Anti-human IgE Research Articles

Identification and Characterization of Major Allergens in Excretory/Secretory Products of the Worm Paragonimus ohirai

Allergens present in the excretory/secretory (ES) products of adult Paragonimus ohirai were biochemically identified. Immunoblot analysis using sera from P. ohirai-infected rats revealed only two allergens to be major proteins in the ES products, with apparent molecular masses (M_r) of 27 and 29 kD. As the ES products contained a high proportion of acidic and neutral cysteine proteinases, we examined whether or not the allergens and the cysteine proteinases were identical. The acidic and neutral cysteine proteinases were biochemically purified from the ES products and showed M_r of 27 and 29 kD, respectively. The two cysteine proteinases had almost identical N-terminal amino acid sequences and were reactive with specific IgE in sera from the infected rats. The allergenicity of the cysteine proteinases was confirmed by 48-hour homologous passive cutaneous anaphylaxis. Immunoblot and immunocapture assays using anti-human IgE monoclonal antibody showed that the proteinase allergens were reactive with specific IgE of patients with paragonimiasis westermani. Also, the cysteine proteinases were reactive with specific IgG of both the infected rats and the patients. Therefore the acidic and neutral cysteine proteinases prepared from the ES products of P. ohirai will be useful allergens and antigens for the immunodiagnosis of paragonimiasis.

IgG Anti-IgE Autoantibodies in Visceral Leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.

Role of protein kinase A in the inhibition of human mast cell histamine release by β-adrenergic receptor agonists

ABSTRACT Background Although β-adrenergic receptor agonists inhibit antigen-induced release of histamine, leukotrienes and prostaglandin D 2 from human lung fragments, dispersed human lung mast cells and human skin mast cells, subcellular mechanisms for the inhibition of histamine release by β-adrenergic receptor agonists are not well delineated. The aim of the present study was to investigate the inhibitory mechanisms of β-adrenergic receptor agonists for human mast cell histamine release using human cultured mast cells. Methods Human mast cells were obtained by culturing umbilical cord blood CD34 + cells in the presence of stem cell factor and interleukin-6. Cultured mast cells were sensitized with human myeloma IgE and stimulated with antihuman IgE. Results Stimulation of mast cells induced the elevation of intracellular cytosolic free Ca 2 + concentrations ([Ca 2 +] i ) and the translocation of protein kinase C (PKC) from the cytosol to the cell membrane, followed by the release of stored histamine. Isoproterenol, salbutamol and dibutyryl cAMP inhibited both the histamine release and PKC translocation, whereas they failed to affect the elevation of [Ca 2 +] i . H-89, a protein kinase A (PKA) inhibitor, abrogated the inhibition. Conclusions The present results suggest that PKA activation induced by β-adrenergic receptor agonists plays a crucial role in inhibiting IgE-mediated histamine release from human cultured mast cells through suppressing PKC translocation.

A molecular model of type I allergy: Identification and characterization of a nonanaphylactic anti-human IgE antibody fragment that blocks the IgE-FcϵRI interaction and reacts with receptor-bound IgE

Background: The IgE-mediated activation of effector cells and antigen-presenting cells through the high-affinity receptor for IgE (FcϵRI) represents a key pathomechanism in type I allergy and many forms of asthma. Objective: We sought to establish an in vitro molecular model for the interaction of human FcϵRI, IgE, and the corresponding allergen and to identify monoclonal anti-human IgE antibodies with a therapeutic profile different from previously established anti-IgE antibodies. Methods: Human FcϵRI α chain, a human monoclonal allergen-specific IgE antibody (chimeric Bip 1), and the corresponding allergen, the major birch pollen allergen Bet v 1, were produced as recombinant proteins and analyzed by means of circular dichroism and native overlays, respectively. Using this molecular model, as well as negative stain immunoelectron microscopic analysis, and in vitro cultivated human basophils, we characterized mouse anti-human IgE antibodies. Results: We established a molecular model for the interaction of human IgE with FcϵRI. Using this molecular model, we identified a nonanaphylactic anti-human IgE antibody fragment (Fab12), which blocked the IgE-FcϵRI interaction and reacted with effector cell–bound IgE. Conclusion: Fab12 represents a candidate molecule for therapy of atopy and asthma because it can be used for the depletion of circulating IgE antibodies, as well as for the depletion of IgE-bearing cells. (J Allergy Clin Immunol 2001;108:409-16.)

