Polarization of endocytosis and receptor topography on cultured macrophages

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular- weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement- opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.     

Transglutaminase and receptor-mediated endocytosis in macrophages and cultured fibroblasts

Summary The function of intracellular transglutaminases remains to be clarified. In fibroblasts the links between the activity of this enzyme and receptor-mediated endocytosis are complex and open to interpretation. However, the issue cannot be firmly laid to rest until the structural specificity of the alkylamine inhibitors of endocytosis is explained. In macrophages, there is substantial evidence that the enzyme plays some role in receptor-mediated phagocytosis, but what this role is and how it might relate to endocytosis in other types of cells is at present an unresolved issue.     

Isolation and characterization of a mannose-specific endocytosis receptor from rabbit alveolar macrophages.

Rabbit alveolar macrophages express a plasma-membrane receptor that recognizes glycoprotein ligands bearing terminal mannose, fucose or N-acetylglucosamine residues. Macrophage membranes were washed extensively with buffers containing high salt and mannose or EDTA to remove endogenously bound ligand, before Triton X-100 extraction. The extracts were chromatographed on mannose-Sepharose. Elution with mannose, followed by dialysis and a second mannose-Sepharose step with EDTA elution, produced a preparation that migrated as single protein band of Mr 175,000 on SDS/polyacrylamide-gel electrophoresis. The purified protein binds mannose-BSA (bovine serum albumin) with a dissociation constant of 1.9 X 10(-8) M. Ligand binding is Ca2+ and pH-dependent, with maximal binding at neutral pH and low binding below pH 6.0. The binding of 125I-mannose-BSA is inhibited by ligands bearing high-mannose oligosaccharides, such as mannan or beta-glucuronidase, as well as the monosaccharides mannose, fucose and N-acetylglucosamine. Galactose, galactosylated BSA, glucose and mannose 6-phosphate are non-inhibitory. Amino acid compositional analyses indicate that the receptor contains high concentrations of aspartate/asparagine and glutamate/glutamine, and low amounts of methionine. The carbohydrate composition was studied by lectin overlays of electrophoretically transferred receptor, and the results indicate the presence of N-linked complex and O-linked sialylated oligosaccharides. A protein of Mr 175,000 was immunoprecipitated from radio-iodinated macrophage membranes with an antibody generated against purified rabbit lung mannose receptor.     

Mannose-specific endocytosis receptor of alveolar macrophages: demonstration of two functionally distinct intracellular pools of receptor and their roles in receptor recycling

Receptor-mediated endocytosis of rat preputial beta-glucuronidase and the glycoconjugate mannose-BSA by rat alveolar macrophages is inhibited by chloroquine and ammonium chloride. We have previously reported that these drugs cause a loss of cell surface binding activity and that they do not inhibit internalization of receptor ligand complexes when incubated with cells at 37 degrees C. In this report we more clearly delineate the intracellular site of weak base inhibition of receptor recycling and the mechanism of that inhibition. From our analysis of the kinetics of ligand transport we conclude that there are two functionally distinct intracellular pools of receptor. One of these, the cycling pool, is not sensitive to the presence of weak bases, and receptor-ligand complexes return from this pool to the cell surface intact. The second pool is responsible for the time-dependent intracellular delivery of ligand to acid vesicles, which is inhibited by weak bases. Chloroquine and ammonium chloride appear to inhibit the dissociation of receptor-ligand complexed in this second pool and thereby the production of free receptors for the continuation of receptor-mediated endocytosis. We examine the internalization and binding of ligand in normal and paraformaldehyde-treated cells and find that these are strongly affected by pH. In particular, the dissociation rate of receptor ligand complexes is enhanced greater than 7.5 fold by lowering the medium pH from 7 to 6. From these results we propose that weak bases raise the pH of acid intracellular compartments, slowing the rate of receptor-ligand dissociation and thereby reducing the cellular pool of free receptors available for further uptake of ligand. In addition, we demonstrate that receptor-ligand complexes cannot return to the cell surface from the amine-sensitive (acid) intracellular pool that led us to call this the nonreleasable pool. This final observation indicates that receptor movements through these two pools are functionally distinct processes.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural analysis of asialoglycoprotein receptor mediated endocytosis in cultured newborn hepatocytes

