The interaction of a lung surfactant protein (SP-A) with macrophages is mannose dependent

Lung surfactant protein A (SP-A) is the main protein component of pulmonary surfactant, which lines the alveolar space. We examined the interaction between recombinant human SP-A and human macrophages or monocytes. Binding and uptake of SP-A adsorbed onto colloidal gold particles was followed by electron microscopy and quantitated on micrographs. SP-A particles were internalized via coated pits/vesicles and transported to secondary lysosomes. Uptake was inhibited in the presence of alpha-D-mannosyl-bovine serum albumin (BSA) but not by beta-D-galactosyl-BSA. Two mannose-dependent recognition mechanisms might mediate SP-A uptake by macrophages. First, as SP-A is a glycoprotein with N-glycosylated glycans it could act as a ligand for the mannose-specific receptor on macrophages. Second, as SP-A is a mannose-specific lectin itself it could bind to mannose residues on the macrophage's cell surface. Activity of the Man-receptor on macrophages was demonstrated with alpha-D-mannosyl-BSA coated onto gold particles. Exposed alpha-D-mannosyl residues on macrophages were identified by Concanavalin A adsorbed onto gold particles. Hence, both mechanisms may be involved in principle. As monocytes have no mannose-specific receptor activity on their cell surface but internalize SP-A gold particles in a mannose-dependent manner, we conclude that at least the second mechanism participates in the recognition of SP-A by macrophages.     

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.     

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)     

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.     

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.     

Pulmonary collectins enhance phagocytosis of Mycobacterium avium through increased activity of mannose receptor

Collectins, including surfactant proteins A (SP-A) and D (SP-D) and mannose binding lectin (MBL), are the important constituents of the innate immune system. Mycobacterium avium, a facultative intracellular pathogen, has developed numerous mechanisms for entering mononuclear phagocytes. In this study, we investigated the interactions of collectins with M. avium and the effects of these lectins on phagocytosis of M. avium by macrophages. SP-A, SP-D, and MBL exhibited a concentration-dependent binding to M. avium. The binding of SP-A to M. avium was Ca(2+)-dependent but that of SP-D and MBL was Ca(2+)-independent. SP-A and SP-D but not MBL enhanced the phagocytosis of FITC-labeled M. avium by rat alveolar macrophages and human monocyte-derived macrophages. Excess mannan, zymosan, and lipoarabinomannan derived from the M. avium-intracellular complex, significantly decreased the collectin-stimulated phagocytosis of M. avium. Enhanced phagocytosis was not affected by the presence of cycloheximide or chelation of Ca(2+). The mutated collectin, SP-A(E195Q, R197D) exhibited decreased binding to M. avium but stimulated phagocytosis to a level comparable to wild-type SP-A. Enhanced phagocytosis by cells persisted even after preincubation and removal of SP-A or SP-D. Rat alveolar macrophages that had been incubated with SP-A or SP-D also exhibited enhanced uptake of (125)I-mannosylated BSA. Analysis by confocal microscopy and flow cytometry revealed that the lung collectins up-regulated the cell surface expression of mannose receptor on monocyte-derived macrophages. These results provide compelling evidence that SP-A and SP-D enhance mannose receptor-mediated phagocytosis of M. avium by macrophages.     

Surfactant protein A inhibits peptidoglycan-induced tumor necrosis factor-alpha secretion in U937 cells and alveolar macrophages by direct interaction with toll-like receptor 2.

Pulmonary surfactant protein A (SP-A) plays an important role in modulation of the innate immune system of the lung. Peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, is known to elicit excessive proinflammatory cytokine production from immune cells. In this study we investigated whether SP-A interacts with PGN and alters PGN-elicited cellular responses. Binding studies demonstrate that PGN is not a ligand for SP-A. However, SP-A significantly reduced PGN-elicited tumor necrosis factor alpha (TNF-alpha) secretion by U937 cells and rat alveolar macrophages. The inhibitory effect on TNF-alpha secretion was dependent upon SP-A concentrations in physiological range. Coincubation of SP-A and PGN with human embryonic kidney 293 cells that had been transiently transfected with the cDNA of Toll-like receptor 2 (TLR2), a cell signaling receptor for PGN, significantly attenuated PGN-induced nuclear factor-kappaB activity. SP-A directly bound to a soluble form of the recombinant extracellular TLR2 domain (sTLR2). Coincubation of sTLR2 with SP-A significantly reduced the binding of sTLR2 to PGN. These results indicate that the direct interaction of SP-A with TLR2 alters PGN-induced cell signaling. We propose that SP-A modulates inflammatory responses against the bacterial components by interactions with pattern-recognition receptors.     

