Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.     

High-performance immunoaffinity chromatography, an immunoaffinity membrane for selective removal of plasma components, and safety evaluation of the latter system.

This article reviews our studies on evaluating the suitability of high-performance immunoaffinity chromatography (HPIAC), an immunoaffinity membrane (IAM) for removing unwanted plasma component, and the safety of the IAM. In an attempt to resolve the problem of amyloid deposition in dialysis patients, we used an HPIAC column, bearing anti-beta 2-microglobulin to remove specifically beta 2-MG from human plasma. The use of a membrane as an affinity ligand support was also studied. A specific antibody immobilized on the membrane was highly effective for the removal of rat immunoglobulin E and of a human serum amyloid P component passed through an extracorporeal circulation (EC) system. Biocompatibility of the specific antibody-bearing IAM was also examined. These techniques should prove useful for medical applications and may have broad applicability to the elimination of any unwanted plasma component. The IAM exerts two functions simultaneously, usual dialysis and elimination by immunoaffinity binding. The rat EC model has been applied as an evaluation system for the safety of medical devices in contact with the blood stream. Combining commonly used hemodialysis (HD) membranes with rat EC, we evaluated the elicitation of immunological responses, as well as the effect of repeated EC. The data suggest that this EC model can reproduce similar immunological responses in HD patients, and can be employed to evaluate medical devices and materials for their delayed, systemic, and repeated exposure effects. The EC system described here can reproduce human HD treatment, remove unwanted substances, and evaluate medical devices and materials for toxicological responses.     

Selective removal of human serum amyloid P component from rat blood by use of an immunoaffinity membrane in an extracorporeal circulation system

We examined the suitability of an immunoaffinity membrane (IAM) bearing specific antibody as a ligand for removing human serum amyloid P component (hSAP) from blood passed through a simple extracorporeal circulation system established in rats. The specific antibody was the most effective of the various ligands tested for removing hSAP from human blood. To determine the value of the hSAP in human or rat plasma, we also developed a simple ELISA. In the rat extracorporeal circulation system, the hSAP level in the inlet blood to the IAM module decreased to 49% of the initial concentration within 60 min. In contrast, the hSAP remained at the initial concentration throughout the study in the module without the IAM. The use of this extracorporeal circulation system in this case allows preclinical evaluation of the ex vivo removal of a human plasma component in an animal model. Biocompatibility of the IAM was also examined. No change in blood cell counts or activation of the coagulation system occurred after contact with the IAM. Non-specific adsorption was not observed, since there was no statistically significant difference in IgG, complement C3, or albumin level between the pre- and post-treatment with this module. The immunological effects of the IAM were also examined using this system. Four weeks after the termination of the extracorporeal circulation, the rats examined showed no detectable antibody titer to the ligand.     

大鼠脑突触质膜糖皮质激素受体的纯化

本文利用抗大鼠肝细胞内糖皮质激素受体的单克隆抗体制备的免疫亲和层析柱,将大鼠脑突触质膜糖皮质激素受体纯化了约1150倍,SDS聚丙烯酰胺簿层梯度凝胶电泳显示,在约67kD处有一较明显的染色条带。     

Titin and myosin, but not desmin, are linked during myofibrillogenesis in postmitotic mononucleated myoblasts

We have used a model system to explore the importance of long-range lateral diffusion of membrane proteins in specific membrane-membrane adhesion. Single, cell-size phospholipid vesicles containing a dinitrophenyl (DNP)-lipid hapten were maneuvered into contact with rat basophilic leukemia (RBL) cells carrying fluorescent anti-DNP IgE in their cell-surface Fc epsilon receptors. Upon cell-vesicle contact the antibody molecules underwent a marked lateral redistribution, accumulating at the site of contact and becoming significantly depleted from noncontacting membrane. As assayed with a micropipette suction method, there was a time-dependent increase in the strength of cell-vesicle adhesion. This development of adhesion paralleled the kinetics of accumulation of the adhesion-mediating antibody molecules at the zone of membrane-membrane contact. Both adhesion and redistribution were absolutely dependent upon a specific interaction of the IgE with the hapten: No redistribution occurred when vesicles lacking the DNP hapten were pushed against IgE-armed RBL cells, and on cells bearing a 1:1 mixture of nonimmune rat IgE and anti-DNP mouse IgE, only the latter underwent redistribution. Vesicles containing DNP-lipids bound to RBL cells carrying anti-DNP IgE but not to cells carrying nonimmune rat IgE. Measurable nonspecific binding did not develop even after 15 min of pushing DNP-bearing vesicles against RBL cells sensitized with nonimmune IgE. Neither redistribution nor adhesion was blocked by metabolic poisons such as NaN3 and NaF. Both redistribution and adhesion occurred in plasma membrane blebs previously shown to lack cytoskeletal filaments. The above observations are consistent with contact-induced redistribution of the IgE being a result of passive diffusion-mediated trapping rather than active cellular responses. Thus, long-range diffusion of specific proteins can in some cases contribute to the formation of stable adhesion between membranes.     

