Rat IgE Research Articles

A single major transcript encodes the membrane-bound form of rat immunoglobulin E.

The primary structure of the membrane-bound form of rat immunoglobulin E was determined by PCR amplification and nucleotide sequence analysis of its mRNA. The sequence was found to correspond to the previously identified membrane exons of the rat epsilon chain gene. The donor splice site in the C4 exon was mapped to a position 35 nt upstream of the stop codon for the secreted form of rat IgE. Therefore, the membrane-bound IgE lacks the 12 C-terminal amino acids present in the secreted form of the protein. Recently, five novel epsilon chain transcripts were isolated from human IgE producing B-cells or B-cell lines. Four of these transcripts encode proteins which differ in their C-terminal ends from the classical membrane or secreted forms of human IgE. To investigate if these transcripts were likely to represent functional mRNAs, their evolutionary conservation was studied by screening a rat IgE producing B-cell line for the expression of similar transcripts. By PCR amplification and cloning of transcripts, containing both the C3 and the M2 exons, approximately 10,000 independent cDNA clones were obtained. These clones were screened with probes directed against regions specific for each of the five novel human epsilon chain mRNAs. However, no evidence was found for the presence of transcripts with a similar structure, indicating that no specific function associated with these transcripts and their corresponding proteins has been conserved between human and rat. The lack of additional M2-containing transcripts in the rat suggest that the novel human IgE transcripts are byproducts of differential splicing and that they most likely encode proteins with no evolutionarily important function.

Purified rat peritoneal mast cells: characteristics of the removal and binding of IgE.

Mast cells were purified by discontinuous density gradient centrifugation to greater than 90% and subsequently exposed to lactic acid in order to dissociate endogenous IgE. Unfortunately no removal of this IgE was achieved, despite previous reports of lactic acid exposure dissociating IgE from human basophils. Only a minimal level of endogenous IgE was found on the surface of mast cells, however, which allowed investigations of IgE binding to proceed without the initial removal of this endogenous IgE. Conditions for binding of normal rat IgE and myeloma IgE were established while efforts to bind human IgE to purified rat mast cells were unsuccessful. Our results suggest that while purified rat mast cells have the potential to play an important role in the study of the IgE requirement of various stimuli including histamine-releasing factor, this role is limited at present.

Demonstration of specific high‐affinity Fcɛ‐receptors on the human basophil‐like leukemia cell line KU812 by flow cytometry

The specificity of IgE binding to a human basophil-like cell line (KU812) was studied by flow cytometry. Four IgE myeloma proteins, representing both light-chain types, one chimeric IgE protein, and polyclonal serum IgE blocked the direct binding of FITC-labeled IgE(DES) myeloma protein to KU812 cells in a dose-dependent and nearly equimolar way. Although not as efficiently as human IgE (from five to eight times less on a molar basis), both rat and mouse IgE blocked IgE(DES)-FITC binding to KU812 cells. In sharp contrast, all four human IgG subclasses, both IgA subclasses, and IgM myeloma proteins, as well as monomeric and heat-aggregated polyclonal human IgG, were unable to block significantly IgE(DES)-FITC binding to KU812 cells (< 0.5% on a molar basis). The cytophilic epitope on IgE was heat-susceptible (56 degrees C, 2 h), lost after reduction alkylation, and resident in the papain-derived Fc epsilon-fragment, but not in the papain-derived Fab epsilon- and Fc'epsilon-fragments nor in the pepsin-derived F(ab')2 epsilon- and Fc"epsilon-fragments. Washing and displacement experiments indicated that a major part of IgE reacted with high affinity to KU812 cells. The results indicate that the binding of IgE to KU812 cells is highly specific and involves the classical high-affinity Fc epsilon RI-receptor. Although the density of receptors is low, this human cell line offers a unique model to study IgE/Fc epsilon RI interactions.

Mechanism of allergic cross-reactions. V. High incidence of unanticipated cross-stimulation by natural allergens of rat basophilic leukemia cells sensitized with monoclonal IgE antibodies.

The incidence of cross-stimulations by natural allergens was investigated using RBL-2H3 cells sensitized with five different mouse monoclonal anti-DNP IgEs and four mercury-induced rat monoclonal IgEs. Cells sensitized with 3 of the 5 monoclonal anti-DNP IgEs (clones SPE-7, SRT-1, LB4) responded by serotonin release upon stimulation by natural allergens such as Dermatophagoides pteronyssinus, horse dander and mugwort extracts. Serotonin release could be inhibited by monovalent DNP-lysine, indicating the involvement of DNP-binding sites of IgEs. Two of the clones (LO-DNP-30 and LA2) were negative on all tests with allergens. All but one (Hg32) of the mercury-induced rat IgE monoclonal antibodies tested positive with DNP-BSA, and with at least one of the six allergen extracts. IgE clone Hg12 mediated serotonin release with 5 of the 6 allergens tested.

