Studies of the cell surface of mouse dendritic cells and other leukocytes

Nussenzweig, M.C., Steinman, R.M., Unkeless, J.C., Witmer, M.D., Gutchinov, B., and Cohn, Z.A. Studies of the cell surface of mouse dendritic cells and other leukocytes. J. Exp. Med. 154: 168-187, 1981https://digitalcommons.rockefeller.edu/historical-scientific-reports/1006/thumbnail.jp

Studies of the cell surface of mouse dendritic cells and other leukocytes

[No abstract available

Effects of immobilized immune complexes on Fc- and complement-receptor function in resident and thioglycollate-elicited mouse peritoneal macrophages

We have examined the Fc- and complement-receptor function of resident and thioglycollate-elicited mouse peritoneal macrophages plated on surfaces coated with rabbit antibody-antigen complexes and with complement. We derive four major conclusions from these studies. (a) The trypsin-resistant Fc receptors of resident and thioglycollate-elicited macrophages are completely modulated when these cells are plated on rabbit antibody-antigen complexes. Residual Fc receptor activity is a result of the incomplete modulation of trypsin-sensitive IgG2a receptors. (b) The complement receptors of thioglycollate-elicited macrophages, but not of resident macrophages, are modulated when these cells are plated on complement-coated surfaces. The capacity of the two cell types to modulate their complement receptors is correlated with their ability to ingest complement-coated erythrocytes. (c) The complement and Fc receptors of both types of macrophages move independently of one another. (d) Complement masks the Fc segments of IgG in immune complexes thereby rendering them ineffective as ligands for macrophage Fc receptors

Modulation of Fc receptors of mononuclear phagocytes by immobilized antigen-antibody complexes. Quantitative analysis of the relationship between ligand number and Fc receptor response

Macrophages plated on surfaces coated with antigen-IgG complexes lose the capacity to bind and ingest IgG-coated particles via their Fc receptors (FcR). Macrophages plated on surfaces containing a similar number of IgG molecules that are not complexed to antigen show little or no decrease in FcR activity. Using a rat monoclonal antibody (2.4G2 IgG) directed against the trypsin-resistant FcR (FcRII) of mouse macrophages we show that the decrease in receptor activity induced by substrate-adherent immune complexes is caused by the physical removal of 60 and 75% of FcRII from the nonadherent membrane surfaces of resident and thioglycollate broth-induced macrophages, respectively. Macrophages maintained on antigen-IgG-coated surfaces for up to 44 h show no recovery in FcRII activity or number, while macrophages on control surfaces exhibit two and threefold increases, respectively, in these parameters. Macrophages maintained for 72 h on antigen-IgG-coated surfaces show a small recovery in FcRII activity, and in the number of FcRII that is accessible to bind 125I-2.4G2 IgG. FcRII modulation, as measured by the binding of 125I-labeled 2.4G2 IgG, is initiated when the number of IgG molecules bound to the substrate is approximately equal to the total number of FcRII on the plasma membranes of all the macrophages on the substrate. FcRII activity and number decrease linearly as the number of substrate-bound IgG molecules increases exponentially, and are maximally reduced when the number of IgG molecules on the substrate is 20-fold greater than the total number of all FcRII on the surfaces of all the macrophages in the culture. Thus there is a stoichiometric relationship between the number of IgG molecules on the substrate and the extent of FcRII modulation

Effects of immobilized immune complexes on Fc- and complement-receptor function in resident and thioglycollate-elicited mouse peritoneal macrophages

We have examined the Fc- and complement-receptor function of resident and thioglycollate-elicited mouse peritoneal macrophages plated on surfaces coated with rabbit antibody-antigen complexes and with complement. We derive four major conclusions from these studies. (a) The trypsin-resistant Fc receptors of resident and thioglycollate-elicited macrophages are completely modulated when these cells are plated on rabbit antibody-antigen complexes. Residual Fc receptor activity is a result of the incomplete modulation of trypsin-sensitive IgG2a receptors. (b) The complement receptors of thioglycollate-elicited macrophages, but not of resident macrophages, are modulated when these cells are plated on complement-coated surfaces. The capacity of the two cell types to modulate their complement receptors is correlated with their ability to ingest complement-coated erythrocytes. (c) The complement and Fc receptors of both types of macrophages move independently of one another. (d) Complement masks the Fc segments of IgG in immune complexes thereby rendering them ineffective as ligands for macrophage Fc receptors

Selective iodination and polypeptide composition of pinocytic vesicles

We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 °C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37° C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 °C followed by washes and iodination (addition of \u27 25 1 and glucose) at 4°C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO and GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, \u3e75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypep tide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-1774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components

Fc receptor modulation in mononuclear phagocytes maintained on immobilized immune complexes occurs by diffusion of the receptor molecule

We describe a method for synchronously assembling antigen-antibody complexes underneath macrophages adherent to an antigen-coated surface. We have used this method to study the mechanism of Fc receptor (FcR) disappearance that occurs when resident and thioglycollate-elicited mouse macrophages are cultured on immune complex-coated surfaces. Erythrocytes opsonized with IgG (E(IgG) and a monoclonal antibody (2.4G2 IgG) directed against the trypsin-resistant FcR (FcRII) were used as indicators of the presence and distribution of FcRII molecules on the macrophage plasma membrane. Inhibitors of aerobic (NaCN) and anerobic (2-deoxyglucose, NaF) glycolysis and pinocytosis, of protein biosynthesis (cycloheximide), and of cytoskeletal function (cytochalasin B and D, colchicine, podophyllotoxin, taxol) did not reduce the rate or extent of FcRII modulation. Moreover, treatment of the macrophages with 0.1-0.5% formaldehyde did not reduce the extent of FcRII modulation as measured by the disappearance of E(IgG) binding sites. FcRII modulation was markedly slowed when the temperature was decreased to 2-4 degrees C. These results prove that FcRII modulation is governed by diffusion of the receptor in the plasma membrane. From the speed of FcRII disappearance from the macrophage's upper surface we calculate that the receptor has a diffusion coefficient at 37 degrees C of 2.5 X 10(-9) cm2/s. This finding indicates that FcRII, in its unligated form, is not linked to the macrophage's cytoskeleton, and that the receptor is capable of accommodating spatially to any distribution of ligands on a particle's surface

Lateral mobility of FcγRIIa is reduced by protein kinase C activation

AbstractThe lateral mobility of membrane proteins can reflect the extent of various protein-protein interactions. Using the fluorescence recovery after photobleaching technique, we have studied the lateral mobility of human FcγRIIa and some FcγRIIa mutants expressed in either P388D1 cells, a mouse macrophagelike cell line, or in Chinese hamster ovary (CHO) cells [1]. After treatment with phorbol myristate acetate (PMA), only the FcγRIIa molecules capable of mediating rapid endocytosis of immune complexes exhibited a reduced lateral diffusion coefficient with respect to untreated controls. Wild type FcγRIIa expressed in CHO cells, and nonfunctional FcγRIIa mutants expressed in P388D1 cells did not show any differences upon PMA treatment. This finding suggests that protein kinase C activation evokes additional protein-protein interactions with the cytoplasmic domain of functional FcγRIIa, which reduced receptor lateral mobility. The identity of these putative interacting proteins and the nature of the interactions remain to be elucidated