We examined the receptor-mediated endocytosis of asialoglycoproteins in thick sections of cultured hepatocytes of newborn rats by high voltage electron microscope. The organelles involved in endocytosis are revealed ultrastructurally using colloidal gold particles coupled to lactosylated bovine serum albumin. Keeping the cells at 4 degrees C the marker binds to the cell surface showing microvilli, and is not internalized. Increasing the temperature to 37 degrees C, we observed that within 5-15 min. the marker enters the intracellular endocytic organelles close to the cell surface. After 60 min. the marker is found in the larger and deeper endocytic organelles and in large lisosome-like vesicles. We find that the process of endocytosis in newborn cultured hepatocytes is similar to that found in cultured cells of adult rats, but the process of internalization is slower.     

Interferon alpha/beta selectively antagonises down-regulation of mannosyl-fucosyl receptors on activated macrophages by interferon gamma

Previous reports have described synergism of various interferon preparations in anticellular and antiviral activity. We report that recombinant interferon (rIFN gamma) and IFN alpha/beta mediate distinct, antagonistic effects on expression of a lectin-like receptor for mannose and fucose (MFR) on mouse peritoneal macrophages (M phi). IFN gamma down-regulates MFR activity, a highly reproducible change in mouse M phi activated to secrete enhanced levels of o-2/H2o2. IFN alpha/beta enhances MFR activity and prevents the action of IFN gamma when added in combination. Antagonism is selective for this M phi activation marker and requires a minimum 4 h exposure period to rIFN gamma, during which IFN alpha/beta can prevent its action.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

Summary The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4° C some LDL-or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8° C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37° C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37° C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4 degrees C some LDL- or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8 degrees C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37 degrees C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37 degrees C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

Pathways of endocytosis in cultured macrophages. Electron microscopic autoradiographic tracing of iodinated plasma membrane proteins

Lactoperoxidase-mediated iodination at 4 degrees C--an established method for covalent labelling of plasma membrane proteins--and quantitative electron microscopic autoradiography were used to follow the pathways of endocytosis in mouse macrophages in vitro. Directly after the labelling, the autoradiographic grains were concentrated to the cell surface. After warming to 37 degrees C, radioactive material was rapidly internalized into cytoplasmic vesicles and subsequently transferred to lysosomes as well as to the Golgi complex. Maximum grain density (% grains/% volume) over the vesicles was observed after 15 min, over the lysosomes after 30 to 45 min and over the Golgi complex after 30 and 90 min. Throughout the experimental period (120 min), the vesicles showed the largest fraction of intracellular grains, but higher grain densities occurred in lysosomes as well as in stacked Golgi cisternae and Golgi-associated vesicles. In spite of the internalization process, the labelling of the cell surface came to a steady state already after 30 min and at all intervals more than 50% of the autoradiographic grains were localized to this compartment. About 25% of the cell-associated radioactivity was lost rapidly with a half-life of 20 to 25 min and the remaining 75% slowly with a half-life of 7 to 9 h. The results indicate that membrane internalized by endocytosis partly follows a route to the lysosomes and that, additionally, there exists a route to and through the Golgi complex. They further support earlier notions of a bidirectional traffic between the surface and interior of the cell and suggest that recycling of membrane components may take place from endocytic vesicles, lysosomes, as well as the Golgi complex.     