Binding and uptake of pulmonary surfactant protein (SP-A) by pulmonary type II epithelial cells

A glycoprotein of Mr 26-36,000 (SP-A) is an abundant phospholipid-associated protein in pulmonary surfactant. SP-A enhances phospholipid reuptake and inhibits secretion by Type II epithelial cells in vitro. We have used two electron microscopic cytochemical methods to demonstrate selective binding and uptake of SP-A by rat pulmonary Type II epithelial cells. Using an immunogold bridging technique, we showed that SP-A binding was selective for Type II cell surfaces. Binding was dose dependent and saturable, reaching maximal binding at approximately 10 ng/ml. On warming to 23 degrees C, SP-A binding sites were clustered in coated pits on the cell surface. To characterize the internalization and intracellular routing of SP-A, we used the biotinyl ligand-avidin-gold technique. Biotinyl SP-A was bound by rat Type II epithelial cells as described above. On warming, biotinyl SP-A was seen in association with coated vesicles and was subsequently located in endosomes and multivesicular bodies. Biotinyl SP-A-gold complexes were seen in close approximation to lamellar bodies 10-60 min after warming. Binding of biotinyl SP-A was inhibited by competition with unlabeled SP-A. These results support the concept that Type II epithelial cells bind and internalize SP-A by receptor-mediated endocytosis. This newly described uptake system may play a role in the recycling of surfactant components or mediate the actions of SP-A on surfactant phospholipid secretion.     

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.     

Binding of surfactant protein A (SP-A) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of SP-A.

Pulmonary surfactant protein A (SP-A) has been shown to act as an opsonin in the phagocytosis of viruses by alveolar macrophages. To determine whether SP-A binds to viral proteins and which part of the SP-A molecule is involved in this interaction, binding studies were undertaken. SP-A was labeled with fluorescein isothiocyanate, and its binding to herpes simplex virus type 1-infected HEp-2 cells, as a model for virus-infected cells in general, was studied using flow cytometry. The binding of SP-A to virus-infected cells was saturable, reversible, and both time- and concentration-dependent, reaching a maximal level after 30 min at an SP-A concentration of 10 micrograms/ml. An approximately 4-fold increase in binding of SP-A to infected cells over control cells was observed. Yeast mannan, a mannose homopolysaccharide, did not influence the binding. However, heparin inhibited binding of SP-A in a concentration-dependent manner. In addition, heparin could also dissociate cell-bound SP-A, indicating that polyanionic oligosaccharides are involved in the binding of SP-A to virus-infected cells. Deglycosylated SP-A, obtained by digestion with N-glycosidase F, did not bind to infected cells. Heparin or deglycosylation of SP-A had no effect on the stimulation of alveolar macrophages by SP-A. It is concluded that the carbohydrate moiety of SP-A is involved in the recognition of viruses by SP-A and may play a role in the antiviral defenses of the lung.     

Dexamethasone increases expression of mannose receptors and decreases extracellular lysosomal enzyme accumulation in macrophages

Macrophages express a mannose-specific pinocytosis receptor that binds and internalizes lysosomal hydrolases. Treatment of rat bone marrow-derived macrophages with dexamethasone resulted in a concentration- and time-dependent increase in mannose-receptor activity. The dexamethasone effect was maximal at 24 h. Half-maximal effects were observed at a dexamethasone concentration of 2.5 X 10(-9) M. With 125I-beta-glucuronidase as ligand, a 2.5-fold increase in uptake rate was observed in dexamethasone-treated cells, with no change in Kuptake (2.5 X 10(-7) M beta-glucuronidase). Cell surface binding (4 degrees C) was elevated 2.6-fold following dexamethasone treatment. The increase in ligand binding appeared to be due to an increase in number of sites with no change in affinity. Cycloheximide suppressed the dexamethasone-mediated rise in receptor number, while cycloheximide alone had little effect on receptor activity over 16 h. These results suggest that dexamethasone stimulates synthesis of mannose receptors in macrophages. Extracellular accumulation of hexosaminidase was sharply reduced by dexamethasone treatment, and corresponded with the rise in mannose-receptor activity. Extracellular levels of hexosaminidase from untreated macrophages were modestly increased by the presence of mannan, while the extracellular activity from dexamethasone-treated cells was increased significantly by mannan. Extracellular hexosaminidase, released from zymosan-treated macrophages, was dramatically reduced by dexamethasone pretreatment. Enzyme released from zymosan-stimulated macrophages was efficiently endocytosed by dexamethasone-treated cells in co-culture experiments, and this endocytosis was blocked by the addition of mannan. These results suggest that the mannose receptor of macrophages may play a role in regulating extracellular levels of lysosomal enzymes via a secretion-recapture mechanism.     

