Electron microscopic evidence for an asialoglycoprotein receptor on kupffer cells: Localization of lectin-mediated endocytosis

Cell, Vol. 29, 859-866.

July 1982.

Copyright

0 1982

by MIT

Electron Microscopic Evidence for an Asialoglycoprotein Receptor on Kupffer Cells: Localization of Lectin-Mediated Endocytosis Victoria Kolb-Bachofen, Jutta Schlepper-Schafer and Wolrad Vogel1 lnstitut fur Biophysik und Elektronenmikroskopie Universitat Dusseldorf Moorenstrasse 5 D-4000 Dusseldorf 1 Federal Republic of Germany Hubert Kolb Diabetes Forschungsinstitut an der Universitat Dusseldorf Auf dem Hennekamp 65 D-4000 Dusseldorf 1 Federal Republic of Germany

Summary Direct evidence is given for the presence of an Nacetyl-o-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-o-galactosamine (25 mM) or EGTA (3 mM), but is not significantly reduced by N-acetyl-o-glucosamine or omannose (25 mfvl). 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. Introduction Conflicting results have been reported on the presence of an asialoglycoprotein receptor on Kupffer cells. Several groups have shown that asialoglycoproteins are taken up by parenchymal and not by sinusoidal cells of the liver (Ashwell and Morell, 1974; Schlesinger et al., 1978; Hubbard and Stukenbrok, 1979; Hubbard et al., 1979; Wall et al., 1980). However, previously we found indirect evidence for an asialoglycoprotein receptor in cell adhesion studies. It was found that a lectin-like receptor on the Kupffer cell surface mediates in vitro the adhesion of neuraminidase-treated erythrocytes and lymphocytes (Kolb and Kolb-Bachofen, 1978; Kolb et al., 1978, 1979; Schlepper-Schafer et al., 1980). The same receptor is responsible for the in vivo homing of desialylated blood cells to the liver (Kolb et al.. 1981; Mijller et al., 1981). The specificity of the Kupffer cell lectin (Kolb et al., 1980) is very similar to the asialo-

glycoprotein receptor on hepatocyte plasma membranes described by Ashwell and Morel1 (1974). Both receptors bind glycoproteins with terminal o-galactosyl residues; among monosaccharides, N-acetyl-o-galactosamine is a better reactant than o-galactose. We now provide direct evidence for the presence of an asialoglycoprotein receptor on isolated Kupffer cells by visualization of asialoglycoprotein binding in electron microscopy. We used the same technique previously to localize the asialoglycoprotein receptor on isolated hepatocytes (Kolb-Bachofen, 1981). Results Localization of Galactose-Specific Lectins on the Kupffer Cell Plasma Membrane Freshly isolated Kupffer cells were incubated with asialofetuin adsorbed onto colloidal gold particles of approximately 17 nm (ASF-gold) for various periods at 4X and subsequently were fixed and prepared for electron microscopy. Considerable binding of ligands is observed after 1 min of incubation time. Maximal binding is seen after 5 min and does not significantly change during the following hour. Increasing or decreasing the ASF-gold concentration by a factor of three did not alter the number of particles bound, indicating that the experiments were performed under saturating conditions. Particles were found binding to the plasma membrane in discrete clusters even when cells were incubated for only 1 min. The clusters were randomly distributed all over the surface, including microvilli or pseudopodia as well as nonvillous membrane areas (Figure 1 A). Carbohydrate inhibition experiments were performed to verify that ASF-gold binding to Kupffer cells is due to galactose-specific membrane lectins. The addition of N-acetyl-D-galactosamine to the cell suspension results in complete inhibition of particle binding (Figure 1 B; Table 11, whereas no significant effect is seen in the presence of N-acetyl-o-glucosamine or o-mannose (quantitative data for o-mannose included in Table 1). Complexing of divalent cations with EGTA abolishes ligand binding. Addition of bovine serum albumin (BSA; 10m4 M) did not interfere with ligand binding. From the data in Table 1, a rough estimate of the total number of particles bound by Kupffer cells was made. The calculation is based on the count of particles on equatorial sections (see Experimental Procedures). An average number of 150 particles bound per cell section (see Table 1) leads to an estimate of 4 x 1 O4 ASF-gold particles bound per Kupffer cell. The screening of equatorial cell sections revealed some heterogeneity among the cells. The major fraction (80%) bound an average of 122 particles per section. A smaller population (20%) showed considerably higher ligand binding, with an average of 306

