Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.

To investigate the antigenic relationship between the macrophage and lymphocyte Fc receptors (FcR), a monoclonal antibody capable of blocking mouse macrophage Fc receptors was selected. Hybrids were formed by fusing the P3U1 mouse myeloma and spleen cells from a rat immunized with the mouse macrophage-like cell lines J774 and P388D1. The Fab fragment of the monoclonal IgG secreted by clone 2.4G2, inhibited the trypsin-resistant Fc receptor II (FcRII), which is specific for immune aggregates of mouse IgG1 and IgG2b, but had no inhibitory effect on the trypsin-sensitive Fc receptor I (FcRI), which binds monomeric IgG2a and erythrocytes coated with IgG2a. Thus, the monoclonal 2.4G2 IgG appeared to be specific for macrophage FcRII. Further evidence that the 2.4G2 IgG was directed against FcRII came from binding studies of the monoclonal antibody to J774 cells and a series of independently isolated variants which do not express FcRII. These variants of J774 bound 5% as much of the monoclonal antibody as the parent line, which bound 600,000 molecules of 2.4G2 IgG per cell. The antigenic relatedness of mouse lymphocyte FcR to mouse macrophage FcRII was demonstrated by the binding of 2.4G2 IgG to FcR-bearing lymphoid cell lines and the inhibition of the lymphocyte FcR by the monoclonal antibody. Preincubation of spleen cells and peritioneal cells with 2.3G2 IgG likewise inhibited rosette formation with ox erythrocytes coated with rabbit IgG. The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex. The 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human FcR-bearing cells.

The presence of two Fc receptors on mouse macrophages: evidence from a variant cell line and differential trypsin sensitivity.

A stable variant of a clone of the P388D1 macrophage line was isolated using four cycles of treatment with mouse IgG2a-rabbit anti-kappa complexes and rabbit complement. The variant had the same Ka and about the same number of sites per cell for IgG2a as the parent line. However, the variant had 10% as many binding sites for rabbit IgG in soluble antigen-antibody complexes, and the affinity of binding was threefold higher. This change in binding of complexes to cells of a cloned line without alternation of IgG2a binding provides evidence for the presence of two distinct Fc receptors. The two receptors could also be distiguished on the P388D1 line and on thioglycollate-induced mouse peritoneal macrophages by differential sensitivity to trypsinization. The receptors that bind monomeric IgG2a, sheep erythrocytes (SRBC) covalently bound with IgG2a or rabbit IgG using glutaraldehyde, and Sephadex beads coupled with IgG2a or rabbit IgG using cyanogen bromide activitation, is sensitive to trypsinization. The receptor that binds soluble rabbit antibody-antigen complexes, trinitrophenyl-SRBC and dinitrophenyl(DNP)-bovine serum albumin Sephadex beads coated with rabbit anti-DNP IgG is trypsin resitant, the observation that uncomplexed rabbit IgG oes not bind to the trypsin-resistant receptor, whereas the same IgG bound to its antigen does, suggests that conformational changes induced by the binding of ligand may be of consequence in macrophage function.

Purificaton of a functional mouse Fc receptor through the use of a monoclonal antibody.

We recently reported the isolation of a rat monoclonal antibody designated 2.4G2 (9) that is directed against the mouse trypsin-resistant Fc receptor (FcR) for IgG2b and IgG1 immune aggregates. We have now utilized the Fab fragment of 2.4G2 as an affinity reagent to purify FcR from the macrophage cell line J774 to apparent homogeneity. The antigen isolated from J774 cells consisted of two general types of polypeptides with broad electrophoretic mobilities of approximately 60,000 and 47,000 mol wt. Similar broad bands ranging from 47,000 to 70,000 mol wt were isolated from various FcR-bearing cell lines of B, T, and null lymphocyte, as well as of macrophage origin. J774 FcR was judged to be a glycoprotein based on the sensitivity of its isoelectric point to neuraminidase digestion, its labeling with galactose oxidase/NaB[3H4], and its binding to concanavalin A-Sepharose. In phosphate-buffered saline, the isolated protein formed large aggregates that were shown to retain FcR activity, albeit with a somewhat altered IgG subclass specificity. The FcR aglutinated erythrocytes that were coated with both IgG2b and IgG2a that did not otherwise hemagglutinate. In addition, iodinated FcR bound to Sephadex beads coated with rabbit IgG, mouse IgG1, IgG2b, and IgG2a, but not to beads coated with mouse IgG3 or rabbit F(ab')2 fragments. The binding of the purified receptor to all IgG classes was inhibited by the Fab fragments of 2.4G2. In contrast, the binding of IgG2a to intact macrophages was inhibited by 2.4G2 Fab by only 15%, whereas rabbit IgG immune aggregate binding was almost completely abolished.

