- Email: [email protected]
Binding of Mannosylated Ferritin to Chicken Bone Marrow Macrophages
Immunobiol., vol. 165, pp. 46-62 (1983)
1 Institut fur Geflugelkrankheiten, Freie Universitiit Berlin, Fachbereich Veteriniirmedizin, Germany 2 Weizmann Institute of Science, Department of Membrane Research, Rehovot, Israel
Binding of Mannosylated Ferritin to Chicken Bone Marrow Macrophages GERTRUD ROSSI! and S. HIMMELHOCH2 Received December 6, 1982 . Accepted March 10, 1983
Abstract Ferritin conjugated to diazotated p-aminophenylmannoside and mannan was used for ultrastructural visualization of binding and endocytosis via the mannose receptor. Conjugates were bound by live macrophages but not by glutaraldehyde-fixed cells. Binding was inhibited by 0.1 mM mannan and 0.2 M a-methylmannoside, and strongly reduced, but not abolished, after trypsin degradation of surface receptors. Binding sites were rapidly assembled on coated pits, which often showed a polar distribution on the cell surface and entered the cell following the direction of microtubuli. Some coated vesicles occasionally kept in touch with the extracellular space. 10-min exposure to the conjugate resulted in changes in the surface morphology, such as a loss of infoldings and increased membrane tension. Ferritin accumulated in large smooth vesicles with coated membrane regions. It was mostly detached from the membrane and agglutinated into big clumps, together with fibrillar material and small vesicles which may derive from the vesicle membrane.
Introduction Membrane lectins have been found on cells with very different biological functions (1-5). The appearance of these specific sugar-binding and cellagglutinating proteins can be limited to a narrow phase of cell development (6, 7); the same lectin may occur at different sites and play different roles during embryonic and adult life (8). The two known functions of membrane lectins are: adhesion, studied in developing muscles and brain (7, 9), in substrate adhesion (10) and cell contacts (11) and mediation of endocytosis, which has been shown for liver cells (12) and macrophages (13). Adhesion is supposed to be the main function of malignant cell-surface lectins during the formation of metastases (14-16). The role of lymphocyte membrane lectins is still not well known. However, there are at least two indications which suggest a possible social function of lectins in immune cells: they are specific markers for subpopulations on lymphocytes (17), while on macrophages their appearance and frequency seem to depend on cell environment (18) and activation (19).
Mannose Binding Sites . 47
Natural glycoproteins (2, 12), as well as glycosylated carrier proteins (20, 21), are used to investigate lectin-mediated binding. Carrier proteins themselves may be bound by cell receptors, as is well known for bovine serum albumin (22) and ferritin (23). However, specific recognition of the sugar as well as its inhibition (2, 4, 15) are facilitated by haptenisation of the sugar. In the present study, ferritin is used as an electron-dense carrier protein to show the early ultrastructural aspect of binding and uptake mediated by mannose-specific receptors. It is glycosylated either with multi moles/mole of a monomer sugar or with 1 mole/mole of a polymer sugar. Chicken bone marrow macrophages of a growth phase, characterized by strong phagocytic activity towards untreated yeast cells (24), are used as a model in this study.
Materials and Methods Reagents
Native ferritin, p-aminophenyl-D-mannopyranoside, ConA, and glutaraldehyde were obtained from Sigma, Munich, F.R.G. Sepharose 4B, ConA sepharose, and lentillectin sepharose were obtained from Pharmacia (Freiburg, F.R.G.). Mannan was prepared from C. albicans, strain 628, Institut Pasteur, using the method of KOCOUREK and BALLOU (25). Polybed 812 kit, obtained from Polyscience, Warrington, USA, was used as embedding material. Agarose L (LKB, Grafelfing, F.R.G.) with low endomosis (m, = -0.02) was used for affinity electrophoresis. Cells
Bone marrow cells were harvested from femur, tibia and radius of SPF chickens "VALO" (Lohmann, Cuxhaven, F.R.G.). Macrophages were obtained by fractionated adherence, as previously described (24). Each fraction was allowed to adhere for 12 hours, and fractions 2 and 3 were used for electron microscopy. Preparation of ferritin conjugates
P-aminophenyl-D-mannopyranoside, dissolved in 0.1 M HCI, was diazotated with 0.05 M sodium nitrite and coupled to ferritin in aqueous solution according to the method of McBROOM et al. (26), in a 66:1 molar ratio. The reaction was allowed to proceed at O°C at a pH of 8.5 to 8.8 for 2 hours and stopped with 0.1 M HCl. Separation of high molecular weight products was performed on Sepharose 4B eluted with 10 mM Tris, 145 mM NaCI, pH 7.1. Unbound ferritin was separated by affinity chromatography on ConA sepharose, in Tris buffer in the presence of 2 mM CaCl2 and 2 mM MnClz, and eluted with 0.3 M a-methylmannoside which was then separated on Sephadex G 25. The conjugate was concentrated with polyethylene glycol 20,000 to a final ferritin content of 6 mg/ml and dialysed against Tris buffer. Mannan was bound to ferritin by the two-step glutaraldehyde method of OTIO et al. (27). Ferritin in 0.1 M sodium phosphate buffer, pH 7, was activated with 2.5 % glutaraldehyde in 0.05 M sodium phosphate buffer to a final concentration of 0.16%, for 2 hours at 37°C. Unbound glutaraldehyde was separated on Sephadex G 25. 140 mg mannan in 0.25 M phosphate buffer was added per 1 mM of activated ferritin. Binding was allowed for 24 hours at 3rC in the presence of NaNJ , and the reaction was stopped with 0.1 M Tris-HCI, pH 7.8.