CE?ll 860

Figure 1. Localization of ASF-Gold Bound to Kupffer Cell Plasma Membrane Cells were incubated with excess ligand for 20 min at 4°C. (A) The electron-dense ASF-gold particles (-17 nm) are bound in clusters thai t are localized in villous as well as in nonvillous membrane areas. (B) Incubation in the presence of N-acetyl-o-galactosamine (25 mM) co ‘mph ztely lateabolishes ASF-gold binding. (C) Particle binding occurs also in clusters on cells that had been fixed with 0.1% glutaraldehyde in phi buffered saline prior to incubation with the ligand. Magnification: 30.000x. Bar = 1 .O pm.

Galactose-Specific 861

Table

Lectin

1. Quantitative

Ligand

on Kupffer

Analysis

Incubation Period at 4’C (min)

Cells

of Particle

Inhibitor

Binding

25 mM

5

None

ASF-gold

5

N-acetyl-o-galactosamine

ASF-gold

20

None

ASF-gold

20

N-acetyl-o-galactosamine

ASF-gold

20

ASF-gold

Particles Bound per Cell Section -e SD. (n) 150 f

50 (20)

2 *

2 (20)

147 f 48 (30) 2 f

1 (20)

Mannose

102 f

32 (21)

124 f

47 (20) 17 (17)

GaCBSA-gold

5

None

Gal-BSA-gold

5

N-acetyl+galactosamine

23 f

BSA-gold

5

BSA-gold

None N-acetyl-o-galactosamine

68 f 26(19)

5

a(n) Number of cell sections examined. The number of bound gold particles was counted sections as described in Experimental Procedures.

85 -c 26 (22)

on equatorial

cell

particles per section. This may represent two populations of Kupffer ceils differing either in their in situ localization or in their state of differentiation. To investigate whether particle binding in clusters is due to rapid receptor aggregation after interaction with ligands or whether free receptors are preclustered in the plasma membrane, cells were prefixed to prevent lateral motion of membrane components. Prefixation results in a 30% reduction of ASF-gold binding without any apparent change in receptor distribution-that is, particles are also seen in small clusters all over the Kupffer cell surface (Figure 1 C). Further control experiments were performed to show the specificity of the ASF-particle binding. Kupffer cells were incubated with gold particles onto which defatted bovine serum albumin was adsorbed (BSA-gold). The observed binding after incubation under identical conditions is not clustered and is not inhibitable by N-acetyl-D-galactosamine (Table 1). However, when BSA was galactosylated (Gal-BSA) prior to adsorption to gold-thus yielding particles with terminal galactosyl groups but otherwise identical to the BSA-gold particles-binding is about as high as with ASF-gold and is inhibited by N-acetyl-o-galactosamine by more than 81% (see Table 1). Lectin-Mediated Endocytosis of ASF-Gold Particles When the interaction of Kupffer cells with ASF-gold was studied at 37°C uptake of particles was observed (Figure 2A). Carbohydrate inhibition experiments were performed to determine whether endocytosis of ASF-gold is lectin-mediated. Ligand uptake is inhibited by 95%-99% in the presence of 25 mM N-

