Membrane-bound IgE Research Articles

THE BASOPHIL ACTIVATION TEST IN IMMEDIATE DRUG ALLERGY

Diagnosis of drug allergy is not always straight forward for several reasons. First, a broad spectrum of drugs can elicit various immune-mediated diseases with distinct pathomechanism, secondly, although exact epitope identification is not mandatory for clinical diagnosis, the epitope that causes the reaction is frequently unknown, thirdly in vitro or in vivo test results might not be predictive of a clinical situation, and finally the gold standard or reference test for diagnosis, the drug challenge, is a complicated and sometimes dangerous endeavour.Upon challenge with specific allergens that cross-link membrane-bound IgE antibodies, basophils upregulate the expression of different activation markers such as CD63 and CD203c. These immunophenotypic alterations can be detected on a single-cell basis by multicolour flow cytometry using specific monoclonal antibodies in the basophil activation test (BAT).This review intends to summarise our current experience with the BAT in the diagnostic management of immediate-type allergy to drugs and related compounds that are generally (but not always) mediated by drug-specific IgE antibodies.

The immunoglobulin tail tyrosine of membrane-bound IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched memory B cells

Improved antibody responses by class-switched memory B cells require enhanced signaling from their antigen receptor (BCR) in a coreceptor-independent manner. However, all BCR classes on newly generated and antigen-experienced B cells utilize the canonical Igα/Igβ subunit for signaling via the immunoreceptor tyrosine-based activation motif (ITAM) in their cytoplasmic domains. We have now identified the signal amplification mechanism of the activated IgG- and IgE-BCR on class-switched B cells. An evolutionary conserved tyrosine residue in the cytoplasmic segments of membrane-bound IgG and IgE heavy chains, named Immunoglobulin Tail Tyrosine (ITT), becomes phosphorylated and recruits the adaptor protein Grb2 in order to prolong activation of protein kinases and sustain the generation of second messengers. Exchange of the ITT for phenylalanine phenocopies the reduced signaling profile of the IgM-BCR expressed on naive B cells. Hence membrane-bound IgG and IgE not only recognize antigen but also exert BCR-intrinsic costimulation to render memory B cells less dependent on T cell help for activation. Moreover, our finding of a signaling competent phospho-ITT confutes the paradigm of BCR tyrosine phosphorylation being confined to ITAM-containing subunits.

Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR

Truncation of the cytoplasmic tail of membrane-bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric epsilon-gamma1 BCR, consisting of the extracellular domains of the epsilon gene and the transmembrane and cytoplasmic domains of the gamma1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the "gamma1-mediated signalling" of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE-secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE-antibody secreting cells with "gamma1-signalling history" migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE-antibody secreting cells with WT "epsilon-signalling history". We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long-lived plasma cell pool than IgG1 plasmablasts.

Membrane Bound IgE: The Key Receptor to Restrict High IgE Levels

The membrane form of immunoglobulins (mIg) is expressed on the surface of B lymphocytes from a very early developmental stage in the bone marrow (pre B cell) until the cell finally differentiates into a plasma cell. mIgs associate with other transmembrane proteins to form the B cell antigen receptor complex (BCR). Numerous studies have confirmed that signaling through the BCR not only steers the B cell through development, but also ensures its survival in the periph- ery as a fully matured cell. During development in the bone marrow, mIg is restricted to the IgM isotype, but as soon as the B cell leaves the bone marrow to populate peripheral lymphoid organs like spleen, lymph nodes, or intestinal mucosal tissue, it starts to express IgD and in later stages of maturation IgG, IgE and IgA. Engagement of the B cell receptor trig- gers signals that control affinity maturation, memory induction, differentiation and other physiological processes in B cells. We showed that truncation of the cytoplasmic tail of mIgE in vivo results in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells, and abrogation of specific secondary responses correlating with a defect in the se- lection of high-affinity antibodies during the germinal centre reaction. We conclude that the BCR is necessary at all times during antibody responses not only for the maturation process, but also for the expansion of antigen specific B cells. As a result of these findings it is obvious that the IgE antigen receptor could be a useful target molecule for therapeutic inter- vention.

