Visualization of the interaction of native and modified low density lipoproteins with isolated rat liver cells

Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.     

The effect of felodipine on the uptake and degradation of acetylated LDL in mouse peritoneal cells and on the distribution of acetylated LDL in macrophage-rich organs of the rat

The effect of felodipine on lipoprotein metabolism ex vivo and in vivo was investigated. In the ex vivo studies mice were given felodipine (40–125 μ mol/kg body weight) or vehicle for one week. Peritoneal macrophages from these animals and controls were isolated and used in binding and degradation studies with human iodinated acetylated LDL (Ac-LDL). Macrophages from felodipine-treated mice showed a significant decrease of binding and degradation of Ac-LDL compared to macrophages from control animals (P<0.05). The in vivo studies were performed in rats pretreated with felodipine or vehicle. To determine the distribution and plasma turnover of LDL and Ac-LDL, ¹²⁵I-tyramine cellobiose labelled LDL or Ac-LDL were given i.v. No differences in the removal rate of Ac-LDL or LDL were observed between felodipine-treated or untreated rats. However, an increased uptake of Ac-LDL could be seen in the liver of the felodipine-treated rats. This increased uptake could be ascribed to the parenchymal cells because no differences in uptake could be seen in the liver endothelial cells. However, a significant decreased uptake was seen in the Kuppfer cells and in the spleen, a macrophage-rich organ, of the felodipine-treated rats. The present study suggests a possible mechanism behind the antiatherogenic effects of calcium antagonists, a decreased uptake of atherogenic modified lipoproteins by peripheral macrophages and an increased uptake by the liver.     

Biochemical and morphological modifications in rabbit Achilles tendon during maturation and ageing.

1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow.     

Characterization of sinusoidal endothelial cells of the liver and bone marrow using an intravital lectin injection method

The vascular endothelia express a variety of structural and biological phenotypes. We used an intravital injection method of plant derived lectins (Lycopersicon esculentum lectin (LEL), Ricinus communis Agglutinin-I (RCA-I), Ulex europaeus Agglutinin-I (UEA-I) and Concanavalin A (ConA)) to elucidate the morphology and function of the sinusoidal endothelium of the liver and bone marrow. All four lectins stained the sinusoidal endothelia of the liver and bone marrow in a patchy granular pattern which differed from the uniform and smooth staining pattern of non-sinusoidal vessels in other organs. By transmission electron microscopy, the granular pattern was identified as internalization of these lectins and subsequent accumulation within the endothelial cells, suggesting their active endocytosis. The endocytosis of these lectins emphasizes the fact that sinusoidal endothelial cells of the liver and bone marrow belong to the reticuloendothelial system (RES), a cell system characterized by internalization of foreign material. We introduce this intravital lectin injection as a useful technique to discriminate sinusoidal endothelial of the liver and bone marrow from other vascular endothelia.     

In vivo binding and uptake of low-density lipoprotein-gold- and albumin-gold conjugates by parenchymal and sinusoidal cells of the fetal rat liver

Summary To elucidate the participation of fetal rat liver cells in the receptor-mediated internalization of low-density lipoproteins (LDL), rat fetuses were injected with either LDL-gold or albumin-gold conjugates. The degree of binding and uptake of LDL-gold and albumin-gold by parenchymal and sinusoidal cells of the fetal rat liver differs markedly. Endothelial cells exhibit low LDL-gold uptake. In contrast, parenchymal cells internalize LDL-gold more actively (45 ± 8 LDL conjugates/100 m² cytoplasm within 60 min). Kupffer cells exceed this value by a factor of 20. The uptake of albumin-gold by endothelial and Kupffer cells is high, whereas it is extremely low in parenchymal cells. Estradiol pretreatment causes a significant doubling (p<0.05) of the LDL-gold particle density/100 m² cytoplasm both in parenchymal and Kupffer cells, whereas estradiol has no effect on the albumin uptake. The results strongly indicate that LDL uptake by parenchymal and Kupffer cells in the fetal rat liver is mediated by estrogen-inducible receptors, which may correspond to B, E receptors in the adult liver.     

