Dissociation between skin test reactivity and anti-aeroallergen IgE: Determinants among urban Brazilian children.

BACKGROUND: The dissociation between specific IgE and skin prick test reactivity to aeroallergens, a common finding in populations living in low and middle-income countries, has important implications for the diagnosis and treatment of allergic diseases. Few studies have investigated the determinants of this dissociation. In the present study, we explored potential factors explaining this dissociation in children living in an urban area of Northeast Brazil, focusing in particular on factors associated with poor hygiene. METHODS: Of 1445 children from low income communities, investigated for risk factors of allergies, we studied 481 with specific IgE antibodies to any of Blomia tropicalis, Dermatophagoides pteronyssinus, Periplaneta americana and Blatella germanica allergens. Data on demographic, environmental and social exposures were collected by questionnaire; serum IgG and stool examinations were done to detect current or past infections with viral, bacterial, protozoan and intestinal helminth pathogens. We measured atopy by skin prick testing (SPT) and specific IgE (sIgE) to aerollergens in serum (by ImmunoCAP). SIgE reactivity to B. tropicalis extract depleted of carbohydrates was measured by an in-house ELISA. Total IgE was measured by in house capture ELISA. SNPs were typed using Illumina Omni 2.5. RESULTS: Negative skin prick tests in the presence of specific IgE antibodies were frequent. Factors independently associated with a reduced frequency of positive skin prick tests were large number of siblings, the presence of IgG to herpes simplex virus, Ascaris lumbricoides and Trichuris trichiura infections, living in neighborhoods with infrequent garbage collection, presence of rodents and cats in the household and sIgE reactivity to glycosylated B. tropicalis allergens. Also, SNP on IGHE (rs61737468) was negatively associated with SPT reactivity. CONCLUSIONS: A variety of factors were found to be associated with decreased frequency of SPT such as unhygienic living conditions, infections, total IgE, IgE response to glycosylated allergens and genetic polymorphisms, indicating that multiple mechanisms may be involved. Our data, showing that exposures to an unhygienic environment and childhood infections modulate immediate allergen skin test reactivity, provide support for the "hygiene hypothesis".

TRIF signaling is essential for TLR4-driven IgE class switching.

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.

Targeting the junction of CɛmX and ɛ-migis for the specific depletion of mIgE-expressing B cells.

Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment. In the long isoform, the CH4 domain is followed by a 67-amino acid-long extracellular portion. Out of these 67 amino acids, the first 52 amino acids following the CH4 domain constitute the CɛmX segment while the rest of the 15 amino acids immediately adjacent to the membrane constitute the ɛ-migis. In the short isoform the CɛmX segment is absent and the CH4 domain is followed only by the 15-amino acid-long ɛ-migis segment. Using antibodies derived from a phage display library, we investigated: (1) ɛ-migis and (2) the junction of CɛmX and ɛ-migis (CɛmX.migis), as potential therapeutic antibody targets. Our results indicate that antibodies obtained from our phage library that target ɛ-migis bind to a variety of human cells irrespective of mIgE expression, possibly due to homology between ɛ-migis and a region of phosphoinositide-binding protein (ARAP3). In contrast, antibodies specific for the CɛmX.migis junctional region, bound specifically to transfected and primary B cells expressing human mIgE and elicited antibody-dependent cellular cytotoxicity and reduction in IgE production. These antibodies did not bind secreted IgE or the mIgE isoform in which CɛmX is absent. These results suggest that CɛmX.migis junctional region is a promising antibody target and the human antibodies we describe warrant further evaluation.

Immature B cells preferentially switch to IgE with increased direct Sµ to Sε recombination.

Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cµ (IgM) constant regions (C(H)) exons with downstream C(H) exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cµ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cµ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cµ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cµ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation.

Protection of IgE-mediated allergic sensitization by active immunization with IgE loops constrained in GFP protein scaffold.