Targeting the Extracellular Membrane-Proximal Domain of Membrane-Bound IgE by Passive Immunization Blocks IgE Synthesis In Vivo

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.

HS1-Associated Protein X-1 Interacts with Membrane-Bound IgE: Impact on Receptor-Mediated Internalization

Engagement of the BCR triggers signals that control affinity maturation, memory induction, differentiation, and various other physiological processes in B cells. In previous work, we showed that truncation of the cytoplasmic tail of membrane-bound Ig (mIg)E in vivo resulted in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells, and the abrogation of specific secondary responses correlating with a defect in the selection of high-affinity Abs during the germinal center reaction. We concluded that the Ag receptor is necessary at all times during Ab responses not only for the maturation process, but also for the expansion of Ag-specific B cells. Based on these results, we asked whether the cytoplasmic tail of mIgE, or specific proteins binding the cytoplasmic tail in vivo commit a signal transduction accompanying the B cell along its differentiation process. In this study, we present the identification of HS1-associated protein X-1 as a novel protein interacting with the cytoplasmic tail of mIgE. ELISA, surface plasmon resonance analysis, and coimmunoprecipitation experiments confirmed the specific interaction in vitro. In functional assays, we clearly showed that HS1-associated protein X-1 expression levels influence the efficiency of BCR-mediated Ag internalization.

Occurrence of IgE in foals: Evidence for transfer of maternal IgE by the colostrum and late onset of endogenous IgE production in the horse

IgE is the key antibody involved in type I allergies. Allergen mediated crosslinking of IgE bound to high affinity Fcɛ-receptors on mast cells and basophils stimulates cellular degranulation and release of inflammatory mediators and cytokines. In this report, we demonstrate that IgE antibodies can be transferred from the mother to offspring in horses via the colostrum. We found a clear correlation between the IgE concentration in colostrum and the total IgE concentration in foal sera on day 2 after birth ( r sp = 0.83). Maternal IgE was detected in foal sera by ELISA and on peripheral blood leukocytes of foals by flow cytometry. Both serum and cell membrane-bound IgE were undetectable in newborn foals before colostrum uptake and peaked on days 2–5 after birth. Cell-bound IgE became undetectable at 2 months after birth. Serum IgE disappeared from the circulation within the first 3–4 months of age. These kinetics suggest that the IgE antibodies which are detectable in foals during the first 4 months after birth are of maternal origin only. The endogenous IgE production was found to begin at 9–11 months of age, when IgE could be detected on peripheral blood leukocytes and in foal sera again. After 18 months of life, the total IgE concentrations in foal sera were comparable to those detected in their dams. The late onset of endogenous IgE production offers an explanation for observations that IgE mediated allergies are generally not observed in horses before puberty. The roles of the passively transferred maternal IgE in newborn foals are not yet known, but could be manifold, ranging from passive immunity and induction of immunoregulatory functions to determinative influences of maternal IgE on the antibody repertoire in the offspring.

Studies on the regulation of IgE expression by the use of “knock in” mice

Rationale The expression of IgE is tightly regulated on different levels. Preliminary data from our laboratories suggest a regulation based on the expression of the membrane mound IgE. Methods A step forward in the analysis of this tight regulation was done with the construction of “knock-in” mice with composite and/or chimeric poly(A) sites influencing the post-transcriptional regulation. Results Expression of mRNA for the secreted form of IgE is favored over that for the membrane form in resting B cells. The ratio between the amount of secreted vs. the amount of membrane-bound Ig that is produced by a single cell is determined by the efficiency with which the internal or external polyadenylation sites are used and by the stability of the ensuing mRNA's. Conclusions Factors that influence the alternative polyadenylation are largely unknown. However, because expression of mIgE is essential for recruitment of IgE-producing cells in the immune response, clarification of this issue is of great importance. So far, IgE regulation is evident on the level of DNA recombination (switch), transcription and RNA processing. It is not inconceivably that also post-translational and post-transcriptional processes may influence the expression of membrane-bound IgE.

