Identification of two types of specific endothelin receptors in rat mesangial cell

Two types of receptors specific for endothelin were identified using cross-linking technique in cultured rat mesangial cells. The molecular weights of these receptors were approximately 58,000 and 34,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The binding of radioiodinated-endothelin to its receptors was inhibited by excess of unlabeled endothelin, but not by nifedipine, nicardipine, verapamil, diltiazem, angiotensin II or [Arg8]-vasopressin. The endothelin binding proteins were solubilized with 1% digitonin and fractionated under non-denaturing conditions by gel filtration. Two endothelin binding peaks eluted at the positions corresponding to the molecular weights of 65,000 and 43,000. These observations indicate that there are two types of specific endothelin receptors in rat mesangial cells which are distinct from voltage dependent L-type calcium channel.     

Characterization of the IgE receptor isolated from human basophils.

The IgE receptor of human basophils was purified by using simple and repetitive affinity chromatography on human IgE-Sepharose. Basophils were partially purified from peripheral blood of patients with chronic myelogenous or basophilic leukemia. Cells were labeled with 125I by using the lactoperoxidase method and were solubilized with nonionic detergent. Elution of IgE-Sepharose with 0.5 N acetic acid, 1% NP-40 allowed recovery of active IgE receptor. Analysis of human IgE receptor by SDS polyacrylamide gel electrophoresis with 10% gels demonstrated one major radioactive peak with an apparent m.w. of 58,000 to 68,000, somewhat larger than rat IgE receptor. The purified human IgE receptor was active since approximately 10 to 42% of labeled receptor could specifically rebind to insolubilized human IgE. Rebinding was blocked by nanomolar concentrations of soluble human IgE or rat IgE but not by human or rat IgG, heat-inactivated human IgE, or heat-aggregated human IgG; thus it appears that rat IgE receptor. The relative abilities of active rat IgE and active human IgE to inhibit human IgE receptor rebinding could not be precisely determined because of the limitations in assessing the proportion of human IgE that retains receptor-binding activity.     

Characterization of the murine T cell receptor for IgE (Fc epsilon RII). Demonstration of shared and unshared epitopes with the B cell Fc epsilon RII

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.     

Labelling of colloidal gold with IgE. A quantitative study using monoclonal IgE anti-beta-lactoglobulin and evaluation of the biological activity of the gold complex with RBL-1 cells

Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal 125I-IgE antibovine milk beta-lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1-8.8 (28 nm in particle diameter). At low coverage of the particles (less that or equal to 5 molecules per particle), the isotherm was independent of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2-5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mapping of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4 degrees C. Surface clusters developed when the cells were incubated at 37 degrees C.     

The Cytoskeleton of Primary Astrocytes in Culture Contains Actin, Glial Fibrillary Acidic Protein, and the Fibroblast-Type Filament Protein, Vimentin

Abstract: Primary astrocytes were cultured from the forebrains of 1-day-old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and >80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton-insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower-molecular-weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL-8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle actin. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.     

Membrane-bound IgE receptor complexes fused with rat basophilic leukemia cells mediate degranulation

The high affinity receptor for IgE on rat basophilic leukemia (RBL) cells mediates antigen-triggered cellular degranulation. Polyethylene glycol-induced membrane fusion methods were used to introduce exogenous IgE receptors into living RBL cells, and these were tested for normal activities. In cell-cell fusion experiments, RBL cells with fluorescein-labeled rat IgE bound to receptors and containing [5-1,2-3H(N)]hydroxytryptamine binoxalate ([3H]5HT) in their secretory granules were fused to cells with receptors occupied by rhodamine-labeled anti-dinitrophenyl mouse IgE. The fused cells showed a uniform surface distribution of both types of IgE, which could be patched independently by anti-IgE or dinitrophenylated bovine gamma globulin (DNP16BGG). [3H]5HT release could be triggered specifically by DNP16BGG. In vesicle-cell fusion experiments, plasma membrane vesicles, with receptors occupied by fluorescein- and 125I-labeled anti-DNP mouse IgE, were fused to RBL cells containing [3H]5HT. The cells showed substantial associated fluorescein fluorescence and 125I counts, and [3H]5HT release could be triggered specifically by DNP16BGG. These experiments indicate that IgE receptors can be dissociated from their natural cellular interactions and retain the ability to reassociate with another cell's components to deliver the transmembrane signal for degranulation.     

