for reactivity with Kupffer cells and interstitial dendritic cells (DCs) in cryostat-cut sections of human liver. Leucocytes with a dendritic cell morphology were identified with CD45 (antileucocyte common) reagents in portal tracts, predominantly around bile ducts, and these cells stained strongly for the HLA-DP, DQ, and DR antigens. Kupffer cells stained less intensely with anti-class-II reagents, particularly anti-HLA-DQ. The interstitial DCs expressed the LFA-1 antigen but failed to stain with CD11b, CD11c, and the defined T and B cell CD antibodies; nor did they stain with antibodies to FcR1, FcR11, FcRIII, or the C3b receptor. Of the myeloid monoclonal antibodies available from the 3rd Leucocyte Differentiation Antigen Workshop, only Y2/131, Ki-M7, Ki-M8, and a minority of CD14 antibodies stained DCs, whereas Kupffer cells showed a wider reactivity with antimacrophage antibodies including those of workshop groups 11, 15, 16, and other unique antibodies. A 2nd probable DC population was identified in the liver capsule that had a similar phenotype to portal interstitial DCs. Although some minor phenotypic differences between liver portal DCs and the phenotypes of Langerhans cells and isolated tonsil DCs were noted, our results support the view that there is a unique hemopoietic lineage of DCs. The presence of DCs, which stimulate strong allogeneic T cell responses, in the portal triads is consistent with the fact that the histologic changes of graft-versus-host disease seen in bone marrow transplantation and the lymphocytic infiltrate in a rejecting liver allograft occur predominantly in the periportal region.

The capacity of freshly isolated endothelial, Kupffer and parenchymal rat liver cells to produce eicosanoids from [1-14C]arachidonic acid was investigated in order to determine the relative importance of these cells to total liver eicosanoid production. Based upon the total formation of [1-14C]arachidonate metabolites in the liver, it can be calculated that Kupffer and endothelial cells are responsible for 65 and 23%, respectively, of the total amount of eicosanoids produced by the liver. Consequently, parenchymal liver cells, representing 92.5% of the total liver mass, contribute only 12% to the total liver production of eicosanoids. The main product of Kupffer cells was prostaglandin D2 (PGD2), representing 55% of the total amount of eicosanoids produced. Liver endothelial cells produced about 4-times less eicosanoids (per mg cell protein) than Kupffer cells, and PGD2 was also the main product of these cells (44%). The production of eicosanoids by parenchymal cells was lower by a factor of 180 (per mg cell protein) than that in Kupffer cells. Besides the ability to form eicosanoids from added 14C-labeled arachidonic acid, Kupffer and endothelial liver cells were also able to produce significant amounts of PGD2 (the main liver prostaglandin) from endogenous arachidonic acid, as determined by a radioimmunoassay. It is concluded that inside the liver, Kupffer cells together with endothelial cells are of major importance in the production of eicosanoids, while the parenchymal cells may be considered metabolic target cells for these products, as indicated by the finding that the major liver prostaglandin, PGD2, could stimulate the glucose output in isolated parenchymal cells.

CD-1 mice inoculated intraperitoneally with Mycobacterium avium, M. bovis, M. microti or M. kansasii showed a persistent peritoneal granulocytosis (above 10(6) cells, i.e. more than 15% of total cells) throughout the 3 month period of infection studied. By contrast, in mice inoculated with the non-pathogenic M. aurum or with heat-killed M. avium the number of granulocytes decreased progressively after the first 15 days. No mycobacteria were found in granulocytes except in the first 2 days of infection. The mycobacteria-induced chronic granulocytosis was accompanied by phagocytosis of granulocytes by macrophages. Throughout the 3 months of infection, macrophages were found to contain intracellular lactoferrin. Macrophages with lactoferrin were also found in subcutaneous infection caused by M. marinum and in systemic infection caused by M. avium or M. kansasii. The in vitro activity of mouse peritoneal macrophages against M. avium and M. microti was increased after ingestion of granulocyte material by macrophages. These results lead us to propose that granulocytes participate in the host response to mycobacterial infections, not as phagocytes but rather through an indirect mechanism, as a source for the macrophages of molecules involved in antimicrobial mechanisms (e.g., lactoferrin and myeloperoxidase) lacking in the mature macrophage.