Monoclonal Antibodies against the CεmX Domain of Human Membrane-Bound IgE and Their Potential Use for Targeting IgE-Expressing B Cells

Background: IgE mediates immediate-type hypersensitivity reactions responsible for various allergic symptoms. It is secreted by IgE-producing plasma cells, which differentiate from B cells expressing membrane-bound IgE (mIgE) on their surface. The Ε-chain of human mIgE contains a membrane-anchoring peptide and an extra 52-amino-acid (a.a.)-long domain (referred to as CΕmX) between the membrane anchor and the CH4 domain. Objective: The study was designed to evaluate the effects of CΕmX-specific monoclonal antibodies (mAbs) to target IgE-expressing B cells and decrease IgE production. Methods: A CΕmX-containing IgG1.Fc fusion protein was produced in CHO cells and used to immunize mice; five hybridoma clones secreting CΕmX-specific mAbs were obtained. Results: Characterization of the mAbs using ELISA, immunoprecipitation, and immunoblotting methods showed that they could bind to both native and denatured forms of CΕmX. The mAbs exhibited mutual inhibition of binding to mIgE. Epitope mapping using synthetic peptides revealed that all five mAbs recognize the same epitope, RADWPGPP, located near the C-terminus of CΕmX . Binding of one of the mAbs to mIgE on SKO-007 cells induced the cross-linking of mIgE molecules on the cell surface, resulting in their patching and capping. In vitro functional analysis revealed that mAbs are able to cause complement-mediated cytotoxicity on transfectants expressing the Fc portion of mIgE. Conclusion: We have prepared several human mIgE-specific mAbs. The potential of the mAbs on targeting mIgE+ B cells was demonstrated by CDC analysis.

Human eosinophils and human high affinity IgE receptor transgenic mouse eosinophils express low levels of high affinity IgE receptor, but release IL-10 upon receptor activation.

FcepsilonRI expressed by human eosinophils is involved in IgE-mediated cytotoxicity reactions toward the parasite Schistosoma mansoni in vitro. However, because receptor expression is low on these cells, its functional role is still controversial. In this study, we have measured surface and intracellular expression of FcepsilonRI by blood eosinophils from hypereosinophilic patients and normal donors. The number of unoccupied receptors corresponded to approximately 4,500 Ab binding sites per cell, whereas 50,000 Ab binding sites per cell were detected intracellularly. Eosinophils from patients displayed significantly more unoccupied receptors than cells from normal donors. This number correlated to both serum IgE concentrations and to membrane-bound IgE. The lack of FcepsilonRI expression by mouse eosinophils has hampered further studies. To overcome this fact and experimentally confirm our findings on human eosinophils, we engineered IL-5 x hFcepsilonRIalpha double-transgenic mice, whose bone marrow, blood, spleen, and peritoneal eosinophils expressed FcepsilonRI levels similar to levels of human eosinophils, after 4 days culture with IgE in the presence of IL-5. Both human and mouse eosinophils were able to secrete IL-10 upon FcepsilonRI engagement. Thus, comparative analysis of cells from patients and from a relevant animal model allowed us to clearly demonstrate that FcepsilonRI-mediated eosinophil activation leads to IL-10 secretion. Through FcepsilonRI expression, these cells are able to contribute to both the regulation of the immune response and to its effector mechanisms.

Allergic rhinitis update: Epidemiology and natural history.