acetyl-o-galactosamine (Figure 2B), whereas the same concentrations of N-acetyl-o-glucosamine or Dmannose were of no effect. To define the uptake mechanism more closely, cells were fixed at various intervals after addition of ASF-gold. After 1 min, gold particles are predominantly bound to the plasma membrane, and some markers are found inside coated membrane structures (Figure 3A). The number of particles binding to these cells (604 + 120 per equatorial cell section) is about three times as much compared to the binding at 4°C (Table 1). The increase of particles bound may be owing to cell recovery and to additional receptors exposed on the plasma membrane during the warmup period prior to ligand addition. After 2 min, the number of particles on the plasma membrane is reduced, and some ASF-gold is now seen in secondary lysosomal vacuoles (Figure 38). After 5 min of incubation, the vast majority of gold particles are found inside the large lysosomal vacuoles, whereas the Kupffer cell membrane is almost devoid of bound ASF-gold (Figure 3C). Lectin activity on the cell surface reappears (as judged from particle binding) after 10 min of incubation; thereafter, ASF-gold always is observed, bound in small clusters randomly distributed on the cell surface, associated with coated membrane structures and within lysosomal vacuoles (see Figure 2A). During the first 10 min of ligand uptake, coated pits of isolated Kupffer cells were located at the base of the microvilli or pseudopodia, as shown in Figure 4A. Cells that have been exposed to ASF-gold for longer periods form channel-like plasma membrane invaginations that often end as coated pits (Figure 48). These structures strongly resemble the “canaliculae vermiformis” described for Kupffer cells in situ (Matter et al., 1968). We did not observe other membrane structures within the cell being associated with ASF-gold other than coated vesicles and lysosomal vacuoles. Because of the irregular shape of the macrophages, coated vesicles seen at the cell periphery (Figure 4C) may as well represent coated pits tangentially cut. However, particle-containing coated vesicles were often seen in the cell’s interior near secondary lysosomes (Figure 4D), and in a few instances we found coated areas of lysosomal vacuoles (Figure 4E), indicating direct transport by coated pits/vesicles of ASF-gold from the plasma membrane to the secondary lysosomes. The intracellular localization of the lysosomal vacuole seen in Figure 4E was verified by examining serial sections of this cell. Discussion In the experiments presented here, the presence of an asialoglycoprotein receptor on Kupffer cells is demonstrated by means of asialofetuin adsorbed onto

Cell 862

Figure

2. Uptake

of ASF-Gold

Cells were incubated (b) and accumulated uptake by 95%-99%.

by Isolated

Kupffer

Cells

with excess ligand for 15 min at 37°C. (A) Particles are seen binding to the cell surface, in coated membrane in lysosomal vacuoles f-1. (B) Incubation in the presence of N-acetyl-o-galactosamine (25 mM) inhibits binding Magnification: 30,000X. Bar = 1 .O Am.

an electron-dense particle (ASF-gold). The asialoglycoprotein-gold complex binds to the Kupffer cell surface at 4°C and endocytosis is seen at 37%. Binding and uptake of ASF-gold is specifically inhibited by Nacetyl-D-galactosamine and is dependent on the presence of divalent cations. We therefore conclude that a lectin-like receptor on Kupffer cells interacts with P-D-galactosyl residues exposed on the desialylated fetuin. To differentiate further between lectin-specific and nonspecific particle binding, we performed parallel tests with Gal-BSA-gold and BSA-gold. Carbohydrate inhibition experiments demonstrated that GalBSA-gold is bound via recognition of o-galactosyl residues. In contrast, binding of BSA particles cannot be inhibited by N-acetyl-D-galactosamine and thus involves a different mechanism.

structures as well as

We therefore conclude that recognition of ASF-gold as well as of Gal-BSA-gold is due to the interaction of a lectin-like receptor on Kupffer cells with D-galactosyl residues expressed on these particles. These findings are in accordance with our previous observation that Kupffer cells adhere to terminal Dgalactosyl residues on desialylated blood cells. Cell contacts could also be inhibited by N-acetyl-o-galactosamine, D-galactose, asialoglycoproteins and by chelators of divalent cations (Kolb and Kolb-Bachofen, 1978; Kolb et al., 1978, 1979, 1980; SchlepperSchafer et al., 1980). The electron microscopic technique described here has also been used for the localization of the asialoglycoprotein receptor on isolated hepatocytes (KolbBachofen, 1981). In this study and in the present

