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.     

The galactose-recognizing system of rat peritoneal macrophages; identification and characterization of the receptor molecule

Resident rat peritoneal macrophages express a galactose-recognizing system, which mediates binding and uptake of cells and glycoproteins exposing terminal galactose residues. Here we describe the identification, isolation, and characterization of the corresponding receptor molecule. Using photoaffinity labelling of adherent peritoneal macrophages with the 4-azido-6-125I-salicylic acid derivative of anti-freeze glycoprotein 8 followed by SDS-PAGE and autoradiography, we identified the receptor of these cells as a protein with an apparent molecular mass of 42 kDa. Furthermore, cell surface receptors were radioiodinated by an affinity-supported labelling technique using the conjugate of asialoorosomucoid and lactoperoxidase, followed by extraction and isolation by affinity chromatography. Finally, the native receptor was isolated and analysed. To estimate its binding activity in solutions, a suitable binding assay was developed, using the precipitation of receptor-ligand complex with polyethylene glycol to separate bound from unbound 125I-asialoorosomucoid, which was used as ligand. It is shown that the isolated receptor binds to galactose-exposing particles and distinguishes between sialidase-treated and -untreated erythrocytes, similar to peritoneal macrophages. The binding characteristics of the membrane-bound and the solubilized receptor are described in the following paper of Lee et al.     

Carbohydrate specificity of the galactose-recognizing receptor of rat peritoneal macrophages

The galactose-recognizing system of rat peritoneal macrophages mediates the binding and uptake of desialylated blood cells and glycoproteins. To characterize the specificity of this receptor, binding studies were performed with various galactose derivatives as competitive inhibitors and sialidase-treated erythrocytes or asialoorosomucoid as ligands for receptors, which were either membrane-bound or isolated after solubilization. From the results obtained it can be concluded that galactose is recognized via its hydrophobic and/or hydrophilic regions, formed by the accumulation of OH-functions on one side and of H-atoms on the other ("side effect"), whereas the binding partner or the anomeric configuration of galactose has no significant influence. Although it became apparent that not a single hydroxyl group of the sugar is responsible for binding, the hydroxyl at C-4 seems to be most important, followed by the OH-group at C-3. Those at C-1, C-2 and C-6 do not play a great role. This order of importance ("position effect") was found with galactose, derivatized by methylation or otherwise, and with diastereomers of galactose. Whereas the recognition of a single galactose residue leads to weak binding only, an appropriate arrangement of several of these ligands in one molecule results in an enormous increase in the binding strength of each galactose residue. This "cluster effect" was observed not only with membrane-bound but also with solubilized receptor. However, the binding of asialoorosomucoid by the latter was better inhibited with free galactose, when compared with the membrane-bound receptor.     

Influence of sialic acids on the galactose-recognizing receptor of rat peritoneal macrophages

The interaction of the galactose-recognizing receptor from rat peritoneal macrophages with ligands containing terminal galactose residues, such as asialoorosomucoid, desialylated erythrocytes or lymphocytes, can be inhibited by free N-acetylneuraminic acid (Neu5Ac) and oligosaccharides or glycoproteins containing this sugar in terminal position. This effect of Neu5Ac on the receptor is specific. The other naturally occurring or most of synthetic neuraminic acid derivatives tested do not exhibit an equivalent inhibitory potency as Neu5Ac. Although free Neu5Ac inhibits 5-fold stronger (K50 = 0.2mM) than free galactose, clustering of Neu5Ac in oligosaccharides and glycoproteins does not lead to stronger inhibition, which is in contrast to galactose-containing ligands. A more branched (triantennary) sialooligosaccharide inhibits less than biantennary and unbranched sialooligosaccharides. This may be the reason, why complex sialic acid-containing ligands like native orosomucoid or blood cells are not bound and internalized by the macrophages. The dissociation of asialoorosomucoid from the receptor is slow under the influence of Neu5Ac and requires relatively high concentrations of this sugar, whereas the dissociation mediated by galactose is rapid and requires lower concentrations. An allosteric influence of Neu5Ac on the binding of galactose by the receptor is discussed.     