48 . GERTRUD ROSS! and S. H!MMELHOCH Purification of the conjugate was performed as above, using lentil-lectin sepharose for affinity chromatography. Binding of sugars to ferritin was controlled by affinity electron microscopy: fixed, ConAcoated cells were labelled with the conjugates and embedded as described below. The absence of native ferritin in the conjugates was confirmed by inhibition of their binding to macrophages in the presence of 0.1-0.2 mM mann an and 0.1-0.2 M a-methylmannoside. Rocket-affinity electrophoresis in ConA agarose (28) (100 [.Ig ConA/ml) was used as a control for the ferritin-mannan conjugate: precipitation obtained with ferritin-mannan and its inhibition by mannan, ferritin-a-mannoside and a-methylmannoside were observed. Electrophoresis was performed for 10 hours at 100 V and 8 mA (2.5 V/cm), using a Tris-Ca-lactate buffer. Treatment and embedding of monolayers
Macrophage monolayers, grown on polystyrene petri dishes (Lux Scient. Coop., Newbury Park, Cal., USA) were washed with serum-free culture medium (RPMI 1640, Flow Laboratories, Bonn, F.R.G.), adjusted to pH 7.1, and labelled with conjugates for 1 or 10 min at 37°C in a 5 % CO 2 in air atmosphere. Specific inhibition of labelling was confirmed by preincubation of monolayers with 0.1 mM mannan or 0.1 M a-methylmannoside and with 0.1 M D-galactose as an unrelated sugar, followed by treatment with conjugates in the presence of the sugar for 1 min. Reduction of labelling due to a degradation of surface proteins was evaluated by treatment of monolayers with 0.02 % trypsin at 37°C for 5 min. The reaction was stopped by washing monolayers with the culture medium. The cells were then incubated for 1 min with conjugate. Control of specific labelling was performed by incubation of glutaraldehyde-fixed monolayers with 0.1 M ConA in Tris-NaCI buffer, pH 7, in the presence of Ca++ and Mn++ for 20 min, followed by 20 min labelling with conjugates. Labelled mono layers were washed with PBS and fixed with 2.5 % glutaraldehyde in PBS with 0.5 % potassium dichromate (29) for 60 min. Post-fixation was done with 1 % osmiumtetraoxide in PBS for 10 min. Plates were dehydrated in graded ethanols and embedded in Polybed 812 (30). Cross- and longitudinal ultra-thin sections were cut on a Sorvall MT 2B
.A FM .+
+
.F
FM FaM + M
Fig. 1. Rocket affinity electrophoresis in ConA agarose. Ferritin-mannan (FM) precipitates, forming a sharp rocket, which is partially inhibited by the addition of ferritin-a-mannoside (FM + FaM) or mannan (FM + M). a-methylmannoside (0.3 M) inhibits only within its diffusion zone. Ferritin-a-mannoside conjugate (FaM) and native ferritin (F) do not precipitate.
Mannose Binding Sites . 49
b
Fig. 2. Treatment of glutaraldehyde-fixed macrophages with ferritin-a-mannoside. a) Cells were incubated with ConA and successively with ferritin-conjugate. Dense labelling is shown on the cell surface (1) and on a filopod (Fp), indicating the distribution of binding sites for ConA. X 45,000. b) Cells were treated with conjugate only. Ferritin-a-mannoside is not bound by glutaraldehyde-fixed cells. X 67,500.
50 .
GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 3. Binding of ferritin-a-mannoside by vital macrophages after treatment with the conjugate for 1 min. a) Most of the ferritin is located on a coated pit (Cp), very few particles are found on the uncoated membrane. X 99,000. b) Coated pits aligned on a membrane section perpendicular to microtubuli (Mt), indicating their polar distribution. X 82,500.
ultramicrotome using a diamond knife (Diatome, Bienne, Switzerland). Sections were stained with 2 % uranyl acetate in water for 30 min, washed and stained with lead citrate (31). Electron micrographs were taken on a Philips 300 at 80 kV.
Mannose Binding Sites
51
Results Affinity electrophoresis was used as a qualitative control for the binding of sugar to ferritin. As seen in Figure 1, ferritin-mannan (FM) precipitated very early, giving a short and sharp rocket. The addition of a small amount of mann an (5 f-tl of a 0.042 mM solution) drastically retarded precipitation (FM + M). A similar elongated rocket was seen in the conjugate which still contained unbound mannan. Addition of a-methylmannoside (0.3 M) induced an inhibition only within the diffusion zone of the sugar, outside this zone, the conjugate was readily precipitated (FM + a MM). When the ferritin-a-mannoside conjugate (FM + FaM) was added, however, precipitation of FM was retarded, giving an indirect proof of binding of the aminophenyl sugar to ferritin. No precipitation was obtained with ferritina-mannoside alone (FaM), as long as it was free of aggregates or high molecular weight products, and none, of course, with native ferritin (F). The binding of p-aminophenylmannoside to ferritin was controlled by affinity electron microscopy on fixed, ConA-coated cells. A dense labelling was obtained both on the cell surface and filopods (Fig. 2a), indicating indirectly the number of binding sites for ConA. Omitting ConA-coating, the ferritin sugar conjugates were never bound to glutaraldehyde-fixed cells, indicating the proteic nature of the mannose receptor and its cross-linking by the fixative (Fig. 2b). Ferritin-a-mannoside was rapidly bound by vital macrophages and appeared clustered in well-defined regions of the cell surface. After treatment of the cells with the conjugate for 1 min, coated pits assembled almost all of the bound material (Fig. 3). Outside the coated pits, very few single black grains were seen scattered over the numerous folds of the surface. Labelled, coated pits were distributed irregularly over the membrane, but appeared more densely packed in certain regions, where microtubuli proceeded perpendicular to the surface (Fig. 3b). In some narrow regions of the cell, an intense uptake of ligand was observed in surface-near coated vesicles as early as after 1 min (Fig. 4). Even fusion of coated and uncoated vesicles occurred. As in Figure 4, microtubuli were found perpendicular to the cell surface, and the coated vesicles appeared to enter the cell following their direction. Rapid invagination of coated pits was found also with ferritin mannan. Deep pits were occasionally still connected with the surface membrane by means of uncoated channels, as shown in Figure Sa. A similar pattern was obtained when macrophages were treated with native ferritin for 1 min (Fig. Sb). However, binding and uptake of both sugar conjugates were almost completely inhibited in the presence of mann an or a-methyl-mannoside. Coated pits and vesicles as well as the uncoated membrane parallel to microtubuli were found without any ferritin (Fig. 6a and b), while binding and uptake of native ferritin were never inhibited in the presence of sugars (Fig. 6c). Galactose failed to inhibit binding and uptake of the conjugates.
52 . GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 4. Uptake of ferritin-a-mannoside in coated vesicles after treatment with the conjugate for 1 min. Coated vesicles (Cv) apparently derive from a narrow space of the cell surface arranged along a microtubulus (Mt). X 99,000.
Treatment with trypsin for 5 min at 37°C induced a strong reduction in surface binding of the sugar conjugates. However, some uptake was observed in coated vesicles, but generally they contained only very few particles. Significant morphological changes occurred in the treated cells,
Mannose Binding Sites . 53
Fig. 5. a) Binding of ferritin-mann an on coated pits (Cp) after treatment with the conjugate for 1 min, in addition, just incorporated coated pits (1). X 82,500. b) Native ferritin bound to coated pits. A pattern similar to that obtained with mannosylated ferritin is recognizable. X 99,000.
such as flattening and vacuolisation. Large vacuoles were occasionally filled with membrane material (Fig. 7a, b). At the end of a lO-min exposure of macrophages to ferritin-a-mannoside, all steps of binding and uptake could be observed (Fig. 8). Coated pits and
54
GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 6. Treatment with ferritin-mannan (a, b) and native ferritin (c) in the presence of 0.3 M a-methylmannoside. a) The uncoated membrane, parallel to the microtubuli (Mt), and coated pits (Cp) show binding of the conjugate. X 32,400. b) The folded membrane and cytoplasmic vesicles are free of ligand. X 99,000. c) Native ferritin binding 0) and uptake in coated vesicles (Cv) is not inhibited in the presence of sugar. X 67,500.