Lymphocytes bearing Fc receptors for IgE. II. Induction of Fcepsilon-receptor bearing rat lymphocytes by IgE.

The proportion of lymphocytes bearing receptors for IgE (FcepsilonR) markedly increased after infection of rats with Nippostrongylus brasiliensis (Nb). The FcepsilonR-bearing lymphocytes from the infected animals bound more IgE-coated erythrocytes in rosette assay than FcepsilonR-bearing cells from normal rats, suggesting that the number of FcepsilonR per cell may also increase following the infection. In contrast, the number of IgE-receptors on peritoneal mast cells did not change after Nb infection. The increase in the proportion of FcepsilonR-bearing lymphocytes in Nb-infected rats is probably due to an increased concentration of IgE in the environment. The proportion of FcepsilonR-bearing cells in normal rat lymphocyte suspensions increased by culture of the cells with rat IgE of 1 microgram/ml or higher concentration. Other immunoglobulins such as rat IgG, human IgE, or rabbit IgG failed to induce either FcepsilonR-bearing cells or FcgammaR-bearing cells. It was also found that induction of Fc receptors by rat IgE is confined to FcepsilonR. Kinetic studies on the induction of FcepsilonR-bearing lymphocytes in vitro showed that the proportion of these cells in lymphocyte suspensions increased within 8 hr incubation with rat IgE but not within 4 hr. Evidence was obtained that both RNA synthesis and protein synthesis, but no DNA synthesis, are required for the induction of FcepsilonR-bearing cells or the expression of the receptors on the cell surface.     

Immunopurification of the blood group RhD protein from human erythrocyte membranes

Rh proteins are membrane proteins encoded by genes at the blood group RH locus. They are of paramount importance in transfusion medicine, but their function is still unknown. Biochemical and biophysical studies of these proteins are scarce since only minute amounts of the very hydrophobic Rh proteins, can be purified from human erythrocytes. Recently, a human monoclonal antibody (LOR-15C9) was described as having the unique property to recognize the Rh30 protein carrying the major blood group D specificity (RhD protein), either in a membrane detergent extract or when blotted on a membrane. In this report, we describe one-step purification of the RhD protein from detergent extracts of red cell membranes, based on immunoaffinity chromatography carried out with immobilized LOR-15C9 IgG. The technique yielded RhD protein with high purity which was devoid of other associated proteins (RhAG, CD47, LW and GPB) that comprise the Rh complex in the erythrocyte membrane. By contrast immunoprecipitation performed with the same antibody led to co-isolation of both RhD and RhAG.     

The fate of IgE bound to rat basophilic leukemia cells. IV. Functional association between the receptors for IgE

Rat basophilic leukemia cells (RBL-2H3) have receptors for immunoglobulin E (IgE) and immunoglobulin G (IgG). These receptors for IgE mediate the endocytosis of chemically or immunochemically cross-linked IgE but not monomeric IgE. However, unoccupied receptors were endocytosed with cross-linked IgE. To further assess the degree and specificity of the observed coendocytosis, we exposed cells carrying monomeric rat IgE and monomeric mouse IgE anti-DNP to a DNP-protein conjugate. We found that up to 30% of the surface-bound monomeric rat IgE redistributed at 0 to 4 degrees C and was then internalized at 37 degrees C with the immunochemically cross-linked mouse IgE. To assess the specificity of the coendocytosis, we exposed cells carrying monomeric rat IgE to immunochemically cross-linked mouse IgG. We found that the binding, patching, and endocytosis of cross-linked mouse IgG had no effect on the monomerically bound rat IgE. The rate of coendocytosis was the same as the rate of endocytosis (t 1/2 3 to 5 min). The extent of coendocytosis depended on the extent of endocytosis but was relatively insensitive to changes in the ratio between mouse and rat IgE over a broad range. These results indicate that some of the receptors for IgE are associated in a specific fashion.     