Interaction of rat peritoneal macrophages with homologous, sialidase-treated lymphocytes in vitro

The interaction in vitro between rat peritoneal macrophages and homologous, sialidase-treated lymphocytes was investigated. Lymphocytes were isolated from blood, thymus, and spleen on a density gradient. Total sialic acids obtained by acid hydrolysis were 10 nmol/10(8) lymphocytes, composed of 29% N-acetyl-neuraminic acid and 71% N-glycoloylneuraminic acid. Sialidase treatment released maximally 33% of membrane sialic acids. Lymphocytes were bound to peritoneal macrophages to an extent which increased in parallel with the amount of sialic acids released, whereas binding of untreated lymphocytes was not significant. This interaction was inhibited by free galactose and substances containing terminal galactose residues. Asialoorosomucoid with its oligoantennary sugar chains proved to be a 10(5) times more potent inhibitor of the interaction than lactose. The addition of homologous serum had no influence on binding. Electron microscopy revealed that vital lymphocytes were tightly bound to macrophages and only damaged lymphocytes appeared to be phagocytozed. The experiments demonstrate that the interaction between rat peritoneal macrophages and sialidase-treated lymphocytes is mediated by a macrophage receptor specific for galactose. This sugar is demasked on the surface of lymphocytes after the removal of terminal sialic acids. The role of this mechanism in cell recognition, elimination and homing of lymphocytes is discussed.     

Studies on the mechanism of uptake of low density lipoprotein-proteoglycan complex in macrophages

Earlier, we (Vijayagopal, P. et al. (1988) Biochim. Biophys. Acta 960, 210) showed that mouse peritoneal macrophages metabolize low density lipoprotein (LDL)-proteoglycan complex by a receptor pathway distinct from the acetyl-LDL receptor. Further studies were conducted to probe further into the mechanism of LDL-proteoglycan complex uptake by macrophages. Both 125I-methyl-LDL-proteoglycan complex and 125I-LDL-proteoglycan complex were taken up and degraded by the cells to the same extent. Similarly, the ability of these ligands to stimulate cholesteryl ester synthesis was also indistinguishable. These results rule out the possibility of apoB,E receptor involvement in the uptake of LDL-proteoglycan complex in macrophages. Sodium fluoride, cytochalasin D and aggregated LDL inhibited degradation of the complex by 24%, 26% and 28%, respectively, indicating that phagocytosis is only a minor pathway for the uptake. Both binding and degradation of the complex were not inhibited by excess hyaluronic acid suggesting that ligand recognition was not through hyaluronic acid binding sites. As compared to acetyl-LDL, the cellular degradation of LDL-proteoglycan complex was retarded. Macrophages exhibited a rapid stimulation of [3H]inositol trisphosphate (IP3) release and diacylglycerol production when incubated with LDL-proteoglycan complex. Furthermore, pertussis toxin produced a 62% inhibition of LDL-proteoglycan complex mediated IP3 release, suggesting that LDL-proteoglycan complex metabolism in macrophages is dependent upon the G-protein coupled signal transduction mechanism. These results show that receptor mediated endocytosis plays a major role in the metabolism of LDL-proteoglycan complex in macrophages.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.     

Binding, uptake, and transcytosis of ligands for mannose-specific receptors in rat liver: an electron microscopic study

