A lectin-like receptor on rat macrophage membranes: colloidal gold probes for detection, localization and description of biological function.
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Animals↗
Biomedical subjects
V Kolb-Bachofen
Publications and source records attributed to V Kolb-Bachofen.
Recognition of xenogeneic erythrocytes: the GalNAc/Gal-particle receptor of rat liver macrophages mediates or participates in recognition.
We studied mechanisms that mediate recognition of human erythrocytes (HRBC) and sheep erythrocytes (SRBC) by rat liver macrophages. We used an in vitro cell binding assay that allows spontaneous formation of cell contacts. Binding of HRBC to rat macrophages shows the following characteristics: inhibition studies with several monosaccharides and oligosaccharides yield complete inhibition of cell contacts with saccharides, which block the GalNAc/Gal-particle receptor on rat liver macrophages. We found the inhibition pattern: N-acetyl-D-galactosamine, lactose greater than D-galactose, D-fucose greater than L-fucose much greater than N-acetyl-D-glucosamine. Cell binding is dependent on the presence of calcium ions, but not influenced by heat-aggregated IgG or gangliosides. The inhibition pattern was the same after treatment of HRBC with neuraminidase. Therefore, binding of HRBC, as well as binding of neuraminidase-treated HRBC, is mediated by the GalNAc/Gal-particle receptor. Binding of SRBC is partly inhibited by galactose-related saccharides. Binding is also partly inhibited by heat-aggregated IgG, gangliosides, and L-fucose. Complete inhibition of cell contacts with SRBC is achieved by combination of all inhibitors. We therefore conclude that binding of SRBC is mediated by several different mechanisms, including the GalNAc/Gal-particle receptor. Binding of neuraminidase-treated SRBC, however, was found to be completely inhibited by saccharides, which block the GalNAc/Gal-particle receptor. We conclude that the GalNAc/Gal-particle receptor mediates or participates in recognition of non-self structures.
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Quantification and localization of galactose-specific binding sites in rat liver during postnatal development.
We investigated the number and distribution of galactose-specific binding sites in developing livers from suckling rats of various ages using Lac-BSA-Au5 (lactosylated bovine serum albumin adsorbed onto colloidal gold particles 5 nm in diameter) as electron-dense ligand, and performing transmission electron microscopy of the specimen. It has been reported that the number of galactose-specific binding sites increases rapidly during organ development post partum (p.p.) and this was ascribed to hepatocyte receptor increase only. We now have investigated in in situ and in vitro experiments whether the binding sites of identical sugar specificity but located on sinusoidal cells show the same increase in expression or are independently regulated. We therefore quantified the number of particles bound by isolated hepatocytes and liver macrophages and found a gradual increase of both binding activities with age, the binding levels of adult liver cells being reached at day 15 p.p. This was confirmed with experiments using in situ prefixed organs thus proving the validity of this finding also for the intact organ. In both sets of experiments--in vitro as well as in vivo--ligand was found binding statistically distributed as single particles on hepatocytes of all ages, whereas on liver macrophages the binding pattern changed during development. On liver macrophages from rats 15 days of age ligand binding occurs in the preclustered pattern described for macrophages from adult rat livers whereas liver macrophages of newborn rats express a different binding pattern: they bind the ligands mostly as single particles with only few and small microaggregates.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
Endocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophages.
We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction.
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Administration of silica or monoclonal antibody to Thy-1 prevents low-dose streptozotocin-induced diabetes in mice.
Multiple injections of low doses of streptozotocin induce an experimental diabetes in mice. We have analyzed in two inbred strains whether the development of hyperglycaemia can be influenced by administration of macrophage-toxic silica particles or by a monoclonal antibody to Thy-1.2. Mice received streptozotocin (30 or 40 mg/kg) on five consecutive days (day 0-day 4) and in addition either silica particles (starting at day 0) or anti-Thy-1.2 (starting at day -2 or -3). In both strains mice receiving streptozotocin alone became hyperglycaemic within two weeks. Additional treatment with silica almost fully prevented diabetes development. Anti-Thy-1.2 administration was similarly effective in C57B1/Ks and partially protective in C57BL/6 mice. Histological analysis of pancreatic islets showed that a large fraction of beta cells had been spared from destruction by this treatment. The data indicate a role for both macrophages and Thy-1 positive cells in the pathogenesis of low-dose streptozotocin-induced diabetes.