Binding of human C-reactive protein to mouse macrophages is mediated by distinct receptors

Human C-reactive protein (CRP) is known to activate mouse macrophages (M phi) to a tumoricidal state and to serve as an opsonin for M phi. Therefore, cell surface receptors for CRP on mouse M phi were characterized and their relationship to the IgG FcR determined. The specific binding of 125I-CRP to resident or elicited mouse M phi was saturable, reversible, and involved both a high and a low affinity receptor population. Binding of CRP to the mouse M phi cell lines PU5 1.8 and J774 was nearly identical to that observed with peritoneal M phi. The high affinity receptor population had a calculated K of 10 nM and a receptor density of approximately 10(5) sites per cell. Mouse Ig of the IgG2a, IgG2b, or IgG1 isotypes inhibited binding of 125I-CRP to PU5 1.8 cells at concentrations five-fold greater than that of the homologous ligand. In the converse experiment, unlabeled CRP failed to inhibit specific binding of 125I-labeled IgG2a, IgG2b or IgG1. Isolation of CRP binding proteins from surface iodinated PU5 1.8 cells by ligand-affinity chromatography or chemical cross-linking yielded a major protein band of 57 to 60 kDa which appeared to be distinct from the IgG1/IgG2b FcR (FcR-II) membrane proteins. Removal of radiolabeled IgG2b/IgG1 binding membrane proteins by affinity chromatography did not remove CRP-binding proteins. The rat mAb 2.4G2 which inhibits binding of radiolabeled mouse IgG2b, did not inhibit the binding of CRP. A rat polyclonal antiserum to CRP-binding membrane proteins of PU5 1.8 cells inhibited 125I-CRP binding, but not 125IgG2b binding. The rat polyclonal antibody reacted with two 57 to 60 kDa membrane proteins from PU5 1.8 cells that appear to be of a similar size on Western blots. The 125I-CRP was internalized via endosomes and intact CRP subunits could be detected intracellularly. The findings suggest that binding of CRP occurs through a receptor that is distinct from the IgG FcRs, but that CRP-R activity may be influenced by an association with an IgG FcR.     

Physicochemical characterization of photoaffinity-labeled angiotensin II receptors

Specific receptor sites for angiotensin II (AII) were analyzed in the adrenal cortex and other target tissues including liver, anterior pituitary gland, and smooth muscle, after covalent labeling with the radioactive photoaffinity analog 125I-[Sar1,(4-N3)Phe8]-AII. The photoreactive AII derivative retained high affinity for adrenal receptors and full steroidogenic activity in adrenal glomerulosa cells. In bovine adrenal cortex, covalent labeling of AII receptors by the photoreactive analog was specifically inhibited by [Sar1]AII with an IC50 of about 5 nM. The Mr of the covalent AII-receptor complex during polyacrylamide gel electrophoresis of the labeled protein under reducing conditions was 58,000. Under non-reducing conditions, a minor band with Mr of 105,000 was also observed. Two labeled species were also found during gel permeation chromatography of the detergent-solubilized complex, with Mrs of 64,000 and 86,000. The pl of the solubilized photolabeled complex was absorbed to wheat germ lectin Sepharose 6MB and could be eluted by N-acetylglucosamine. The Mrs of specific AII-binding sites in several target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed target tissues, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed significant differences within and between species. The most striking differences were between rat adrenal cortex (79,000) and both rat liver (60,000) and bovine adrenal cortex (58,000). After enzymatic deglycosylation, the Mr of the major component present in the bovine and rat adrenal cortex decreased by 40% and 55% to 35,000 and 34,000, respectively, suggesting that variations in carbohydrate content contribute to the physical heterogeneity of AII receptors in individual target tissues.     

Comparison of the Fc receptors for IgE on human lymphocytes and monocytes

Fc receptors for IgE (Fc epsilon R) on human peripheral blood lymphocytes and monocytes and cultured lymphoblastoid and macrophage-like cell lines were compared with respect to: 1) binding affinity for radiolabeled IgE, 2) inhibition of IgE-specific rosette formation and inhibition of binding of radiolabeled IgE by an antiserum raised against Fc epsilon R isolated from a lymphoblastoid cell line, and 3) m.w. of radiolabeled cell surface proteins precipitated with the anti-Fc epsilon R serum. Scatchard analysis of 125I-IgE binding to lymphocytes, monocytes, and their corresponding cell lines showed biphasic binding curves with all cell types, from which 2 binding affinities were calculated to be KA = 6.2 +/- 1.1 and 2.0 +/- 0.5 x 10(7) M-1. The anti-Fc epsilon R serum inhibited both IgE rosette formation and binding of radiolabeled IgE by lymphocytes and monocytes but did not inhibit IgE rosettes formed by basophils. The inhibitory activity of the anti-Fc epsilon R serum could be absorbed with Fc epsilon R(+) but not with Fc epsilon R(-) cell lines. The anti-Fc epsilon R serum precipitated 2 peptides having m.w. of approximately 47,000 and 23,000 daltons from lysates of both cell surface-labeled lymphocyte and macrophage cell lines. These data indicate that Fc epsilon R on normal lymphocytes and monocytes, as well as on cultured lymphoblastoid and macrophage-like cells, are related structurally, since they share antigenic determinants, bind IgE with a similar affinity, and have similar m.w. However, they differ in all 3 parameters from Fc epsilon R on basophilic granulocytes.     

