The human mast cell receptor binding site maps to the third constant domain of immunoglobulin E.

The characterization of the site on the IgE molecule which accommodates the high affinity receptor for IgE (Fc epsilon RI) should allow the design of IgE analogues which can be utilized to block allergic responses. Using chimeric human IgE molecules in which different constant region domains were exchanged with their murine homologues, we demonstrate here that the C epsilon 3 in its native configuration is essential for the binding to the alpha subunit of the human Fc epsilon RI. Deletion of the human C epsilon 2 from such chimeric molecules did not impair their ability to interact with the Fc epsilon RI, indicating that C epsilon 2 is not directly involved in the human Fc epsilon RI binding site and that C epsilon 3 alone is necessary and sufficient to account for most of the human Fc epsilon RI-binding capacity.     

Systemic administration of an Fcgamma-Fc(epsilon)-fusion protein in house dust mite sensitive nonhuman primates

Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response. The treatment effect lasted up to 4 weeks and was associated with reduction in the number of circulating basophils and decreased expression of Fc(epsilon)RI on repopulating basophils. Repeat hGE2 dosing induced production of serum antibodies against human Fcgamma and Fc(epsilon) domains and acute anaphylaxis-like reactions. Immune serum induced histamine release from human IgE or hGE2-treated cord blood-derived mast cells and basophils in vitro. These results indicate that repeat administration with hGE2 induced an antibody response to the human molecule that resulted in activation rather than inhibition of allergic responses.     

Genetically engineered negative signaling molecules in the immunomodulation of allergic diseases

PURPOSE OF REVIEW: This review summarizes current knowledge regarding the control of human mast cell and basophil signaling and recent developments using a new therapeutic platform consisting of a human bifunctional gamma and epsilon heavy chain (Fc gamma-Fc epsilon) protein to inhibit allergic reactivity. RECENT FINDINGS: Crosslinking of Fc gamma RIIb to Fc epsilon RI on human mast cells and basophils by a genetically engineered Fc gamma-Fc epsilon protein (GE2) leads to the inhibition of mediator release upon Fc epsilon RI challenge. GE2 protein was shown to inhibit cord blood-derived mast cell and peripheral blood basophil mediator release in vitro in a dose-dependent fashion, including inhibition of human IgE reactivity to cat. IgE-driven mediator release from lung tissue was also inhibited by GE2. The mechanism of inhibition in mast cells included alterations in IgE-mediated Ca mobilization, spleen tyrosine kinase phosphorylation and the formation of downstream of kinase-growth factor receptor-bound protein 2-SH2 domain-containing inositol 5-phosphatase (dok-grb2-SHIP) complexes. Proallergic effects of Langerhan's like dendritic cells and B-cell IgE switching were also inhibited by GE2. In vivo, GE2 was shown to block passive cutaneous anaphylaxis driven by human IgE in mice expressing the human Fc epsilon RI and inhibit skin test reactivity to dust mite antigen in a dose-dependent manner in rhesus monkeys. SUMMARY: The balance between positive and negative signaling controls mast cell and basophil reactivity, which is critical in the expression of human allergic diseases. This approach using a human Fc gamma-Fc epsilon fusion protein to co-aggregate Fc epsilon RI with the Fc gamma RII holds promise as a new therapeutic platform for the immunomodulation of allergic diseases and potentially other mast cell/basophil-dependent disease states.     

Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with Fc epsilon RI alpha

Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine Fc epsilon RI alpha. beta-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.     

Specific inhibition of immunoglobulin E-mediated allergic reaction using antisense Fc epsilon RI alpha oligodeoxynucleotides.

We have investigated the ability of an antisense immunoglobulin E (IgE) receptor alpha-subunit oligodeoxynucleotide (Fc epsilon RI alpha ODN) specifically to inhibit IgE-mediated allergic reactions in the mouse. Synthetic antisense Fc epsilon RI alpha ODN dose-dependently inhibited passive cutaneous anaphylaxis and histamine release from the mouse peritoneal mast cells (MPMC) activated by anti-dinitrophenyl (DNP) IgE. Northern blot analysis showed that the mast cells treated with antisense Fc epsilon RI alpha ODN exhibited no detectable levels of L-histidine decarboxylase mRNA after anti-DNP IgE stimulation, whereas the cells treated with sense Fc epsilon RI alpha ODN possessed significant amounts of this mRNA. Examination of the elevation of cAMP levels in MPMC following the activation with anti-DNP IgE demonstrated a significant rise in activated cells, but not in the antisense Fc epsilon RI alpha ODN-treated cells. Moreover, antisense Fc epsilon RI alpha ODN had a significant inhibitory effect on anti-DNP IgE-induced tumour necrosis factor-alpha production. Our results demonstrated that antisense Fc epsilon RI alpha ODN inhibited the IgE-mediated allergic reaction in vivo and in vitro.     