Binding of monomeric immunoglobulins to Fc receptors of mouse macrophages.

The binding properties of surface receptors of immunoglobulins on mouse macrophages were studied with mouse myeloma proteins and normal peritoneal macrophages, thioglycollate-stimulated macrophages, and a macrophage cell line, P388D1. Primary cultures of mouse embryo fibroblasts served as controls. IgG2a proteins were bound strongly;IgG2b was bound weakly (one-twentieth as well as IgG2a);IgM, IgA, and IgG1 were not bound significantly. The number of binding sites per cell for IgG2a was 4 X 10(5) for thioglycollate-stimulated cells and 1 X 10(5) for normal and P388D1 cells. Binding was exothermal: with decreasing temperature the equilibrium (association) constants increased and dissociation rate constants decreased (at 37degreesC the respective values were 2 X 10(7) M-1 and 0.26 min-1, the latter value corresponds to a half time for dissociation of 2.6 min). From the rapidity of association and dissociation, it appears that the surface of the macrophage is in a dynamic equilibrium with IgG2a molecules in the cell's immediate microenvironment. The receptors for IgG2a are clearly specific for determinants in the immunoglobulin constant domain: two IgG2a proteins with greatly different isoelectric points (determined by isoelectric focusing) were bound with the same affinity to the same receptors; moreover, the Fc fragment was bound and Fab fragments were not. The Fc receptors for IgG2a proteins were readily eliminated by exposing macrophages briefly to trypsin. The receptors were regenerated during subsequent cultivation in serum-free medium; regeneration was inhibited totally by cycloheximide and partially by actinomycin D.

Secretion of plasminogen activator by stimulated macrophages.

Cultured thioglycollate-stimulated peritoneal macrophages synthesize, accumulate, and continuously release high levels of plasminogen activators for at least 4 days whereas cultures of unstimulated macrophages do not; the higher specific catalytic activity of released vs. cell-associated enzyme suggests that the plasminogen activators are actively secreted. The major macrophage plasminogen activator is a serine protease of mol wt 48,000, and thus resembles the comparable enzyme released by virally transformed fibroblasts. Macrophages release a second plasminogen activator of mol wt 28,000 that is also a serine enzyme. The secretion products released by stimulated and unstimulated macrophages have been compared by SDS-polyacrylamide gel electrophoresis after chemical labeling with (3)H-DFP or biosynthetic labeling with (14)C-amino acids. These procedures show that some proteins are formed in both cultures, whereas others are uniquely secreted by each type of macrophage. The serine enzymes released by the two kinds of macrophages differ in specificity and electrophoretic mobility.

Induction of macrophage plasminogen activator by endotoxin stimulation and phagocytosis: evidence for a two-stage process.