A Comparison of FcεRI-Mediated RANTES Release from Human Platelets between Allergic Patients and Healthy Individuals

Background: Recently some studies have suggested that human platelets may play an important role in allergic inflammation through the high affinity IgE receptor (FcΕRI), the low affinity IgE receptor (FcΕRII/CD23) and the low affinity IgG receptor (FcγRIIA/CD32) expressed on the cell surface. We reported that human platelets via the FcΕRI induced the release of the chemical mediator serotonin and the chemokine RANTES (regulated upon activation, normal T expressed and presumably secreted), but the biological implication of human platelets in type I allergy has not yet been understood clearly. Methods: We compared the levels of RANTES release from platelets obtained from allergic patients and healthy individuals, stimulated with monoclonal antibody (Ab) to human FcΕRI α-chain, or human myeloma IgE and anti-human IgE Ab. Results: We confirmed that the level of RANTES release from platelets of allergic patients stimulated with human IgE and anti-human IgE was significantly higher than that of healthy individuals. Conclusions: We demonstrated that the surface expression levels of FcΕRI on the platelets from allergic patients and healthy individuals were not significantly different, but that the platelets of allergic patients were more activated by the IgE-FcΕRI pathway than those of healthy individuals. Taken together, these results suggest a novel and important role for human platelets in perpetuating allergic inflammation through the IgE and FcΕRI.

Envelope gp120 of HIV-1 binds to and activates human basophils

Envelope glycoprotein gp120 isolated from different clades of HIV-1 is a potent stimulus for IL-4 and IL-13 release from basophils purified from healthy donors seronegative for antibodies to HIV-1 and HIV-2. The expression of IL-4 mRNA, constitutively present in basophils, was increased after stimulation with gp120 and was inhibited by cyclosporin A and tacrolimus. A significant correlation was found between the maximum release of IL-4 induced by gp120 and by an antibody anti-human IgE. IgE removal from basophils by brief exposure to lactic acid abolished IL-4 release in response to both gp120 and anti-IgE. Preincubation of gp120 with three different human monoclonal IgM VH3+ but not with monoclonal IgM VH6+ inhibited gp120-induced secretion of IL-4 from basophils. Preincubation of basophils with synthetic peptides distant from the NH2 and COOH-termini of gp120MN resulted in the inhibition of gp120MN induced IL-4 release. These results indicate that gp120 acts as a viral immunoglobulin superantigen by interacting with the VH3 region of IgE and induces the expression and the release of IL-4 and IL-13 from human basophils.

Synergistic antiallergic activity of combined histamine H 1- and cysteinyl leukotriene 1-receptor blockade in human bronchus

Mast cell histamine (HA) and cysteinyl leukotrienes (CysLT) account for most of the early phase bronchospasm in asthma. However, activation of the smooth muscle CysLT 1-receptor plays a major role in asthmatic bronchospasms. CysLT-receptor antagonists or CysLT-synthesis inhibitors are efficacious in asthma but do not completely abolish asthmatic bronchospasms. A recent clinical study showed that combined antagonists loratadine (H 1) and zafirlukast (CysLT 1) were more effective against allergic bronchospasms than either drug alone. We examined the combined efficacy of H 1- and CysLT 1-receptor antagonists in allergic human bronchus. The H 1- and CysLT 1-receptor antagonists chlorpheniramine (CTM; 1 μM) and MK-571 (0.03 μM), were tested alone and in combination, against anti-human IgE antibody (Ab)-induced contractions of passively sensitized isolated human bronchus. Ab-induced allergic contractions were reduced 15% and 36% by CTM (1 μM) and MK-571 (0.03 μM), respectively. Combined CTM (1 μM) and MK-571 (0.03 μM) significantly inhibited the Ab response by 87%. Mechanistic investigations in isolated human bronchus and cultured human cord blood mast cells suggest that H 1- and CysLT-receptor interactions likely occur at the airway smooth muscle level. CTM and MK-571 synergistically inhibited human allergic bronchospasm in the present in vitro model. The mechanism underlying this synergistic activity requires further investigation.