W&ctose-Specific

Figure

3. Kinetics

Lectin

on Kupffer

of Ligand

Uptake

Cells

by Isolated

Kupffer

Cells

The macrophages were incubated with ASF-gold particles at 37’C. and the reaction was stopped at various times. (A) After incubation for 1 ligands are found predominantly binding to the plasma membrane; some are localized inside coated membrane structures 0). (6) After 2 m in of incubation, few particles are localized at the plasma membrane, the majority are found inside coated membrane structures (b) and some ! are inside the lysosomal vacuoles (+). (C) After 5 min at 37°C. most of the particles have accumulated inside lysosomal structures (-+), the pls lsma membrane being entirely devoid of particles. Magnification: 40.000x Bar = 0.5 pm.

Cell 864

Figure

4. Demonstration

of Ligand

Uptake

and Transport

to Lysosomal

Vacuoles

via Coated

Vesicles

(A) Macrophages incubated with the ligand for 1 min show coated pits 0) filled with particles at the base of pseudopodial cell projections. (B) Longer incubation periods (here, 20 min) result in the formation of particle-filled channel-like structures that often end as coated pits 0). (C) Coated vesicles near the cell surface containing ASF-gold (,) may as well represent coated pits tangentially cut. (D) However, coated membrane structures (b) filled with ASF-gold were found in the cell’s interior in close proximity to lysosomal vacuoles(J), indicating the existence of coated vesicles. (E) In a few cases, fusion of coated membranes (,) with secondary lysosomes is observed. The rareness of this structure indicates that the fusion is a rapid process. Magnification: (A. C and D) 70.000x; (B) 50,000x; (E) 84.000x. Bars = 0.1 pm. experiments, found

to be

glycoprotein-gold ligands

with

complexes very

low

nonspecific

have

been binding

properties (less than 5% specific binding), and therefore they are most suitable for an ultrastructural analysis of lectin-glycoconjugate interactions. When the interaction of ASF-gold with Kupffer cells of is followed at 4’C, binding but no endocytosis particles is seen. Binding of ASF-gold occurs in disCrete clusters that are randomly distributed all over the cell surface. Aggregation of receptors is not Iigand-induced, since the same result is seen with

prefixed

Kupffer

cells.

Clusters

are

also

seen

when

Gal-BSA-gold is used as ligand. More important, clusters are not observed with BSA-gold, a particle that is bound by a o-galactose independent mechanism. Electron microscopic examination of the standard ASF-gold solution (see Experimental Procedures) did not reveal clustering of particles prior to the interaction with Kupffer cells, in accordance with other reports (Horisberger and Rosset, 1977). The observation of receptor aggregates is in marked contrast to our previous demonstration of single-particle binding to

g&ctose-Specific

Lectin

on Kupffer

Cells

the galactose receptors on isolated hepatocytes (Kolb-Bachofen, 19811, where only prolonged incubation led to microaggregation of ligand-receptor complexes. In this study, liver cells were isolated following the same protocol; it therefore seems improbable that the cell isolation procedure may have caused receptor patching. In addition, there is evidence that the receptor of C3b is also preclustered on the macrophage plasma membrane (Petty et al., 1980). When the interaction between Kupffer cells and ASF-gold is followed at 37’C, endocytosis of particles is seen. Carbohydrate inhibition experiments show that this process also is lectin-mediated. Studying the kinetics of the endocytotic process, we found that in the presence of excess ligand a synchronous burst of endocytotic activity starts. The ligand needs 2-5 min for transport into the secondary lysosome, and it takes more than 5 min until new lectin activity appears on the plasma surface-that is, between the second and fifth minute the macrophage appears unable to bind additional ligand to the cell surface. This observation is in accordance with recent data of Weigel (1981) concerning the galactose receptor on hepatocytes where Weigel found a time lag between beginning endocytosis and reappearance of free receptors on the hepatocyte plasma membrane. Along with Weigel, we also favor the interpretation that this lag is due to receptor internalization and its recycling to the cell surface. However, we cannot rule out the possibility that the receptor does not leave the plasma membrane during the process of ligand endocytosis, but temporarily acquires an inactive state. The membrane structures involved in endocytosis are clearly clathrin-coated. The isolated Kupffer cells showed coated pits only at the base of microvilli or pseudopodia. Because of the irregular shape of the macrophage, it is difficult to differentiate between a coated vesicle and a tangentially sectioned pit. However, coated vesicles containing gold particles were often seen in the cell’s interior in close association to secondary lysosomes, and in a few instances we observed “coated pits” in secondary lysosomes, indicating fusion of coated vesicles. We therefore assume that coated membrane structures are involved in all phases of the transport of ASF-gold from the plasma membrane to the secondary lysosomes. In another system, the pathway of receptor-mediated endocytosis appears to involve an additional vesicle type-the “receptosome” -intermediary between coated pits and lysosomes (Willingham and Pastan, 1980; Wehland et al., 1981). The present demonstration of lectin-dependent endocytosis of asialofetuin-gold particles by Kupffer cells is of specific significance in light of previous investigations on soluble asialoglycoprotein uptake that find the Kupffer cells unable to ingest molecular asialoglycoprotein (Ashwell and Morell, 1974; Schles-