Chylomicron metabolism. Chylomicron uptake by bone marrow in different animal species

Previously it was shown in rabbits that 20-40% of the injected dose of chylomicrons was cleared from the plasma by perisinusoidal bone marrow macrophages. The present study was undertaken to determine whether the bone marrow of other species also cleared significant amounts of chylomicrons. Canine chylomicrons, labeled in vivo with [14C]cholesterol and [3H] retinol, were injected into marmosets (a small, New World primate), rats, guinea pigs, and dogs. Plasma clearance and tissue uptake of chylomicrons in these species were contrasted with results obtained in rabbits in parallel studies. The chylomicrons were cleared rapidly from the plasma in all animals; the plasma clearance of chylomicrons was faster in rats, guinea pigs, and dogs compared with their clearance from the plasma of rabbits and marmosets. The liver was a major site responsible for the uptake of these lipoproteins in all species. However, as in rabbits, the bone marrow of marmosets accounted for significant levels of chylomicron uptake. The uptake by the marmoset bone marrow ranged from one-fifth to one-half the levels seen in the liver. The marmoset bone marrow also took up chylomicron remnants. Perisinusoidal macrophages protruding through the endothelial cells into the marrow sinuses were responsible for the accumulation of the chylomicrons in the marmoset bone marrow, as determined by electron microscopy. In contrast to marmosets, chylomicron clearance by the bone marrow of rats, guinea pigs, and dogs was much less, and the spleen in rats and guinea pigs took up a large fraction of chylomicrons. The uptake of chylomicrons by the non-human primate (the marmoset), in association with the observation that triglyceride-rich lipoproteins accumulate in bone marrow macrophages in patients with type I, III, or V hyperlipoproteinemia, suggests that in humans the bone marrow may clear chylomicrons from the circulation. It is reasonable to speculate that chylomicrons have a role in the delivery of lipids to the bone marrow as a source of energy and for membrane biosynthesis or in the delivery of fat-soluble vitamins.     

Receptor-mediated and bulk-phase endocytosis cause macrophage and cholesterol accumulation in Niemann-Pick C disease

These studies explored the roles of receptor-mediated and bulk-phase endocytosis as well as macrophage infiltration in the accumulation of cholesterol in the mouse with Niemann-Pick type C (NPC) disease. Uptake of LDL-cholesterol varied from 514 microg/day in the liver to zero in the central nervous system. In animals lacking LDL receptors, liver uptake remained about the same (411 microg/day), but more cholesterol was taken up in extrahepatic organs. This uptake was unaffected by the reductive methylation of LDL and consistent with bulk-phase endocytosis. All tissues accumulated cholesterol in mice lacking NPC1 function, but this accumulation was decreased in adrenal, unchanged in liver, and increased in organs like spleen and lung when LDL receptor function was also deleted. Over 56 days, the spleen and lung accumulated amounts of cholesterol greater than predicted, and these organs were heavily infiltrated with macrophages. This accumulation of both cholesterol and macrophages was increased by deleting LDL receptor function. These observations indicate that both receptor-mediated and bulk-phase endocytosis of lipoproteins, as well as macrophage infiltration, contribute to the cholesterol accumulation seen in NPC disease. These macrophages may also play a role in parenchymal cell death in this syndrome.     