Cytofluorometric quantification of IgE and IgE receptors on individual mast cells

Information about the IgE receptor and IgE content of mast cells under different conditions in vivo is essential for further understanding the functions of the mas cell-IgE system. A cytofluorometric method for measuring cell-bound IgE on individual mast cells was therefore explored using peritoneal mast cells of Sprague-Dawley rats infected with the nematode N. brasiliensis and rat basophilic leukaemia cells (RBL-1) as experimental models. We systematically studied the effects of variables such as fixation, incubation time, temperature and concentrations of antibody on the fluorescence emission of the mast cells. Optimum conditions were selected for the quantitative measurement of IgE at the single-cell level using direct labelling with FITC-conjugated goat anti-rat IgE(Fc). Polystyrene beads with a defined fluorophor content and a fluorescent uranyl glass were used to standardise the measurement procedure. A linear relatioship between fluorescence intensity and IgE concentration was obtained by fluorescence measurements on RBL-1 cells incubated in rat IgE. In the case of rat peritoneal mast cells it was possible to saturate the IgE receptors by incubating the mast cells in rat IgE. By measuring the mast cells before and after IgE incubation, the relative content of IgE, the relative number of IgE receptors and the degree of receptor saturation could be estimated. In this way we measured the IgE content of peritoneal mast cells of Sprague-Dawley rats maintained under pathogen-free conditions. The distribution of IgE content in the mast-cell populations was extremely variable. Up to 30% of the mast cells in individual populations contained little or no IgE, but very few if any of the cells lacked IgE receptors. On average, 60–70% of the receptors available for binding were occupied by IgE in these normal rats.

Application of an immunocolloid gold technique for the ultrastructural demonstration of IgE-receptor complexes on rat mast cells.

In previous works using cytofluorometry, we demonstrated a broad range of IgE and IgE-receptor levels within individual mast cell populations with a 60 to 80% occupancy of the IgE receptors on mast cells by native IgE. This study was performed in order to confirm our previous findings using an independent method and to visualize the distribution of IgE-receptor complexes on mast cells at an ultrastructural level. For this purpose an indirect immunocolloidal gold-labelling technique has been applied. By counting the number of labelled gold particles, a relative measure of IgE-receptor surface expression and IgE occupancy of the receptors could be obtained. With respect to mast cell morphology and anti-IgE binding specificity criteria, 1% glutaraldehyde + 4% paraformaldehyde (1:1, vol/vol) was found to be the best of the seven fixatives applied in this study. This technique revealed numerous gold particles on the surface of mast cells from barrier-maintained rats (26 +/- 11 per mast cell section, mean +/- SD). Increased numbers of gold particles were counted if the mast cells were incubated with rat myeloma IgE (20 micrograms/ml) (46 +/- 33 per mast cell section, mean +/- SD). There were significantly increased numbers of gold particles on the mast cells of rats infected with N. brasiliensis (126 +/- 30 per mast cell section, mean +/- SD). This indicates that some of the IgE receptors (about 50% of the total number of IgE receptors in this case) on mast cells were occupied by native IgE and that parasite infection significantly increased the number of IgE molecules on the surface of the mast cells. These results correspond with the findings we have made using the cytofluorometric technique and confirm the large individual variations in the density of IgE receptors and IgE among the mast cells of a given cell population. Macrophages, lymphocytes and eosinophils, carrying the low-affinity IgE receptors (Fc epsilon RII), contained less than 5 (normal rats after incubation in rat IgE) or 10 (nematode-infected rats) gold particles per cell section. We also observed some non-granulated lymphocyte-like cells which bound a large number of gold particles after incubation with rat myeloma IgE (20 micrograms/ml), indicating that they contained IgE receptors Fc epsilon RI). They were interpreted as mast cell precursors which have previously been shown to exist in the peritoneal cavity.