The galactose-specific receptor system in rat liver during development

The number and distribution of galactose-specific binding sites were investigated in rat liver cells during perinatal development. Ligand binding to hepatocytes, macrophages and endothelial cells was followed with in vitro and in situ experiments by electron microscopy, using lactosylated bovine serum albumin adsorbed onto 5 nm colloidal gold particles as ligand. Binding capacity, starting at a late stage of fetal development, is very low both on the hepatocyte and on the macrophage surface, which show single particles statistically distributed. By contrast, bound particles are absent from fetal endothelial cells, which also lack the typical coated regions. In vivo, experiments at 37 degrees C show that endocytosis occurs to some extent in prenatal life. These results indicate that the expression of galactose-specific receptors' activity on the different liver cell types follows different developmental patterns, which are independently modulated.     

Biochimica et biophysica acta,vol. 646 (1981)

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for d-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, galactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Dissé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2–5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

Hepatic receptor for asialo-glycoproteins. Ultrastructural demonstration of ligand-induced microaggregation of receptors

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for D-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, glactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Disé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2-5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

Binding and internalization of gold-labeled IFN-gamma by human Raji cells

Binding and internalization of gold-labeled IFN-gamma (IFN-gamma/Au) by human Raji cells was examined by scanning and transmission electron microscopy. For SEM, visualization of gold particles was enhanced by the silver enhancement technique and by backscattered electron imaging. Binding studies revealed distinct labeling of microvilli-bearing cells after incubation with at least 10 U/ml IFN-gamma/Au, whereas cells with a smooth surface showed substantially lower labeling. After application of higher IFN-gamma (greater than 200 U/ml) concentrations, labeling intensity remained constant, which is consistent with the concentration of radiolabeled IFN-gamma required for saturating receptors on Raji cells. The specificity of IFN-gamma/Au binding was demonstrated by complete displacement with unlabeled IFN-gamma and by partial inhibition of labeling with a monoclonal anti-IFN-gamma R antibody. Thus, colloidal gold represents a valuable tag for visualizing the interaction of IFN-gamma with its receptor. Internalization of IFN-gamma/Au was initiated by accumulation of gold particles in coated pits which occurred within 10 min after warming of Raji cells. Additional incubation at 37 degrees C (up to 2 h) led to the appearance of gold particles in endocytic vesicles and lysosomes. Thus, our studies indicate that IFN-gamma/Au enters the Raji cells via the typical endocytotic pathway.     

Interaction of high density lipoproteins with cholesteryl ester-laden macrophages: biochemical and morphological characterization of cell surface receptor binding, endocytosis and resecretion of high density lipoproteins by macrophages

Morphological and biochemical experiments were carried out to investigate the interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages. It is demonstrated that resident mouse peritoneal macrophages express HDL receptors. Subsequent to receptor-mediated binding, HDL are internalized and intracellularly transported into endosomes. These endosomes do not fuse with the lysosomal compartment but interact with the margin of intracellular plasma lipid droplets. Macrophages do not degrade, but rather resecrete internalized HDL particles as described for the transferrin-receptor pathway. HDL binding to freshly isolated macrophages is saturable at a concentration of approximately 320 ng HDL-protein/mg cell protein and a Scatchard plot indicates the presence of some 130 000-190 000 receptors/cell with a Kd of approximately 9 X 10(-7) M. Binding of HDL on the macrophage surface is significantly enhanced in cholesterol-laden macrophages, whereas the increase in the rate of uptake and secretion is less pronounced. Within the HDL fraction the HDL2 subclass showed higher binding, uptake and secretion activity as compared with HDL3. From these experimental data we postulate that cholesterol uptake from macrophages is mediated by HDL particles which interact with these cells via a receptor-mediated retroendocytosis pathway.     

