A novel family of hairpin peptides that inhibit IgE activity by binding to the high-affinity IgE receptor

A family of structured peptides that bind to FcepsilonRIalpha, the alpha-chain of the high-affinity receptor for IgE, has been identified. Binding selections using FcepsilonRIalpha and polyvalent peptide-phage libraries yielded a dominant 18-residue peptide-phage clone, as well as related sequences that did not resemble fragments of IgE. Synthetic peptides based on these sequences inhibited IgE binding to its receptor, and were found by NMR analysis to adopt a stable beta-hairpin structure in solution. Optimized peptides with micromolar receptor affinity exhibited high stability in biological fluids and inhibited cellular histamine release in an in vitro bioassay of IgE activity. The structure-activity relationships of these peptides, which are less than 1% of the size of IgE, suggest an overlap between their binding site and that of IgE on FcepsilonRI. Thus, the peptides demonstrate that blocking a small epitope on this receptor chain is sufficient to block IgE activity. Such structured peptides represent a possible starting point for the design of novel antagonists, and offer the potential for testing in vivo a new approach for treating allergic disease.     

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

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

Mutagenesis within human FcepsilonRIalpha differentially affects human and murine IgE binding

Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.     

Different stabilities of the structurally related receptors for IgE and IgG on the cell surface are determined by length of the stalk region in their alpha-chains

A variant of the high affinity IgE receptor FcepsilonRI, which is composed of alpha- and gamma-chains without the beta-chain, is expressed on human APC, such as dendritic cells, and has been suggested to facilitate Ag uptake through IgE and hence to facilitate Ag presentation to T cells. The level of FcepsilonRI on these cells is correlated with the serum IgE concentration, suggesting IgE mediates the up-regulation of the alphagamma2-type FcepsilonRI. The IgE-mediated FcepsilonRI up-regulation on mast cells and basophils has been shown to enhance the ability of these cells to release chemical mediators and cytokines that are responsible for allergic inflammatory reactions. Here, to elucidate the mechanism controlling FcepsilonRI expression, we compared two structurally related Ig receptors, human FcepsilonRI and FcgammaRIIIA, which carry different alpha-chains but the same gamma-chains. The half-life of FcepsilonRI on the cell surface was short unless it bound IgE, whereas FcgammaRIIIA was stably expressed without IgG binding. Shuffling of the non Ig-binding portions of the FcepsilonRIalpha and FcgammaRIIIAalpha chains revealed that the stalk region was critical in determining the difference in their stability and ligand-induced up-regulation. Unexpectedly, analyses with added or deleted amino acids in the stalk region strongly suggested that the length rather than the amino acid sequence of the stalk region was of major importance in determining the different stabilities of FcepsilonRI and FcgammaRIIIA on the cell surface. This finding provides new insights into the mechanism regulating surface FcepsilonRI expression.     

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.     

The transcription factors Elf-1 and GATA-1 bind to cell-specific enhancer elements of human high-affinity IgE receptor alpha-chain gene.

Key regulatory regions necessary for the expression of the gene encoding FcepsilonRI alpha-chain, a component of the high-affinity IgE receptor primarily responsible for IgE-dependent allergic response, were investigated. Two regions, -74/-69 and -55/-47, which contained binding motifs for proteins belonging to the Ets family and the GATA family, respectively, were shown to be necessary for the activation of the alpha-chain promoter. Both the regulatory elements enhanced the promoter activity only in alpha-chain-producing cells PT18 and RBL-2H3 (mast cell lines), indicating that the elements required specific trans-acting proteins present in the alpha-chain-producing cells. EMSA using nuclear extracts and in vitro-translated proteins revealed that Elf-1 and GATA-1 bound to the enhancer elements. This is the first report describing the regulation in the expression of the FcepsilonRI alpha-chain.     

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.     