Mannose Binding Sites . 55
Fig. 7. Binding of ferritin-a-mannoside after treatment of cells with 0.02 % trypsin for 5 min at a) Uncoated membrane, microvilli (Mv), and filopods (Fp) are free of ferritin, which, however, appears in a coated vesicle (Cv). X 32,400. b) The same as a). In addition, coated pits (Cp) are seen free of ferritin. Vacuole (V) filled with membrane material. X 32,400. c) Some ferritin particles are bound on a flat pit (1) and are occasionally seen in coated vesicles (Cv). Clear area (A) limited by vesicles and tubuli. X 64,800.
3rc.
56
. GERTRUD ROSSI
and S.
HIMMElHOCH
Fig. 8. Uptake of mannosylated ferritin by macrophages treated with ferritin-a-mannoside for 10 min at The ligand appears in coated vesicles (Cv) and in large smooth vesicles (Sv), where it is partially detached from the membrane and agglutinated to big clumps (X) together with fibrillar material and small vesicles (v). The smooth vesicles are surrounded by small electron-dense vesicles (ev) and secretory granules (Sg) some of which contain ferritin (1). The inset shows different steps of ligand uptake. Coated vesicle in contact with surface membrane by means of an uncoated channel (1). X 60,000.
3rc.
Mannose Binding Sites . 57
Fig. 9. Cross-section of a macrophage treated with ferritin-a-mannoside for 1 min. On both sides of the cell the ligand appears mainly on coated pits (Cp), the surface membrane shows many infoldings. X 48,000.
vesicles close to the cell surface did not occur more frequently than after 1 min. Large amounts of ferritin accumulated in large smooth vesicles, detached from the membrane and agglutinated into big clumps, together with amorphous or fibrillar material and with small vesicles. Small electron-
58 .
GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 10. Cross-section of a cell after treatment with ferritin-a-mannoside for 10 min. Ferritin is bound to microvilli; furthermore, the ligand is located in Cv, Sv and in small electron-dense vesicles. Part of the Sv membrane is coated. Coated pits are scarce, one Cp is located at the basis of a microvillus (Mv). The surface is unfolded. X 60,000.
dense Golgi vesicles surrounded the large smooth vesicles and occasionally contained some ferritin grains or seemed to be in contact with the vesicle membrane. Some ferritin was even found in secretory granules.
Mannose Binding Sites . S9
Comparison of the cross-sections of two macrophages treated for 1 min (Fig. 9) and for 10 min (Fig. 10) with ferritin-a-mannoside gives a general impression of the different location sites of the conjugate. After short-term treatment, it was restricted to the surface which appeared rough with small folds, coated pits and vesicles. After long-term treatment, ferritin was located deep in the cell within small dense vesicles and large vesicles agglutinated with fibrillar or round structures. The large vesicles were sometimes incomplete and fused with the cytoplasm. The surface of the long-term treated cells appeared unfolded, smooth and with straight short microvilli which bound ferritin in a diffuse manner. Coated pits were scarce and mostly located near the basis of microvilli.
Discussion
Ultrastructural visualization of binding sites for mannose allows, more than their quantitative estimation, an evaluation of their surface distribution and behaviour as true receptors. This can be achieved by following an electron-dense mannosylated protein during its binding to coated pits and uptake in coated vesicles. Due to its size, ferritin is considered a fine membrane marker, but the protein itself is bound to cell receptors (23). Glycosylation is aimed to cover the carrier protein. Its recognition as being «mannosylated» and the specific inhibition of its binding are achieved by glycosylation either with the highly branched polymannose mannan or by a high number of moles/mole of p-aminophenylmannopyranoside. A dense labelling of ConA-coated cells is achieved with both conjugates. Affinity electrophoresis in ConA agarose is a sensitive control method for ferritin-mannan, but it is less effective as a control of ferritin mannoside, which inhibits precipitation of ferritin mannan but does not precipitate itself. Mannosylated ferritin is bound by in vitro differentiating bone marrow macrophages of young chicken on receptor sites, which are found assembled on coated pits in less than 1 min. Coated pits are not equally distributed over the whole cell surface, but they appear more concentrated in well-defined membrane regions. A similar polarisation is observed also for the endocytic activity: pinocytosis is found much more advanced in one narrow section of a cell where all the coated vesicles seem to enter the cell following the direction of microtubuli (see Fig. 4a). Surface-associated ferritin conjugate has never been found clustering over the surface. It is assembled as single points aligned on coated pits, suggesting that the binding molecule is not moving over the membrane, but within the bilayer like an integral membrane protein (32) able to interact with clathrin (33). The proteic nature of its binding is apparent by its inactivation with glutaraldehyde, more so than with the short-term degradation caused by trypsin treatment, since in this case degraded surface receptors may be
60 . GERTRUD ROSSI and S. HIMMELHOCH
readily replaced from inner stores (13). However, significant morphological changes which occur in trypsin-treated cells may be considered as an injury. Once collected in smooth vesicles and detached from the vesicle membrane, the endocytosed glycoprotein appears clustered in big clumps. This agglutination may be due to pH-values of the vesicle content which may be near the isoelectric point of the protein, or to detachment of receptor-ligand complexes or membrane pieces: at least the ferritin particles appear associated to amorphous or fibrillar material or to small vesicle-like structures. Coated membrane sections of the smooth vesicles may indicate their origin from fusion of coated vesicles with preformed smooth vesicles (34). Degradation of the glycoprotein cannot be demonstrated by morphological investigations. The ferritin particles remain visible even when part of the vesicle membrane appears to be missing and the cytoplasm seems to fuse with the vesicle content. Due to the rapid internalization of coated pits, the cell surface is never saturated with ligand. Binding and uptake continue over the observed period of time, while the surface remains nearly free, indicating that the macrophage uses the surface receptor predominantly for endocytosis. The continuous internalization of membrane areas causes morphological changes, which become recognizable after 10 min exposure to the conjugate. The fine membrane infoldings disappear, and the membrane shows a major tension. Straight microvilli bind ferritin particles in a diffuse manner. The picture suggests that microvilli continue binding the ligand, which is moved to coated pits at their bases, but uptake slowly decreases since coated pits are less available on the remainder surface. Acknowledgements The authors are indebted to Profs. H.-J. MERKER and R. GOSSRAU for their advice and fruitful discussions as well as their generous help in making their EM facilities available for this study. They are also grateful to Dr. R. GOLDMAN for stimulating discussions and for having placed her laboratory facilities at their disposal. - This study was supported by the Minerva Stiftung.