Solubilization and purification of rat liver 5''-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.     

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.     

Cytophilic binding of IgE to the macrophage. I. Binding characteristics of IgE on the surface of macrophages in the rat.

Involvement of IgE antibody in macrophage cytotoxicity against Schistosoma mansoni schistosomula suggests a cytophilic interaction of this class of antibody with the membrane of macrophages. Peroxidase-labeled monoclonal IgE protein was used to investigate IgE-macrophage interaction in the rat. Benzidine-aggregated rat IgE bound to the surface of peritoneal macrophages under experimental conditions, preventing endocytosis of the labeled aggregates. Binding was inhibited by preincubation with unlabeled IgE (aggregated). When unaggregated IgE was used, labeling of the macrophage surface, even when endocytosis was inhibited, was also observed at 37 °C but not at 4 °C. This result indicated different binding characteristics than reported for cytophilic IgG. Radiolabeled monoclonal IgE (deaggregated by ultracentrifugation after labeling) bound to peritoneal rat macrophages at 37 °C with a maximum between 10 and 20 min and a progressive shedding thereafter, whereas no change in bound radioactivity was observed at 4 °C or after preincubation with unlabeled IgE. Radiolabeled bovine serum albumin as a control did not interact significantly with the macrophages at both temperatures in these experimental conditions. The use of ?-mono-specific rabbit anti-rat IgE allowed the identification of IgE on the surface of peritoneal macrophages from rats infected with S. mansoni.     

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.     

Azelastine and suplatast shorten the distribution half-life of IgE in rats

We aim to clarify whether suplatast and azelastine (anti-allergic drugs) can shorten the half-life of imnunoglobulin E (IgE) in the circulating blood. Thirty Wistar rats were divided into six groups. Distilled water or anti-allergic drugs were given orally for 6 days after the first sensitization. Two milligrams of monoclonal dinitrophenyl (DNP)-specific rat IgE was administered to the rats, which had been given suplatast or azelastine orally. The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. The elimination half-life of rat IgE was about 12 h irrespective of the sensitized state. The intercompartmental rate constants (Kct and Ktc) in the suplatast-administered or azelastine-administered group were larger than those of the distilled water-administered group under non-sensitized conditions. These findings suggested that the anti-allergic drugs used in the present study facilitated the excretion of IgE from the circulation in rats.     

Determination of deoxynivalenol (DON) in blood, bile, urine and excrement samples from swine using immunoaffinity chromatography and LC-UV-detection

A work up procedure is described by which DON concentrations in blood, bile, urine and excrements from swine can be quantified by HPLC and UV- detection at λ = 220 nm. The central step thereby is the purification and concentration of DON by means of an immunoaffinity column. While, in our experiments, the quantification of DON in blood and urine was straightforward an additional purification step by a preparative HPLC run prior to immunoaffinity chromatography was needed when bile and excrements were investigated. However, when low DON concentrations in blood and urine are expected, a preparative HPLC run prior to immunoaffinity chromatography is recommended as well, because larger amounts of sample materials should be analyzed and more impurities interfere with the column proteins. In our study, using spiked samples, recoveries ranged from 75—90% and limits of detection were 0.01 to 0.02 μg/ml.     