Regulation of mast cell survival by IgE.

Mast cells play critical roles in hypersensitivity and in defense against certain parasites. We provide evidence that mouse mast cell survival and growth are promoted by monomeric IgE binding to its high-affinity receptor, Fc epsilon RI. Monomeric IgE does not promote DNA synthesis but suppresses the apoptosis induced by growth factor deprivation. This antiapoptotic effect occurs in parallel with IgE-induced increases in Fc epsilon RI surface expression but requires the continuous presence of IgE. This process does not involve the FasL/Fas death pathway or several Bcl-2 family proteins and induces a distinctly different signal than Fc epsilon RI cross-linking. The ability of IgE to enhance mast cell survival and Fc epsilon RI expression may contribute to amplified allergic reactions.     

The high-affinity receptor for immunoglobulin E: a target for therapy of allergic diseases.

The high-affinity receptor for IgE (Fc epsilon RI) on mast cells and basophils is a tetrameric complex, alpha beta gamma 2. Here we summarize the latest developments on the structure and function of this receptor. By genetic transfer, we have engineered a cell line secreting substantial amounts of a peptide containing exclusively the extracellular domain of the alpha-subunit. This domain by itself is sufficient to mediate high-affinity binding of IgE. Glycosylation and the presence of the other subunits are not necessary for the binding function. The gamma-subunit of Fc epsilon RI is part of other receptors such as Fc gamma RIII and the T cell receptor, and therefore is likely to play an important although still undefined functional role. A detailed knowledge of how the receptor interacts with IgE and induces cellular degranulation may lead to the design of new therapeutic approaches to allergic diseases. The potential strategies are discussed.     

Proximal signaling events in Fc epsilon RI-mediated mast cell activation

Mast cells are central mediators of allergic diseases. Their involvement in allergic reactions is largely dependent on activation through the specific receptor for IgE (Fc epsilon RI). Cross-linking of Fc epsilon RI on mast cells initiates a cascade of signaling events that eventually results in degranulation, cytokine/chemokine production, and leukotriene release, contributing to allergic symptomology. Because of the importance of IgE in allergy, much focus has been placed on deciphering the signaling events that take place downstream of Fc epsilon RI. Studies have identified spleen tyrosine kinase as a key proximal regulator of Fc epsilon RI-mediated signaling. In this review, we discuss the multiple pathways that diverge from spleen tyrosine kinase with emphasis on the role of adapter molecules to orchestrate these signaling events. Understanding the molecular mechanisms underlying mast cell activation ideally will provide insights into the development of novel therapeutics to control allergic disease.     

Roles of the transmembrane domain and the cytoplasmic domain of Fc epsilon RI alpha in immunoglobulin E-mediated up-regulation of surface Fc epsilon RI expression.

BACKGROUND: Human mast cells express both Fc epsilon RI alpha and Fc gamma RI alpha. IgE up-regulates Fc epsilon RI alpha expression, but IgG1 does not up-regulate Fc gamma RI alpha expression. The transmembrane domain (TM) of Fc gamma RI alpha determines the stability of cell surface expression of this receptor. OBJECTIVE: The aim of this study was to clarify the roles of the TM and cytoplasmic domain (CY) of Fc epsilon RI alpha in IgE-mediated Fc epsilon RI up-regulation. METHODS: Chimeric receptors created by domain shuffling between Fc epsilon RI alpha and Fc gamma RI alpha were transduced into human mast cell line HMC-1. Cell surface expression of the chimeric receptors and the effect of IgE or IgG1 on chimeric receptor expression were examined by FACS. The association of the chimeric receptors with FcR gamma was investigated by immunoprecipitation. RESULTS: The results showed that the TM and CY of Fc epsilon RI alpha are not essential for IgE-mediated up-regulation of surface Fc epsilon RI. CONCLUSION: The extracellular domain of each Fc receptor determines the diversity of Ig-regulated Fc receptor expression.     