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Galactose-specific receptors on liver cells. I. Hepatocyte and liver macrophage receptors differ in their membrane anchorage.
Colloidal gold particles coated with asialoglycoproteins are bound by hepatocytes as well as by liver macrophages. Binding by both cell types is inhibited by N-acetylgalactosamine and related saccharides and is dependent on the presence of Ca2+. We have now performed an electron microscopic study on receptor anchorage in the plasma membranes. Cells with prebound ligand were treated with 20 mM EDTA at 4 degrees C, washed free of chelator and tested for residual galactose-specific receptor activity. Whereas hepatocytes preserve binding activity (73% of untreated control), liver macrophages lose galactose-specific receptor activity (12% of untreated control). Liver macrophages regain binding activity after a 2 min incubation at 37 degrees C allowing for receptor recycling. If the macrophages were fixed with low glutaraldehyde concentration prior to EDTA treatment they fully retained their receptor activity (74% of control). Ligands were also removed from both cell types by incubation with 80 mM N-acetylgalactosamine. After washing the cells free of the competing monosaccharide, both the hepatocytes as well as the macrophages show full binding activity (120% and 85% of untreated controls). Therefore, membrane anchorage sites of the macrophage receptors are not identical to ligand-binding sites. These results suggest a Ca2+-Mg2+-dependent receptor anchorage on the macrophage plasma membrane. As shown in the accompanying paper (Roos, P.H., Hartmann, H.J., Schlepper-Schäfer, J., Kolb, H. and Kolb-Bachofen, V. (1985) Biochim. Biophys. Acta 847, 115-121), EDTA-induced dissociation from the membrane can be used for isolation of the galactose-specific receptors of liver macrophages.
Acetylgalactosamine↗
Galactose-specific receptors on liver cells. II. Characterization of the purified receptor from macrophages reveals no structural relationship to the hepatocyte receptor.
We have isolated a galactose-specific receptor protein from rat liver macrophages by three techniques, all using EDTA extraction and subsequent affinity chromatography. The purified receptor has an apparent molecular mass of 30 kDa and exhibits hemagglutinating activity. Monospecific receptor-antisera produce one precipitation line with the macrophage receptor in Ouchterlony double diffusion but show no cross-reaction with the hepatocyte receptor. Sinusoidal cells, but not hepatocytes, are stained with monoclonal antibodies to the macrophage receptor, whereas anti-hepatocyte receptor antibodies stain hepatocyte surfaces but not sinusoidal cells. We conclude that the galactose-specific receptor from liver macrophages is structurally different from the hepatocyte receptor, although the two lectins share a similar binding specificity.
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Chronic inflammatory polyneuropathy. Reduction of nerve conduction velocities in monkeys by systemic passive transfer of immunoglobulin G.
In chronic (relapsing) inflammatory polyneuropathy (CRIP), successful treatment with plasma exchange has led to the concept of pathogenic humoral factors. In 6 patients with CRIP, 5 of whom improved after plasma exchange, the potential pathogenic role of circulating immunoglobulin (Ig) fractions was tested by applying the systemic passive transfer model to marmoset monkeys. After continuous treatment with intramuscular injections for 2-8 weeks, monkeys injected with the crude immunoglobulin fractions or with purified IgG from 5 of the 6 patients showed a significant and partially reversible reduction of the motor nerve conduction velocity (mean 34%, P less than 0.001) when compared with pre-treatment values. In control animals the reduction was 4%. Morphological examination revealed only minor ultrastructural changes of the myelin sheath. Immunocytochemistry revealed that human IgG was able to cross the blood-nerve barrier. It is concluded that the circulating IgG-fraction of patients with CRIP contains a factor that may contribute to the disordered nerve function after crossing the blood-nerve barrier. It may be the removal of this particular factor which is responsible for the rapid recovery of nerve conduction in patients after plasma exchange.