The injection of thioglycollate medium into the peritoneal cavity of the mouse induces high levels of macrophage fibrinolytic activity due to the production and secretion of a plasminogen activator, a trypsinlike serine protease, which is absent in unstimulated macrophages. Intraperitoneal injection of endotoxin or mineral oil can stimulate only a fraction (<10%) of the fibrinolytic activity of thioglycollate cells, similar to the partial stimulation (<10%) seen 1-2 days after phagocytosis of latex or SRBC by unstimulated macrophages. The endotoxin-stimulated macrophages contain and release relatively low levels of plasminogen activator, but these primed cells can be triggered to produce and secrete high levels of enzyme, by phagocytosis of latex. Under conditions where the plasminogen activator is induced and secreted, there are no effects on the production and/or release of lysozyme or intracellular acid hydrolases, Discovery of a two-stage procedure for inducing macrophage plasminogen activator made it possible to study the role of cell priming and phagocytosis separately. Endotoxin was a more effective priming agent, weight for weight, than lipid A:BSA complex. Secretion of the plasminogen activator was induced only by thioglycollate, or endotoxin and latex. In situ fibrinolysis was induced by these agents and mineral oil, BCG, and fetal calf serum, in decreasing order of effectiveness. Phagocytosis of latex in all cases except thioglycollate stimulation, increased fibrinolytic activity from three- to sixfold. Latex and a variety of other particles such as M. lysodeikticus, aggregated gamma-globulin and immune complexes showed dose-dependent stimulation of fibrinolysis by endotoxin-primed macrophages. Although the initial phagocytic trigger was not specific for the substance employed, the ability to induce a sustained response depended on the persistence of the phagocytized particle within the cell. Fibrinolysis and secretion of plasminogen activator continued at high levels for at least 9 days after uptake of latex, a nondigestible particle, whereas plasminogen activator was secreted only transiently after ingestion of rapidly digested M. lysodeikticus. The induction of plasminogen activator secretion provides a mechanism by which the activated macrophage can exert a selective effect on its extracellular environment.

Cell surface expression of murine, rat, and human Fc receptors by Xenopus oocytes.

We report that Xenopus laevis oocytes can efficiently translate and insert heterologous membrane receptors into the oocyte plasma membrane, where they can be detected by the binding of either monoclonal antibodies or ligands. Thus, oocytes injected with mRNA from the mouse J774 macrophage-like cell line, the rat RBL-1 basophilic leukemia, and the U937 promonocyte cell line, bound 2.4G2 Fab, rat IgE, and mouse IgG2a, respectively. The increase in the high avidity Fc gamma R observed after gamma-interferon induction of U937 cells was also observed after injection of mRNA from gamma-interferon-induced U937 cells into oocytes. This suggests either much greater message stability or a greater rate of transcription of Fc gamma Rhi mRNA in the gamma-interferon-induced cells. The assay affords a sensitive method for the detection of rare mRNA species that code for plasma membrane proteins.

Expression and characterization of a truncated murine Fc gamma receptor.

We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these 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.

Identification of soluble Fc receptors in mouse serum and the conditioned medium of stimulated B cells.

We have evaluated the expression of surface Fc gamma 2b/gamma 1R by lipopolysaccharide (LPS)-activated murine spleen cells, the release of soluble Fc gamma 2b/gamma 1R by activated spleen cells, and the presence of circulating Fc gamma 2b/gamma 1R in mouse serum. LPS activation of murine spleen cells and a cloned B cell line, BCL-1 CW 13.20-3B3, resulted in a 5-10-fold increase in surface Fc gamma 2b/gamma 1R and the concominant appearance in the culture medium of a soluble molecule that is antigenically related to the Fc gamma 2b/gamma 1R. The increase in cell-associated and soluble Fc gamma 2b/gamma 1R after LPS activation is attributable primarily to B cells. Circulating Fc gamma 2b/gamma 1R was also detected in normal mouse serum at a concentration of 10(-9) to 10(-8) M. Levels of circulating Fc gamma 2b/gamma 1R increase with the age of the animals, and were low in adult germ-free mice and elevated in young mice with certain autoimmune diseases. The circulating Fc gamma 2b/gamma 1R bound to IgG-Sepharose, and was partially purified by affinity chromatography on 2.4G2 Fab-Sepharose. After radiolabeling and immunoprecipitation with rabbit anti-Fc gamma 2b/gamma 1R serum, one component of Mr 48,000, was detected.

Autoimmune mice make anti-Fc gamma receptor antibodies.

