Structural heterogeneity of endocytic membranes in macrophages as revealed by the cholesterol probe, filipin

1981 ◽  
Vol 51 (1) ◽  
pp. 95-107
Author(s):  
R. Montesano ◽  
P. Vassalli ◽  
L. Orci
Keyword(s):  
Structural Heterogeneity ◽  
Cholesterol Content ◽  
Peritoneal Macrophages ◽  
Con A ◽  
Coated Pits ◽  
Mechanisms Of Formation ◽  
Smooth Membrane ◽  
Endocytic Vesicles ◽  
Cytochemical Technique ◽  
Mouse Peritoneal Macrophages

The polyene antibiotic, filipin, by specifically interacting with cholesterol, produces approximately 25-nm protuberances (filipin-sterol complexes) in freeze-fractured membranes, and the addition of filipin to aldehyde fixatives has been recently introduced as a cytochemical technique for the localization of cholesterol in cell membranes. In a previous study we showed that, in fibroblasts filipin-sterol complexes are absent from endocytic coated pits. To establish whether the absence of filipin-sterol complexes is a phenomenon restricted to coated pits or is correlated with endocytosis in general, we applied the filipin probe to cultured mouse peritoneal macrophages, in which different forms of endocytosis take place. The macrophages were incubated with bovine albumin or concanavalin A (Con A) to induce pinocytosis, and with heat-killed straphylococci or opsonized erythrocytes to induce phagocytosis, then fixed in glutaraldehyde/filipin and freeze-fractured. Filipin-sterol complexes were plentiful on the plasma membrane, on the smooth-membrane invaginations and vesicles induced by albumin, on the large endocytic vacuoles induced by Con A, and on the membrane of phagosomes but, in contrast, they were absent from coated pits and vesicles, as well as from coated segments of invagination or vesicles. These results indicate that the membranes involved in different types of endocytosis do not react in the same way with filipin and may, therefore, have a different cholesterol content. This could reflect different mechanisms of formation for the various types of endocytic vesicles.

scholarly journals EFFECTS OF CONCANAVALIN A ON MOUSE PERITONEAL MACROPHAGES

1974 ◽  
Vol 140 (5) ◽  
pp. 1364-1386 ◽  
Author(s):  
Paul J. Edelson ◽  
Zanvil A. Cohn
Keyword(s):  
Concanavalin A ◽  
Peritoneal Macrophages ◽  
Surface Marker ◽  
Marker Enzyme ◽  
Apparent Effect ◽  
Con A ◽  
Cytoplasmic Vesicles ◽  
Secondary Lysosomes ◽  
Mouse Peritoneal Macrophages ◽  
Pinocytic Vesicles

Concanavalin A (Con A) binds to saccharide residues on the mouse peritoneal macrophage plasma membrane and stimulates extensive pinocytic interiorization of the membrane. The overall pinocytic rate is increased 3.5–4.5 times by the addition of Con A, and the surface marker enzyme adenosine triphosphatase can be identified histochemically in association with the cytoplasmic vesicles generated after exposure of the cells to Con A. Once formed, these pinocytic vesicles may persist for several days and fail to show morphologic evidence of fusion with primary or preformed secondary lysosomes. There is no apparent effect on the capacity of the macrophage to ingest either latex particles or IgG-coated SRBC administered either simultaneously with or subsequent to the Con A.

scholarly journals THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

1972 ◽  
Vol 55 (1) ◽  
pp. 186-204 ◽  
Author(s):  
Ralph M. Steinman ◽  
Zanvil A. Cohn
Keyword(s):  
Horseradish Peroxidase ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
Wide Range ◽  
Mouse Macrophages ◽  
Secondary Lysosomes ◽  
Endocytic Vesicles ◽  
In Vitro Interaction ◽  
Mouse Peritoneal Macrophages

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 106 cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-125I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-125I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.

scholarly journals THE CYTOTOXIC EFFECT OF MOUSE MACROPHAGES ON SYNGENEIC AND ALLOGENEIC ERYTHROCYTES

1973 ◽  
Vol 137 (3) ◽  
pp. 807-820 ◽  
Author(s):  
H. Melsom ◽  
R. Seljelid
Keyword(s):  
Cytotoxic Effect ◽  
Peritoneal Macrophages ◽  
Target Cells ◽  
Activated Macrophages ◽  
Con A ◽  
Osmotic Effect ◽  
Prolonged Cultivation ◽  
Cytotoxic Reaction ◽  
Mouse Macrophages ◽  
Mouse Peritoneal Macrophages

A cytotoxic effect of mouse peritoneal macrophages against syngeneic and allogeneic erythrocytes was demonstrated by isotope release and release of hemoglobin. The cytotoxic effect was dependent on the contact between viable, activated macrophages and target cells. Activation was accomplished by prolonged cultivation of macrophages and by the presence of Zn++ and Con-A. Immunization did not prove necessary. Morphological observations as well as experiments with various salt concentrations indicate that the cytotoxic reaction may involve some kind of osmotic effect upon the target cells.

scholarly journals Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors.