inger et al., 1978; Hubbard and Stukenbrok, 1979; Hubbard et al., 1979; Steer and Clarenburg, 1979; Wall et al., 1980). It appears that the interaction of Kupffer cell lectins with single asialofetuin molecules is not a sufficient trigger of endocytosis, whereas the reaction with a multivalent particle induces a physiological response. For this discrimination of ligand size, the clustering of receptors may be of importance. The signal for uptake of a ligand, then, is only triggered when more than a few receptors per cluster are occupied, a condition that is fulfilled best by particles with multiple binding sites, but not by soluble glycoproteins. Experimental

Procedures

Livers of male Wistar rats weighing 150-l 80 g were perfused with buffered collagenase solution (Boehringer-Mannheim), and the resulting liver cell suspension was enriched for Kupffer cells, as described by Schlepper-Schafer et al. (1980). The resulting cell suspension contained about 70% Kupffer cells, 10%-200/a hepatocytes and IO%-20% endothelial cells. Preparation of the Electron-Dense Ligand Asialofetuin adsorbed onto colloidal gold particles was used as ligand in electron microscopic studies. Gold granules of approximately 17 nm diameter were prepared as described by Horisberger and Rosset (1977) by reducing a 0.01% AuCI, solution with 1% sodium citrate. Asialofetuin was a gift from C. Bauer (Free University, Berlin). It was prepared from fetal calf fetuin (Grade Ill; Sigma; Munich) by acid hydrolysis (Bauer et al., 1976). Bovine serum albumin (essentially free of fatty acids: Sigma: Munich) was galactosylated as described by Kolb-Bachofen (1981), yielding a glycoprotein with five molecules galactose per one molecule BSA. The protein-gold complex was prepared by incubating the gold sol with protein (13 pg ASF, BSA or Gal-BSA per milliliter gold sol) at a pH of 6.0-6.5 in the presence of 0.01% polyethylene glycol (Carbowax 20 M; Union Carbide) for 30 min at 4°C. Stabilization of the protein-gold complex was scored visually after addition of NaCl to a sample of 50 pg (Horisberger and Rosset. 1977). About 5 gg ASF/ml gold sol was the lowest protein concentration to prevent flocculation, in good accordance with data found for the coupling of protein A (Slot and Geuze. 1981), which is of about the same molecular weight. From this protein concentration and the number of particles/ml sol (Horisberger and Rosset, 1977), the number of molecules bound of ASF/l7 nm particle was calculated as 80 molecules per particle. This calculation does not correlate to experiments of Kent and Allen (1981). who found 3 x 1 O3 antibody molecules (IgG) binding to one particle. The protein-gold complexes were washed twice by centrifugation at 32,000 x g for 30 min at 4OC and finally suspended in phosphatebuffered saline containing 0.02% polyethylene glycol. A concentrated stock solution of all three ligands (particle content between 4-6 x 1 O’*/ml) was either used 1-2 hr after preparation or stored for up to 3 weeks at 5°C (100 units of penicillin per milliliter of streptomycin added). Differences between storage periods were not found in the amount of nonspecific binding, nor did we observe a decrease of specific particle binding that might occur because of desorption of protein during storage (Goodman et al., 1981). A standard concentration of 10” particles/ml was used in experiments. The particle concentration of the protein-gold suspension was determined by photometry at 520 or 540 nm wavelength. Extinction was correlated to particle content by counting the number of particles in electron micrographs of carbon-coated grids onto which 2 pl of protein-gold solution had been dried. Electron Freshly