Green fluorescent protein (GFP) exhibits a rigid central beta-barrel, formed by eleven beta-strands with floppy loops spanning between the stands. Herein, we evaluate whether the rigid beta-barrel may serve as a scaffold that can constrain the loops of a foreign protein, and thus its antigenicity. The spanning loops, site 6 of GFP, were engineered with RE cloning sites for inserting oligonucleotides corresponding to FcepsilonRI-binding sequence of human IgE. In a high-throughput format, shortened oligonucleotides encoding eight amino acid residues of the receptor-binding regions were inserted into site 6 of GFP by PCR, followed by enabling sequences for in vitro transcription and translation at the 5' end. Antigenized C2-3 linker (C2-3L) was shown by immuno-blots with polyclonal anti-IgE under native gel electrophoresis and transfer. Recombinant antigenized GFP was expressed and purified to homogeneity by metal affinity column, followed by Sephacryl S-200 high resolution gel filtration. Hyperimmune sera from mice immunized with C2-3L antigenized GFP contain anti-IgE reactive with JW8 murine/human chimeric IgE. Further, elevated serum anti-C2-3L and affinity pure antibodies effectively inhibits binding of JW8 IgE to recombinant FcepsilonRIalpha, and desensitizes JW8 to rat RBL-2H3 transfected with human FcepsilonRIalpha. This observation raised the possibility that active IgE vaccine may be employed in raising active protective anti-IgE in allergic patients as an alternative to passive immunization with MAb-E25 anti-IgE. Taken together, GFP appears suitable protein scaffold for spanning/constraining the C2-3L of human IgE as active vaccine; and this technique may be generally employed for eliciting antibodies to specific B-cell epitopes of other proteins.

Detection of epsilon class switching and IgE synthesis in human B cells.

We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.

Role of the FcepsilonRI beta-chain ITAM as a signal regulator for mast cell activation with monomeric IgE.

The beta-chain of the high-affinity receptor for IgE (FcepsilonRI) plays a crucial role for amplification of the intracellular signaling in mast cells upon FcepsilonRI cross-linking by IgE*antigen complexes (IgE*Ag). Some monomeric IgE as well as IgE*Ag stimulate FcepsilonRI-signaling pathways, leading to cell activation, whereas the biological functions of the beta-chain in the monomeric IgE-mediated mast cell signaling and responses are largely unknown. In the present study, FcepsilonRI is reconstituted with either wild-type beta-chain or mutated beta-chain immunoreceptor tyrosine-based activation motif (ITAM) employing retrovirus-mediated gene transfer into the FcepsilonRI beta-chain-/- mast cells. We demonstrated that the transfectants with mutated beta-chain ITAM stimulated with monomeric IgE sufficiently produce inflammatory cytokines, although degranulation, intracellular Ca(2+) mobilization and leukotriene C(4) synthesis are significantly reduced. Furthermore, analyses of molecular mechanisms of the signaling revealed that the expression of cytokine genes and activation of extracellular signal-regulated kinase 1/2 and protein kinase C were significantly delayed in the beta-chain ITAM mutant cells stimulated with monomeric IgE, suggesting that the beta-chain ITAM regulates kinetics of gene transcriptions and signaling pathways for cytokine production. These findings for the first time revealed the unique functions of the beta-chain ITAM in both chemical mediator release and cytokine production of mast cells upon monomeric IgE stimulation.

Differential activation of signal transducer and activator of transcription 6 in B cells from allergic children and their non-allergic siblings.

BACKGROUND: The germline (GL) epsilon promoter is regulated by IL-4 and is essential for class switching to IgE. IL-4-induced gene expression is largely mediated through activation of latent transcription factor STAT6 (signal transducer and activator of transcription 6). OBJECTIVE: We investigated whether increased levels of IgE in allergic individuals may be associated with alteration in the level or activation of STAT6 and subsequent increase in GL epsilon promoter activity. METHODS: Electrophoretic mobility shift assay and Western blotting assays were used to investigate the level of expression and activation of STAT6 in Epstein-Barr virus (EBV)-transformed B cell lines from children with birch pollen allergy and their non-allergic siblings. The activity of the GL epsilon promoter was tested in a transient transfection assay. RESULTS: STAT6 was expressed at the same level in all B cell lines tested. In two out of five sibling pairs STAT6 was activated by IL-4 more efficiently in the allergic individuals but in the three other pairs the opposite was found. In transient transfections, no difference in IL-4-induced GL epsilon promoter function was detected, although basal promoter activity varied between allergic and healthy siblings in two out of five pairs. CONCLUSIONS: We demonstrate for the first time that upon IL-4 signalling STAT6 transcription factor activation differs in B cells from different individuals. Although we did not find any association between STAT6 activation and allergy, we do not exclude a possibility that stronger activation of this transcription factor is associated with an expression of allergic phenotype.