Production of humanized antibody against human high-affinity IgE receptor in a serum-free culture of CHO cells, and purification of the Fab fragments.

We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (Fc epsilonRIalpha) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-Fc epsilonRIalpha antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 microg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.

Piezoelectric quartz crystal based label-free analysis for allergy disease

This work presents a piezoelectric (Pz) quartz crystal based label-free quantification of total IgE and allergen-specific IgE in human sera for allergy testing. An evaluation of the different brands of crystals was first initiated with respect to variability in mass sensitivity, frequency measurement reliability and stability, and surface roughness. Thereafter, for total IgE quantification, a direct assay format was adopted. By means of thioctic acid (TA) and coupling reagents, anti-human IgE antibodies were immobilized on AT-cut Pz crystals (10 MHz). The modified crystals could detect serum IgE directly corresponding to a downward frequency shift. The results showed that silver-coated crystals as compared with their gold-coated counterparts provided approximately 1.5 times higher mass detection sensitivity for total IgE in the range of 5–300 IU/ml with a linear regression line, y=1.8957 x+1.5603, R 2=0.995. For the detection of allergen-specific IgE, a sandwiched assay format was used. As the allergen-modified sensor surface captured various classes of associated antibodies (IgE, IgG, etc) and interfering serum proteins as well, the initial frequency shift downwards caused by sera sample incubation would not be proportional to specific IgE levels. Thus, following sample incubation, a second incubation step with secondary anti-human IgE was added to recognize IgE from other bound substances. The frequency shift after secondary antibody binding reflected the amount of allergen-specific IgE proportionally. Compared with 10 MHz crystals, the 20 MHz counterparts provided approximately four times higher mass detection sensitivity for allergen specific IgE in the range of 0.15–17.5 IU/ml with a linear regression line, y=50.525 x+107.777, R 2=0.954. Total IgE and allergen specific IgE assay results of real patients’ sera using the Pz sensors agreed well with those obtained by commercially available test kits with correlation coefficient 0.96–0.98. The possibility of regenerating the quartz crystals for further re-use was also dealt with.

Detection of Multiple Allergen-specific IgEs on Microarrays by Immunoassay with Rolling Circle Amplification

First described in 1967, the radio allergo sorbent test (RAST) has been the standard technique for measuring allergen-specific IgE antibodies in serum (1). An updated version of the RAST test, termed CAP (Pharmacia), has been introduced (2). In clinical practice, CAP results must be interpreted with care. The diagnostic performance of CAP varies in an allergen-specific manner, and CAP scores do not always correlate with clinical severity (3)(4). CAP sensitivity, specificity, and positive predictive values agree well with skin prick tests (SPTs) for house dust mites and grasses, but poorly with tests for cat dander and peanuts (5). Microarray technology potentially offers advantages in diagnostic applications such as allergy testing because the amount of reagent required, and thus the cost per assay, is greatly reduced (6). This approach has been difficult to reduce to practice, however, because the extremely small volumes (∼0.5–5 nL) of sample used to create spots on these microarrays require extremely sensitive methods of analyte detection (7). We have used rolling circle amplification (RCA) (8) for the detection of antibody bound to antigen (9). In this “immunoRCA”, the 5′ end of a RCA primer is attached to an antibody; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of circular DNA sequence copies that remain attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes. ImmunoRCA, therefore, represents a novel approach for signal amplification of antibody-antigen recognition events on microarrays. ImmunoRCA can detect IgE in a format using high-density microarrays of anti-human IgE printed on glass slides by a pin-tool type microarraying robot (9). Here, we describe the production of microarrays of multiple allergens and demonstrate the utility of these microarrays in combination with immunoRCA to simultaneously detect …