The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface. Following this clustering event, the ligand is internalized into a noncoated intracellular vesicle, the receptosome, which selectively avoids fusion with lysosomes and moves toward the Golgi region of the cell by saltatory motion. Using alpha 2-macroglobulin as the ligand and electron microscopic cytochemical methods, we have shown the unusual appearance of this previously undescribed organelle and have followed the labeled ligand in these vesicles in the cytoplasm. To accomplish this, cells were incubated with immunolabeled alpha 2-macroglobulin at 4C under conditions where ligand-receptor complexes cluster into coated pits on the cell surface. Formation of the receptosome occurs between 2 and 5 min after raising the temperature of cells to 37C. These labeled receptosomes were seen to associate with many small vesicular elements in the cytoplasm, and were often found near the Golgi or GERL region after 15 min. Between 15 and 30 min a significant transfer of labeled ligand occurred from the receptosomal population to a population of small uniform lysosomes. By 60 min, all of the label was contained in these small lysosomes. Immunocytochemical studies showed that the receptosomes were not associated with clathrin, actin, myosin or tubulin. This unique, short-lived, specialized organelle selectively delivers the products of receptor-mediated endocytosis to intracellular sites.     

Expression of cycloheximide resistance in carrot somatic hybrids and their segregants

Cycloheximide resistance (CH^r) was shown to be a function expressed in differentiated plant tissues, but not in unorganized callus tissues. A variant, WCH105, expressing CH^r in the callus, as well as in regenerated plantlets, was isolated from a cell line derived from a wild carrot plant. The plantlets regenerated from WCH105 are green, but do not produce normal, dissected leaves. Protoplasts of WCH105 were fused with that of a cycloheximidesensitive (CH^s) cell line derived from an albino, domesticated carrot. Hybrid selection was based on (1) irreversible growth inhibition of WCH105 protoplasts by iodacetamide, and (2) restoration of green plants producing dissected leaves.——Analysis of the CH^r trait as an unselected marker in the callus cells of the somatic hybrids indicated that it behaved as a recessive. The combined recessive and resistant phenotype of this trait allowed the recovery of CH^r segregants from CH^s hybrids at a frequency of 10^-4, 1000 times higher than the spontaneous frequency of CH^r. The recovery of CH^r somatic segregants confirmed the recessiveness of the CH^r trait.     

Temperature effect on endocytosis and exocytosis by rabbit alveolar macrophages

Endocytosis of 125I-mannose-bovine serum albumin (BSA) and exocytosis of 125I-mannose-poly-D-lysine by rabbit alveolar macrophages were examined as a function of temperature. A plot for total ligand uptake (cell-associated ligand plus degraded ligand) versus time shows a single inflection point at 20 degrees C. Ligand degradation does not occur below 20 degrees C. Internalization of surface-bound 125I-mannose-BSA is negligible below 10 degrees C. The rate constant for internalization increases dramatically above 20 degrees C: 0.02 min-1 at 20 degrees C, 0.05 min-1 at 25 degrees C, 0.13 min-1 at 30 degrees C, and 0.29 min-1 at 35 degrees C. 125I-Mannose-N-acetyl-poly-D-lysine preloaded in lysosomes is exocytosed in a temperature and time-dependent fashion. Even at lower temperatures (2-10 degrees C), secretion of 125I-mannose-N-acetyl-poly-D-lysine was detected, indicating that movement of lysosomal content to plasma membrane and beyond cannot be suppressed at these temperatures. Thus, the temperature dependence of exocytosis of an 125I-labeled ligand is quite different from that of endocytosis, suggesting that the two processes are controlled by different mechanisms. Stimulation of secretion of preloaded 125I-mannose-N-acetyl-poly-D-lysine by mannose-BSA was more pronounced at lower temperatures with a sharp inflection point at 10 degrees C. These findings suggest that endosomes containing newly internalized mannose-BSA interact with the exocytosis pathway and enhance secretion of 125I-mannose-N-acetyl-poly-D-lysine from lysosomes.     