We have investigated the initial distribution of mannose-specific binding sites in rat liver as well as the uptake and transcytosis pathways of ligands for this receptor in in situ and in vivo experiments. As ligands we used mannan adsorbed onto colloidal gold particles of sizes 5, 17, and 35 nm (Man-Au5, Man-Au17, or Man-Au35). The in situ binding pattern of Man-Au5 in the prefixed liver is identical to the one described earlier for galactose-exposing ligands in the same organ. With the exception of the binding by hepatocytes, where only scarce binding of Man-Au5 was observed, ligands were found adhering in a preclustered pattern all over the cell surface of liver macrophages and binding in aggregates over the coated pits of endothelial cells. In double-labeling experiments different particle sizes were used for glycoproteins with terminal mannosyl or galactosyl residues. This simultaneous localization of the two binding activities revealed that on endothelial cells the two activities are always found to be present in the same coated pit. On liver macrophages the clustered binding occurred at different membrane areas. Uptake and transcytosis of Man-Au5, 17, 35 were studied after their injection into the tail vein. Three and fifteen minutes after injection most of the Man-Au5 and all of Man-Au17 or Man-Au35 was found in sinusoidal liver cells, i.e., macrophages and endothelial cells. One hour after injection, endocytosed ligand is redistributed from large--presumably lysosomal--vacuoles to small noncoated vesicles that are localized predominantly near the space of Dissé. Between 1 and 40 h after injection, ligands of all sizes are transcytosed and found in the hepatocytes. No ligand accumulation is observed in hepatocytes as an indirect indication for secretion into bile. With this investigation we give evidence for transcytotic activity not only of liver endothelium but also of the resident liver macrophages.     

Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells.

Increased levels of agalactosyl IgG (G0 IgG) are found in several autoimmune diseases, including rheumatoid arthritis, in which they are correlated with severity of the disease. To investigate whether structural alteration of IgG may lead to aberrant processing and presentation of IgG peptides as autoantigens, we have studied uptake of G0 IgG by human dendritic cells and macrophages cultured from PBMC. We found that enzymatic removal of terminal galactose residues, which exposes N-acetylglucosamine residues, increases uptake of soluble IgG mediated by mannose receptor on macrophages and dendritic cells. Efficient uptake appears to require recycling of the receptor, can be blocked by saccharides or Abs reactive with mannose receptor, and is dependent upon the state of maturation of the dendritic cells. No differences between IgG isotypes in ability to be internalized by APC were identified, suggesting that uptake would not be limited to a particular subset of Abs. These results suggest a novel pathway by which Abs or Ag-Ab complexes can be taken into dendritic cells and macrophages, and potentially generate epitopes recognized by T cells. These findings may have particular relevance for autoimmune disorders characterized by high levels of G0 IgG.     

Macrophage recognition of LDL modified by levuglandin E2, an oxidation product of arachidonic acid

Levuglandin (LG) E₂, a secoprostanoic acid levulinaldehyde derivative, is a product of free radical oxidation that forms covalent adducts with lysyl residues on proteins. Treatment of LDL with LGE₂ leads to uptake and degradation by mouse peritoneal macrophages. Oxidized LDL, but not acetyl LDL efficiently competed for binding and uptake of LGE₂-modified ¹²⁵I-LDL. This result suggests that LGE₂-modified LDL was recognized by a class of scavenger receptor that demonstrated ligand specificity for oxidized LDL but not for acetyl LDL.     

Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation

The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.     

Monensin inhibits recycling of macrophage mannose-glycoprotein receptors and ligand delivery to lysosomes.

Binding studies with cells that had been permeabilized with saponin indicate that alveolar macrophages have an intracellular pool of mannose-specific binding sites which is about 4-fold greater than the cell surface pool. Monensin, a carboxylic ionophore which mediates proton movement across membranes, has no effect on binding of ligand to macrophages but blocks receptor-mediated uptake of 125I-labelled beta-glucuronidase. Inhibition of uptake was concentration- and time-dependent. Internalization of receptor-bound ligand, after warming to 37 degrees C, was unaffected by monensin. Moreover, internalization of ligand in the presence of monensin resulted in an intracellular accumulation of receptor-ligand complexes. The monensin effect was not dependent on the presence of ligand, since incubation of macrophages with monensin at 37 degrees C without ligand resulted in a substantial decrease in cell-surface binding activity. However, total binding activity, measured in the presence of saponin, was much less affected by monensin treatment. Removal of monensin followed by a brief incubation at pH 6.0 and 37 degrees C, restored both cell-surface binding and uptake activity. Fractionation experiments indicate that ligands enter a low-density (endosomal) fraction within the first few minutes of uptake, and within 20 min transfer to the lysosomal fraction has occurred. Monensin blocks the transfer from endosomal to lysosomal fraction. Lysosomal pH, as measured by the fluorescein-dextran method, was increased by monensin in the same concentration range that blocked ligand uptake. The results indicate that monensin blockade of receptor-mediated endocytosis of mannose-terminated ligands by macrophages is due to entrapment of receptor-ligand complexes and probably receptors in the pre-lysosomal compartment. The inhibition is linked with an increase in the pH of acid intracellular vesicles.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

