High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies.

The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha-subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha-protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha-protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha-protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE.     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.     

Murine follicular dendritic cells and low affinity Fc receptors for IgE (Fc epsilon RII).

The present study was undertaken to determine whether mouse follicular dendritic cells (FDC) bear Fc epsilon RII (CD23) and whether IgE-immune complexes are retained by FDC. Mouse Fc epsilon RII was localized by both L and electron microscopy using the mAb B3B4. In lymph nodes of normal mice, Fc epsilon RII was low but detectable on FDC. By 14 days after Nippostrongylus brasiliensis infection, the level of Fc epsilon RII increased on B lymphocytes located in the cortex of draining mesenteric lymph nodes. However, the Fc epsilon RII level on FDC remained low. Although numerous IgE-producing plasma cells were seen at day 14, very little IgE was associated with FDC. By 26 days after infection, Fc epsilon RII was observed on FDC in increased levels and IgE binding was clearly associated with FDC. Unexpectedly, FDC of control mice immunized with albumin in CFA to elicit an IgG response showed intense labeling for Fc epsilon RII. In contrast, the B cells exhibited very little Fc epsilon RII. IgE immune complexes were observed in association with FDC in the CFA-immunized mice. When mice were given a hapten-specific monoclonal of the IgE isotype, hapten carrier complexes were trapped and retained on Fc epsilon RII-bearing FDC. In conclusion, FDC were clearly one of the major murine cell types bearing Fc epsilon RII. IgE immune complexes were found in association with FDC and Fc epsilon RII appeared to play a major role in trapping and retaining IgE immune complexes. FDC Fc epsilon RII was subject to regulatory control, but the Fc epsilon RII level on FDC was regulated very differently from the Fc epsilon RII level on B cells.     

A cell surface glycoprotein of rat basophilic leukemia cells close to the high affinity IgE receptor (Fc epsilon RI). Similarity to human melanoma differentiation antigen ME491.

A monoclonal antibody (mAb), AD1, was isolated that recognized a cell surface protein on rat basophilic leukemia cells (RBL-2H3). At high concentration, this antibody inhibited IgE-mediated but not calcium ionophore-induced histamine release (49% inhibition at 100 micrograms/ml). The mAb AD1 did not inhibit the binding of IgE or of several antibodies directed to the high affinity IgE receptor (Fc epsilon RI). Likewise, IgE did not inhibit mAb AD1 binding. However, several anti-Fc epsilon RI antibodies did inhibit mAb AD1 binding as intact molecules but not as Fab fragments. Therefore, the sites on the cell surface to which mAb AD1 binds are close to Fc epsilon RI. The mAb AD1 immunoprecipitated a broad, 50-60-kDa band from 125I-surface-labeled RBL-2H3 cells that upon peptide N-glycosidase F treatment was transformed into a sharp 27-kDa band. A similar 27-kDa protein was immunoprecipitated from surface-radiolabeled cells after culture with tunicamycin. Thus, the protein recognized by mAb AD1 is highly glycosylated with predominantly N-linked oligosaccharides. The N-terminal sequence of 43 amino acids was found to be different from any subunit of Fc epsilon RI but nearly identical to that of the human melanoma-associated antigen ME491. Therefore, mAb AD1 binds to a surface glycoprotein on RBL-2H3 cells sterically close to the Fc epsilon RI but distinct from the recognized subunits of the receptor.     

The analysis of the human high affinity IgE receptor Fc epsilon Ri alpha from multiple crystal forms

We have solved the structure of the human high affinity IgE receptor, Fc epsilon RI alpha, in six different crystal forms, showing the structure in 15 different chemical environments. This database of structures shows no change in the overall shape of the molecule, as the angle between domains 1 and 2 (D1 and D2) varies little across the ensemble. However, the receptor has local conformational variability in the C' strand of D2 and in the BC loop of D1. In every crystal form, a residue inserts between tryptophan residues 87 and 110, mimicking the position of a proline from the IgE ligand. The different crystal forms reveal a distribution of carbohydrates lining the front and back surfaces of the structure. An analysis of crystal contacts in the different forms indicates regions where the molecule interacts with other proteins, and reveals a potential new binding site distal to the IgE binding site. The results of this study point to new directions for the design of molecules to inhibit the interaction of Fc epsilon RI alpha with its natural ligand and thus to prevent a primary step in the allergic response.     

Regulation of rat basophilic leukemia-2H3 mast cell secretion by a constitutive Lyn kinase interaction with the high affinity IgE receptor (Fc epsilon RI)

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the Fc epsilonRI beta subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on Fc epsilonRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of Fc epsilonRI beta and gamma subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating Fc epsilonRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the Fc epsilonRI beta subunit is a crucial step in the initiation of Fc epsilonRI signaling and that Lyn is limiting for Fc epsilonRI-induced secretion of inflammatory mediators.     

Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI.     

Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells

The high affinity receptor for IgE (Fc epsilon RI) found on mast cells and basophils is a tetrameric complex of a single alpha subunit, a single beta subunit, and two identical gamma subunits. The genes for the three subunits of mouse Fc epsilon RI have now been cloned from the mast cell line, PT18. When compared at the DNA level, the rat and mouse subunits are similarly conserved. However, at the protein level the homology between mouse and rat alpha is surprisingly low (71% identities) especially in the cytoplasmic regions (57% identities) which are of different length (25 and 20 residues, respectively). By contrast the beta and gamma are homogeneously conserved between mouse and rat (83 and 93% identities, respectively). The consensus amino acid sequence of the alpha subunit derived from three species (rat, mouse, and human) shows that the cytoplasmic tail diverges to the same extent as the leader peptide. Conversely, the transmembrane domain of the alpha is highly conserved and contains 10 consecutive residues that are identical. Comparisons between mouse Fc epsilon RI and other mouse proteins reveal regions of high homology between the alpha subunit and Fc gamma RIIa and between the gamma subunit and the zeta chain of the T cell receptor. Cells transfected with the alpha gene express the alpha subunit on their surface very inefficiently. Efficient expression is only achieved after co-transfection of the three rodent genes or of the human alpha gene together with the rodent gamma without apparent need for beta. The subunits are completely interchangeable upon transfection so that various chimeric mouse-rat-human receptors can be expressed.     

Characterization of the family of dimers associated with Fc receptors (Fc epsilon RI and Fc gamma RIII).

The receptor for IgE (Fc epsilon RI) is a multimeric complex containing one alpha chain, one beta chain with four transmembrane domains and one homodimer of disulfide-linked gamma-chains. The Fc epsilon RI gamma-chains form additional disulfide-linked dimers with the homologous zeta- and eta-chains, as part of the TCR complex. The low affinity receptor for IgG (Fc gamma RIII)2 on NK cells is also associated with zeta-chains. Here we show that the gamma-chain is expressed in NK cells both as a group of heterogenous gamma gamma homodimers and also as a heterodimer bound to zeta. Fc gamma RIIIA is associated with three types of dimers zeta zeta, gamma zeta, and notably gamma gamma as well. In fact, gamma gamma appears to be the predominant species associating with Fc gamma RIIIA. The surface expressed Fc epsilon RI also associates with the same group of heterogenous gamma gamma homodimers. We also show that there is no C-terminal posttranslational cleavage of gamma occurring before its insertion into the plasma membrane as previously suggested. Thus, like the TCR, Fc gamma RIIIA may form a variety of receptor isoforms, though at present we do not understand the functional implications of these structures.     

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.     

Characterization of the murine T cell receptor for IgE (Fc epsilon RII). Demonstration of shared and unshared epitopes with the B cell Fc epsilon RII

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.     

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.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.