Allergic rhinitis is the most common chronic condition, with an estimated prevalence in the United States of 5-22%, which increases from infancy, peaks in childhood and adolescence, and decreases in the elderly. As a major cause of morbidity, absenteeism, and restricted activity in both children and adults, allergic rhinitis, similar to asthma, appears to be increasing with time. Allergic rhinitis is commonly defined as seasonal or perennial, depending upon whether symptoms are manifested at defined yearly intervals or throughout the year, respectively. While trees, grasses, weeds, and molds are the most frequent causes of seasonal allergic rhinitis, dust mites and molds are the major contributors to perennial allergic rhinitis. The pathogenesis of allergic rhinitis is based upon interactions of allergen with membrane-bound allergen-specific IgE on the surface of mediator cells, i.e., basophils and mast cells, leading to the release of allergic mediators (both performed and newly synthesized) including histamine, leukotrienes, and eosinophil cationic protein (ECP). These are responsible for both immediate allergic responses characteristic of acute allergic rhinitis and the late inflammatory reactions responsible for chronic allergic rhinitis. The evaluation of rhinitis should include a detailed patient history, a careful physical examination, and appropriate diagnostic tests including skin prick tests or serum assays for allergen-specific IgE. Seasonal allergic rhinitis is readily distinguished from perennial allergic rhinitis by history and confirmed by positive skin tests to causative aeroallergens. It is important to differentiate seasonal rhinitis from non-allergic disorders including infectious rhinitis, structural or anatomic problems such as nasal polyps or septal deviation, rhinitis medicamentosa (due to the overuse of topical vasoconstrictors), hormonal rhinopathy (e.g., pregnancy, hypothyroidism), non-allergic vasomotor rhinopathy, non-allergic inflammatory rhinitis with eosinophils (NARES), or rarely, a neoplasm. A knowledge of the epidemiologic and clinical presentation of allergic rhinitis together with these pathophysiologic mechanisms is essential for a modern-day diagnostic and therapeutic approach to the patient who suffers from allergic rhinitis.

High-Order Autocorrelation with Imaging Fluorescence Correlation Spectroscopy: Application to IgE on Supported Planar Membranes

The use of high-order autocorrelation with imaging fluorescence correlation spectroscopy is described. Fluorescently labeled, antitrinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The IgE-coated membranes were illuminated with a laser beam that was totally internally reflected at the substrate/solution interface. The evanescently excited fluorescence arising from the membrane-bound IgE was measured with a CCD camera. The images were corrected for background and for the elliptically Gaussian spatial dependence of the evanescent excitation intensity. A series of high-order pixel-to-pixel spatial fluorescence fluctuation autocorrelation functions was calculated from the images. The autocorrelation functions generated multiple independent parameters which were used to characterize the nonuniform spatial distributions of the membrane-bound IgE. These parameters varied with the IgE density and also changed significantly when the IgE-coated membranes were further treated with unlabeled, polyclonal anti-IgE. The high-order autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Images of fluorescent beads were used to demonstrate the principles and the methods.

Responsiveness of human skin mast cells to repeated activation: an in vitro study.

To assess human mast-cell (MC) behavior after repetitive activation, we cocultured human foreskin MC (SMC) with human foreskin fibroblasts (F). Under these conditions, we have previously demonstrated that SMC keep their viability and functional activity for up to 8 days. SMC were presensitized with atopic serum and repeatedly activated by consecutively increasing concentrations of anti-IgE antibodies (alpha-IgE, 0.0002-0.1%). This treatment, which mimics the "rush desensitization" procedure, led to complete SMC unresponsiveness to activation by alpha-IgE at optimal concentrations, as evaluated by histamine release. However, presensitization of SMC with IgE antibodies before exposure to alpha-IgE restored their sensitivity to this stimulus. These data indicate that desensitization was probably due to lack of membrane-bound IgE rather than to downregulation of intracellular mechanisms. In fact, SMC challenged by an optimal concentration of alpha-IgE could release histamine upon a second activation by 2 h after the first activation, if the cells had been presensitized before the second challenge. SMC incubation with increasing concentrations of compound 48/80 (0.2-10 micrograms/ml) led to MC unresponsiveness to an optimal concentration of this stimulus. Furthermore, SMC activated by an optimal concentration of compound 48/80 and rechallenged with the same agent were insensitive to the second activation for at least 24 h. In summary, we have shown that it is possible to induce "desensitization" in SMC to both IgE-dependent and IgE-independent stimuli by incubating the cultures with consecutively increasing concentrations of the activator. SMC can release histamine when reactivated with alpha-IgE antibodies after presensitization by 2 h after the first challenge, while they reacquire their susceptibility to reactivation with compound 48/80 in only 2-3 days.