References 1. TEICHBERG, J. V., J. SII.MAN, D. D. BETTSCH, and G. RESHEFF. 1975. A j3-galactoside binding protein from electric organ tissue of Electrophorus electricus. Proc. Nat!. Acad. Sci. USA 72(4): 1383. 2. KAWASAKI, T., and G. ASHWELL. 1977. Isolation and characterization of an avian hepatic binding protein specific for N-acetylglucosamin-terminated glycoprotein. J. BioI. Chern. 252(18): 6536. 3. GARTNER, T. K., D. C. WILLIAMS, F. C. MINIClN, and D. R. PHIJ.TPS. 1978. Thrombin induced platelet aggregation is mediated by a platelet plasma membrane-bound lectin. Science 200: 1281. 4. GREMO, F., D. KOBILER, and S. H. BARONDES. 1978. Distribution of an endogenous lectin in the developing chick optic tectum. J. Cell BioI. 79: 491.
Mannose Binding Sites . 61 5. STAHL, P. D., J. S. RODMAN, M. J. MILl.FR, and P. H. SCHLESINGER. 1978. Evidence for receptor-mediated binding of glycoproteins, glycoconjugates and lysosomal glycosidases by alveolar macrophages. Proc. Natl. Acad. Sci. USA 75(3): 1399. 6. NOWAK, T. P., P. L. HAYWOOD, and S. H. BARONDES. 1976. Developmentally regulated lectin in embryonic chick muscle and a myogenic cell line. Biochem. Biophys. Res. Comm. 68(3): 650. 7. KOBILER, D., E. C. BEYER, and S. H. BARONDES. 1978. Developmentally regulated lectins from chick muscle, brain and liver have similar chemical and immunological properties. Dev. BioI. 64: 265. 8. BFYER, E. c., K. T. TOKUYASU, and S. H. BARONDFS. 1979. Localisation of an endogenous lectin in chicken liver, intestine and pancreas. J. Cell BioI. 82: 565. 9. GARTNFR, T. K., and T. R. PODLESKI. 1976. Evidence that the types and specific activity of lectins control fusion and L6 myoblasts. Biochem. Biophys. Res. Comm. 70: 1142. 10. WFI(;rT, P. H. 1980. Rat hepatocytes bind to synthetic galactoside surface via a patch of asialoglyco-protein receptors. J. Cell BioI. 87: 855. 11. KOLIl, H., and V. KOLB-BACHOfFN. 1978. A lectin like receptor on mammalian macrophages. Biochem. Biophys. Res. Comm. 85: 678. 12. BAENZIGER, J. U., and D. FIETE. 1980. Galactose and N-acetylgalactosamine specific endocytosis of glycopeptides by isolated rat hepatocytes. Cell 22: 611. 13. STAHL, P., P. H. SCHLESINGER, E. SIGARDSON, J. S. ROllMAN, and Y. C. Ln:. 1980. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell 19: 207. 14. GRAllEL, L. B., S. D. ROSEN, and G. R. MARTIN. 1979. Teratocarcinoma stem cells have a cell surface carbohydrate-binding component implicated in cell-cell adhesion. Cell 17: 477. 15. RAZ, A., and R. LOTAN. 1981. Lectin-like activities associated with human and murine neoplastic cells. Cancer Research 41: 3642. 16. RAZ, A., and R. LOTAN. 1982. On the possible role of tumor-associated lectins in metastasis in «Membranes in Tumor Growth». GALEOTTI, T., G. NERI, and S. PAPA, eds. Elsevier/North Holland, Biomedical Press. 17. BARZILAY, M., M. MONSIGNY, and N. SHARON. 1982. Interaction of soybean agglutination with human peripheral blood lymphocyte subpopulations: evidence for the existence of a lectin-like substance on the lymphocyte surface. In: «Proceedings of Fourth Lectin Meeting in Copenhagen». T. C. BOG-HANSEN, ed. Walter de Gruyter, Berlin, in press. 18. ROSSI, G., H. WEILER, and S. GRUND. 1982. Phagocytic activity of chicken macrophages towards untreated yeast cells as influenced by cell interactions. In: «Proceedings of the VIIIth ISHAM Congress, Palmerston North». M. Baxter, ed., in press. 19. EZEKOWITZ, R. A. B., J. AUSTYN, P. D. STAHL, and S. GORDON. 1981. Surface properties of bacillus Calmette-Guerin-activated mouse macrophages. J. Exp. Med. 154: 60. 20. KOLll-BACHOFEN, V., J. SCHLEPPER-SCHAFER, and W. VO(;EII.. 1982. Electron microscopic evidence for an asialoglycoprotein receptor on Kupffer cells: localisation of lectinmediated endocytosis. Cell 29: 859. 21. KIEllA, c., A. C. ROCHE, F. DELMOTTE, and M. MONSIGNY. 1979. Lymphocyte membrane lectins. Direct visualisation by the use of fluoresceinyl-glycosylated cytochemical markers. FEBS Letters 99: 329. 22. MEHL, T. D., and D. LAGUNOFl'. 1975. Uptake of aggregated albumin by rat macrophages in vitro: affinities of cells for monomeric and aggregated bovine serum albumin. J. Reticuloendoth. Soc. 18(2): 125. 23. POIYCARD, A., and H. BESSJS. 1962. Micropinocytosis and rhopheocytosis. Nature 194: 110. 24. ROSSI, G., and F. TURBA. 1981. Phagozytose unterschiedlich behandelter Candidazellen durch Knochenmarks- und Peritonealmakrophagen vom Huhn. Mykosen 24: 684. 25. KOCOUREK, J., and C. E. BALl.OU. 1969. Methods for fingerprinting yeast cell wall mannans. J. Bacteriol. 100(3): 1175.
62 . GERTRUD ROSSI and S. HIMMELHOCH 26. McBROOM, C. R., C. H. SAMANEN, and 1. J. GOl.DSTEIN. 1972. Carbohydrate antigens: coupling of carbohydrates to proteins by diazonium and phenylisothiocyanate reactions. Methods in Enzymology XXVIII, Complex Carbohydrates Part B. V. GINSBUR(;, ed., Academic Press. 27. OTTO, H., H. TAKAMIYA, and A. VOGT. 1973. A two-stage method for cross-linking antibody globulin to ferritin by glutaraldehyde. Comparison between the one-stage and the two-stage method. J. Immunol. Methods 3: 137. 28. OWEN, P., J. D. OPPENHEIM, M. S. NACHBAR, and R. E. KESSl.ER. 1977. The use of lectins in the quantitation and analysis of macromolecules by affinity electrophoresis. Anal. Biochem. 80: 446. 29. DALTON, A. J. 1955. A chrome-osmium fixative for electron microscopy. Anat. Rec. 121: 281. 30. Luf'T, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409. 31. REYNOl.DS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell BioI. 17: 208. 32. NICOl.SON, G. L. 1976. Transmembrane control of the receptors on normal and tumor cells. 1. Cytoplasmic influence over cell surface components. Biochem. Biophys. Acta 475: 57. 33. PEARSE, B. M. F. 1976. Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Cell BioI. 73(4): 1255. 34. PASTAN, J. H., and M. C. WILLINGHAM. 1981. Journey to the center of the cell: role of the receptosomes. Science 214: 504. Dr. G. ROSSI, Institut fur Geflugelkrankheiten, Freie Universitat Berlin, KoserstraBe 21, D-I000 Berlin 33, Germany
1 Institut fur Geflugelkrankheiten, Freie Universitiit Berlin, Fachbereich Veteriniirmedizin, Germany 2 Weizmann Institute of Science, Department of Membrane Research, Rehovot, Israel
Binding of Mannosylated Ferritin to Chicken Bone Marrow Macrophages GERTRUD ROSSI! and S. HIMMELHOCH2 Received December 6, 1982 . Accepted March 10, 1983
Abstract Ferritin conjugated to diazotated p-aminophenylmannoside and mannan was used for ultrastructural visualization of binding and endocytosis via the mannose receptor. Conjugates were bound by live macrophages but not by glutaraldehyde-fixed cells. Binding was inhibited by 0.1 mM mannan and 0.2 M a-methylmannoside, and strongly reduced, but not abolished, after trypsin degradation of surface receptors. Binding sites were rapidly assembled on coated pits, which often showed a polar distribution on the cell surface and entered the cell following the direction of microtubuli. Some coated vesicles occasionally kept in touch with the extracellular space. 10-min exposure to the conjugate resulted in changes in the surface morphology, such as a loss of infoldings and increased membrane tension. Ferritin accumulated in large smooth vesicles with coated membrane regions. It was mostly detached from the membrane and agglutinated into big clumps, together with fibrillar material and small vesicles which may derive from the vesicle membrane.