Inhibition of IgE-dependent histamine release from human peripheral blood basophils by humanized Fab fragments that recognize the membrane proximal domain of the human Fc epsilon RI alpha-chain

BACKGROUND: Inhibition of the interaction between IgE and the alpha-chain of Fc epsilon RI (Fc epsilon RI alpha) is a straightforward strategy to develop therapeutic reagents for IgE-mediated allergic diseases. OBJECTIVE: The purpose of this study is the humanization of CRA2 and/or CRA4, mouse anti-human Fc epsilon RI alpha monoclonal antibodies (mAbs) which recognize the IgE-binding membrane proximal immunoglobulin-like domain of Fc epsilon RI alpha. METHODS: The two mAbs were humanized by CDR grafting onto human V region frameworks encoded by human germline V and J genes. The activities of the recombinant antibodies to bind Fc epsilon RI alpha and inhibit IgE binding to Fc epsilon RI alpha were analyzed by flow cytometry and ELISA. Human peripheral blood basophils were pretreated with the Fab fragments of the humanized CRA2 and stimulated with IgE and an anti-IgE polyclonal antibody. The released histamine was measured. RESULTS: The humanized CRA2 had almost the same activities of binding and inhibition of IgE binding to Fc epsilon RI alpha as the original mouse CRA2. Although the Fc epsilon RI-binding activity was maintained following humanization of the CRA4 light chain V region, it was lost by the humanization of the CRA4 heavy chain V region. Pretreatment of human peripheral blood basophils with the Fab fragments of the humanized CRA2 inhibited their subsequent degranulation activated by cross-linking of the Fc epsilon RI. CONCLUSION: In the humanized CRA2, all amino acid residues except CDR are replaced with the residues encoded by human germline genes. The humanization of CRA2 might be an important step in the development of immunotherapy to manipulate the IgE network in which mast cells, basophils, and various types of Fc epsilon RI alpha expressing cells are involved.     

Inhibition of human cord blood-derived mast cell responses by anti-Fc epsilon RI mAb 15/1 versus anti-IgE Omalizumab

Aggregation of the alpha-chain of the high affinity IgE receptor (Fc epsilon RI alpha) on mast cells or basophils after cross-linking of receptor-bound IgE by its antigen or an anti-IgE antibody results in cell activation and release of inflammatory mediators. Omalizumab (Xolair), Novartis Pharmaceuticals; Genentech Inc.) is a recombinant humanized anti-IgE mAb developed for the treatment of severe allergic asthma. It complexes with free serum IgE, which prevents its binding to Fc epsilon RI and thereby interrupts the allergic cascade. Administration of an inhibitory anti-Fc epsilon RI alpha mAb may represent an alternative strategy to neutralize IgE-mediated receptor activation. In the present report, for the first time, we have performed direct side of side comparison between the inhibitory anti-Fc epsilon RI alpha mAb designated 15/1 and Omalizumab for their effects on human cord blood-derived mast cells. We provide the first evidence that both 15/1 mAb and Omalizumab efficiently inhibit Fc epsilon RI-mediated human mast cell responses in vitro (degranulation, activation, release of IL-8 and IL-13, phosphorylation of Akt) and that mAb 15/1 is a non-anaphylactogenic antibody, which compared to Omalizumab, displays markedly higher inhibitory potency in the presence of high IgE levels.     

Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with Fc epsilon RII and Fc epsilon RI.

Three rat monoclonal antibodies specific for mouse IgE (C12B9, 23G3, and B1E3) were established by using monoclonal anti-DNP mouse IgE (mIgE) as immunogen. These antibodies, as well as a fourth, (R1E4) were characterized. It was found that one antibody (C12B9) recognizes an allotypic determinant (Igh-7a) found on the C epsilon chain of mIgE. Antibody cross-blocking studies and epitope mapping studies using recombinant mIgE indicated that 3 antibodies (C12B9, R1E4 and 23G3) were directed against the C epsilon 3 domain while one (B1E3) was directed against the C epsilon 4 domain. A highly specific sandwich RIA for mIgE was developed using these antibodies. Use of these monoclonal anti-mIgE antibodies in conjunction with recombinant chimeric mIgE-human IgG1 molecules, demonstrated that the C epsilon 3 domain is important in the binding of mIgE to the murine B cell Fc epsilon RII as well as to the murine mast cell F epsilon RI. The presence of the C epsilon 4 domain influenced the binding of the recombinant IgE to the Fc epsilon RII; in contrast to the C epsilon 4 domain had no effect on binding to the Fc epsilon RI.     