Monoclonal antibodies against the C(epsilon)mX 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 epsilon-chain of human mIgE contains a membrane-anchoring peptide and an extra 52-amino-acid (a.a.)-long domain (referred to as C(epsilon)mX) between the membrane anchor and the CH4 domain. OBJECTIVE: The study was designed to evaluate the effects of C(epsilon)mX-specific monoclonal antibodies (mAbs) to target IgE-expressing B cells and decrease IgE production. METHODS: A C(epsilon)mX-containing IgG1.Fc fusion protein was produced in CHO cells and used to immunize mice; five hybridoma clones secreting C(epsilon)mX-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 C(epsilon)mX. 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 C(epsilon)mX. 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.

Generation of therapeutic antibody responses against IgE through vaccination.

IgE is the central mediator in atopic allergies such as hay fever, eczema, and asthma; therefore, it is a prime target in the development of allergen-independent preventive treatments. We describe an active immunization strategy that has the potential to reduce IgE to a clinically significant extent. The active vaccine component is a chimeric IgE molecule, Cepsilon2-Cepsilon3-Cepsilon4. The receptor-binding target domain, Cepsilon3, is derived from the recipient species, whereas the flanking domains, Cepsilon2 and Cepsilon4, are derived from an evolutionarily distant mammal. The flanking domains have dual functions, acting both as structural support for the Cepsilon3 domain and to break T cell tolerance by providing foreign T cell epitopes. The efficacy of the vaccine was studied in an ovalbumin-sensitized rat model. Vaccination resulted in antibody responses against IgE in all rats and in a substantial reduction in serum IgE levels in three out of four strains. The skin reactivity upon allergen challenge was significantly reduced in vaccinated animals. The vaccine appears to be safe to use as an antigen. No cross-linking activity was observed in sera of vaccinated animals, and the response to vaccination was reversible with time. Our results suggest that active immunization against IgE has the potential to become a therapeutic method for humans.

B cells with the guts to switch.

Expression of mIgM was thought to be esential for the differentiation of B cells expressing antibodies of other classes. New evidence suggests isotype class switching to IgA can occur in the absence of mIgM.

Sequence-specific antibodies against human IgE isoforms induced by an epitope display system.

BACKGROUND: Unlike other immunoglobulin isotypes, the human C epsilon gene generates by alternative splicing two types of secretory and two types of membrane epsilon chains. The two secreted epsilon heavy chains, epsilon(S1) and epsilon(S2), differ only in the sequence of the last eight C-terminal amino acids, being epsilon(S2) six amino acids longer. The two types of membrane isoforms differ in the extracellular membrane proximal domain, with the longer variant, epsilon(mL), containing 52 extra amino acids which are absent in the shorter epsilon(mS) isoform. OBJECTIVES: We wished to produce quality antibody reagents that specifically detect epitopes that are epsilon isoform-specific. STUDY DESIGN: Short sequences of seven or ten amino acids were chosen as target epitopes and expressed as part of the highly immunogenic loops of deletion variants of engineered Flock House Virus capsid protein RNA2. Chimeric proteins were expressed in E. coli, and used to immunize rabbits. Antisera were screened by immunoblotting of purified IgE isoforms expressed by murine transfectomas. RESULTS: Chimeric proteins expressing epsilon isoform-specific epitopes proved to be strong immunogens in vivo and induced highly specific rabbit antisera. Two antisera so obtained recognize specifically the IgE-S2 isoform. A third one recognizes the long membrane variant m(L)IgE and a fourth one detects an epitope specific to m(S)IgE. CONCLUSION: Here we describe a simplified and efficient protocol of immunization which does not require peptide synthesis and conjugation to carrier protein. Our results show that short peptides of unknown immunogenicity, when genetically introduced into the modified Flock House Virus epitope display system, successfully induced IgE isoform-specific polyclonal antisera in rabbits. These are valuable tools to specifically identify secretory and membrane isoforms of human IgE, and the method is potentially applicable to other variant isoforms or mutants of a given protein.