Introduction Membrane lectins have been found on cells with very different biological functions (1-5). The appearance of these specific sugar-binding and cellagglutinating proteins can be limited to a narrow phase of cell development (6, 7); the same lectin may occur at different sites and play different roles during embryonic and adult life (8). The two known functions of membrane lectins are: adhesion, studied in developing muscles and brain (7, 9), in substrate adhesion (10) and cell contacts (11) and mediation of endocytosis, which has been shown for liver cells (12) and macrophages (13). Adhesion is supposed to be the main function of malignant cell-surface lectins during the formation of metastases (14-16). The role of lymphocyte membrane lectins is still not well known. However, there are at least two indications which suggest a possible social function of lectins in immune cells: they are specific markers for subpopulations on lymphocytes (17), while on macrophages their appearance and frequency seem to depend on cell environment (18) and activation (19).
Mannose Binding Sites . 47
Natural glycoproteins (2, 12), as well as glycosylated carrier proteins (20, 21), are used to investigate lectin-mediated binding. Carrier proteins themselves may be bound by cell receptors, as is well known for bovine serum albumin (22) and ferritin (23). However, specific recognition of the sugar as well as its inhibition (2, 4, 15) are facilitated by haptenisation of the sugar. In the present study, ferritin is used as an electron-dense carrier protein to show the early ultrastructural aspect of binding and uptake mediated by mannose-specific receptors. It is glycosylated either with multi moles/mole of a monomer sugar or with 1 mole/mole of a polymer sugar. Chicken bone marrow macrophages of a growth phase, characterized by strong phagocytic activity towards untreated yeast cells (24), are used as a model in this study.
Materials and Methods Reagents
Native ferritin, p-aminophenyl-D-mannopyranoside, ConA, and glutaraldehyde were obtained from Sigma, Munich, F.R.G. Sepharose 4B, ConA sepharose, and lentillectin sepharose were obtained from Pharmacia (Freiburg, F.R.G.). Mannan was prepared from C. albicans, strain 628, Institut Pasteur, using the method of KOCOUREK and BALLOU (25). Polybed 812 kit, obtained from Polyscience, Warrington, USA, was used as embedding material. Agarose L (LKB, Grafelfing, F.R.G.) with low endomosis (m, = -0.02) was used for affinity electrophoresis. Cells
Bone marrow cells were harvested from femur, tibia and radius of SPF chickens "VALO" (Lohmann, Cuxhaven, F.R.G.). Macrophages were obtained by fractionated adherence, as previously described (24). Each fraction was allowed to adhere for 12 hours, and fractions 2 and 3 were used for electron microscopy. Preparation of ferritin conjugates
P-aminophenyl-D-mannopyranoside, dissolved in 0.1 M HCI, was diazotated with 0.05 M sodium nitrite and coupled to ferritin in aqueous solution according to the method of McBROOM et al. (26), in a 66:1 molar ratio. The reaction was allowed to proceed at O°C at a pH of 8.5 to 8.8 for 2 hours and stopped with 0.1 M HCl. Separation of high molecular weight products was performed on Sepharose 4B eluted with 10 mM Tris, 145 mM NaCI, pH 7.1. Unbound ferritin was separated by affinity chromatography on ConA sepharose, in Tris buffer in the presence of 2 mM CaCl2 and 2 mM MnClz, and eluted with 0.3 M a-methylmannoside which was then separated on Sephadex G 25. The conjugate was concentrated with polyethylene glycol 20,000 to a final ferritin content of 6 mg/ml and dialysed against Tris buffer. Mannan was bound to ferritin by the two-step glutaraldehyde method of OTIO et al. (27). Ferritin in 0.1 M sodium phosphate buffer, pH 7, was activated with 2.5 % glutaraldehyde in 0.05 M sodium phosphate buffer to a final concentration of 0.16%, for 2 hours at 37°C. Unbound glutaraldehyde was separated on Sephadex G 25. 140 mg mannan in 0.25 M phosphate buffer was added per 1 mM of activated ferritin. Binding was allowed for 24 hours at 3rC in the presence of NaNJ , and the reaction was stopped with 0.1 M Tris-HCI, pH 7.8.
48 . GERTRUD ROSS! and S. H!MMELHOCH Purification of the conjugate was performed as above, using lentil-lectin sepharose for affinity chromatography. Binding of sugars to ferritin was controlled by affinity electron microscopy: fixed, ConAcoated cells were labelled with the conjugates and embedded as described below. The absence of native ferritin in the conjugates was confirmed by inhibition of their binding to macrophages in the presence of 0.1-0.2 mM mann an and 0.1-0.2 M a-methylmannoside. Rocket-affinity electrophoresis in ConA agarose (28) (100 [.Ig ConA/ml) was used as a control for the ferritin-mannan conjugate: precipitation obtained with ferritin-mannan and its inhibition by mannan, ferritin-a-mannoside and a-methylmannoside were observed. Electrophoresis was performed for 10 hours at 100 V and 8 mA (2.5 V/cm), using a Tris-Ca-lactate buffer. Treatment and embedding of monolayers
Macrophage monolayers, grown on polystyrene petri dishes (Lux Scient. Coop., Newbury Park, Cal., USA) were washed with serum-free culture medium (RPMI 1640, Flow Laboratories, Bonn, F.R.G.), adjusted to pH 7.1, and labelled with conjugates for 1 or 10 min at 37°C in a 5 % CO 2 in air atmosphere. Specific inhibition of labelling was confirmed by preincubation of monolayers with 0.1 mM mannan or 0.1 M a-methylmannoside and with 0.1 M D-galactose as an unrelated sugar, followed by treatment with conjugates in the presence of the sugar for 1 min. Reduction of labelling due to a degradation of surface proteins was evaluated by treatment of monolayers with 0.02 % trypsin at 37°C for 5 min. The reaction was stopped by washing monolayers with the culture medium. The cells were then incubated for 1 min with conjugate. Control of specific labelling was performed by incubation of glutaraldehyde-fixed monolayers with 0.1 M ConA in Tris-NaCI buffer, pH 7, in the presence of Ca++ and Mn++ for 20 min, followed by 20 min labelling with conjugates. Labelled mono layers were washed with PBS and fixed with 2.5 % glutaraldehyde in PBS with 0.5 % potassium dichromate (29) for 60 min. Post-fixation was done with 1 % osmiumtetraoxide in PBS for 10 min. Plates were dehydrated in graded ethanols and embedded in Polybed 812 (30). Cross- and longitudinal ultra-thin sections were cut on a Sorvall MT 2B
.A FM .+
+
.F
FM FaM + M
Fig. 1. Rocket affinity electrophoresis in ConA agarose. Ferritin-mannan (FM) precipitates, forming a sharp rocket, which is partially inhibited by the addition of ferritin-a-mannoside (FM + FaM) or mannan (FM + M). a-methylmannoside (0.3 M) inhibits only within its diffusion zone. Ferritin-a-mannoside conjugate (FaM) and native ferritin (F) do not precipitate.