Down-regulation of human basophil IgE and FC epsilon RI alpha surface densities and mediator release by anti-IgE-infusions is reversible in vitro and in vivo

Previously, infusions of an anti-IgE mAb (rhumAb-E25) in subjects decreased serum IgE levels, basophil IgE and FcepsilonRIalpha surface density, and polyclonal anti-IgE and Ag-induced basophil histamine release responses. We hypothesized that these effects would be reversed in vivo by discontinuation of infusions and in vitro by exposing basophils to IgE. Subjects received rhumAb-E25 biweekly for 46 wk. Blood samples taken 0-52 wk after rhumAb-E25 were analyzed for serum IgE and basophil expression of IgE, FcepsilonRIalpha, and CD32. Basophil numbers were unaffected by infusions. Eight weeks after infusions, free IgE levels rose in vivo but did not reach baseline. Basophil IgE and FcepsilonRIalpha rose in parallel with free IgE while CD32 was stable. FcepsilonRI densities, measured by acid elution, returned to 80% of baseline, whereas histamine release responses returned to baseline. Basophils cultured with or without IgE or IgG were analyzed for expression of IgE, FcepsilonRIalpha, and CD32. By 7 days with IgE, expression of IgE and FcepsilonRIalpha rose significantly, whereas cultures without IgE declined. IgE culture did not effect CD32. IgG culture did not effect expression of any marker. The present results strongly suggest that free IgE levels regulate FcepsilonRIalpha expression on basophils.     

Efficient folding of the FcepsilonRI alpha-chain membrane-proximal domain D2 depends on the presence of the N-terminal domain D1

Human high affinity receptor for IgE is a membrane glycoprotein multichain complex presenting two extracellular Ig modules in its alpha-chain (D1D2). The receptor IgE binding region is located within the membrane-proximal module D2, while the N-terminal module D1 appears to promote an optimal receptor conformation for IgE binding. To understand the structural relationship between the two modules, we dissected FcepsilonRI alpha-chain into its discrete Ig units and expressed them in mammalian cells. Unexpectedly, D2 was secreted as a disulphide-linked dimer, while D1 was monomeric. Active secretion and full glycosylation of dimeric D2 suggest a native-like conformation of the protein, justifying the escape from the endoplasmic reticulum/Golgi quality control systems. We then propose a domain-swapping model for D2, in which two interdigitated polypeptide chains assume the overall conformation of two Ig modules, as observed for rat CD2 N-terminal domain. Fusion of an unrelated Ig fold moiety at the N terminus of D2 did not interfere with its dimerisation. While D1D2 assumes a correct fold, co-expression of both isolated domains in the same cell did not restore monomeric folding of D2. Thus, D1 appears to assist the appropriate folding of FcepsilonRI alpha-chain, acting as an uncleavable intramolecular chaperone-like block towards D2.     

Drastic up-regulation of Fcepsilonri on mast cells is induced by IgE binding through stabilization and accumulation of Fcepsilonri on the cell surface.

It has been shown that IgE binding to FcepsilonRI on mast cells results in increased FcepsilonRI expression, which in turn enhances IgE-dependent chemical mediator release from mast cells. Therefore, prevention of the IgE-mediated FcepsilonRI up-regulation would be a promising strategy for management of allergic disorders. However, the mechanism of IgE-mediated FcepsilonRI up-regulation has not been fully elucidated. In this study, we analyzed kinetics of FcepsilonRI on peritoneal mast cells and bone marrow-derived mast cells. In the presence of brefeldin A, which prevented transport of new FcepsilonRI molecules to the cell surface, levels of IgE-free FcepsilonRI on mast cells decreased drastically during culture, whereas those of IgE-bound FcepsilonRI were stable. In contrast, levels of FcgammaRIII on the same cells were stable even in the absence of its ligand, indicating that FcepsilonRI alpha-chain, but not beta- and gamma-chains, was responsible for the instability of IgE-free FcepsilonRI. As far as we analyzed, there was no evidence to support the idea that IgE binding to FcepsilonRI facilitated synthesis and/or transport of FcepsilonRI to the cell surface. Therefore, the stabilization and accumulation of FcepsilonRI on the cell surface through IgE binding appears to be the major mechanism of IgE-mediated FcepsilonRI up-regulation.     