5428133 Chimeric anti-human igemonoclonal antibody which binds to secreted IgE and membrane-bound IgE expressed by IgE-expressing B cells but notto IgE bound to fc receptors on basophils : Chang Tse-wen Houston, TX, United States Assigned to Tanox Biosystems Inc

5428133 Chimeric anti-human igemonoclonal antibody which binds to secreted IgE and membrane-bound IgE expressed by IgE-expressing B cells but notto IgE bound to fc receptors on basophils : Chang Tse-wen Houston, TX, United States Assigned to Tanox Biosystems Inc

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.

Characterization and expression of alternatively spliced IgE heavy chain transcripts produced by peripheral blood lymphocytes.

We have investigated the IgE heavy chain isoforms produced in vivo by analyzing the epsilon mRNA species present in unstimulated PBL and expressing them individually in a myeloma cell line. Seven epsilon mRNA species were identified by using reverse transcription-PCR, cloning, and sequencing analysis. These species included the classical secreted (epsilon CH4-S) and membrane-bound (epsilon CH4-M1'-M2) IgE and five alternatively spliced epsilon transcripts. At the protein level, the five alternatively spliced epsilon transcripts (epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, epsilon CH4'-1-M2, and epsilon CH3-13-CH4) corresponded to four epsilon heavy chain isoforms, in which various parts of the CH4 domain were replaced by new stretches of amino acids at the carboxyl termini. The same epsilon mRNA species also were present in the IgE producing myeloma cell line U266. However, except for the classical membrane and secreted IgE, the corresponding proteins could not be identified. To further characterize the epsilon CH4-S, epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, and epsilon CH4-M1'-M2 species, we expressed them as chimeric mouse/human anti-4-hydroxy-5-iodo-3-nitrophenacetyl Abs in a mouse myeloma cell line. Only the classical secreted and membrane isoforms were found to be secreted or expressed on the cell surface, respectively, and the other forms were retained within the cells and degraded. These data suggest that some of the epsilon mRNA isoforms produced by PBL are aberrantly spliced mRNAs, the protein products of which are eliminated by post-translational events.

The Absence of Correlation among IL-3, IL-8 and Histamine-Releasing Factors with Oral Prausnitz–Küstner Reaction in Food Allergy

The oral Prausnitz-Küstner (oP-K) reaction is closely associated with severe IgE-mediated allergy to food, but its mechanisms are not well defined. Our objectives were to determine the serum factor(s) involved in the oP-K reaction. The sera were obtained from 12 patients with food allergies (9 systemic urticaria/angioedema, 3 atopic dermatitis). The serum concentrations of IL-3 and IL-8 were measured by means of an enzyme-linked immunosorbent assay (ELISA). To evaluate histamine-releasing serum factor (HRSF) activity, basophils were isolated from nonallergic peripheral blood, stripped of membrane-bound IgE with lactic acid, and then passively sensitized with sera. Histamine release from these cells was determined in the presence or absence of an anti-IgE antibody or an anti-Fc∈ RI antibody. Six of the 12 patients showed a positive oP-K reaction. The results as to serum concentrations of IL-3 and IL-8 and the activity of HRSF from patients with a positive oP-K reaction were not different statistically from those for patients with a negative oP-K one. IL-3, IL-8, and HRSF are not crucial to development of the oP-K reaction in patients with food allergies. The heterogeneity of IgE protein may explain the difference between a positive oP-K reaction and a negative oP-K one.

Origin and fate of IgE-bearing lymphocytes. II. Gut-associated lymphoid tissue as sites of first appearance of IgE-bearing B lymphocytes and hapten-specific IgE antibody-forming cells in mice immunized with benzylpenicilloyl-keyhole limpet hemocyanin by various routes: relation to asialo GM1 ganglioside+ cells and IgE/CD23 immune complexes.