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GalNAc/Gal-specific rat liver lectins: their role in cellular recognition.
By investigating the presence and distribution of GalNAc/Gal-specific receptors on liver cells in vitro and in vivo, we provided evidence that the hepatocyte is not the only liver cell expressing receptor activity but that receptors of similar specificity are found on liver macrophages and also on endothelial cells. The receptor distribution in the plasma membrane is strinkingly different between the three cell types, as judged from the binding pattern of colloidal gold particles coated with asialofetuin or lactosylated serum albumin. Binding to hepatocytes occurs as single particles statistically distributed, binding to liver macrophages in a clustered arrangement all over the cell membrane and binding to endothelial cells also in a clustered arrangement but restricted to coated pits only. The different receptor distribution results in different binding and uptake abilities. Whereas hepatocytes bind and take up molecules and small particles (5 nm) only, the clustered receptor arrangement of endothelial cells and macrophages enables them to effectively bind and ingest larger particles. Ligands larger than 35 nm can be taken up by the macrophages only. The different receptor arrangement results also in different capacities of cell contact formation. Although in vitro liver macrophages and hepatocytes can both bind desialylated cells the macrophage needs much less galactosyl groups exposed on erythrocytes to establish stable contacts than the hepatocyte. The contacts formed by hepatocytes stay reversible for 30 min at 37 degrees C, whereas the contacts formed by the liver macrophages become irreversible after 10 min at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts.
The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.
Acetylgalactosamine↗
Cell mediated immunity to islet cells: lessons from animal studies.
As a pathogenic factor of type I (insulin-dependent) diabetes cell mediated immunity to pancreatic islet cells, i.e. lymphocytic insulitis has been studied in mice with spontaneous lupus-like autoimmune disease, in mice with experimentally induced immune dysregulation and in mice treated with multiple low doses of streptozotocin or with alloxan. The following conclusions have been reached: Autoimmunity to B-islet cells requires a disturbed immune system. Autoimmune insulitis requires altered B-islet cells. Cellular immunity to B-islet cells apparently involves two different mechanisms: The intrainsular invasion of single lymphocytes and macrophages and the mostly periinsular/periductular infiltration of large numbers of lymphocytes and macrophages. Some observations indicate a primary role of helper and suppressor T lymphocytes as well as of macrophages in B-islet cell destruction. In this paper an attempt will be made to combine observations on the induction and course of insulitis in several animal models in a general scheme of pathological events. Several conclusions on the mechanism of islet autoimmunity have been reached. At the time being however, these "lessons" must be regarded as hypotheses, which may be helpful in understanding the pathogenesis of human type I diabetes.
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Identification of a receptor for senescent erythrocytes on liver macrophages.
Isolated rat liver macrophages adherent to culture dishes phagocytize selectively senescent rat erythrocytes as well as in vitro aged rat erythrocytes but not young or freshly isolated erythrocytes. Since in vitro aged erythrocytes lack cell bound antibodies and phagocytosis occurs in serum free medium, antibodies do not appear to play a role. Phagocytosis as well as binding of old erythrocytes to liver macrophages is inhibited by N-acetyl-D-galactosamine and related monosaccharides (1-25 mM range) and by corresponding synthetic glycoproteins (10(-6)-10(-10) M range) but not by D-mannose or N-acetyl-D-glucosamine. We conclude that recognition and phagocytosis of senescent and in vitro aged erythrocytes is mediated by the galactose particle receptor on rat liver macrophages.
Acetylgalactosamine↗
Galactose-particle receptor on liver macrophages. Quantitation of particle uptake.