1993 ◽  
Vol 123 (1) ◽  
pp. 89-97 ◽  
Author(s):  
S H Hansen ◽  
K Sandvig ◽  
B van Deurs
Keyword(s):  
High Efficiency ◽  
De Novo ◽  
Coated Vesicles ◽  
Transferrin Receptors ◽  
Con A ◽  
Coated Pits ◽  
Late Endosomes ◽  
Endocytic Vesicles ◽  
Endocytic Compartments ◽  
K Depletion

We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin-dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)-depleted cells with Con A-gold for 15 min, approximately 75% of Con A-gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)-depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.

scholarly journals EFFECTS OF CONCANAVALIN A ON MOUSE PERITONEAL MACROPHAGES

1974 ◽  
Vol 140 (5) ◽  
pp. 1387-1403 ◽  
Author(s):  
Paul J. Edelson ◽  
Zanvil A. Cohn
Keyword(s):  
Preceding Paper ◽  
Peritoneal Macrophages ◽  
Anomalous Behavior ◽  
Parasitic Infections ◽  
Half Time ◽  
Lysosomal Hydrolases ◽  
Con A ◽  
Inner Surface ◽  
Mouse Peritoneal Macrophages

The half-time for the degradation of horseradish peroxidase (HRP) is increased from 14 h to 37 h in Con A-treated cells, while the half-time for the degradation of [125I]BSA is increased from 5.4 h to 14.8 h. This supports prior microscopic observations which suggested that Con A pinosomes showed a marked impairment in their ability to form phagolysosomes. Artifacts due to anomalous behavior of HRP-Con A complexes, or to inhibition of lysosomal hydrolases by Con A, could be excluded. These indications of impaired phagolysosome formation, as well as those described in the preceding paper, could be reversed by postincubation of the cells in mannose, but not in galactose. This reversal is accompanied by a dissociation of Con A-FITC from the inner surface of the pinosome membrane, into the vesicle contents. These observations may be relevant to the ability of Con A to affect several membrane characteristics, and are also of interest in relation to the impaired formation of phagolysosomes which has been described in certain in vitro parasitic infections of macrophages or other cells.

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages

1990 ◽  
Vol 93 (4) ◽  
pp. 443-446 ◽  
Author(s):  
Th. Peschke ◽  
L. Wollweber ◽  
A. Gabert ◽  
K. Augsten ◽  
R. Stracke
Keyword(s):  
Peritoneal Macrophages ◽  
Con A ◽  
Surface Receptors ◽  
Mouse Peritoneal Macrophages

scholarly journals Modulation of apoprotein E secretion in response to receptor-mediated endocytosis in resident and inflammatory macrophages.

1984 ◽  
Vol 159 (1) ◽  
pp. 167-178 ◽  
Author(s):  
R Takemura ◽  
Z Werb
Keyword(s):  
Dextran Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cholesterol Content ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Activated Macrophages ◽  
Receptor Mediated Endocytosis ◽  
Inflammatory Macrophages ◽  
Dose Dependent ◽  
Mouse Peritoneal Macrophages

We have determined the effect of various endocytic ligands on the secretion of ApoE by macrophages. ApoE was a major secreted protein of resident macrophages, but BCG-activated macrophages secreted little ApoE and periodate-elicited macrophages secreted intermediate amounts of ApoE. Resident, periodate-elicited, and BCG-activated mouse peritoneal macrophages were incubated with AcLDL, EIgG, EIgMC, dextran sulfate, latex, or zymosan, and the resulting protein secretion patterns were analyzed by [35S]methionine labeling and SDS-polyacrylamide gel electrophoresis. AcLDL increased total [35S]methionine incorporation into secreted proteins. Although AcLDL increased the secretion of ApoE by resident macrophages less than or equal to fivefold in a dose-dependent manner, with maximal stimulation at 4.8 micrograms/ml, it decreased the secretion of ApoE by periodate-elicited macrophages to almost nothing and did not affect the low rate of secretion of ApoE by BCG-activated macrophages. However, EIgG, which increases cellular cholesterol content of macrophages as AcLDL does, did not increase ApoE secretion, and dextran sulfate, which is recognized by the same receptor as AcLDL, also did not increase ApoE secretion. The binding and uptake of EIgG, dextran sulfate, zymosan, latex, and EIgMC all decreased the secretion of ApoE. These endocytic ligands also altered the pattern of secreted and cellular proteins other than ApoE. The pattern of response was ligand-specific. However, increased secretion of polypeptides of Mr 62,000 and 68,000 was common to many stimuli. We conclude that receptor-mediated endocytosis modulates the secretion of ApoE and other proteins pleiotypically in resident, inflammatory, and activated macrophages.

scholarly journals Specialization of the macrophage plasma membrane at sites of interaction with opsonized erythrocytes.

1983 ◽  
Vol 96 (5) ◽  
pp. 1227-1233 ◽  
Author(s):  
R Montesano ◽  
A Mossaz ◽  
P Vassalli ◽  
L Orci
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Receptor Interaction ◽  
Coated Pits ◽  
Electron Dense Material ◽  
Functional Studies ◽  
Structural Specialization ◽  
Mouse Peritoneal Macrophages

We incubated mouse peritoneal macrophages for 3-8 min at 37 degrees C with antibody-coated sheep erythrocytes and examined regions of close interaction between the two cell types by electron microscopy. At sites of focal macrophage-erythrocyte contact we observed a distinctive specialization of the macrophage plasma membrane consisting of a prominent subplasmalemmal band of electron-dense material, approximately 25-35 nm in thickness. In many instances, this band showed a periodic substructure similar to that seen in clathrin coats. Moreover, many slender erythrocyte processes penetrated into invaginations of the macrophage surface which were bristle-coated at their blind extremity. As previously shown for clathrin-coated pits, the segments of the macrophage plasma membrane beneath which the defense material was found were selectively resistant to the membrane-perturbing effect of the antibiotic, filipin. This structural specialization of the macrophage plasma membrane at sites of ligand-receptor interaction during immune phagocytosis of antibody-coated erythrocytes may represent the morphological counterpart of the zipper mechanism of phagocytosis previously demonstrated by functional studies.

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