Surfactant protein A binds to IgG and enhances phagocytosis of IgG-opsonized erythrocytes

Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein C1q interact. Since complement protein C1q binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to C1q, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to C1q. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding.     

Endocytic pathway for surfactant protein A in human macrophages: binding, clathrin-mediated uptake, and trafficking through the endolysosomal pathway

In the noninflamed lung, surfactant protein A (SP-A) acts as an anti-inflammatory molecule through its effects on macrophage (MPhi) function, modulating cytokine and reactive oxygen and nitrogen intermediate production. The receptors responsible for these effects of SP-A on human MPhi are not clear, although SP-A binding to several proteins has been described. In this study, we demonstrate high-affinity specific binding of SP-A to primary human MPhi. SP-A binding was inhibited by EGTA, indicating calcium dependence. However, mannan did not inhibit SP-A binding, suggesting that binding is mediated by a direct protein-protein interaction that does not involve carbohydrate recognition. Our laboratory has previously shown that SP-A is rapidly endocytosed by human MPhi into discrete vesicles. Although previous work indicates that SP-A is ultimately degraded by murine MPhi over time, the trafficking pathway of SP-A through MPhi after uptake has not been reported and is of potential biological importance. We examined trafficking of SP-A in human MPhi by electron and confocal microscopy and show for the first time that SP-A is endocytosed by primary human MPhi through clathrin-coated pits and colocalizes sequentially over time with the early endosome marker EEA1, late endosome marker lamp-1, and lysosome marker cathepsin D. We conclude that SP-A binds to receptor(s) on human MPhi, is endocytosed by a receptor-mediated, clathrin-dependent process, and trafficks through the endolysosomal pathway. These studies provide further insight into the interactions of SP-A with the MPhi cell surface and intracellular compartments that play important roles in SP-A modulation of lung MPhi biology.     

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-Sch?fer, 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.     

Chemical modification of surfactant protein A alters high affinity binding to rat alveolar type II cells and regulation of phospholipid secretion

Alveolar type II cells express a high affinity receptor for pulmonary surfactant protein A (SP-A), and the interaction of SP-A with these cells leads to inhibition of surfactant lipid secretion. We have investigated the binding of native and modified forms of SP-A to isolated rat alveolar type II cells. Native and deglycosylated forms of SP-A readily competed with 125I-SP-A for cell surface binding. Alkylation of SP-A with excess iodoacetamide yielded forms of SP-A that did not inhibit surfactant lipid secretion and did not compete with 125I-SP-A for cell surface binding. Reductive methylation of SP-A with H2CO and NaCNBH3 yielded forms of SP-A with markedly reduced receptor binding activity that also exhibited significantly reduced capacity to inhibit lipid secretion. Modification of SP-A with cyclohexanedione reversibly altered cell surface binding and the activity of SP-A as an inhibitor of lipid secretion. Two monoclonal antibodies that block the function of SP-A as an inhibitor of lipid secretion completely prevented the high affinity binding of SP-A to type II cells. A monoclonal antibody that recognizes epitopes on SP-A but failed to block the inhibition of secretion also failed to completely attenuate high affinity binding to the receptor. Concanavalin A inhibits phospholipid secretion of type II cells by a mechanism that is reversed in the presence of excess alpha-methylmannoside. Concanavalin A did not block the high affinity binding of 125I-SP-A to the receptor. Neither the high affinity binding nor the inhibitor activity of SP-A was prevented by the presence of mannose or alpha-methylmannoside. The SP-A derived from humans with alveolar proteinosis is a potent inhibitor of surfactant lipid secretion but failed to completely displace 125I-SP-A binding from type II cells. From these data we conclude that: 1) cell surface binding activity of rat SP-A is directly related to its capacity to inhibit surfactant lipid secretion; 2) monoclonal antibodies directed against SP-A can be used to map binding domains for the receptor; 3) the lectin activity of SP-A against mannose ligands does not appear to be essential for cell surface binding; 4) concanavalin A does not compete with SP-A for receptor binding; and 5) the human SP-A derived from individuals with alveolar proteinosis exhibits different binding characteristics from rat SP-A.