Profound reduction in allergen sensitivity following treatment with a novel allergy vaccine

A novel approach is described for the treatment of IgE-mediated allergic reactions which is based on the induction of a strong anti-IgE response in the host. Vaccination of ovalbumin-sensitized rats with constant domains two and three of rat IgE coupled to a heterologous carrier protein resulted in a profound decrease in serum levels of IgE, and later in a nearly complete block of histamine release from mast cells and basophils upon challenge with either a cross-linking polyclonal anti-IgE antiserum or a specific allergen.

Identification of sites on the human Fc epsilon RI alpha subunit which are involved in binding human and rat IgE.

The high affinity IgE Fc receptor (Fc epsilon RI), found on mast cells and basophils, is a tetrameric receptor complex. The extracellular portion of the Fc epsilon RI alpha subunit consists of two immunoglobulin-like domains and binds IgE in the absence of the other subunits. To localize the high affinity IgE binding site within the Fc epsilon RI alpha subunit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substituted with those from the human Fc gamma RIIIA alpha or the rat Fc epsilon RI alpha subunit. The chimeric receptors were monitored for their capacity to bind human and rat IgE, and their reactivity with different antireceptor antibodies. Domain I substitutions maintained high affinity human IgE binding. Domain II substitutions resulted in a total loss of both human and rat IgE binding. Single-domain alpha subunits could not bind IgE, suggesting that both extracellular domains are required for proper protein folding or IgE binding. To further localize the IgE binding sites, homolog-scanning mutagenesis was performed. At least three independent regions of domain II encompassing residues 118-129, 136-150, and 148-162 were required for IgE binding. Our results suggest that domain II of the human Fc epsilon RI alpha confers most of the important contributions to the binding of the human IgE Fc molecule, whereas domain I of the rat Fc epsilon RI alpha makes important contributions to the binding of rat IgE.

スギ花粉アレルゲン免疫ラットにおけるIgE抗体応答

We established an IgE-capture ELISA for rat IgE antibody, and examined rat strain differences in the antibody responses against the major allergen of Japanese cedar pollen (Cry j I). First, IgE-capture ELISA and passive cutaneous anaphylaxis (PCA) method were compared. IgE-capture ELISA was found to be, in sensitivity, about 10 times higher than, and, in specificity, equal to, PCA. Second, seven strains of rat were immunized three times at days 0, 14 and 28 by intraperitoneal injections of 1 or 10 micrograms Cry j I mixed with 4 mg aluminium hydroxide gel. Anti-Cry j I IgE was detected by IgE-capture ELISA. Anti-Cry j I IgG and IgM responses were found not significantly different among different strains, but IgE responses in Brown-Norway (BN) rat were about 10 times higher than in other strains of rats. These results suggest that BN rat may be a useful animal model for studying physiological and pathological changes in the nose after pollen challenge to that organ.

Co-crosslinking Fc epsilon RII/CD23 and B cell surface immunoglobulin modulates B cell activation.

Previous studies have shown that a highly multivalent from of anti-IgD or anti-IgM, prepared by conjugating the respective antibodies to dextran, causes extensive B cell proliferation with ng/ml concentrations of the anti-immunoglobulin (Ig). A modification of this system has been exploited to investigate the effect of co-crosslinking the Fc epsilon RII and surface Ig by binding DNP to the dextran backbone (DNP-dextran) and employing a DNP-specific monoclonal IgE of either rat or mouse origin. Addition of anti-IgD-(H delta a/1)[DNP-dextran] or anti-IgM-[DNP-dextran] to purified, resting murine B cells resulted in B cell proliferation over a broad dose (0.03-30 micrograms/ml). Addition of DNP-specific rat or mouse IgE dramatically modulated the proliferative response. Proliferation in response to doses greater than 0.3 microgram/ml H delta a/1-[DNP-dextran] was consistently reduced in a dose-dependent manner in the presence of increasing amounts of IgE while proliferation to lower concentrations of H delta a/1-[DNP-dextran] was slightly enhanced or not influenced at all by the IgE anti-DNP. Interleukin-4 (IL-4) significantly increased the IgE effect, in line with its known enhancing effects on Fc epsilon RII levels. Experiments measuring Ig production rather than proliferation demonstrated that in the presence of IgE anti-DNP, B cells produced lower amounts of immunoglobulin (IgG1 or IgM) in response to an anti-Ig signal. Control experiments demonstrated that the IgE effect on proliferation was blocked by monoclonal anti-Fc epsilon RII, but not anti-Fc gamma RII, thus demonstrating the necessity for IgE/Fc epsilon RII interaction. In addition, the necessity for co-crosslinking was shown by the inability of IgE anti-DNP to affect the proliferative response to H delta a/1-dextran even in the presence of various doses of DNP-dextran. These results demonstrate that co-crosslinking of sIg and the Fc epsilon RII results in an altered B cell response to anti-Ig mediated activation. IL-4 does not ablate this inhibition, in contrast to the effect of co-crosslinking Fc gamma RII and surface Ig, suggesting a model whereby IgE can modulate its own production.