The phagocytic activity of the reticuloendothelial system (RES) in the liver is important in host resistance to shock. Fibronectin is a large molecular weight glycoprotein which influences particulate uptake by phagocytic cells. This study addressed the effect of repeated low-dose endotoxin challenge on immunoreactive fibronectin and reticuloendothelial phagocytic function in rats. Intravenous Escherichia coli endotoxin increased circulating immunoreactive fibronectin by 100% within 24 hr; normalization was within 96 hr. Elevated fibronectin levels at 48 hr were associated with increased plasma opsonic activity as tested by liver slice phagocytic assay and RES stimulation, and in vitro uptake of gelatinized target particles by Kupffer cells in liver slices from endotoxin treated rats was significantly increased. Endotoxin tolerance was produced by repeated low dose challenge with endotoxin for 7 days and was associated with RES stimulation, even though the circulating fibronectin concentrations had returned to normal. By immunofluorescence, insoluble fibronectin was widely distributed in the liver in a pattern analogous to the sinusoidal vascular network. We suggest that increased RES phagocytic activity after low dose endotoxin challenge is due to early elevation of plasma fibronectin and cellular stimulation of phagocytic function followed by a sustained stimulation of Kupffer cells in the presence of normal fibronectin levels. Both cellular and humoral factors may contribute to increased Kupffer cell phagocytic activity during endotoxin tolerance.

It has been postulated that the selective accumulation of circulating desialylated cells in the mammalian liver results from the binding of desialylated glycoproteins on surfaces of the cells to asialoglycoprotein receptors in the liver. Since circulating cells in the liver are in contact predominantly with sinusoidal lining cells (Kupffer cells and endothelial cells), this postulate requires the presence of asialoglycoprotein receptors on the luminal surface of the sinusoidal lining cells. Whether the receptor is present on these cells, however, remains controversial. To clarify this issue, we used an indirect immunoelectron microscopic method to determine the distribution of the receptor on the surfaces of hepatic cells accessible to the circulation. F(ab')2 fragments of antireceptor antibodies were perfused in situ via the portal vein prior to tissue fixation. After perfusion fixation, sections were reacted with peroxidase-labeled antibodies to the antireceptor F(ab')2. The plasma membranes of Kupffer cells, endothelial cells, and fat-storing cells were devoid of the asialoglycoprotein receptor. In contrast, the receptor was associated with hepatocytes, where it was present diffusely on the sinusoidal plasma membranes and concentrated within coated pits. We conclude that it is unlikely that circulating desialylated cells bind to the asialoglycoprotein receptor in the liver unless a breach in the continuity of sinusoidal lining cells exists.

Collagenase perfusion of the liver followed by pronase treatment of the cell suspension thus obtained gave a quantitative recovery of viable nonparenchymal liver cells (NPC). From these NPC, Kupffer (K) cells can be purified by attachment to tissue culture dishes. Tail vein injection of carbon 1-2 h before liver perfusion permitted stepwise calculation as well as visualization of carbon-containing K cells. When these K cells have been put into tissue culture medium with serum and incubated overnight, they exhibit typical macrophage characteristics. Phase-contrast and transmission electron microscopy showed typical macrophage morphology and scanning electron microscopy revealed well-spread cells with cytoplasmic projections and ruffled membranes. Endocytosis studies using radioactive colloidal gold and inert latex particles also indicated that these cells are highly active in pinocytosis and phagocytosis. Further characterization of K cells is the identification of Fc receptor on their membranes. Studies on lysosomal enzymes showed that purified K cells possess higher specific activities in beta-glucuronidase, acid DNase, and cathepsin D than in purified parenchymal cells.