NOVEL IMMUNOTHERAPEUTIC APPROACHES FOR THE TREATMENT OF ALLERGIC DISEASES

Advances in the knowledge of the cellular and molecular basis of immunity have led to an enhanced thorough understanding of the immunologic features that characterize an allergic response. A hallmark of an allergic response is a persistently elevated level of specific IgE antibodies to a variety of antigens, also referred to as allergens, to which the affected individual is regularly exposed by inhalation, ingestion, or contact with the skin. 48 Allergic sensitization involves processing of the antigen by an antigen-presenting cell (APC) and presentation, in association with a class II major histocompatibility complex (MHC) protein, to a T-cell receptor. Whereas dendritic cells (and macrophages) predominantly act as APCs in primary immunization, B cells may also participate in secondary immune responses to allergens. Activation of T cells requires the first signal from this trimolecular interaction and an additional costimulatory signal involving the ligation of B-7 on an APC (CD80/86) with CD28 or CTLA4 on a T cell. 27 When stimulated by antigens, helper T cells produce an array of specific cytokines, all of which have been designated as either T H 1 cytokines (interleukin [IL]-2, interferon [IFN]-γ, and tumor necrosis factor [TNF]-β) or T H 2 cytokines (IL-4, IL-5, IL-9, IL-10, and IL-13). The state of T-cell activation depends on several factors. 27,85 The first of these factors is the affinity of the interaction between the antigen and the T cell. The site of the antigen recognized by the T cell is termed the epitope. The affinity of epitope–T cell interaction is affected by the concentration of the antigen and the type of APC. 80 It also depends on the cytokine milieu of the T cell during antigen interaction. Thus, IFN-γ and IL-12 promote a T H 1-like response, whereas IL-4 promotes a T H 2-like response. 12 Additionally, host immune response genes may bias the overall immune responsiveness of an individual to favor a T H 1- or T H 2-like response. A T H 2-like cytokine profile is associated with the induction of IgE antibody (Ab) production in vitro and in vivo. 73 Specifically, IL-4 favors the development of T H 2-like cells from uncommitted T cells, and both IL-4 and IL-13 play a role in IgE antibody production. Manifestation of an allergic reaction depends on the specific IgE levels and the amount of exposure at the time of the reaction. Although an allergic condition is a risk factor for asthma, about 20% to 30% of asthmatics do not show positive skin tests to allergens. In general terms, asthma is defined as an inflammatory disease where not only lymphocytes, but also other cells, such as mast cells, basophils, eosinophils, and epithelial cells, play a role. Studies to date suggest that T H 2-like cytokines, such as IL-4 and IL-5, also play an important role in nonatopic asthma. 3,85 Developing immunotherapeutic approaches against allergic diseases, including asthma, may be pragmatically divided into two categories based on how soon they may make an inroad to the clinic, as shown in Figure 1. The first category includes therapies that are already in phase II/phase III clinical trials and are expected to be available in clinics within the next 1 to 3 years; such therapies include antihuman IgE antibodies and the soluble IL-4 receptor. The second category includes experimental approaches that are mainly at the stage of preclinical research in model systems and may proceed to clinical trials and the clinic within the next 5 to 10 years; these therapies include various allergen-specific and allergen-nonspecific approaches.

Mimicry of human IgE epitopes by anti-idiotypic antibodies

According to Jerne’s network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunisation, and confirm Jerne’s hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.

Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells.

Flavonoids have a variety of activities including anti-allergic activities, and are known to inhibit histamine release from human basophils and murine mast cells. The effects of luteolin, a flavone, on the immunoglobulin (Ig) E-mediated allergic mediator release from human cultured mast cells (HCMCs) were investigated and compared with those of baicalein and quercetin. HCMCs were sensitized with IgE, and then treated with flavonoids before challenge with antihuman IgE. The amount of released mediators was determined as was mobilization of intracellular Ca2+ concentration, protein kinase C (PKC) translocation and phosphorylation of intracellular proteins were detected after anti-IgE stimulation. Luteolin, baicalein and quercetin inhibited the release of histamine, leukotrienes (LTs), prostaglandin D2 (PGD2), and granulocyte macrophage-colony stimulating factor (GM-CSF) from HCMC in a concentration-dependent manner. Additionally, the three flavonoids inhibited A23187-induced histamine release. As concerns Ca2+ signalling, luteolin and quercetin inhibited Ca2+ influx strongly, although baicalein did slightly. With regard to PKC signalling, luteolin and quercetin inhibited PKC translocation and PKC activity strongly, although baicalein did slightly. The suppression of Ca2+ and PKC signallings might contribute to the inhibition of mediator release. The activation of extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinase (JNK), that were activated just before the release of LTs and PGD2 and GM-CSF mRNA expression in IgE-mediated signal transduction events, were clearly suppressed by luteolin and quercetin. In contrast, the flavonoids did not affect the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathway. These results indicate that luteolin is a potent inhibitor of human mast cell activation through the inhibition of Ca2+ influx and PKC activation.