Mannose Binding Sites . 49
b
Fig. 2. Treatment of glutaraldehyde-fixed macrophages with ferritin-a-mannoside. a) Cells were incubated with ConA and successively with ferritin-conjugate. Dense labelling is shown on the cell surface (1) and on a filopod (Fp), indicating the distribution of binding sites for ConA. X 45,000. b) Cells were treated with conjugate only. Ferritin-a-mannoside is not bound by glutaraldehyde-fixed cells. X 67,500.
50 .
GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 3. Binding of ferritin-a-mannoside by vital macrophages after treatment with the conjugate for 1 min. a) Most of the ferritin is located on a coated pit (Cp), very few particles are found on the uncoated membrane. X 99,000. b) Coated pits aligned on a membrane section perpendicular to microtubuli (Mt), indicating their polar distribution. X 82,500.
ultramicrotome using a diamond knife (Diatome, Bienne, Switzerland). Sections were stained with 2 % uranyl acetate in water for 30 min, washed and stained with lead citrate (31). Electron micrographs were taken on a Philips 300 at 80 kV.
Mannose Binding Sites
51
Results Affinity electrophoresis was used as a qualitative control for the binding of sugar to ferritin. As seen in Figure 1, ferritin-mannan (FM) precipitated very early, giving a short and sharp rocket. The addition of a small amount of mann an (5 f-tl of a 0.042 mM solution) drastically retarded precipitation (FM + M). A similar elongated rocket was seen in the conjugate which still contained unbound mannan. Addition of a-methylmannoside (0.3 M) induced an inhibition only within the diffusion zone of the sugar, outside this zone, the conjugate was readily precipitated (FM + a MM). When the ferritin-a-mannoside conjugate (FM + FaM) was added, however, precipitation of FM was retarded, giving an indirect proof of binding of the aminophenyl sugar to ferritin. No precipitation was obtained with ferritina-mannoside alone (FaM), as long as it was free of aggregates or high molecular weight products, and none, of course, with native ferritin (F). The binding of p-aminophenylmannoside to ferritin was controlled by affinity electron microscopy on fixed, ConA-coated cells. A dense labelling was obtained both on the cell surface and filopods (Fig. 2a), indicating indirectly the number of binding sites for ConA. Omitting ConA-coating, the ferritin sugar conjugates were never bound to glutaraldehyde-fixed cells, indicating the proteic nature of the mannose receptor and its cross-linking by the fixative (Fig. 2b). Ferritin-a-mannoside was rapidly bound by vital macrophages and appeared clustered in well-defined regions of the cell surface. After treatment of the cells with the conjugate for 1 min, coated pits assembled almost all of the bound material (Fig. 3). Outside the coated pits, very few single black grains were seen scattered over the numerous folds of the surface. Labelled, coated pits were distributed irregularly over the membrane, but appeared more densely packed in certain regions, where microtubuli proceeded perpendicular to the surface (Fig. 3b). In some narrow regions of the cell, an intense uptake of ligand was observed in surface-near coated vesicles as early as after 1 min (Fig. 4). Even fusion of coated and uncoated vesicles occurred. As in Figure 4, microtubuli were found perpendicular to the cell surface, and the coated vesicles appeared to enter the cell following their direction. Rapid invagination of coated pits was found also with ferritin mannan. Deep pits were occasionally still connected with the surface membrane by means of uncoated channels, as shown in Figure Sa. A similar pattern was obtained when macrophages were treated with native ferritin for 1 min (Fig. Sb). However, binding and uptake of both sugar conjugates were almost completely inhibited in the presence of mann an or a-methyl-mannoside. Coated pits and vesicles as well as the uncoated membrane parallel to microtubuli were found without any ferritin (Fig. 6a and b), while binding and uptake of native ferritin were never inhibited in the presence of sugars (Fig. 6c). Galactose failed to inhibit binding and uptake of the conjugates.
52 . GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 4. Uptake of ferritin-a-mannoside in coated vesicles after treatment with the conjugate for 1 min. Coated vesicles (Cv) apparently derive from a narrow space of the cell surface arranged along a microtubulus (Mt). X 99,000.
Treatment with trypsin for 5 min at 37°C induced a strong reduction in surface binding of the sugar conjugates. However, some uptake was observed in coated vesicles, but generally they contained only very few particles. Significant morphological changes occurred in the treated cells,
Mannose Binding Sites . 53
Fig. 5. a) Binding of ferritin-mann an on coated pits (Cp) after treatment with the conjugate for 1 min, in addition, just incorporated coated pits (1). X 82,500. b) Native ferritin bound to coated pits. A pattern similar to that obtained with mannosylated ferritin is recognizable. X 99,000.
such as flattening and vacuolisation. Large vacuoles were occasionally filled with membrane material (Fig. 7a, b). At the end of a lO-min exposure of macrophages to ferritin-a-mannoside, all steps of binding and uptake could be observed (Fig. 8). Coated pits and
54
GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 6. Treatment with ferritin-mannan (a, b) and native ferritin (c) in the presence of 0.3 M a-methylmannoside. a) The uncoated membrane, parallel to the microtubuli (Mt), and coated pits (Cp) show binding of the conjugate. X 32,400. b) The folded membrane and cytoplasmic vesicles are free of ligand. X 99,000. c) Native ferritin binding 0) and uptake in coated vesicles (Cv) is not inhibited in the presence of sugar. X 67,500.