We demonstrate, using a recombinant truncated Fc gamma RII molecule as a probe, the presence of anti-Fc gamma R antibodies in several strains of autoimmune mice. Affinity chromatography on a truncated Fc gamma R column of pooled sera from aged NZB females resulted in isolation of 16 micrograms of IgM per ml of serum, approximately 2% of the total IgM; no anti-Fc gamma R IgM was found in sera from C58/J mice. Mice with high titers of anti-Fc gamma R IgM also had anti-Fc gamma R IgG. Affinity-purified anti-Fc gamma R IgG bound to Fc gamma R-bearing cells. A good correlation was found between the presence of anti-Fc gamma R Ig and impaired phagocytosis of immune complexes in autoimmune strains such as NZB or NZB/NZW F1. Sera with high titers of anti-Fc gamma R Ig from NZB and motheaten mice inhibited the binding of soluble immune complexes. Furthermore, BXSB, a lupus-prone mouse strain that does not produce anti-Fc gamma R Ig, shows normal macrophage binding and phagocytosis of immune complexes. A set of four IgM mAbs that bind to Fc gamma R was identified. These antibodies were polyspecific; some were directed against DNA, and others recognized a wide variety of antigens including histones, thyroglobulin, and transferrin, but all anti-Fc gamma R IgM antibodies effectively inhibited the binding of IgG1 anti-DNP/DNP20BSA complexes to J774 macrophages. The role of anti-Fc gamma R Ig in autoimmunity remains to be established. It may act to crosslink and activate Fc gamma Rs on neutrophils, macrophages, NK, and mesangial cells, or it may desensitize Fc gamma R function of Fc gamma R-bearing cells.

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.

Characterization of a membrane pore-forming protein from Entamoeba histolytica.

We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica. The formation of ion channels by PFM was examined in three systems. (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by [3H]TPP+ uptake. (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles. (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl. Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer. Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8. PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E. coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min. High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000. This protein may play a role in the cytotoxicity mediated by E. histolytica.

An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. II. Mammalian fibroblast cultures transformed by DNA and RNA tumor viruses.

Chick, hamster, mouse, and rat embryo fibroblast cultures, transformed by either DNA or RNA viruses, show fibrinolytic activity under suitable conditions of growth and in appropriate media; normal counterpart cultures do not. The fibrinolysin is produced by the interaction of two protein factors: one of these, a cell factor, is released by transformed cells and accumulates in the medium when cultures are incubated in the absence of scrum. The second factor, the serum factor, is a specific protein that is present in sera of many avian and mammalian species, including man. Not all sera yield fibrinolysin on interaction with any given transformed cell factor, and the spectrum of activating sera is distinctive for each cell factor. This pattern appears to be determined by the cell type, rather than by the transforming virus. An important role for the fibrinolysin in oncogenic transformation is suggested by the following correlations. (a) The initial appearance of fibrinolysin precedes the morphological change after the transfer to permissive temperatures of chick fibroblast cultures infected with a temperature-sensitive mutant of RSV. (b) The initiation of fibrinolysis and of morphological change both require the synthesis of new protein, but not the synthesis of either DNA or rRNA. (c) The activity of the fibrinolysin is correlated with the retention of abnormal morphology in hamster cells transformed by SV-40. (d) The sera of normal chicks effectively activate fibrinolysis with the cell factor from transformed chick cells. In contrast the sera of chicks with RSV tumors do not; these contain an inhibitor of the fibrinolytic activity.

An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. I. Chick embryo fibroblast cultures transformed by avian RNA tumor viruses.

Chick embryo fibroblast cultures develop fibrinolytic activity after transformation by Rous sarcoma virus (RSV). This fibrinolytic activity is not present in normal cultures, and it does not appear after infection with either nontransforming strains of avian leukosis viruses or cytocidal RNA and DNA viruses. In cultures infected with a temperature sensitive mutant of RSV the onset of fibrinolysis appears after exposure to permissive temperatures and precedes by a short interval the appearance of morphological evidence of transformation. See PDF for Structure The rate of fibrinolysis in transformed cultures depends on the nature of the serum that is present in the growth medium: some sera (e.g., monkey or chicken serum) promote high enzymatic activity, while others (calf, fetal bovine) do not. Some sera contain inhibitors of the fibrinolysin. Based on the effect of a small number of known inhibitors, at least one step of the fibrinolytic process shows specificity resembling that of trypsin. The sera of sarcoma-bearing chickens contain an inhibitor of the fibrinolysin, whereas normal chicken sera do not. For general discussion, conclusions, and summary see the accompanying paper, part II, (J. Exp. Med. 137:112).

IgM anti-Fc gamma R autoantibodies trigger neutrophil degranulation.