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.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

Studies on the mechanism of enkephalin receptor down regulation

The association of [3H] [D-Ala2, D-Leu5] enkephalin ([3H]DADLE]) with mouse neuroblastoma cells (N4TG1) was investigated. Under identical conditions the time course, dose response curve and temperature dependence for ligand uptake were similar to those for ligand-induced receptor loss (down regulation). Uptake of [3H]DADLE was inhibited by opiate ligands as well as by the metabolic inhibitors sodium azide and 2,4 dinitrophenol. Comparison of the effects of these inhibitors on receptor binding, ligand uptake and receptor loss indicated that these cells accumulate [3H]DADLE in excess of their surface receptor number. The data suggest that receptor recycling occurs and that ligand is internalized via receptor mediated endocytosis.     

Generation of a soluble IFN-gamma inducer by oxidation of galactose residues on macrophages

Depletion of macrophages from human peripheral blood mononuclear cells (PBMC) caused a marked decrease in galactose oxidase and sodium periodate, but not a calcium ionophore, stimulated Interferon-gamma (IFN-gamma) production. Reconstitution of such depleted cultures with galactose oxidase treated macrophages, but not lymphocytes, restored IFN-gamma levels to those of control nonfractionated PBMC. Thus, galactose oxidase seemed to act on macrophages which in turn stimulated lymphocyte production of IFN-gamma. Unlike human cells which have terminal galactose residues on glycoproteins, murine cell glycoproteins terminate their oligosaccharide component in the order N-acetyl-neuraminic acid followed by D-galactose, N-acetyl-glucosamine, and glycoprotein. Galactose oxidase or sodium periodate only activated murine macrophages to stimulate lymphocyte IFN-gamma production after exposing D-galactose residues by the removal of the terminal N-acetyl-neuraminic acid residues with neuraminidase. Removal of such exposed terminal galactose residues with beta-galactosidase inhibited the effect of galactose oxidase on murine macrophages. Taken together, these results strongly suggest that oxidation of terminal galactose residues on macrophages is the initial site of action of galactose oxidase and sodium periodate. Studies with Boyden chambers have shown that galactose oxidase-treated macrophages released a soluble factor which stimulates lymphocyte production of IFN-gamma. Based on these findings, it appears that the oxidation of terminal galactose residues on the surface of macrophages leads to the induction and transmission of a soluble signal for lymphocyte production of IFN-gamma.     

Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins

Long term incubation of proteins with glucose, named the Maillard reaction (Maillard, L. C. (1912) C. R. Acad. Sci. (Paris) 154, 66-68), gives rise to advanced glycosylation end product (AGE) with fluorescence, color, as well as cross-linked properties. The receptor-mediated endocytosis of AGE-proteins by macrophages was reported (Vlassara, H., Brownlee, M., and Cerami, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5588-5592). The present study on the binding of AGE-bovine serum albumin (BSA) to rat peritoneal macrophages and sinusoidal liver cells demonstrated the presence of a saturable, high affinity receptor for AGE-BSA with Kd = 2.4 x 10(-7) M (macrophages) and 2.1 x 10(-7) M (sinusoidal cells). The cellular binding of AGE-BSA and its endocytic uptake by these cells were competitively inhibited by BSA preparations modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, ligands known to be specific for a scavenger receptor for aldehyde-modified proteins (Horiuchi, S., Murakami, M., Takata, K., and Morino, Y. (1986). J. Biol. Chem. 261, 4962-4966). These ligands also had a profound in vivo effect on the plasma clearance of 125I-AGE-BSA as well as its hepatic uptake. Thus, endocytic uptake of AGE-proteins by macrophages appeared to be mediated by a scavenger receptor for aldehyde-modified proteins. This provides evidence for the biological importance of the scavenger receptor in eliminating senescent macromolecules from the circulation.