The expression and characterization of rat IgE produced by construction of the epsilon-heavy chain gene from exon modules.

A system of exon "modules" was produced from the functionally rearranged epsilon-heavy gene isolated from the rat IgE-secreting immunocytoma IR162. The five individual exons, encoding the variable and constant region domains, were isolated and subcloned into the multiple cloning site of a pair of plasmid vectors with opposed orientation multiple cloning sites. The use of opposed orientation multiple cloning sites and the flanking restriction enzyme sites contained therein allows for the modular manipulation of the gene. These exon modules were initially used to reconstruct the epsilon-heavy chain gene into the native configuration to demonstrate the efficacy of the modular system for synthesis of IgE. Upon transfection into the rat myeloma cell line Y3, the reconstructed gene produced a polypeptide that associated with the endogenous light chain polypeptide and was secreted from the cell as tetrameric IgE. All physical and functional characterizations indicate that the IgE molecule produced is indistinguishable from native IR162 IgE. This modular system of exons will facilitate the manipulation of IgE structure through the systematic assembly of different epsilon-heavy chain mutant constructions. The resulting novel IgE proteins will be very useful to study the molecular nature of the interaction of IgE with its Fc receptor.

Experimental arthritis in rats induced by intra-articular injection of IgE aggregates: evidence for arthritogenic role of complexed IgE.

An experimental arthritis model in the rat was used to study the arthritogenic potential of complexed IgE. IgE aggregates were produced in vitro by cross linking monoclonal rat IgE by dimethyl suberimidate and were injected into the knee joints. Animals which had not been injected and animals injected with phosphate buffered saline served as controls. The concentration of histamine in tissues, diffusion into the joint of bovine serum albumin labelled with iodine-125 injected intravenously, and the histology of the joints were studied. There was a significant decrease in the concentration of histamine in synovial tissue 8 and 24 hours after the injection of the IgE aggregates. A decreased number of stainable mast cells were found 8, 24, and 48 hours after exposure. A moderate hyperplasia of the synovial lining layer was also noted. These results provide further evidence for the arthritogenic potential of complexed IgE, especially in the initiation of arthritis through activation of mast cells.

Concanavalin A-induced activation of hamster mast cells: morphological changes and histamine secretion.

Peritoneal mast cells of Syrian hamsters release histamine to the action of concanavalin A (Con A) in dose-dependent fashion. The rate of release was very rapid in the first seconds of cell activation and completed in 60 s after the challenge. Morphological changes concomitant to the lectin treatment, followed by electron microscopy, show that early signs of exocytosis are seen after 10 s. The process starts in peripherally located granules which swell, have a decreased density and form pores by fusion of the cellular membrane and the perigranular membranes. Then it spreads toward the cell interior by fusion of granules and forming intracytoplasmic cavities. Some extruded granules are also observed. Preincubation of lectin with rat IgE or with rat serum induced an inhibition of its histamine releasing action. Immunization increased the Con A-induced histamine release in young but not in older hamsters. An IgE-mediated mechanism is suggested for the parallel ultrastructural changes and histamine release effects induced by Con A on the hamster mast cell.