Depolarization of rat basophilic leukemia cells inhibits calcium uptake and exocytosis

We have investigated the unusual observation that depolarization of rat basophilic leukemia cells in high potassium not only fails to induce secretion, but also inhibits the secretion induced when receptors for IgE are aggregated by antigen. Antigen-stimulated 45Ca uptake and the rise in cytoplasmic free ionized calcium measured with the fluorescent indicator quin2 were both inhibited in depolarized cells. 45Ca efflux, on the other hand, was unaffected, which confirms that IgE receptor activation was not impaired in high potassium. Unlike the large increase in total cell calcium seen when cells in normal saline solution were stimulated with antigen, there was a decrease in total cell calcium when depolarized cells were stimulated. This is consistent with our finding that 45Ca uptake was inhibited while 45Ca efflux was unaffected. Inhibition of 45Ca uptake and secretion closely paralleled the decrease in membrane potential, and could be overcome by increasing the extracellular calcium concentration. We conclude that changes in the electrochemical gradient for calcium are important in determining calcium influx and the magnitude of antigen-stimulated secretion from rat basophilic leukemia cells, while the release of calcium from intracellular stores is unaffected.     

Purification and characterisation of MHC class I antigen from rat liver with monoclonal antibody

We have described three monoclonal antibodies (HAM1, HAM2, and HAM3) to rat liver cell membrane glycoproteins. Recently also we reported another monoclonal antibody (HAM4) to rat hepato-renal membrane antigen. Using these monoclonal antibodies, it is possible to purify membrane antigens. This paper describes the details of the purification and the nature of the antigen purified with one of the monoclonal antibodies (HAM2) to rat liver cell membrane glycoproteins. Antigen was purified with immunoaffinity column. The amino acid composition was determined and compared with those of mice MHC class I antigen (H-2) and with the rat lymphocyte membrane antigens which were purified with monoclonal antibodies and of which amino acids compositions were determined.     

Structural mapping of Fc receptor bound immunoglobulin E: proximity to the membrane surface of the antibody combining site and another site in the Fab segments

Resonance energy-transfer methods have been used to investigate the structure of immunoglobulin E (IgE) bound to its high-affinity receptor on plasma membrane vesicles derived from rat basophilic leukemia cells. The structural mapping of receptor-bound IgE was initiated in an earlier study [Holowka, D., & Baird, B. (1983) Biochemistry 22, 3475], and it is based on measuring the minimal distance from IgE sites that are selectively labeled with donor probes to a plane of amphipathic acceptors at the membrane surface. This paper describes the use of monoclonal IgE specific for 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) to place a donor probe, DNS-L-Lys, in the antibody combining sites. The distance from these sites to the membrane surface was determined to be greater than 100 A with two different amphipathic acceptor probes. Another site in the Fab segments of monoclonal IgE (anti-dinitrophenyl) could be labeled selectively with N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]maleimide (CPM) in the absence of reducing agents [CPM(-)], and the reaction could not be blocked by prereaction with N-ethylmaleimide. The pattern of CPM(-)-labeled proteolytic fragments and the lack of fluorescence quenching by (trinitrophenyl)lysine in the antibody combining sites suggested the CPM(-)-labeled site to be in the C epsilon 1 domain of IgE. The distance between this site on receptor-bound IgE and the membrane surface was determined to be 75-87 A with two different amphipathic acceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haplorchis taichui: worm recovery rate and immune responses in infected rats (Rattus norvegicus)

Worm recovery rate, mucosal mast cells (MMCs), eosinophils and serum IgE concentration in rats were investigated after orally feeding 300 Haplorchis taichui metacercariae to male rats. The duodenal, jejunal and ileal tissue sections were stained with 1% alcian blue and 0.5% safranin-O for MMC count. Eosinophil count and the serum IgE concentration assay were measured from cardiac puncture blood. The average worm recovery rates were 20.00%, 13.00%, 0.67%, 1.67% and 0.00% on day 3, 7, 14, 21 and 28 post-infection (PI), respectively. The number of MMCs in the infected rats were significantly higher than in the controls (P < 0.01), reaching a peak on day 21 PI. They decreased thereafter, with the decline in worm recovery. Eosinophil count and Serum IgE concentration were also increased but not significantly higher than the controls. However, they showed a positive relationship to worm recovery. It could be concluded from the results that MMCs, eosinophils and IgE may play an important role in the expulsion of H. taichui from rat intestine. However, the mechanism by which the MMC result in the helminth expulsion still need to be understood, and it is recommended that other cells such as goblet cells be studied further.     

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.