Receptor-mediated endocytosis of lactate dehydrogenase M4 by liver macrophages: a mechanism for elimination of enzymes from plasma. Evidence for competition by creatine kinase MM, adenylate kinase, malate, and alcohol dehydrogenase

We have previously shown that the rapid clearance of intravenously injected lactate dehydrogenase M4 from plasma is mainly due to endocytosis by macrophages in liver, spleen, and bone marrow. We have now studied endocytosis of lactate dehydrogenase M4 in detail, using freshly isolated rat liver macrophages (Kupffer cells) in vitro. 125I-lactate dehydrogenase M4 rapidly accumulated in the cells and was subsequently degraded to trichloroacetic acid-soluble material. Degradation was inhibited by leupeptin, an inhibitor of lysosomal proteases. Breakdown of the protein was also greatly diminished by treatment of the cells with chloroquine, a weak base which inhibits proteolysis by raising the pH in endosomes and lysosomes. High concentrations of chloroquine inhibited uptake. Lactate dehydrogenase M4 was not endocytosed by liver endothelial cells, although, under the same conditions, these cells were shown to accumulate horse radish peroxidase via a mannose-specific receptor. Uptake of lactate dehydrogenase M4 by Kupffer cells was strongly reduced after pretreatment of the cells with low concentrations of proteases. Endocytosis of lactate dehydrogenase M4 exhibited saturation kinetics (Km = 0.8 microM) and was competitively inhibited by mitochondrial and cytosolic malate dehydrogenase, alcohol dehydrogenase, adenylate kinase, and creatine kinase MM, enzymes which are rapidly cleared in vivo. Enzymes with long half-lives in plasma, namely lactate dehydrogenase H4, alanine aminotransferase, and cytosolic aspartate aminotransferase did not compete at concentrations up to 10 microM. Our results indicate that Kupffer cells contain a receptor that is involved in the clearance of lactate dehydrogenase M4 and a number of other tissue-derived enzymes from plasma. Uptake of lactate dehydrogenase M4 does not occur via a receptor that recognizes carbohydrate residues, for the enzyme is not a glycoprotein.     

In vivo and in vitro catabolism of native and biologically modified LDL

Incubation of human low density lipoprotein (LDL) at 37 degrees C in the presence of human umbilical-vein endothelial cells (EC) causes a time-dependent shift in the charge and density of LDL. After intravenous injection into rats, native LDL is merely cleared from the circulation by Kupffer cells while EC-modified LDL is rapidly cleared by endothelial liver cells. The uptake of native LDL by Kupffer cells and EC-modified LDL by endothelial cells in vivo can be explained by the presence of two different specific receptors on these cell types. It is concluded that the liver endothelial cells form an important protection against a possible atherogenic action of EC-modified LDL.     

Preclustered receptor arrangement is a prerequisite for galactose-specific clearance of large particulate ligands in rat liver

We had hypothesized that preclustered arrangement of galactose-specific receptor activity on rat liver macrophages enables these cells to internalize multivalent, particulate ligands in contrast to the clearance of molecules mediated by statistically distributed receptors on hepatocytes. We now took advantage of the nonclustered receptor distribution in newborn rat liver macrophages to study the in vivo clearance of particulate ligands. Gold particles 5, 17, and 50 nm in diameter (Au5, Au17, Au50), coated with lactosylated bovine serum albumin (LacBSA), were injected into the vena cava and livers were perfusion fixed after allowing for binding and uptake for 3 min. In sinusoidal cells from rats 15 days old LacBSA-Au5 and LacBSA-Au17 were taken up by endothelial cells and all sizes by liver macrophages. In newborn rat liver no LacBSA-Au50 or LacBSA-Au17 was retained in liver macrophages. Uptake of LacBSA-Au5 by sinusoidal cells was significant. LacBSA-Au17 was taken up in significant amounts by endothelial cells of newborn rats which correlates to the findings that galactose-specific binding sites on endothelial cells were found to localize as clusters over coated pits irrespective of age. These results demonstrate the crucial role of clustered receptors in binding and uptake of larger particulate ligands via this lectin-like binding activity.     

Quantitative role of parenchymal and non-parenchymal liver cells in the uptake of [14C]sucrose-labelled low-density lipoprotein in vivo.