Kinetics of ligand binding to the type 1 Fc.epsilon. receptor on mast cells

Rates of association and dissociation of several specific monovalent ligands to and from the type I Fc epsilon receptor (Fc epsilon RI) were measured on live mucosal type mast cells of the rat line RBL-2H3. The ligands employed were a monoclonal murine IgE and Fab fragments prepared from three different, Fc epsilon RI-specific monoclonal IgG class antibodies. These monoclonals (designated H10, J17, and F4) were shown previously to trigger mediator secretion by RBL-2H3 mast cells upon binding to and dimerization of the Fc epsilon RI. Analysis of the kinetics shows that the minimal mechanism to which all data can be fitted involves two consecutive steps: namely, ligand binding to a low-affinity state of the receptor, followed by a conformational transition into a second, higher affinity state h of the receptor-ligand complex. These results resolve the recently noted discrepancy between the affinity of IgE binding to the Fc epsilon RI as determined by means of binding equilibrium measurements [Ortega et al. (1988) EMBO J. 7, 4101] and the respective parameter derived from the ratio of the rate constant of rat IgE dissociation and the initial rate of rat IgE association [Wank et al. (1983) Biochemistry 22, 954]. The probability of undergoing the conformational transition differs for the four different Fc epsilon RI-ligand complexes: while binding of Fab-H10 and IgE favors the h state, binding of Fab-J17 and Fab-F4 preferentially maintains the low-affinity 1 state (at 25 degrees C). The temperature dependence of the ligand interaction kinetics with the Fc epsilon RI shows that the activation barrier for ligand association is determined by positive enthalpic and entropic contributions. The activation barrier of the 1----h transition, however, has negative enthalpic contributions counteracted by a decrease in activation entropy. The h----1 transition encounters a barrier that is predominantly entropic and similar for all ligands employed, thus suggesting that the Fc epsilon RI undergoes a similar conformational transition upon binding any of the ligands.

Serum IgE levels in rats infected with Paragonimus westermani

Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20-25 metacercariae of P. westermani from Cambaroides similis. Before and after infection (1, 2, 3, 4, 6, 8 weeks) of P. westermani, the blood was collected from the retro-orbital venous plexus of rats and kept serum at -70 degrees C. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0.18 +/- 0.042 at 2 weeks, 0.28 +/- 0.151 at 4 weeks and 0.43 +/- 0.055 at 8 weeks after infection. The absorbances of non-infected rats ranged 0.07 +/- 0.021-0.12 +/- 0.025. 2. Specific IgG values were slightly increased at 3 weeks (0.20 +/- 0.032) and gradually increased up to 8 weeks (0.31 +/- 0.067) after infection. The absorbances of non-infected rats ranged 0.11 +/- 0.035-0.18 +/- 0.019. The present results suggested that P. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.

Role of CD8 T Cells in Rat IgE Responses

The role of the cytokines interleukin-4 and interferon-gamma in the regulation of IgE responses in the mouse and man have focused on the role of CD4 T cells. In the rat, antigen-specific CD8 T cells, generated following inhalation of antigen, have been shown to be capable of suppressing IgE responses. Repeated intraperitoneal injections of 1 ng ricin and 1 microgram antigen established a long-lived IgE response in both low- and high-IgE responder rat strains (Wistar and Brown Norway). The duration of the IgE antibody response was 204 and 248 days, respectively. Total IgE levels rose from 30 +/- 20 to 39,000 +/- 7,500 ng/ml in the Wistar rat and from 120 +/- 100 to 47,000 +/- 8,000 ng/ml in the Brown Norway rat. An even greater (10(4)-fold) increase was seen in antigen-specific IgE antibody levels. Ricin alone had no effect and concomitant or prior stimulation with antigen was required. The proportion of CD4+ and CD8+ cells present in the spleen at the peak of the IgE response was markedly increased compared with animals given ricin or antigen alone. Furthermore, CD8 T cells were approximately 100 times more sensitive to ricin than CD4 T cells. These data suggest that enhancement of IgE responses in ricin-treated animals results from the selective deletion of T cells which suppress IgE and are of the CD8 phenotype.

A model for the solution conformation of rat IgE

Conference Article| October 01 1990 A model for the solution conformation of rat IgE KENNETH G. DAVIS; KENNETH G. DAVIS *Department of Applied Biochemistry and Food Science, Nottingham University Agricultural School, Sutton Bonington, Loughborough LE12 5RD, U.K. Search for other works by this author on: This Site PubMed Google Scholar MARTIN GLENNIE; MARTIN GLENNIE †Tenovus Research Laboratory, Southampton General Hospital, Southampton SO9 4XY, U.K. Search for other works by this author on: This Site PubMed Google Scholar STEPHEN E. HARDING; STEPHEN E. HARDING *Department of Applied Biochemistry and Food Science, Nottingham University Agricultural School, Sutton Bonington, Loughborough LE12 5RD, U.K. Search for other works by this author on: This Site PubMed Google Scholar DENNIS R. BURTON DENNIS R. BURTON ‡Krebs Institute of Biomolecular Science, Department of Molecular Biology and Biotechnology, Sheffield University, Western Bank, Sheffield S10 2TN, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1990) 18 (5): 935–936. https://doi.org/10.1042/bst0180935 Article history Received: March 07 1990 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation KENNETH G. DAVIS, MARTIN GLENNIE, STEPHEN E. HARDING, DENNIS R. BURTON; A model for the solution conformation of rat IgE. Biochem Soc Trans 1 October 1990; 18 (5): 935–936. doi: https://doi.org/10.1042/bst0180935 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1990 Biochemical Society1990 Article PDF first page preview Close Modal You do not currently have access to this content.