Receptor-mediated endocytosis of fucosylated neoglycoprotein by macrophages

The characteristics of the recognition system involved in the receptor mediated endocytosis of the neoglycoprotein, fucose human serum albumin (HSA) were studied. It was found that (i) fucose-HSA showed strong affinity binding and uptake by various macrophages; (ii) binding was specific for L-fucose and D-mannose; (iii) binding was found to be inhibited by oxidant like H₂O₂ and swainsonine whereas it was elevated by dexamethasone; (iv) clearance of¹²⁵I-fucose-HSA was rapid and strongly inhibited by unlabelled fucose-HSA. Greater than 70% of fucose-HSA was found in liver and more than 60% of this was found in liver lysosomes; (v) uptake of fucose-HSA was thirty-fold more efficient in liver macrophages (Kupffer cells) than in hepatocytes; (vi) moreover, mannose-HSA and ovalbumin which are potent inhibitors of mannose/N-acetylglucosamine receptors inhibited clearance and uptake of fucose-HSA by liver as well as by isolated Kupffer cells suggesting the involvement of both fucose and mannose receptors or a single type of receptor having greater affinity for fucose-HSA than for mannose-HSA. These results emphasize the important role of fucose-terminated glycoproteins in site-specific drug targeting.     

Androgen metabolism and inhibition of interleukin-1 synthesis in primary cultured human synovial macrophages

The presence of androgen receptors on synovial macrophages in human normal and rheumatoid synovial tissues has been described previously. It is now reported that primary cultured human macrophages obtained from normal and rheumatoid synovia express functional androgen receptors. We have investigated the capacity of cultured macrophages to metabolize androgens and have found that these cells were capable of metabolizing testosterone to the bioactive metabolite dihydrotestosterone. Therefore, macrophages contain the key enzymes of steroidogenesis, in particular the 5alpha-treductase. Furthermore, interleukin-1beta production by primary cultured rheumatoid macrophages was analysed, following exposure to physiological concentrations of testosterone (10(-8) M). A significant decrease of IL-1beta levels in conditioned media after 24 h (p < 0.05) was observed. It is concluded that androgens may act directly on human macrophages and may interfere with some of their functions via receptor-dependent mechanisms.     

Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages

The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.     

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

Heparan sulfate proteoglycan as a cell-surface endocytosis receptor

How various macromolecules are exchanged between cells and how they gain entry into recipient cells are fundamental questions in cell biology with important implications e.g. non-viral drug delivery, infectious disease, metabolic disorders, and cancer. The role of heparan sulfate proteoglycan (HSPG) as a cell-surface receptor of diverse macromolecular cargo has recently been manifested. Exosomes, cell penetrating peptides, polycation–nucleic acid complexes, viruses, lipoproteins, growth factors and morphogens among other ligands enter cells through HSPG-mediated endocytosis. Key questions that partially have been unraveled over recent years include the respective roles of HSPG core protein and HS chain structure specificity for macromolecular cargo endocytosis, the down-stream intracellular signaling events involved in HSPG-dependent membrane invagination and vesicle formation, and the biological significance of the HSPG transport pathway. Here, we discuss the intriguing role of HSPGs as a major entry pathway of macromolecules in mammalian cells with emphasis on recent in vitro and in vivo data that provide compelling evidence of HSPG as an autonomous endocytosis receptor.     

Expression of differentiated properties in fetal liver cells and their somatic cell hybrids

A line of murine fetal liver cells is described and characterized. Hybrids between these differentiated cells and a non-differentiated fibroblast line were isolated and studied for the expression of the differentiated characteristics of the parent liver line. These include expression and inducibility for the enzyme tryptophan pyrrolase, ability to accumulate glycogen granules, and a characteristic morphology and growth rate, all of which were suppressed in the hybrid. The results obtained from sequential chromosome counts of the hybrid indicate that the modal number drops from about 100 to about 72 in 5 months, a fact that may be related to the extreme differences in generation times of the two parents.