The distribution of gold in Kupffer cells and in subcellular fractions of rat liver was studied at intervals following intraperitoneal injection of 195Au sodium thiomalate (Myochrysine). Kupffer cells, isolated by digestion of whole liver with Pronase, had radioactive gold counts per milligram of protein that were twice the counts in the digested liver supernatant. After fractionation of liver cells by differential centrifugation, radiogold was found predominantly in the nuclear, mitochondrial, and lysosomal fractions. When the distribution of isotope was related to the protein content, the highest gold concentration was found in the lysosomal fraction, where it was 28 times that in the soluble fraction. Most radiogold was nondialyzable, probably a result of binding to larger intracellular compounds. Approximately 95% of the nondialyzable gold was in the organelle membrane of attached to membrane-adsorbed material, as determined by treatment of dialyzed mitochondrial and lysosomal fractions with Triton X-100. These data suggest that the intracellular locus of gold action may reside in organelle membranes.

The site of erythropoietin (Ep) production and/or storage in the rat liver was determined. A guinea pig anti-Ep was produced against purified rat Ep (64,096 +/- 4j064 IU/mg). This antibody was found to be highly specific using rocket immunoelectrophoresis, Ouchterlony gel diffusion methods, and immunoprecipitin reactions as well as Ep neutralization tests (capable of completely neutralizing up to 2,000 IU Ep/mg). This anti-Ep was labeled with either fluorescein for light microscopic study or ferritin for electron microscopy. Kupffer cells showed varying degrees of labeling after hepatectomy alone or hepatectomy combined with nephrectomy and/or hypoxia. Greatest labeling was seen in Kupffer cells of rats that were nephrectomized 48 hr posthepatectomy and kept at ambient pressure. No labeling of hepatocytes or vascular and bile duct endothelium was noted.

Receptor-dependent uptake mechanisms for low-density lipoprotein (LDL) were studied in rabbit liver parenchymal and non-parenchymal cells. Hybridization studies with a cDNA probe revealed that mRNA for the apo (apolipoprotein) B,E receptor was present in endothelial and Kupffer cells as well as in parenchymal cells. By ligand-blotting experiments we showed that apo B,E-receptor protein was present in both parenchymal and non-parenchymal cells. Studies of binding of homologous LDL in cultured rabbit parenchymal cells suggested that about 63% of the specific LDL binding was mediated via the apo B,E receptor. Approx. 47% of the specific LDL binding was dependent on Ca2+, suggesting that specific Ca2+-dependent as well as Ca2+-independent LDL-binding sites exist in liver parenchymal cells. Methylated LDL bound to the parenchymal cells in a saturable manner. Taken together, our results showed that apo B,E receptors are present in rabbit liver endothelial and Kupffer cells as well as in the parenchymal cells, and that an additional saturable binding activity for LDL may exist on rabbit liver parenchymal cells. This binding activity was not inhibited by EGTA or reductive methylation of lysine residues in apo B. LDL degradation in parenchymal cells was mainly mediated via the apo B,E receptor.

Using an indirect immunoperoxidase technique on frozen sections with the monoclonal antibody 96.5, we investigated the in situ distribution of melanotransferrin, a transferrin (Tf) and transferrin receptor (TfR) related glycoprotein, in human liver. Specimens included normal liver, liver in iron overload, hepatocellular carcinoma, angioma and foetal liver. On light microscopy, immunoreactivity was almost exclusively present on sinusoidal lining cells, apparently endothelial cells; the pattern was similar in normal and in iron-loaded liver. A gradient of more enhanced staining in acinar zone II and III was observed. The endothelial localization of the staining was supported by the positivity of the central vein endothelium and of the angiomas. Immunoelectron microscopy on three liver specimens showed positivity on sinusoidal endothelial cells but not on Ito and Kupffer cells. In addition, positivity on rough endoplasmic reticulum vesicles of some hepatocytes was also present. Four hepatocellular carcinomas showed an intense staining in tumour cells, 3 were weakly positive and 3 were negative. In the foetal livers, the central vein endothelium was positive from 21 weeks of gestation onward and additional positivity of zone III sinusoidal endothelial cells was present from 27 weeks on. The present results show that in the liver melanotransferrin has a localization different from Tf and the TfR. These latter molecules are predominantly localized in parenchymal cells. In addition, there does not appear to be a coordinate regulation secondary to iron storage, between melanotransferrin, Tf and the TfR. The observed gradient in the staining pattern in foetal and adult liver specimens further supports the heterogeneity of the endothelial cell population in the liver and suggests a developmental relationship between endothelial cells of sinusoids and central vein.

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.