Molecular basis of IgE cross-reactivity between human β-casein and bovine β-casein, a major allergen of milk

Twenty patients allergic to cow’s milk proteins and with high levels of specific IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human β-casein. Highly purified human and bovine β-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the five N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine β-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained specific IgE directed against human β-casein. Inhibition studies using native human and bovine β-caseins as well as bovine β-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in different parts of the molecule were shared by the two homologous proteins.

Immunosensors based on supported lipid membranes, protein films and liposomes modified by antibodies

The comparison of physical properties, sensitivity and reproducibility of various detection parameters of several newly developed affinity biosensors for determination of human IgE and herbicide 2,4-D is presented. The protein film based biosensors were composed of antibody (swine anti-human IgE (Q-SwaHU/IgE) or monoclonal antibody (MAb) against herbicide 2,4-D) attached to thin gold support through cysteamine or cysteamine–bovine serum albumin. The antigen (Ag)–antibody (Ab) interaction was detected by measurement of conductivity. The detection limit for human IgE was ∼1 nM, however, the sign of response depends on the method of antibody attachment to the gold support. This type of design and detection method was not appropriate for detecting small molecules, like 2,4-D. In the case of metal (stainless steel or gold) supported lipid films (s-BLM), the sensor was constructed by means of binding of avidin-modified antibody to s-BLM contained biotinylated phospholipids. Measurement of electrical capacitance, C, and elasticity modulus in direction perpendicular to the membrane plane, E ⊥, allowed to detect the Ag–Ab reaction, that resulted in decrease of C and increase of E ⊥. Most reproducible results have been obtained with lipid films supported on thin gold layers with detection limit of determination of 2,4-D∼1 μM. The best reproducibility and sensitivity (0.1–1 nM) have been obtained in liposome immunoassay. The Ag–Ab reaction was monitored by means of measurement the changes of ultrasound velocity in liposome suspension.

Self-Assembled Monolayer-Based Piezoelectric Crystal Immunosensor for the Quantification of Total Human Immunoglobulin E

This work presents a piezoelectric (Pz) immunosensor for the quantification of total human IgE in serum samples. The anti-human IgE is deposited on the surface of the 10 M Hz AT-cut gold coated crystal resonator by self-assembled technique, and serves as a receptor layer. The highly ordered self-assembled monolayers (SAMs) ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The fabricated Pz sensor can quantitatively detect human serum IgE in the range of 5–300 IU/ml with high precision (CV < 8%). A total of 28 patient serum samples are detected by the Pz sensor, and the results agree well with those given by two commercially provided test kits (Total IgE FAST Test, Pharmacia-CAP). The correlation coefficients are 0.94 between FAST and Pz sensor, and 0.90 between CAP and Pz sensor, respectively. After regeneration with urea and glycine buffer the coated crystal can be reused five times without appreciable loss of activity. Compared with conventional cross-linking methods, nonspecific binding caused by the SAM binding method is three to five times less.