Temperature effect on IgE binding to CD23 versus Fc epsilon RI

A chimeric soluble CD23, consisting of the extracellular domain of mouse CD23 and a modified leucine zipper (lz-CD23), has been shown to inhibit IgE binding to the FcepsilonRI. A similar human CD23 construct was also shown to inhibit binding of human IgE to human FcepsilonRI. In both systems, the inhibition was found to be temperature dependent; a 10-fold molar excess of lz-CD23 gave 90-98% inhibition at 4 degrees C, dropping to 20-30% inhibition at 37 degrees C. Surface plasmon resonance analysis of lz-CD23 binding to an IgE-coated sensor chip suggested that the effective concentration of lz-CD23 was lower at the higher temperatures. Analysis of (125)I-IgE binding to CD23(+)-Chinese hamster ovary cells also indicated that increased temperature resulted in a lower percentage of IgE capable of interacting with CD23. In contrast, IgE interacts more effectively with FcepsilonRI(+)-rat basophilic leukemia cells at 37 degrees C compared with 4 degrees C. The results support the concept that the open and closed IgE structures found by crystallography interact differently with the two IgE receptors and suggest that temperature influences the relative percentage of IgE in the respective structural forms. Changes in CD23 oligomerization also plays a role in the decreased binding seen at physiological temperatures.     

Export of the high affinity IgE receptor from the endoplasmic reticulum depends on a glycosylation-mediated quality control mechanism

The high affinity IgE receptor (FcepsilonRI) is a multisubunit complex comprised of either alphagamma(2) or alphabetagamma(2) chains. The cotranslational assembly of the IgE-binding alpha-chain with a dimer of gamma-chains occurs in a highly controlled manner and is proposed to involve masking of a dilysine motif present at the cytoplasmic C terminus of the FcepsilonRI alpha-chain that targets localization of this subunit to the endoplasmic reticulum (ER). Here, we show that ER quality control modulates export from the ER of newly synthesized alphagamma(2) and alphabetagamma(2) receptors. We demonstrate that the presence of untrimmed N-linked core glycans (Glc(3)Man(9)GlcNAc(2)) on the FcepsilonRI alpha-chain activates the ER quality control mechanism to retain this subunit in the ER, despite the presence of gamma-chains. At the same time, the untrimmed, ER-localized alpha-chain exhibits IgE-binding activity, suggesting that FcepsilonRI alpha-chain folding occurs before constitutive glucose trimming. In additional experiments, we demonstrate that cell surface expression of an alpha-chain C-terminal truncation mutant is also dependent on glucose trimming, but not on gamma-chain coexpression. We suggest that glucosidase trimming of terminal glucose residues is a critical control step in the export of FcepsilonRIalpha from the ER. Finally, we show that the constitutive ER FcepsilonRI alpha-chain, expressed in the absence of the other FcepsilonRI subunits, associates with the ER lectin-like chaperone calnexin, but not the structurally similar ER chaperone calreticulin, presumably through interaction with monoglucosylated alpha-chain ER glycoforms.     

Epitope analysis and primary structures of variable regions of anti-human FcepsilonRI monoclonal antibodies, and expression of the chimeric antibodies fused with human constant regions

The structural analysis of monoclonal antibodies (mAbs) against the alpha subunit of the high affinity IgE receptor (FcepsilonRIalpha) is an alternative approach to obtaining information for the design of inhibitors that will block complementary interaction between IgE and FcepsilonRIalpha and to analyzing the various biological effects induced by anti-FcepsilonRIalpha autoantibodies in chronic urticaria. In this study, epitopes for mouse anti-human FcepsilonRIalpha mAbs and primary structures of variable regions of the mAbs were analyzed. Three mAbs inhibitory for IgE-binding reacted to the deletion mutants of FcepsilonRIalpha containing the whole second immunoglobulin-like domain as well as IgE did. On the other hand, two uninhibitory mAbs reacted to those containing the whole first immunoglobulin-like domain. The cDNAs for variable regions of the five mAbs were cloned and sequenced. Two inhibitory mouse/human chimeric antibodies were expressed in COS7 cells and bound to Chinese hamster ovary transfectant cells expressing FcepsilonRI (CHO/alphabetagamma), and these inhibited the binding of IgE to CHO/alphabetagamma cells.     