Anti-Fc gamma R IgM monoclonal antibodies (mAbs) isolated from lipopolysaccharide-stimulated spleen cells from tightskin (TSK) mice were found to be polyspecific, reacting with a wide variety of molecules, including double-stranded DNA, topoisomerase, RNA polymerase, and different collagen types. Approximately 60% of the polyspecific IgM mAbs have anti-Fc gamma R specificity. These anti-Fc gamma R mAbs induce the release of hydrolases from both azurophil and specific granules of human neutrophils. 25-45% of the total cellular content (determined in Nonidet P-40 lysates) of neutrophil elastase, 10-25% of beta-glucuronidase, and 30-50% of alkaline phosphatase was released after incubation with the mAbs. The degranulation process was accompanied by dramatic morphological changes shown by scanning and transmission electron microscopy. The release of hydrolytic enzymes stimulated by the IgM anti-Fc gamma R mAbs was inhibited by preincubation of neutrophils with Fab fragments of either anti-human Fc gamma RII (IV.3) or anti-human Fc gamma RIII (3G8) mAbs. The binding of the anti-Fc gamma R TSK mAbs to human neutrophils was inhibited by Fab fragments of mAb 3G8. However, we found that the TSK anti-Fc gamma R mAbs do not bind to human Fc gamma RII expressed in either CHO cells or the P388D1 mouse macrophage cell line. Since the enzyme release could be inhibited by Fab fragments of mAb IV.3, we suggest that the signal transduction may require Fc gamma RII activation subsequent to crosslinking of the glycan phosphatidyl inositol-anchored Fc gamma RIII-1. These data demonstrate for the first time that polyspecific autoantibodies with Fc gamma R specificity can trigger neutrophil enzyme release via human Fc gamma RIII-1 in vitro and indicate a possible role for such autoantibodies in autoimmune inflammatory processes.

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

The surface of dendritic cells (DC) has been analyzed by means of monoclonal antibodies (Ab) and lactoperoxidase (LPO)-mediated radioiodination. Antigens and other exteriorily disposed polypeptides of purified spleen DC were compared with those of tissue macrophages (Mphi), monocytes, and other bone marrow-derived elements. Quantitative binding studies and autoradiography with (125)I-Ab established that DC expressed high levels of I-A and H-2D, 2 x 10(5) and 1 x 10(5) Ab binding sites per cell, respectively. DC from conventional, germ-free, and specific pathogen-free mice were all rich in Ia. Expression of Ia on B cells was 5-10 percent of that on DC and increased fivefold during lipopolysaccharide mitogenesis. More than 70-90 percent of purified Mphi and monocytes from specific pathogen-free mice were Ia negative, but increased levels of Ia were noted on cells from mice reared under conventional conditions. Thus large amounts of Ia on DC is a constitutive trait, whereas the expression of Ia by other cell types may be governed by the environmental and immunological status of the host. The 2.4G2 Fc receptor Ag was not detected on DC. Peritoneal and spleen Mphi had 10(5) 2.4G2 binding sites/cell, whereas monocytes and lymphocytes were less reactive (1 x 10(4)-3 x 10(4) binding sites/cell). Four other Mphi-related antigens were evaluated. Each had a distinctive tissue distribution and none bound exclusively to Mphi and monocytes. Neither 1.21J (Mac-1) nor F4/80 reacted with DC. Immunoprecipitation studies of externally ((125)I) and biosynthetically ([(35)S]methionine)dabeled cells confirmed the binding data. Sensitive binding assays with (125)I-Ab confirmed previous observations that DC lack Ig and Thy-1. Lyt-1 was also not found on DC, but 5-12 percent of the cells in purified DC preparations expressed both Lyt-2 and Ia. All DC expressed the leukocyte common antigens at levels similar to other leukocytes. The spectrum of surface polypeptides labeled by LPO-mediated iodination was different on Mphi, DC, and lymphocytes. Polypeptides migrating at molecular weights of 155,000, 85,000, and 62,000 appeared to be restricted to DC. These observations establish that the cell surface of DC differs considerably from other leukocytes, including the blood monocyte, and suggest that the DC is part of a unique Ia-rich leukocyte differentiation pathway.

Blockade of clearance of immune complexes by an anti-Fc gamma receptor monoclonal antibody.

Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcgammaR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.

Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis.

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG-coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.