Cow’s milk casein, a hidden allergen in natural rubber latex gloves

Background: We have previously noted that one natural rubber latex (NRL) glove brand used for skin prick testing in the diagnosis of NRL allergy contained cow’s milk casein. Objective: We sought to examine whether other commonly marketed NRL glove brands contain cow’s milk casein. Methods: The casein content of 30 NRL glove extracts (1:5 wt/vol) was measured by RAST inhibition and by rocket immunoelectrophoresis and rocket radioimmunoelectrophoresis by using casein-specific rabbit antiserum, a serum pool from patients with cow’s milk allergy, and radiolabeled anti-human IgE. The NRL allergen content was measured by ELISA inhibition. Results: The casein content of the glove used in NRL allergy screening (Triflex, Baxter) was rather high (ie, about 400 μg/g of glove). Its total protein content was 1000 μg/g of glove. Rocket radioimmunoelectrophoresis detected distinct amounts of casein in 8, minimal amounts in 7, and no casein in the remaining 15 NRL glove brands. Conclusions: Several brands of NRL gloves were found to contain casein, implying that extracts prepared from such gloves can cause false-positive skin prick test reactions when diagnosing NRL allergy. The use of casein as a stabilizer in glove manufacture without appropriate labeling should be stopped because it can also cause contact urticaria syndrome in individuals with cow’s milk allergy. (J Allergy Clin Immunol 1999;104:177-80.)

Does IgE Bind to and Activate Eosinophils from Patients with Allergy?

Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcepsilonRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcepsilonRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcgammaRII, CD32), but no expression of FcepsilonRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcepsilonRI mAb, suggesting that eosinophils do express minimal amounts of FcepsilonRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express approximately 0.5% of the FcepsilonRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.

An in vitro Model for the Allergen–IgE–FcεRI Interaction

Background: The interaction of immune complexes consisting of allergens and allergen–specific IgE with the high–affinity Fcε receptor represents the key event in the induction of symptoms in type I allergic individuals. Immediate–type symptoms result from the release of biological mediators due to allergen–induced cross–linking of FcεRI receptors on mast cells and basophils, whereas FcεRI–mediated presentation of allergen–IgE complexes may contribute to late–phase symptoms through enhanced T cell activation. The interaction of allergens/allergen–specific IgE/FcεRI represents, therefore, an important target for therapeutic intervention strategies in type I allergy. Methods and Results: A molecular model of the allergen–IgE–FcεRI interaction was established. It consists of recombinant purified Bet v 1, the major birch pollen allergen, a chimeric Bet v 1 specific monoclonal IgE antibody, and the baculovirus–expressed purified human alpha chain of FcεRI. The chimeric Bet v 1–specific IgE antibody consists of the light chain and the heavy chain variable region of a mouse monoclonal Bet v 1 specific antibody, Bip 1, and the constant region of human IgE. The interaction of rBet v 1, chimeric Bip 1, and human alpha chain was investigated by overlay experiments. Nitrocellulose–immobilized recombinant alpha chains was incubated with chimeric Bip 1 and, for control purposes, with mouse–derived Bip 1. Bound chimeric Bip 1 was detected with ¹²⁵I–labeled rBet v 1. The specific interaction of rBetv 1, chimeric Bip 1, and recombinant human alpha chain is demonstrated. We thus establish a molecular model of the allergen/IgE/alpha chain interaction. The usefulness of the described in vitro system is exemplified by the identification of a mouse monoclonal antihuman IgE antibody which blocked the IgE–alpha chain interaction. Conclusions: The module system consisting of rBet v 1, chimeric Bip 1, and recombinant alpha chain may be used for the identification of competitors of the allergic effector reaction by means of high throughput screening of compounds or by combinatorial chemistry.

IgE Cross-reactivity with Caseins from Different Species in Humans Allergic to Cow's Milk

Fifty-eight sera from humans allergic to cow's milk proteins were analysed for the specificity of their IgE response to the whole casein fraction of milk from different ruminant and nonruminant species (e.g. cow, sheep, goat, rabbit and rat). IgE-specific responses were determined by an enzyme allergosorbent test using the purified casein fractions as immobilized antigen and an anti-human IgE monoclonal antibody labelled with acetylcholinesterase. Co-and/or cross-sensitizations to caseins of the different ruminant species occurred extensively, though IgE responses to ovine and caprine casein appeared to be lower than that obtained with bovine casein. Cross-reactivity is suggested by the significant reactivity of rat and rabbit casein toward human IgE. In terms of specificity and intensity, the IgE response to caseins demonstrates a great variability. Structural homologies in caseins of such different species, that can share common epitopes for the IgE of some patients, suggest that prevention of cow's milk allergy cannot be achieved by using milk from other species as substitutes.