Fine structure analysis of interaction of FcepsilonRI with IgE

The high affinity receptor for IgE (FcepsilonRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcepsilonRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the alpha-subunit (FcepsilonRIalpha). In this study, the IgE binding site of human FcepsilonRIalpha has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcepsilonRIalpha and the functionally distinct but structurally homologous low affinity receptor for IgG (FcgammaRIIa) have been used to localize two IgE binding regions of FcepsilonRIalpha to amino acid segments Tyr129-His134 and Lys154-Glu161. Both regions were capable of independently binding IgE upon placement into FcgammaRIIa. Molecular modeling of the three-dimensional structure of FcepsilonRIalpha-D2 has suggested that these binding regions correspond to the "exposed" C'-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129-His134 and Lys154-Glu161 regions of FcepsilonRIalpha was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in the B-C loop region, which lies adjacent to the C'-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcepsilonRIalpha-IgE interaction for the possible treatment of IgE-mediated allergic disease.     

Early divergence of Fc epsilon receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production, and survival

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Monomeric IgE binding to its high affinity receptor (FcepsilonRI) results in a number of biological outcomes in mouse mast cells, including increased surface expression of FcepsilonRI and enhanced survival. IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic IgEs cause extensive FcepsilonRI aggregation, leading to potent enhancement of survival and other activation events, whereas poorly cytokinergic IgEs can do so less efficiently. In this study, we demonstrate that IgE-induced receptor up-regulation is not sensitive to monovalent hapten, which can prevent receptor aggregation induced by IgE, whereas other activation events such as receptor internalization, degranulation, IL-6 production, and survival are sensitive to monovalent hapten. IgE-induced receptor up-regulation is also unique in that no Src family kinases, Syk, or Btk are required for it. By contrast, highly cytokinergic IgE-induced receptor internalization is dependent on Lyn, but not other Src family kinases, Syk, or Btk, whereas degranulation, IL-6 production, and survival require Syk. Weak to moderate stimulation with IgE plus anti-IgE or IgE plus Ag enhances survival, while stronger signals are required for degranulation and IL-6 production. Collectively, signals emanated from IgE-bound FcepsilonRI for receptor up-regulation and internalization are shown to diverge at the receptor or receptor-proximal levels from those for other biological outcomes.     

IL-6 enhances IgE-dependent histamine release from human peripheral blood-derived cultured mast cells

We examined whether interleukin (IL)-6 exerts the stimulatory effects on the secretion of histamine from human mast cells triggered by crosslinking of the high affinity IgE receptor (FcepsilonRI) with IgE and anti-IgE. As target cells, we used peripheral blood-derived cultured mast cells grown with SCF, because they were superior in FcepsilonRIalpha expression to cord blood-derived mast cells. Incubation with SCF+IL-6 for 1 week increased the IgE-dependent release as well as intracellular content of histamine in the cultured mast cells, as compared with the values obtained by incubation with SCF alone. The magnitude of these increases was higher than that for priming with SCF+IL-4. A striking difference was also found in the expression of FcepsilonRIalpha between the two-factor combinations. The addition of IL-6 during FcepsilonRI crosslinking with IgE/anti-IgE in the presence of SCF did not influence histamine secretion. When SCF, IL-6 and IL-4 were used together, a further increase was observed in the anti-IgE-dependent liberation of histamine from the cultured mast cells, compared with the two-factor combinations. These results suggest that IL-6 functions as a secretagogue for the inflammatory mediator of human mast cells in the presence of SCF.