Microscopy of Ligand Binding isolated Kupffer cells (5 X lo5

cells

in 0.2

ml of Eagle’s

Cell 866

medium) were mixed with 0.1 ml of thestandard protein-gold solution. The interaction of Kupffer cells with protein-gold was either followed at ice bath temperature or at 37X. Carbohydrate inhibition of ligand binding or uptake was performed by adding monosaccharides to the Kupffer cell suspension 5 min prior to protein-gold. The final saccharide concentration in all assays was 25 mM. Preincubation of Kupffer cells with EGTA (Titriplex VI; Merck: Darmstadt) was performed in the same way: the final EGTA concentration was 3 mkt. In a few experiments, cells were prefixed by incubation with 0.1% glutaraldehyde (Serva; Heidelberg) in phosphate-buffered saline for 2 min at ice bath temperature, followed by washing and resuspension in Eagle’s medium. In all experiments, the binding reaction was stopped by the addition of an equal volume of ice-cold 0.2% glutaraldehyde in S-collidine buffer (0.1 M S-collidine, 0.1 M saccharose, 0.1 M NaCl and 2 mM CaCI,). Cells were immediately spun down at 400 x g and washed once with Eagle’s medium to remove excess gold particles. The cell pellet was fixed following the method of Franke et al. (1976) with the variations described by Kolb-Bachofen (1981). Samples were dehydrated with ethanol and embedded in epoxy resin according to the method of Spurr (1969). Electron microscopy was performed on greyish sections f-60 nm) contrasted with aqueous uranyl acetate for 5 min and lead citrate (Venable and Coggeshall. 1985) for 1 min. Estimation of the Number of Particles Bound on Kupffer Cells From light microscopic photographs, the mean diameter of freshly isolated rat Kupffer cells was determined to be about 12-l 4 pm. Random sections of embedded material were examined in the electron microscope. Sections through Kupffer cells with a diameter of 10 pm or more were defined as sections through the cell’s equator. The number of particles bound to the plasma membrane of these Kupffer cell sections was counted. Assuming the thickness of the examined sections as 0.06 pm, an estimate of the number of the bound particles per Kupffer cell could be calculated. Acknowledgments We thank Ulla Lammersen. Brigitte Renn and Andrea Schlomer for expert technical assistance and Sabine Wenzel-Unger for photographic work. We wish to thank Dr. F. A. Gries for his friendly interest in our studies. These studies were supported by a grant from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

December

3. 1981;

revised

April 2, 1982

Kent, S. P. and Allen, F. B. (1981). Antibody containing radioactive gold in the demonstration ecules. Histochemistry 72, 83-90. Kolb, H. and Kolb-Bachofen. mammalian macrophages. 878-683.

coated gold particles of cell surface mol-

V. (1978). A lectin-like Biochem. Biophys. Res.

Ashwell, G. and Morell. A. G. (1974). The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. 41, 99-128. metabolism

Franke, W. W., Luder. M. R.. Kartenbeck. J.. Zerban, H. and Keenan, T. W. (1976). Involvement of vesicle coat material in casein secretion and surface regeneration. J. Cell Biol. 69, 173-l 95. Goodman, S. L.. Hodges, G. M.. Trejdosiewicz, L. K. and Livingston, D. C. (1981). Colloidal gold markers and probes for routine application in microscopy. J. Microscopy 123, 201-213.

receptor Comm.

on 85,

Kolb. H., Kriese. A., Kolb-Bachofen. V. and Kolb. H. A. (1978). Possible mechanism of entrapment of neuraminidase-treated lymphocytes in the liver. Cell. Immunol. 40, 457-462. Kolb. H.. Kolb-Bachofen, V. and Schlepper-Schafer. contacts mediated by ogalactose-specific lectins Cell. 36, 301-308.