Neuron localization and neuroblastoma cell expression of brain-derived growth factor

Bovine brain-derived growth factor (BDGF) is a approximately 16-17 kD polypeptide mitogen with a broad spectrum of cell specificity. Using a highly specific mouse polyclonal anti-BDGF antiserum for indirect immunoperoxidase and immunofluorescent stainings, BDGF was found to be specifically localized in the neurons of bovine brain cortex. The indirect immunofluorescent staining was blocked by the presence of excess purified BDGF. Human neuroblastoma cells showed cytoplasmic staining with anti-BDGF antiserum. The cell lysates of neuroblastoma cells elicited a BDGF-like activity which could be completely inhibited by preincubation with anti-BDGF antiserum.     

Characterization of the target cell receptor for IgE. II. Polyacrylamide gel analysis of the surface IgE receptor from normal rat mast cells and from rat basophilic leukemia cells.

Purified rat peritoneal mast cells (RMC) and cultured rat basophilic leukemia (RBL) cells were surface labeled with 125I by using lactoperoxidase, incubated with unlabeled rat monoclonal IgE and subjected to solubilization by treatment with Nonidet P-40 (NP-40). With both cell types significant amounts of radioiodinated material could be specifically precipitated by a "sandwich" system consisting of rabbit anti-rat epsilon-chain and goat anti-rabbit Ig. The precipitates were dissociated with sodium dodecyl sulfate (SDS) and urea and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. With RMC three radioactive bands were seen. One corresponded to IgE present on the RMC at the time of isolation. A small band migrating in the region of light chain was seen with both sepcific (anti-IgE) and control precipitates. It showed no demonstrable relationship to IgE. The major radioactive band corresponded to a m.w. of 62,000. This band was dependent upon the presence of IgE and was not found when non-IgE binding control cells were used. With RBL cells, only the IgE-dependent 62,000 dalton peak was present. Saturation of the IgE receptor sites of the RMC or RBL cells before lactoperoxidase labeling almost totally eliminated this radioactive band, indicating that cell-bound IgE rendered this membrane component inaccessible to the radiolabel. These results strongly suggest that this cellular component is identical, at least in part, with the target cell surface receptor for reaginic antibody. The data also further support the hypothesis that the neoplastic RBL cells have a normal surface receptor for IgE.     

Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

Summary To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Cells were studied in Passages 2 to 8. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue. Supported in part by grant DK 31063 from the National Institutes of Health, Bethesda, MD.     

Surface Proteins of Cultured Mouse Cerebellar Cells

Surface proteins of cultured monolayer cells from embryonic and early postnatal C57BL/6J mouse cerebella were identified by a lactoperoxidase-catalysed ¹³¹iodine labelling technique. Major iodinated polypeptides have molecular weights of approximately 200, 145, 120, 100, 85, 65, 50, and 30 X 10³ (P200, P145, ?) as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis. Membrane glycoproteins, of apparent molecular weights 200, 145, 100, 85, and 50 X 10³, are detected by biosynthetic labelling with [³H]fucose. The two major iodinated proteins are the glycoproteins P200 and P145. P145 is released from the cells into the medium together with other surface proteins. No changes in the patterns of labelled cerebellar cell surface proteins are detectable between embryonic day 17 and postnatal day 10. A pattern similar to the one seen with cerebellum is obtained with embryonic day 12 and 17 cerebral cortex. Cultured retinal cells from 2-day-old mice, skin fibroblasts, and l -cells display a distinctly different pattern, which does not contain P145 as a major iodinated component. In granule cell-enriched fractions of cerebellar cells the two glycoproteins P200 and P145 are proportionately increased, while three proteins, P100, P85, and P50, are more abundant in the glial cell-enriched fraction. These three polypeptides are also enriched in cells obtained from staggerer mutant mice. An antiserum against 4-day-old cerebellar cells (anti-NS-4) precipitates the 145 and 200 X 10³ molecular weight proteins, from lysates of both embryonic cerebral and postnatal cerebellar cells. From lysates of mouse retinal cells, anti-NS-4 antiserum precipitates two proteins with molecular weights of 140 and 210 X 10³. Rohrer H. and Schachner M. Surface proteins of cultured mouse cerebellar cells. J. Neurochem. 35, 792–803 (1980).     

Partial purification of a receptor for the adipokinetic hormones from Musca autumnalis face flies.