Liver macrophages have been shown previously to bind and ingest gold particles coated with asialoglycoproteins via a N-acetyl-D-galactosamine / D-galactose-specific lectin (Kolb-Bachofen, V., Schlepper-Schäfer, J., Vogell, W. and Kolb, H. (1982) Cell 29, 859-866). We present here a quantitative analysis of lectin-dependent particle endocytosis. We used a conjugate of asialofetuin with colloidal gold as ligand, the cellular uptake of which could be followed by spectrophotometry. Freshly isolated Kupffer cells from the rat liver ingest asialofetuin at a rate of approx. 4200 particles/cell per min. Uptake is inhibited by saccharides related in structure to D-galactose and depends on the presence of Ca2+. The rate of endocytosis is zero below 10 degrees C, shows a modest increase until 20 degrees C and a steep increase between 20 and 37 degrees C. Uptake is energy-dependent and strongly inhibited by cytochalasin B but only slightly by colchicine.
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Two galactose-specific receptors in the liver with different function.
In the rat liver both hepatocytes and macrophages have been shown to express on the surface lectins with similar binding specificity for galactose residues. Functionally the two lectins differ in the uptake of ligands. Whereas the hepatocytes ingest molecules and small particles (less than 10 nm), the macrophages take up particles only. Antisera raised against hepatic galactose-specific receptor failed to react with the macrophage lectin but blocked ligand binding to the hepatocyte only, indicating either a different antigenic structure or membrane localization of the two lectins.
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Receptor-mediated particle uptake by liver macrophages. The galactose-particle receptor mediates uptake via coated and also non-coated structures.
The endocytosis pathways of particles with terminal beta-D-galactosyl groups were studied in isolated rat Kupffer cells by electron microscopy. Colloidal gold particles of sizes 5, 17 and 50 nm were coated with asialofetuin (ASF) and isolated liver macrophages were allowed to bind (at 4 degrees C) or take up (at 37 degrees C) these ligands. Particles of all three sizes were bound via the galactose-particle receptor as shown by carbohydrate inhibition experiments and were ingested effectively. But, whereas ASF-gold particles of sizes 5 and 17 nm are taken up via the coated pit/coated vesicle pathway, the 50 nm particles are not. These enter the cell via non-coated endocytic vacuoles. All three particle sizes are transported to the same lysosomal compartment. These observations demonstrate that at least in macrophages one receptor is capable to mediate endocytosis via two different pathways depending on ligand size and/or valency.
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A membrane lectin on macrophages: localization and demonstration of receptor recycling.
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Animals↗
Electron microscopic evidence for an asialoglycoprotein receptor on Kupffer cells: localization of lectin-mediated endocytosis.
Direct evidence is given for the presence of an N-acetyl-D-galactosamine-specific lectin on the Kupffer cell surface by visualization of ligand binding in electron microscopy. When freshly isolated Kupffer cells are incubated with asialofetuin adsorbed onto colloidal gold particles (ASF-gold), binding and endocytosis of ligand are seen. Recognition of ASF-gold by Kupffer cells is completely abolished in the presence of N-acetyl-D-galactosamine (25 mM) or EGTA (3 mM), but is not significantly reduced by N-acetyl-D-glucosamine or D-mannose (25 mM). ASF particles are endocytosed via the coated pit/vesicle pathway and appear to be transported to the secondary lysosomes by coated vesicles, as shown by the occurrence of coated areas in the secondary lysosome membrane. These observations demonstrate the presence of an asialoglycoprotein receptor on Kupffer cells; therefore, the hepatocyte is not the only cell in the rat liver with D-galactose receptor activity.
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Does the D-galactose receptor on Kupffer cells recycle?
Kupffer cells carry on the cell surface a lectin-like receptor with specificity for D-galactose/D-glucose residues. After tryptic digestion of receptors, new lectin activity appears on the plasma membrane within 15 min. Protein biosynthesis de novo is not involved. This indicates the transfer of new lectin molecules stored within the cell to the cell surface. Kupffer cells can enter a physiological state where receptor recycling does not take place.
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