J. (1979). on liver cells.

Cell Biol.

Kolb, H., Vogt. D., Herbertz. L.. Corfield, A., Schauer, R. and Schlepper-Schafer. J. (1980). The galactose-specific lectins on rat hepatocytes and Kupffer cells have identical binding characteristics. HoppeSeyler’s Z. Physiol. Chem. 361, 1747-l 750. Kolb, H., Friedrich. E. and Siiss. R. (1981). Lectin mediates homing of neuraminidase-treated erythrocytes to the liver as revealed by scintigraphy. Hoppe-Seyler’s Z. Physiol. Chem. 362, 1609-1614. Kolb-Bachofen, V. (1981). Hepatic receptor for asialo-glycoproteins. Ultrastructural demonstration of ligand-induced microaggregation receptors. Biochim. Biophys. Acta 645, 293-299.

of

Matter, A., Orci, L., Forssmann, W. G. and Rouiller. C. (1968). The stereological analysis of the fine structure of the “micropinocytosis vermiformis” in Kupffer cells of the rat. J. Ultrastruct. Res. 23, 272-279. Muller, E.. Franco, M. W. and Schauer, R. (1981). Involvement of membrane galactose in the in viva and in vitro sequestration of desialylated erythrocytes. Hoppe-Seyler’s Z. Physiol. Chem. 362, 1615-l 620. Petty, H., Smith, L.. Fearon, D. and McConnel, H. (1982). Lateral distribution and diffusion of the C3b receptor of complement, HLA antigens, and lipid probes in peripheral blood leukocytes. Proc. Nat. Acad. Sci. USA 77,6587-6591. Schlepper-Schafer, J., Kolb-Bachofen. V. and Kolb, H. (1980). Analysis of lectin-dependent recognition of asialo-erythrocytes by Kupffer cells. Biochem. J. f86, 827-831. Schlesinger, P. H.. Doebber, T. W.. Mandell, B. F.. White, R., Deschryver, C.. Rodman, J. S.. Miller, M. J. and Stahl, P. (1978). Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Biochem. J. 176, 103-l 09. Slot, W. J. and Geuze. H. J. (1981). Sizing of protein A-colloidal gold probes for immunelectron microscopy. J. Cell Biol. 90, 533-538. Spurr, A. (1969). A low-viscosity epoxy resin embedding electron microscopy. J. Ultrastruct. Res. 26, 31-43.

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Venable. J. H. and Coggeshall. R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25. 407-408. Wall, D. A., Wilson, G. and Hubbard, A. L. (1980). The galactosespecific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell 27, 79-93. Wehland, J.. Willingham, M. C., Dickson, R. and Pastan, I. (1981). Microinjection of anticlathrin antibodies into fibroblasts does not interfere with the receptor-mediated endocytosis of up-macroglobulin. Cell 25. 105-l 19.

Horisberger, M. and Rosset, J. (1977). Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytochem. 25, 295-305.

Weigel, P. H. (1981). Evidence that hepatic asialoglycoprotein receptor is internalized during endocytosis and that receptor recycling can be uncoupled from endocytosis at low temperature. Biochem. Biophys. Res. Commun. 107, 1419-1425.

Hubbard, A. and Stukenbrok, H. (1979). autoradiographic study of the carbohydrate rat liver. II. J. Cell Biol. 83, 65-81.

Willingham, M. C. and Pastan. I. (1980). The receptosome: an intermediate organelle of receptor-mediated endocytosis in cultured fibroblasts. Cell 21, 67-77.

An electron microscope recognition system in the