Partial purification of the receptors for the neurohormones, diptera corpora cardiaca factors 1 and 2 (DCC1 and DCC2) was achieved. Receptor proteins were obtained from the abdomens of face fly, Musca autumnalis De Geer. Purification methods included detergent solubilization, affinity chromatography, and polyacrylamide gel electrophoresis. Analysis by gel electrophoresis has identified two proteins from this partial purification with relative molecular weights of 45 and 90 kD. A crude receptor preparation was used to develop a ligand binding assay with radiolabeled (tritiated and iodinated) DCC1. Ligand binding was inhibited by 90% when excess unlabeled DCC1 was added to the assay mixture. Ligand binding was optimum at pH 7.5. Binding saturation occurred at approximately 12 picomole radiolabeled ligand concentration. Because DCC1 and DCC2 have been shown to effect the lipid and trehalose levels in the insect an understanding of the neuropeptide-receptor interaction is important for the development of new methods of control of dairy and poultry muscoid flies.     

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Natural cytotoxic (NC) cell activity in basophilic cells: release of NC-specific cytotoxic factor by IgE receptor triggering

A murine interleukin 3 (IL 3)-dependent basophilic mast cell line, PT-18 (A17), and a rat basophilic leukemic cell line, RBL-2H3, were shown to be capable of selective natural cytotoxic (NC) but not natural killer (NK) cell activity. The basophilic cell types could also be augmented in their NC activity by bridging of their surface IgE receptors. IgE-mediated triggering of the basophilic cells was accomplished by coating the cells with IgE and exposing the IgE-bound cells to specific antigen or to anti-IgE monoclonal antibody. Another method of triggering was by direct binding of basophilic cells to anti-IgE receptor monoclonal antibody. Basophilic cells, triggered by these methods, not only displayed increased NC activity but also released a soluble factor capable of selectively lysing NC tumor targets, WEHI-164, but not three of the NK-sensitive targets, YAC-1, RLM1, and RBL-5. Normal C3H/HeJ mouse embryonic fibroblasts were also not lysed. Dose response and time course of the cytotoxic factor release from triggered RBL-2H3 cells were similar to those of tritiated serotonin release. As with serotonin or histamine release, the NC-specific cytotoxic factor (NCCF) was not released in the absence of extracellular calcium. Therefore, NCCF appears to be released along with other mediators during the triggering of basophilic cells by bridging of IgE receptors. The m.w. of the native form of this factor, determined by a gel filtration method, was about 43,000.     

Identification of a macrophage-specific cell surface antigen.

A mouse-specific macrophage antigen (MSMA) was identified in NP-40 extracts of 125I-radiolabeled mouse preitoneal macrophages by using a rabbit anti-mouse macrophage serum (AMS) and SDS-polyacrylamide gel electrophoresis. The antigen was shown to have a m.w. of 83,000 daltons and was present on both normal and "activated" peritoneal macrophages. MSMA was also present on syngeneic adherent spleen cells, allogeneic peritoneal macrophages, a mouse macrophage cell line (P388D1), and exhibited some cross-reactivity with peritoneal macrophages from closely related species (rats and hamsters). MSMA was not present on nonadherent peritoneal exudate cells, spleen cells, erythrocytes, thymocytes, or bone marrow cells. Extensive absorptions of AMS with thymocytes and erytrocytes from mice were necessary to remove other antibodies that reacted with other mouse membrane antigens. An antiserum directed against a specific membrane antigen has great potential in elucidating structure-function relationships with regard to a number of macrophage activities.     

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.     

Labelling of colloidal gold with IgE

Summary Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal ¹²⁵I-IgE antibovine milk -lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1–8.8 (28 nm in particle diameter). At low coverage of the particles (5 molecules per particle), the isotherm was independant of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2–5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mappings of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4°C. Surface clusters developed when the cells were incubated at 37°C.     

Mast cell membrane antigens and Fc receptors in anaphylaxis: II. Functionally distinct receptors for IgG and for IgE on mouse mast cells

The present work was aimed at analyzing the functional relationships between mouse mast cell receptors for IgG and IgE antibodies. It was based on a study of the inhibition of IgG1-and IgE-induced passive mast cell degranulation produced by various immunoglobulin preparations capable of interfering with Fc receptors. Rat myeloma IgE, a high-affinity ligand for IgE receptors, was used to search for a possible participation of IgE receptors in IgG1-dependent degranulation. Mouse myeloma IgG, which inhibited only weakly IgG1-mediated reactions, had no chance to compete successfully with high-affinity IgE antibodies, but aggregated HGG was found to behave as a high-affinity ligand for IgG receptors. This enabled us to search for a possible participation of IgG receptors in IgE-dependent degranulation. The results show that rat myeloma IgE and aggregated HGG specifically inhibited IgE-induced and IgG1-induced reactions, respectively, but failed to inhibit reactions not requiring free Fc receptors. The conclusion was that receptors for IgG and for IgE are functionally independent on mouse mast cells, and are both expressed on the same cells.