Omalizumab-induced reductions in mast cell Fce psilon RI expression and function

BACKGROUND: By design, omalizumab binds free IgE in the circulation and prevents its attachment to the surface of mast cells and basophils, thereby preventing them from responding to allergens. Previously, omalizumab rapidly reduced free IgE levels, as well as basophil high-affinity IgE receptors, leading to significant reductions in basophil mediator response to allergen. It is assumed that tissue mast cells are similarly altered in their Fc epsilon RI density and function. OBJECTIVE: We examined the phenotypic shift of skin mast cells in parallel to that of blood basophils in 3 subjects infused with omalizumab. METHODS: Three subjects with allergic rhinitis underwent intradermal skin test titration with house dust mite antigen at days 0, 7, 70, and 196 of omalizumab treatment. As control subjects, 5 untreated subjects with allergic rhinitis were evaluated at similar time points. All subjects underwent skin biopsy 18 to 24 hours later at the site of allergen injection. Biopsy specimens were characterized by means of immunohistochemisty for tryptase and Fc epsilon RI alpha immunoreactivity, as well other markers (CD3, CD45RO, CD68, cutaneous lymphocyte antigen, and major basic protein). RESULTS: Omalizumab recipients, but not control subjects, demonstrated reductions in Fc epsilon RI alpha immunoreactivity at days 70 and 196 in parallel with reductions in the acute wheal response to allergen. However, no reductions in tryptase-positive cells were noted at these time points. CONCLUSION: Reductions in free IgE levels by omalizumab leads to a rapid reduction in basophil Fc epsilon RI receptor expression. In contrast, the time course for the decrease of Fc epsilon RI expression in skin mast cells is slower and associated with decreased acute allergen wheal size.     

Kinetic analysis of the interaction between recombinant human Fc(epsilon)RIalpha and serum IgEs from allergic patients

Binding of IgE to the high-affinity IgE receptor (Fc(epsilon)RI) is the essential event for allergic reaction. Although there are many reports on binding kinetics between myeloma IgE and Fc(epsilon)RI, little is known about the kinetics between heterogeneous polyclonal IgE in the serum and Fc(epsilon)RIalpha. To elucidate the binding characteristics of heterogeneous serum IgE, we measured kinetic parameters of binding between IgE from allergic patients and a recombinant ectodomain of the human Fc(epsilon)RIalpha subunit by real-time interaction analysis based on surface plasmon resonance. Purified IgE monomer from the plasma of allergic patients displayed kinetics for the interaction with Fc(epsilon)RIalpha similar to those of myeloma IgE. In the case of crude IgE samples from allergic patients, one of seven specimens showed significantly higher affinity than highly purified IgE, suggesting that it is possible for IgEs in this specimen to form complexes of higher molecular weight.     

Isolation and characterization of IgE receptors from rat intestinal mucosal mast cells

High-(Fc epsilon RI) and low-(Fc epsilon RII) affinity IgE receptors were isolated from surface radioiodinated, Nonidet-P40-solubilized rat intestinal mucosal mast cells (IMMC) and compared with those on rat peritoneal mast cells (PMC) and rat basophilic leukemia (RBL) cells. Fc epsilon RII were isolated by affinity chromatography using IgE-Sepharose or by anti-Fc epsilon RII antisera and protein A-Sepharose. The surface-exposed, IgE-binding alpha subunits of Fc epsilon RI [Fc epsilon RI alpha] were isolated by affinity chromatography using IgE and anti-IgE-Sepharose. Fc epsilon RI alpha on IMMC had an apparent molecular mass of 59 kDa, somewhat larger than that of PMC (51 kDa), RBL-2H3 cells (51 kDa) or RBL-CA10.7 cells (46 kDa). Brief (45 s) incubation of IMMC or PMC in glycine-HCl, pH 3, prior to iodination removed much of the surface-bound IgE. This permitted more thorough labeling of the receptors, but had no affect on the estimate of receptor size. Surprisingly and in contrast to acid-treated PMC, upon anti-IgE-Sepharose isolation acid-treated IMMC yielded an intensely radioactive Fc epsilon RI alpha band in the absence of added IgE. Such a finding suggests that IMMC, more so than PMC, may have an intracellular store of IgE, as has been suggested by many others. IMMC also differed from PMC in the number of forms of Fc epsilon RII isolated; 50-kDa and 58-kDa forms of Fc epsilon RII were obtained from IMMC, whereas PMC yielded most often a single 56-kDa Fc epsilon RII band. These results were mimicked by the two RBL cell sublines: RBL-2H3 cells yielded two Fc epsilon RII (46 kDa and 55 kDa), but only one form of Fc epsilon RII (54-kDa) was obtained from RBL-CA10.7 cells. Thus, the two subtypes of rat mast cells, which have previously been shown to differ in mediator profile and responsiveness to secretagogues and antiallergic drugs, are also distinguished by differences in IgER profile.     