Mannose Binding Sites . 55
Fig. 7. Binding of ferritin-a-mannoside after treatment of cells with 0.02 % trypsin for 5 min at a) Uncoated membrane, microvilli (Mv), and filopods (Fp) are free of ferritin, which, however, appears in a coated vesicle (Cv). X 32,400. b) The same as a). In addition, coated pits (Cp) are seen free of ferritin. Vacuole (V) filled with membrane material. X 32,400. c) Some ferritin particles are bound on a flat pit (1) and are occasionally seen in coated vesicles (Cv). Clear area (A) limited by vesicles and tubuli. X 64,800.
3rc.
56
. GERTRUD ROSSI
and S.
HIMMElHOCH
Fig. 8. Uptake of mannosylated ferritin by macrophages treated with ferritin-a-mannoside for 10 min at The ligand appears in coated vesicles (Cv) and in large smooth vesicles (Sv), where it is partially detached from the membrane and agglutinated to big clumps (X) together with fibrillar material and small vesicles (v). The smooth vesicles are surrounded by small electron-dense vesicles (ev) and secretory granules (Sg) some of which contain ferritin (1). The inset shows different steps of ligand uptake. Coated vesicle in contact with surface membrane by means of an uncoated channel (1). X 60,000.
3rc.
Mannose Binding Sites . 57
Fig. 9. Cross-section of a macrophage treated with ferritin-a-mannoside for 1 min. On both sides of the cell the ligand appears mainly on coated pits (Cp), the surface membrane shows many infoldings. X 48,000.
vesicles close to the cell surface did not occur more frequently than after 1 min. Large amounts of ferritin accumulated in large smooth vesicles, detached from the membrane and agglutinated into big clumps, together with amorphous or fibrillar material and with small vesicles. Small electron-
58 .
GERTRUD ROSSI
and S.
HIMMELHOCH
Fig. 10. Cross-section of a cell after treatment with ferritin-a-mannoside for 10 min. Ferritin is bound to microvilli; furthermore, the ligand is located in Cv, Sv and in small electron-dense vesicles. Part of the Sv membrane is coated. Coated pits are scarce, one Cp is located at the basis of a microvillus (Mv). The surface is unfolded. X 60,000.
dense Golgi vesicles surrounded the large smooth vesicles and occasionally contained some ferritin grains or seemed to be in contact with the vesicle membrane. Some ferritin was even found in secretory granules.
Mannose Binding Sites . S9
Comparison of the cross-sections of two macrophages treated for 1 min (Fig. 9) and for 10 min (Fig. 10) with ferritin-a-mannoside gives a general impression of the different location sites of the conjugate. After short-term treatment, it was restricted to the surface which appeared rough with small folds, coated pits and vesicles. After long-term treatment, ferritin was located deep in the cell within small dense vesicles and large vesicles agglutinated with fibrillar or round structures. The large vesicles were sometimes incomplete and fused with the cytoplasm. The surface of the long-term treated cells appeared unfolded, smooth and with straight short microvilli which bound ferritin in a diffuse manner. Coated pits were scarce and mostly located near the basis of microvilli.
Discussion
Ultrastructural visualization of binding sites for mannose allows, more than their quantitative estimation, an evaluation of their surface distribution and behaviour as true receptors. This can be achieved by following an electron-dense mannosylated protein during its binding to coated pits and uptake in coated vesicles. Due to its size, ferritin is considered a fine membrane marker, but the protein itself is bound to cell receptors (23). Glycosylation is aimed to cover the carrier protein. Its recognition as being «mannosylated» and the specific inhibition of its binding are achieved by glycosylation either with the highly branched polymannose mannan or by a high number of moles/mole of p-aminophenylmannopyranoside. A dense labelling of ConA-coated cells is achieved with both conjugates. Affinity electrophoresis in ConA agarose is a sensitive control method for ferritin-mannan, but it is less effective as a control of ferritin mannoside, which inhibits precipitation of ferritin mannan but does not precipitate itself. Mannosylated ferritin is bound by in vitro differentiating bone marrow macrophages of young chicken on receptor sites, which are found assembled on coated pits in less than 1 min. Coated pits are not equally distributed over the whole cell surface, but they appear more concentrated in well-defined membrane regions. A similar polarisation is observed also for the endocytic activity: pinocytosis is found much more advanced in one narrow section of a cell where all the coated vesicles seem to enter the cell following the direction of microtubuli (see Fig. 4a). Surface-associated ferritin conjugate has never been found clustering over the surface. It is assembled as single points aligned on coated pits, suggesting that the binding molecule is not moving over the membrane, but within the bilayer like an integral membrane protein (32) able to interact with clathrin (33). The proteic nature of its binding is apparent by its inactivation with glutaraldehyde, more so than with the short-term degradation caused by trypsin treatment, since in this case degraded surface receptors may be
60 . GERTRUD ROSSI and S. HIMMELHOCH
readily replaced from inner stores (13). However, significant morphological changes which occur in trypsin-treated cells may be considered as an injury. Once collected in smooth vesicles and detached from the vesicle membrane, the endocytosed glycoprotein appears clustered in big clumps. This agglutination may be due to pH-values of the vesicle content which may be near the isoelectric point of the protein, or to detachment of receptor-ligand complexes or membrane pieces: at least the ferritin particles appear associated to amorphous or fibrillar material or to small vesicle-like structures. Coated membrane sections of the smooth vesicles may indicate their origin from fusion of coated vesicles with preformed smooth vesicles (34). Degradation of the glycoprotein cannot be demonstrated by morphological investigations. The ferritin particles remain visible even when part of the vesicle membrane appears to be missing and the cytoplasm seems to fuse with the vesicle content. Due to the rapid internalization of coated pits, the cell surface is never saturated with ligand. Binding and uptake continue over the observed period of time, while the surface remains nearly free, indicating that the macrophage uses the surface receptor predominantly for endocytosis. The continuous internalization of membrane areas causes morphological changes, which become recognizable after 10 min exposure to the conjugate. The fine membrane infoldings disappear, and the membrane shows a major tension. Straight microvilli bind ferritin particles in a diffuse manner. The picture suggests that microvilli continue binding the ligand, which is moved to coated pits at their bases, but uptake slowly decreases since coated pits are less available on the remainder surface. Acknowledgements The authors are indebted to Profs. H.-J. MERKER and R. GOSSRAU for their advice and fruitful discussions as well as their generous help in making their EM facilities available for this study. They are also grateful to Dr. R. GOLDMAN for stimulating discussions and for having placed her laboratory facilities at their disposal. - This study was supported by the Minerva Stiftung.
References 1. TEICHBERG, J. V., J. SII.MAN, D. D. BETTSCH, and G. RESHEFF. 1975. A j3-galactoside binding protein from electric organ tissue of Electrophorus electricus. Proc. Nat!. Acad. Sci. USA 72(4): 1383. 2. KAWASAKI, T., and G. ASHWELL. 1977. Isolation and characterization of an avian hepatic binding protein specific for N-acetylglucosamin-terminated glycoprotein. J. BioI. Chern. 252(18): 6536. 3. GARTNER, T. K., D. C. WILLIAMS, F. C. MINIClN, and D. R. PHIJ.TPS. 1978. Thrombin induced platelet aggregation is mediated by a platelet plasma membrane-bound lectin. Science 200: 1281. 4. GREMO, F., D. KOBILER, and S. H. BARONDES. 1978. Distribution of an endogenous lectin in the developing chick optic tectum. J. Cell BioI. 79: 491.