In order to assess the relative importance of the receptor for low-density lipoprotein (LDL) (apo-B,E receptor) in the various liver cell types for the catabolism of lipoproteins in vivo, human LDL was labelled with [14C]sucrose. Up to 4.5h after intravenous injection, [14C]sucrose becomes associated with liver almost linearly with time. During this time the liver is responsible for 70-80% of the removal of LDL from blood. A comparison of the uptake of [14C]sucrose-labelled LDL and reductive-methylated [14C]sucrose-labelled LDL ([14C]sucrose-labelled Me-LDL) by the liver shows that methylation leads to a 65% decrease of the LDL uptake. This indicated that 65% of the LDL uptake by liver is mediated by a specific apo-B,E receptor. Parenchymal and non-parenchymal liver cells were isolated at various times after intravenous injection of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL. Non-parenchymal liver cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells when expressed per mg of cell protein. This factor is independent of the time after injection of LDL. Taking into account the relative protein contribution of the various liver cell types to the total liver, it can be calculated that non-parenchymal cells are responsible for 71% of the total liver uptake of [14C]sucrose-labelled LDL. A comparison of the cellular uptake of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL after 4.5h circulation indicates that 79% of the uptake of LDL by non-parenchymal cells is receptor-dependent. With parenchymal cells no significant difference in uptake between [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL was found. A further separation of the nonparenchymal cells into Kupffer and endothelial cells by centrifugal elutriation shows that within the non-parenchymal-cell preparation solely the Kupffer cells are responsible for the receptor-dependent uptake of LDL. It is concluded that in rats the Kupffer cell is the main cell type responsible for the receptor-dependent catabolism of lipoproteins containing only apolipoprotein B.     

Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits. Factors that modify tissue-specific uptake

The metabolism of [14C]cholesterol- and [3H]retinol-labeled chylomicrons obtained from canine thoracic duct or rabbit mesenteric lymph was investigated in normal fasted rabbits. Typically, 70-80% of the chylomicrons injected into the rabbits were cleared from the plasma in 20 min, and their uptake was accounted for principally by the liver and the bone marrow. Surprisingly, the bone marrow was a major site of uptake; the uptake ranged from about half that of the liver to a nearly equal amount. The importance and specificity of chylomicron-chylomicron remnant uptake by the bone marrow were established by demonstrating that (a) bone marrow throughout the body accumulated these lipoproteins, (b) the level of uptake was consistent regardless of how the values were calculated or how the chylomicrons were prepared, (c) the uptake represented specific binding, and (d) radiolabeled intestinal lipoproteins induced in vivo delivered cholesterol and retinol to the marrow. Electron microscopic examination of the rabbit bone marrow established that perisinusoidal macrophages uniquely accounted for the uptake of the chylomicrons. Whereas liver cleared a variety of both triglyceride-rich lipoproteins (chylomicrons, chylomicron remnants, and very low density lipoproteins) and cholesterol-rich lipoproteins (beta-very low density lipoproteins and high density lipoproteins containing apolipoprotein E), bone marrow uptake appeared to be restricted to the triglyceride-rich lipoproteins. More chylomicron remnants (generated in a hepatectomized rabbit) were cleared by the liver than by the bone marrow, and the addition of excess apolipoprotein E to chylomicrons resulted in their preferential uptake by the liver. The role of chylomicron-chylomicron remnant delivery of lipids or lipid-soluble vitamins to rabbit bone marrow is open to speculation, and whether triglyceride-rich lipoprotein uptake occurs to a significant extent in the bone marrow of humans remains to be determined.     

Histochemical localization of autometallographically detectable mercury in tissues of the immune system from mice exposed to mercuric chloride

Summary The distribution of mercury in the spleen, liver, lymph nodes, thymus and bone marrow was studied by autometallography in mice exposed to mercuric chloride intraperitoneally. Application of immunofluorescence histochemistry and an autometallographic silver amplification method was employed to the same tissue section. Mercury was not only detected in macrophages marked by the antibody M1/70 but also in macrophage-like cells, which were either autofluorescent or devoid of fluorescent signals. These two cell types were identified as macrophages at the electron microscopical level. Autometallographically stained macrophages were observed in the spleen, lymph nodes, thymus and in Kupffer cells of the liver. Furthermore, mercury was observed in endothelial cells. No obvious pathological disturbances were observed at light and electron microscopical level. At the subcellular level mercury was localized in lysosomes of macrophages and endothelial cells.     