Characterization of two dog IgE-specific antibodies elicited by different recombinant fragments of the epsilon chain in hens.

Two recombinant [His]6-tagged fragments of the canine immunoglobulin E (IgE) heavy chain (second domain: IgEf2 and third and fourth domains: IgEf3/4) were cloned, expressed in Escherichia coli (E. coli) as [His]6-tagged proteins, and affinity-purified over nickel-nitrilotriacetic acid columns. The recombinant proteins were used to immunize hens. The raised and affinity-purified chicken antibodies (Ab) isolated from egg yolk exhibited specific binding to the respective recombinant canine IgE fragment (IgEf) on immunoblots and displayed high titers against the IgEf in ELISA. Immunoblotting of canine serum separated by PAGE under native conditions with the IgEf2- and IgEf3/4-specific Ab resulted in staining of a protein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab further recognized an 80 kD protein in IgEf3/4-specific Ab affinity-enriched dog serum separated under denaturing conditions. In an ELISA for the detection of antigen-specific IgE in dog serum, reduced binding of the IgEf-specific Ab was observed after heat treatment of the dog serum. The reactivity of both of the raised chicken Ab was only present in postimmune reagents and could only be inhibited by preincubation with the IgEf used for immunization and not with dog immunoglobulin G, E. coli extract, or with a nonrelevant recombinant [His]6-tagged protein. In immunohistochemistry, the IgEf3/4-specific Ab specifically recognized cells in paraffin-embedded tissue sections of lymph nodes. Furthermore, both of the IgEf-specific Ab elicited positive immediate type 1 skin reactions in dogs. Semiquantitative assessment of total serum IgE in dogs was developed using IgEf2-specific Ab as coating reagent and the biotinylated IgEf3/4-specific Ab as developing Ab in ELISA. In conclusion, both IgEf-specific Ab recognize native dog IgE with the advantages that they are directed against different and known constant domains of the IgE molecule, and that they can be used for immunohistochemistry on paraffin-embedded tissue. The two dog IgE-specific Ab could initiate clinical research on the involvement of immediate-type hypersensitivity reactions in dogs.

Interaction of the low-affinity receptor CD23/Fc epsilonRII lectin domain with the Fc epsilon3-4 fragment of human immunoglobulin E.

CD23/Fc epsilonRII, the low-affinity receptor for IgE, is a multifunctional protein of importance in blood cell development and the immune system. We have studied the interaction of CD23 with IgE in solution using hydrodynamic methods applied to recombinant fragments of both ligands: sCD23, corresponding to the soluble lectin domain of CD23, and Fc epsilon3-4, a dimer of the C epsilon3-C epsilon4 sequence of IgE. The hydrodynamic, spectroscopic, and biological properties of these fragments suggest that they have a fully native structure. Sedimentation equilibrium studies on mixtures of sCD23 and Fc epsilon3-4 indicate that IgE has two binding sites for CD23, each characterized by affinities of approximately 10(5) M(-1). Analysis of the sedimentation as a function of temperature allows conclusions to be drawn about the thermodynamics of binding at the two sites. Binding at the first site is characterized by large changes in enthalpy (delta H(degree)To = -2.1 +/- 3.3 kcal mol(-1)) and heat capacity (delta Cp(degree) = -320 +/- 320 cal mol(-1) K(-1)), whereas binding at the second site is characterized by small changes in enthalpy (delta H(degree)To = 0.1 +/- 5.6 kcal mol(-1)) and heat capacity (delta Cp(degree) = -140 +/- 550 cal mol(-1) K(-1)). In native CD23, there are two or three lectin domains, associated through an alpha-helical coiled-coil stalk. The predicted structure of the CD23 oligomers and symmetry considerations rule out the possibility of two lectin domains from one oligomer binding to identical sites in IgE. The notion of two types of interaction in the 2:1 complex between CD23 and IgE is consistent with the thermodynamic data presented.