Mannose Binding Sites . 61 5. STAHL, P. D., J. S. RODMAN, M. J. MILl.FR, and P. H. SCHLESINGER. 1978. Evidence for receptor-mediated binding of glycoproteins, glycoconjugates and lysosomal glycosidases by alveolar macrophages. Proc. Natl. Acad. Sci. USA 75(3): 1399. 6. NOWAK, T. P., P. L. HAYWOOD, and S. H. BARONDES. 1976. Developmentally regulated lectin in embryonic chick muscle and a myogenic cell line. Biochem. Biophys. Res. Comm. 68(3): 650. 7. KOBILER, D., E. C. BEYER, and S. H. BARONDES. 1978. Developmentally regulated lectins from chick muscle, brain and liver have similar chemical and immunological properties. Dev. BioI. 64: 265. 8. BFYER, E. c., K. T. TOKUYASU, and S. H. BARONDFS. 1979. Localisation of an endogenous lectin in chicken liver, intestine and pancreas. J. Cell BioI. 82: 565. 9. GARTNFR, T. K., and T. R. PODLESKI. 1976. Evidence that the types and specific activity of lectins control fusion and L6 myoblasts. Biochem. Biophys. Res. Comm. 70: 1142. 10. WFI(;rT, P. H. 1980. Rat hepatocytes bind to synthetic galactoside surface via a patch of asialoglyco-protein receptors. J. Cell BioI. 87: 855. 11. KOLIl, H., and V. KOLB-BACHOfFN. 1978. A lectin like receptor on mammalian macrophages. Biochem. Biophys. Res. Comm. 85: 678. 12. BAENZIGER, J. U., and D. FIETE. 1980. Galactose and N-acetylgalactosamine specific endocytosis of glycopeptides by isolated rat hepatocytes. Cell 22: 611. 13. STAHL, P., P. H. SCHLESINGER, E. SIGARDSON, J. S. ROllMAN, and Y. C. Ln:. 1980. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell 19: 207. 14. GRAllEL, L. B., S. D. ROSEN, and G. R. MARTIN. 1979. Teratocarcinoma stem cells have a cell surface carbohydrate-binding component implicated in cell-cell adhesion. Cell 17: 477. 15. RAZ, A., and R. LOTAN. 1981. Lectin-like activities associated with human and murine neoplastic cells. Cancer Research 41: 3642. 16. RAZ, A., and R. LOTAN. 1982. On the possible role of tumor-associated lectins in metastasis in «Membranes in Tumor Growth». GALEOTTI, T., G. NERI, and S. PAPA, eds. Elsevier/North Holland, Biomedical Press. 17. BARZILAY, M., M. MONSIGNY, and N. SHARON. 1982. Interaction of soybean agglutination with human peripheral blood lymphocyte subpopulations: evidence for the existence of a lectin-like substance on the lymphocyte surface. In: «Proceedings of Fourth Lectin Meeting in Copenhagen». T. C. BOG-HANSEN, ed. Walter de Gruyter, Berlin, in press. 18. ROSSI, G., H. WEILER, and S. GRUND. 1982. Phagocytic activity of chicken macrophages towards untreated yeast cells as influenced by cell interactions. In: «Proceedings of the VIIIth ISHAM Congress, Palmerston North». M. Baxter, ed., in press. 19. EZEKOWITZ, R. A. B., J. AUSTYN, P. D. STAHL, and S. GORDON. 1981. Surface properties of bacillus Calmette-Guerin-activated mouse macrophages. J. Exp. Med. 154: 60. 20. KOLll-BACHOFEN, V., J. SCHLEPPER-SCHAFER, and W. VO(;EII.. 1982. Electron microscopic evidence for an asialoglycoprotein receptor on Kupffer cells: localisation of lectinmediated endocytosis. Cell 29: 859. 21. KIEllA, c., A. C. ROCHE, F. DELMOTTE, and M. MONSIGNY. 1979. Lymphocyte membrane lectins. Direct visualisation by the use of fluoresceinyl-glycosylated cytochemical markers. FEBS Letters 99: 329. 22. MEHL, T. D., and D. LAGUNOFl'. 1975. Uptake of aggregated albumin by rat macrophages in vitro: affinities of cells for monomeric and aggregated bovine serum albumin. J. Reticuloendoth. Soc. 18(2): 125. 23. POIYCARD, A., and H. BESSJS. 1962. Micropinocytosis and rhopheocytosis. Nature 194: 110. 24. ROSSI, G., and F. TURBA. 1981. Phagozytose unterschiedlich behandelter Candidazellen durch Knochenmarks- und Peritonealmakrophagen vom Huhn. Mykosen 24: 684. 25. KOCOUREK, J., and C. E. BALl.OU. 1969. Methods for fingerprinting yeast cell wall mannans. J. Bacteriol. 100(3): 1175.
62 . GERTRUD ROSSI and S. HIMMELHOCH 26. McBROOM, C. R., C. H. SAMANEN, and 1. J. GOl.DSTEIN. 1972. Carbohydrate antigens: coupling of carbohydrates to proteins by diazonium and phenylisothiocyanate reactions. Methods in Enzymology XXVIII, Complex Carbohydrates Part B. V. GINSBUR(;, ed., Academic Press. 27. OTTO, H., H. TAKAMIYA, and A. VOGT. 1973. A two-stage method for cross-linking antibody globulin to ferritin by glutaraldehyde. Comparison between the one-stage and the two-stage method. J. Immunol. Methods 3: 137. 28. OWEN, P., J. D. OPPENHEIM, M. S. NACHBAR, and R. E. KESSl.ER. 1977. The use of lectins in the quantitation and analysis of macromolecules by affinity electrophoresis. Anal. Biochem. 80: 446. 29. DALTON, A. J. 1955. A chrome-osmium fixative for electron microscopy. Anat. Rec. 121: 281. 30. Luf'T, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409. 31. REYNOl.DS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell BioI. 17: 208. 32. NICOl.SON, G. L. 1976. Transmembrane control of the receptors on normal and tumor cells. 1. Cytoplasmic influence over cell surface components. Biochem. Biophys. Acta 475: 57. 33. PEARSE, B. M. F. 1976. Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Cell BioI. 73(4): 1255. 34. PASTAN, J. H., and M. C. WILLINGHAM. 1981. Journey to the center of the cell: role of the receptosomes. Science 214: 504. Dr. G. ROSSI, Institut fur Geflugelkrankheiten, Freie Universitat Berlin, KoserstraBe 21, D-I000 Berlin 33, Germany