Production of Tumor Necrosis Factor with IgE Receptor Triggering from Sensitized Lung Tissue

Mast cells and basophils have been known to play a central role in allergic inflammation through the release of chemical mediators by cross-linkage of IgE receptors. The IgE receptor triggering and calcium ionophore A23187 have also been shown to induce gene expression and production of tumor necrosis factor (TNF) by rat basophilic leukemia cells. In the present study, we examined whether IgE receptor triggering could induce gene expression and production of TNF in rat lung tissue. The lung tissue released not only histamine but also cytotoxic activity on L929 cells 2 and 4 h after incubation with dinitrophenyl conjugated to ovalbumin (DNP-OVA) following passive sensitization with anti-DNP monoclonal rat IgE antibody, whereas neither DNP-OVA nor anti-DNP IgE antibody could induce the cytotoxic activity when used solely. Calcium ionophore A23187 also could induce both histamine release and cytotoxic activity. These activities induced by IgE receptor triggering, A23187, and lipopolysaccharide were completely neutralized by preincubation with anti-mouse TNF-rabbit serum, but not with normal rabbit serum. Northern blot analysis using cDNA probe of mouse TNF demonstrated expression of TNF gene as early as 2 h after IgE receptor triggering. These data demonstrating that IgE receptor triggering induced gene expression and production of TNF in lung tissue suggest the participation of TNF in the pathogenesis of late asthmatic response through its biologic activities such as the attraction and activation of neutrophils and eosinophils.

The effects of the protein kinase C inhibitors staurosporine and H7 on the IgE dependent mediator release from RBL 2H3 cells

RBL 2H3 cells, a model for mast cell function, sensitized with rat IgE, released histamine and peptidoleukotrienes (LT) in response to rabbit anti-rat IgE in a concentration-dependent manner. The calcium ionophore, A23187 also stimulated the release of both mediators but to a greater extent. The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) failed to influence mediator release when added alone, but when added with either A23187 or anti-IgE, TPA significantly enhanced the release of both histamine and LT. The effects of anti-IgE, TPA and A23187 were completely inhibited by prior addition of the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) but not by N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide dihydrochloride (HA1004), a compound which has similar potency to H7 as an inhibitor of some protein kinases but is less potent as a protein kinase C inhibitor. Although other explanations are possible, these results support the hypothesis that the release of histamine and leukotrienes from RBL 2H3 cells resulting from the cross bridging of the IgE receptors, is dependent on activation of protein kinase C.

Molecular structure and expression of the murine lymphocyte low-affinity receptor for IgE (Fc epsilon RII).

The cDNA encoding the murine low-affinity receptor for IgE (Fc epsilon RII) has been isolated from a cDNA library prepared from B cells activated with lipopolysaccharide and interleukin 4. It encodes a 37-kDa protein of 331 amino acids with two potential N-linked glycosylation sites. Analogous to its human counterpart, there is no signal sequence and the putative transmembrane region is close to the amino terminus, indicating an inverse membrane orientation with the carboxyl terminus at the cell exterior. The predicted murine Fc epsilon RII amino acid sequence demonstrates a 57% identity with its human counterpart. The murine sequence has an additional internal repeat motif of 21 amino acids giving four repeats as compared to three in the human sequence. Furthermore, the murine Fc epsilon RII is truncated at the carboxyl terminus and the Arg-Gly-Asp sequence, a common recognition site of integrin receptors, which is found in the reverse configuration in human Fc epsilon RII, is missing. B cells activated with interleukin 4 and lipopolysaccharide have an increased amount of Fc epsilon RII mRNA as compared with resting or lipopolysaccharide-stimulated B cells. Con A-activated normal T cells, the TH-2 cell line D10, as well as the macrophage cell line J774 have no detectable Fc epsilon RII mRNA. Expression analysis using transiently transfected COS cells revealed that recombinant murine Fc epsilon RII binds anti-Fc epsilon RII as well as mouse and rat IgE but does not bind human IgE or mouse IgG. Fc epsilon RII expressed in COS cells has a molecular mass of 45 kDa whereas the Fc epsilon RII from B-cell lines is a 49-kDa protein.