Formaldehyde treated albumin contains monomeric and polymeric forms that are differently cleared by endothelial and Kupffer cells of the liver: evidence for scavenger receptor heterogeneity.

Formaldehyde treated albumin (F-HSA) was found to consist of a monomeric and a polymeric fraction. Both fractions were primarily endocytosed by rat liver sinusoidal cells. However, immunohistochemical staining of endocytosed material showed that the relative contribution of the endothelial and Kupffer cells in uptake of the monomer and the polymer differed significantly, with the monomer mainly having an endothelial cell- and the polymer predominantly having a Kupffer cell pattern of distribution. To directly confirm these heterogeneous patterns, we injected in vivo the 125I-labeled F-HSA fractions and isolated the endothelial and Kupffer cells by centrifugal elutriation. 73.7% of the monomeric F-HSA was found in endothelial cells and only 14.9% was found in Kupffer cells. In contrast, the polymeric F-HSA (1500 kD) was mainly endocytosed by Kupffer cells (71%), whereas the endothelial cells contributed only for 24% in hepatic uptake. In vivo studies and isolated perfused rat liver experiments showed that endocytosis of both monomer and polymer was inhibited by co-administration of polyinosinic acid, a well known inhibitor for scavenger receptors, indicating that these receptors on endothelial and Kupffer cells are mainly involved in this uptake process.     

Uptake and degradation of human very-low-density lipoproteins by rat liver endothelial cells in culture

Rat liver endothelial cells in primary cultures take up and degrade 125I-labelled human very-low-density lipoproteins (VLDL) in a saturable fashion at physiological triacylglycerol concentrations. The iodinated VLDL are readily taken up by the freshly isolated endothelial cells and degradation products appear in the medium about 30 min after the addition of VLDL to the cultures. Uptake and degradation at 37 degrees C are effectively inhibited by unlabelled human VLDL, low-density lipoproteins (LDL), high-density lipoproteins and lymph chylomicrons, but only modestly by acetylated LDL. Purified apolipoproteins E and C-III:1 also compete with the uptake of iodinated VLDL, but when degradation was studied for longer periods of time, such a competition could not be demonstrated. This may be due to the fact that the added apolipoproteins become associated with the lipoproteins. In binding experiments at 7 degrees C, iodinated apolipoprotein C III:1 bound to the liver endothelial cells in a manner characteristic of receptor binding with a dissociation constant of 0.5 microM. This binding could not only be inhibited by unlabelled apolipoprotein C-III:1 but also by unlabelled apolipoprotein E. The results indicate that rat liver endothelial cells carry receptors for VLDL and that these recognize the apolipoproteins E, C-III and B on the lipoprotein surface. Considering the large endothelial surface and high blood flow through the liver, significant quantities of lipoproteins can be taken up and degraded, thus influencing the levels of circulating lipoproteins in the in vivo situation.     

Fluid endocytosis by rat liver and spleen. Experiments with 125I-labelled poly(vinylpyrrolidone) in vivo.

1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.     

Kupffer cells support extramedullary erythropoiesis induced by nitrogen-containing bisphosphonate in splenectomized mice

Our previous study indicated that injecting nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. This was due to the depletion of BM-resident macrophages, which support erythropoiesis. In this study, we examined NBP treatment-induced extramedullary hematopoiesis in splenectomized mice, focusing on hepatic hematopoiesis. NBP-treated mice did not display anemia or significant change in erythropoietin production, while megakaryopoiesis and erythropoiesis were constantly observed in the liver. Erythroblastic islands were detected in the sinusoidal lumen. Kupffer cells expressed VCAM-1 following NBP treatment, which is an important factor for erythroblast differentiation. Cl₂MBP-liposome treatment depleted the erythroblastic islands, and decreased the number of hematopoietic cells in the liver, as determined by colony forming assays. Together, these results indicate that Kupffer cells support erythropoiesis, acting as stromal cells in the liver, and that they might act as a niche for hematopoietic precursor cells in an emergency.     