Role of the extracellular domain of Fc epsilon RI alpha in intracellular processing and surface expression of the high affinity receptor for IgE Fc epsilon RI

The high affinity receptor for immunoglobulin E, Fc epsilon RI, is a critical component of IgE-mediated allergic reactions. It is expressed as a tetramer (alphabetagamma(2)) made of an IgE-binding alpha chain and a signaling module formed by the beta chain and a dimer of gamma chains. It is expressed in humans and rodents on basophils and mast cells at a high level, and, upon activation, it induces the liberation of allergy mediators. In humans a trimeric form lacking the beta chain also exists (alphagamma(2)). This trimeric form is expressed on antigen presenting cells where it acts to facilitate antigen presentation via IgE. Both the expression and the signaling capacity of the trimer are lower than those of the tetramer. The differences between human (tetrameric and trimeric) and murine (tetrameric only) expression is explained in part by the fact that mouse alpha cannot be expressed at the cell surface in the absence of beta, while human alpha can. Here we demonstrate that the capacity of human alpha to be expressed at the cell surface in the absence of beta is encoded entirely in its extracellular domain. These findings show that the extracellular domain of the type I transmembrane protein Fc epsilon RI alpha plays a role in Fc epsilon RI intracellular processing and expression at the cell surface.     

Activation of mast cells by immunoglobulin E-receptor cross-linkage,but not through adenosine receptors,induces A1 expression and promotes survival

BACKGROUND: Mast cells are a potent source of mediators that regulate the inflammatory response in allergy and asthma. Mast cells can be activated through different receptors, for example, via cross-linkage of the high-affinity IgE receptor (Fc epsilon RI) and by adenosine acting on specific receptors. We have recently described mast cell survival of an IgE receptor activation by up-regulation of the anti-apoptotic gene A1. OBJECTIVE: To compare mast cell survival and expression of A1 after activation through the Fc epsilon RI and by an adenosine agonist. METHODS: Bone marrow-derived, cultured mouse mast cells (BMCMC) were activated either with IgE+antigen or with the adenosine receptor agonist 5'-N-ethylcarboxamido adenosine (NECA). Release of beta-hexosaminidase, cell viability, phosphorylation of Akt and IkB-alpha, and expression of pro-survival and pro-apoptotic genes were measured after activation. RESULTS: Activation of BMCMC with NECA caused the release of beta-hexosaminidase, although to a lesser extent than after Fc epsilon RI activation (33% and 98%, respectively). Activation by both NECA and Fc epsilon RI stimulated phosphorylation of Akt (Ser473 and Thr308) and IkB-alpha (Ser32), both of which are implicated in the regulation of cell survival. However, only cells that were activated through Fc epsilon RI, but not by NECA, expressed A1 and exhibited an increased survival rate compared to the control. CONCLUSION: These results show that adenosine receptor activation of BMCMC does not induce the same survival programme in mast cells as does activation through Fc epsilon RI. These findings may be important for understanding the role that mast cells play in asthma provoked by different stimuli.     

Mapping of murine IgE epitopes involved in IgE-Fc epsilon receptor interactions

The generation of anti-IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (Fc epsilon R) interaction site has been determined using cross-inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the Fc epsilon R interaction site and the binding sites of various anti-IgE mAb, we employed site-directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant epsilon heavy chain (C epsilon) gene bearing a (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH region. Several site-specific mutants in the C epsilon 3 and C epsilon 4 domains of this recombinant C epsilon gene were prepared and expressed by transfection into the light chain-producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti-IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the C epsilon 3 domain, a mutant with a truncated C epsilon 4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C epsilon 3, and a chimeric human C epsilon in which the human C epsilon 3 was replaced by the homologous mouse C epsilon 3 domain. These mutants have permitted the localization, to the C epsilon 3 domain, of the epitopes recognized by the 84.1C and 95.3 anti-IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the Fc epsilon R binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc epsilon R binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE-Fc epsilon R interaction, has been mapped to the C epsilon 4 domain. When tested for binding to the Fc epsilon R on RBL-2H3 cells, the point mutant bound to the Fc epsilon R with twofold reduced affinity, while the C epsilon 3 deletion mutant and the mutant truncated in C epsilon 4 lost all receptor binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)