Chicken antibodies to a recombinant fragment of the equine immunoglobulin epsilon heavy-chain recognising native horse IgE.

An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, nucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a [His]6-tag and purified over a Ni-NTA column. The recombinant protein was used to immunise hens. Testing of the raised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA revealed high titres against the recombinant equine IgE fragment (reqIgEf). The reqIgEf-specific IgG was successfully affinity-purified on an unconventional affinity matrix: the [His]6-tagged recombinant IgE fragment was bound to Ni-NTA agarose and used to adsorb specific immunoglobulins. In Western-blot of ammonium sulphate precipitated horse serum and bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing-reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa. A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar fluid (BALF) was separated under non-reducing conditions. These reactions could be inhibited by preincubation of the immune IgG with reqIgEf, while preincubation with horse IgG did not inhibit the reaction. Antibody-affinity chromatography of horse serum with the reqIgEf-specific chicken IgG resulted in an enrichment of the 80 kDa protein in denaturing Western-blot. Determination of the amino acid composition of this protein and comparison with the equine IgE heavy- chain sequence strongly indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE. The reqIgEf-specific chicken IgG was further characterised in an ELISA for the detection of allergen-specific horse IgE. It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG. The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner. In addition, preincubation with horse IgG, a nonrelevant [His]6-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characteristic. This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases.

[The use of monoclonal antibodies and a biotin-streptavidin system of amplification for the quantitative determination of human IgE]. / Ispol'zovanie monoklonal'nykh antitel i biotin-streptavidinovoi sistemy usileniia dlia kolichestvennogo opredeleniia IgE cheloveka.

A "sandwich"-variant of the solid phase immunoenzyme method of quantitative determination of human IgE in biological liquids has been developed on the basis of two monoclonal antibodies to various epitopes of Fc-fragment of E-chain of human IgE and biotin-streptavidin system of the signal amplification. The total analysis time is 3 h: the range of determined concentrations of IgE is 1-200 kE/l; sensitivity-1 kE/l; the type of calibrating curve is linear. To make an analysis twice 10 microliters of the tested sample are required. Reproducibility of the suggested method is not below 5%.

The recognition of a recombinant human Fc epsilon fragment by the subclasses of IgG autoanti-IgE: disproportional subclasses distribution of complexed autoantibody.

Circulating IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma where it is suggested to be potentially involved in the removal of IgE-allergen complexes. Since such a putative role will largely be determined by the subclass profile of complexed (i.e. IgE-bound) IgG anti-IgE, a study was undertaken to determine the subclass distribution of complexed IgG anti-IgE antibody in the sera of asthmatic patients. The study exploits the heat-labile property of IgE by heating (30 min at 56 degrees C) serum to liberate bound anti-IgE, pre- and post-heated sera are then assayed for IgG subclass anti-recombinant human Fc epsilon (rFc epsilon) activities by ELISA and any heat-induced increase in antibody activity is taken as a measure of complexed anti-IgE. This has revealed a disproportionately high amount of IgG4 in complexed (but not free) IgG anti-IgE. The propensity of IgG4 to form complexes with IgE has important biological consequences, particularly with regard to the activation of C1q and Fc gamma R by other subclasses.

Elucidation of the epitope locations of human autoanti-IgE: recognition of two epitopes located within the C epsilon 2 and the C epsilon 4 domains.

IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma, where it is suggested to be potentially involved in modulating IgE-mediated hypersensitivity. Using a series of overlapping recombinant human epsilon-chain peptides, we have shown that circulating IgG anti-IgE antibodies recognise at least 2 epitopes located within the C epsilon 2 and the C epsilon 4 domains, respectively. The C epsilon 2 recognition site is located within the C-terminal portion of the C epsilon 2 domain (i.e. aa301-339) which is thought to contribute residues to the Fc epsilon RI-binding site on IgE. The recognition by autoanti-IgE of an effector function site of such pivotal importance in IgE-mediated hypersensitivity suggests that it plays a possible modulatory role during mast cell and basophil activation.