Elimination of porcine hemopoietic cells by macrophages in mice.

The difficulty in achieving donor hemopoietic engraftment across highly disparate xenogeneic species barriers poses a major obstacle to exploring xenograft tolerance induction by mixed chimerism. In this study, we observed that macrophages mediate strong rejection of porcine hemopoietic cells in mice. Depletion of macrophages with medronate-encapsulated liposomes (M-liposomes) markedly improved porcine chimerism, and early chimerism in particular, in sublethally irradiated immunodeficient and lethally irradiated immunocompetent mice. Although porcine chimerism in the peripheral blood and spleen of M-liposome-treated mice rapidly declined after macrophages had recovered and became indistinguishable from controls by wk 5 post-transplant, the levels of chimerism in the marrow of these mice remained higher than those in control recipients at 8 wks after transplant. These results suggest that macrophages that developed in the presence of porcine chimerism were not adapted to the porcine donor and that marrow-resident macrophages did not phagocytose porcine cells. Moreover, M-liposome treatment had no effect on the survival of porcine PBMC injected into the recipient peritoneal cavity, but was essential for the migration and relocation of these cells into other tissues/organs, such as spleen, bone marrow, and peripheral blood. Together, our results suggest that murine reticuloendothelial macrophages, but not those in the bone marrow and peritoneal cavity, play a significant role in the clearance of porcine hemopoietic cells in vivo. Because injection of M-liposomes i.v. mainly depletes splenic macrophages and liver Kupffer cells, the spleen and/or liver are likely the primary sites of porcine cell clearance in vivo.     

Uptake of LDL in parenchymal and non-parenchymal rabbit liver cells in vivo. LDL uptake is increased in endothelial cells in cholesterol-fed rabbits.

1. Hepatic uptake of low-density lipoprotein (LDL) in parenchymal cells and non-parenchymal cells was studied in control-fed and cholesterol-fed rabbits after intravenous injection of radioiodinated native LDL (125I-TC-LDL) and methylated LDL (131I-TC-MetLDL). 2. LDL was taken up by rabbit liver parenchymal cells, as well as by endothelial and Kupffer cells. Parenchymal cells, however, were responsible for 92% of the hepatic LDL uptake. 3. Of LDL in the hepatocytes, 89% was taken up via the B,E receptor, whereas 16% and 32% of the uptake of LDL in liver endothelial cells and Kupffer cells, respectively, was B,E receptor-dependent. 4. Cholesterol feeding markedly reduced B,E receptor-mediated uptake of LDL in parenchymal liver cells and in Kupffer cells, to 19% and 29% of controls, respectively. Total uptake of LDL in liver endothelial cells was increased about 2-fold. This increased uptake is probably mediated via the scavenger receptor. The B,E receptor-independent association of LDL with parenchymal cells was not affected by the cholesterol feeding. 5. It is concluded that the B,E receptor is located in parenchymal as well as in the non-parenchymal rabbit liver cells, and that this receptor is down-regulated by cholesterol feeding. Parenchymal cells are the main site of hepatic uptake of LDL, both under normal conditions and when the number of B,E receptors is down-regulated by cholesterol feeding. In addition, LDL is taken up by B,E receptor-independent mechanism(s) in rabbit liver parenchymal, endothelial and Kupffer cells. The non-parenchymal liver cells may play a quantitatively important role when the concentration of circulating LDL is maintained at a high level in plasma, being responsible for 26% of hepatic uptake of LDL in cholesterol-fed rabbits as compared with 8% in control-fed rabbits. The proportion of hepatic LDL uptake in endothelial cells was greater than 5-fold higher in the diet-induced hypercholesterolaemic rabbits than in controls.