The organs in which B cells bearing membrane-bound IgE (sIgE+) and benzylpenicilloyl (BPO)-specific IgE antibody-forming cells (AFC) first appeared were determined in BALB/c mice given BPO-keyhole limpet hemocyanin (10 micrograms) in aluminum hydroxide by various routes (i.p, gavage, s.c., i.v., or i.m.). In mice immunized by the i.p. route, the numbers and location of sIgE+ B cells and asialo GM1 ganglioside (AsGm1+) cells, the location of IgE/CD23 immune complexes, and the numbers of BPO-specific IgE AFC in lymphoid organs were determined. With all routes of immunization, no sIgE+ B cells or BPO-specific IgE AFC were ever detected in any organ before day 8. On day 8, with the s.c., i.v., or i.m. routes, sIgE+ B cells and IgE AFC appeared exclusively in Peyer's patches (PP); with the i.p. or gavage routes, sIgE+ B cells simultaneously appeared in both PP and mesenteric lymph nodes, whereas IgE AFC appeared only in PP. In mice immunized by the i.p. route, IgE/CD23 immune complexes and strikingly increased numbers of AsGm1+ cells transiently appeared only in PP after the appearance and preceding the "disappearance" of the sIgE+ B cells and IgE AFC. The data suggest that specific IgE responses originate in gut-associated lymphoid tissue and appear later in spleen. The data also associate the appearance of IgE/CD23 immune complexes and AsGm1+ cells with the "disappearance" of sIgE+ B cells and IgE AFC from PP.

Measurement of histamine-releasing factor activity in individual nasal washings: Relationship with atopy, basdphil response, and membrane-bound IgE

We collected individual pools of nasal washings (NWs) from 15 allergic and 15 nonallergic subjects to determine histamine-releasing factor (HRF) activity and to ascertain the relationship of these cytokines with atopic status, basophil releasability, and cell membrane-bound IgE. NWs were concentrated, dialyzed, and assayed with basophils from a single donor. Samples from 12 of 15 allergic subjects and from all the nonallergic subjects revealed ⪰15% histamine release (HR), 33.5% ± 21.3% (mean ± SD) and 38.6% ± 19.6%, respectively ( p > 0.05). When we assayed the same samples with autologous basophils, the allergic group demonstrated higher HR than the nonallergic group (31.9% ± 19.7% versus 4.8% ± 4.3%; p < 0.001). A standard lot of mononuclear cell-derived HRFs was also screened with basophils from both groups. Means for HR from basophils of allergic and nonallergic subjects were 51.9% ± 16.7% versus 26.3% ± 8.2%, respectively ( p < 0.001). Pretreatment of basophils with lactic acid led to abrogation of sensitivity to HRF. Acid-stripped cells incubated with sera from patients with asthma regained their capacity to release histamine. We found that HRF activity can be detected in NWs of most donors, and there is no difference among allergic and nonallergic subjects. Our results suggest that the capacity of these cytokines to induce HR depends on several factors: atopic status, basophil releasability, and membrane-bound IgE.

A new isoform of human membrane-bound IgE.

The epsilon-chain of membrane-bound IgE on the surface of B lymphocytes is known to contain a membrane-anchoring peptide segment that is encoded by two membrane exons, me.1 and me.2. In analyzing pertinent segments in mRNA from human IgE-expressing B cells by using PCR methods and Northern blotting analyses, we have identified three species of mRNA of epsilon-chain with variations in the splicing of the membrane exons. The conventional species (m/s) contains the predicted me.1 and me.2; species m/1 harbors 156 extra nucleotides 5' of me.1 with unaltered reading frame; species s/t lacks me.1 and hence the segment encoding the hydrophobic transmembrane stretch and contains a shifted me.2 reading frame. Rabbit antibodies, which were prepared by immunization using a peptide of 36 amino acid residues representing an encoded segment unique to mRNA species m/l, could specifically bind to human IgE-expressing B cell lines and react with an epsilon-chain on Western immunoblots. These results indicate that there exists a previously unidentified isoform of human membrane-bound IgE.