Fc Epsilon RI Alpha Research Articles

Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor

The Fc-region of immunoglobulin E (IgE) comprising C epsilon 2, C epsilon 3, and C epsilon 4 domains is sufficient for binding to the alpha chain of the high affinity IgE-Fc receptor (Fc epsilon RI alpha). In order to identify the smallest Fc fragment capable of binding to the Fc epsilon RI alpha with high affinity, various regions of the IgE-Fc molecule were expressed in COS cells and investigated for their ability to bind Fc epsilon RI alpha. The smallest fragment that showed Fc epsilon RI alpha binding activity spans amino acids 329-547 and lacks the entire C epsilon 2 domain. Two active fragments, viz. Fc epsilon(315-547) (containing Cys328 which is responsible for interchain S-S bonding) and Fc epsilon(329-547), have been overexpressed in CHO cells and purified to homogeneity. The purified proteins bind to the Fc epsilon RI alpha with high affinity, similar to native IgE. SDS-polyacrylamide gel electrophoresis analyses indicate that Fc epsilon(315-547) is an S-S-linked dimer of apparent molecular mass of 68 kDa. Fc epsilon(329-547) appears on SDS-gel as three distinct bands at approximately 32 kDa, both under reducing and nonreducing conditions. However, size exclusion chromatography and analytical ultracentrifugation studies suggest that Fc epsilon(329-547) also remains associated as a dimer. The presence of N-linked glycosylation was detected in both proteins. The deglycosylated form of Fc epsilon(315-547) was isolated after Endo F/N-glycosidase F digestion and demonstrated to have binding activity comparable to that of the mock-digested protein. These results suggest that the presence of N-linked sugars is not necessary for Fc epsilon RI alpha binding. Both proteins blocked the release of histamine from RBL cells expressing human Fc epsilon RI alpha in a dose-dependent manner. The availability of these recombinant IgE-Fc proteins will be helpful in elucidating the key epitopes essential for the binding of IgE to its high affinity receptor.

Soluble human high-affinity receptor for IgE abrogates the IgE-mediated allergic reaction.

The high-affinity receptor for IgE (Fc epsilon RI) has a tetrameric structure structure composed of one alpha, one beta, and two disulfide-linked gamma subunits, of which the alpha subunit binds IgE with high affinity. A recombinant soluble form of the ectodomain of the human Fc epsilon RI alpha subunit (rsFc epsilon RI alpha) was recently generated by gene engineering and was verified to bind IgE with an affinity as high as that of native Fc epsilon RI on the cell surface. rsFc epsilon RI alpha was prepared on a large scale in order to analyze its biological function. rsFc epsilon RI alpha completely inhibited IgE binding to the cell surface, resulting in abrogation of the chemical mediator release from RBL-2H3 cells. Furthermore it completely abolished the passive cutaneous anaphylaxis (PCA) response by trapping IgE specifically when it was administered into rats prior to IgE sensitization. Even after IgE sensitization, treatment of rsFc epsilon RI alpha substantially reduced the PCA response. It was finally shown that rsFc epsilon RI alpha inhibited IgE binding to human peripheral blood basophils and the histamine release from them. In this paper we address the ability of rsFc epsilon RI alpha to specifically prevent the IgE-mediated allergic reaction.

Conservation of signal transduction mechanisms via the human Fc epsilon RI alpha after transfection into a rat mast cell line, RBL 2H3.

The high affinity receptor for IgE (Fc epsilon RI) is present on mast cells and basophils, and the aggregation of IgE-occupied receptors by Ag is responsible for the release of allergic mediators. The Fc epsilon RI is composed of at least three different subunits, alpha, beta, and gamma, with the alpha subunit binding IgE. The series of biochemical events linking receptor aggregation to the release of mediators has not been fully delineated. As a step towards understanding these processes, and for the development of functional cell lines, we have transfected the human Fc epsilon RI alpha subunit into the rat mast cell line RBL 2H3. These human Fc epsilon RI alpha-transfected cell lines have been characterized with respect to the association of the human alpha subunit with endogenous rat beta and gamma subunits and the ability of aggregated Fc epsilon RI alpha subunits to mediate a variety of biochemical events. The signal transduction events monitored include phosphoinositide hydrolysis, Ca2+ mobilization, tyrosine phosphorylation, histamine release, and arachidonic acid metabolism. In all cases, the events mediated by aggregating human Fc epsilon RI alpha subunits were indistinguishable from those produced via the rat Fc epsilon RI alpha. These results demonstrate that the human Fc epsilon RI alpha subunit can functionally substitute for the rat Fc epsilon RI alpha subunit during signal transduction. The availability of this cell line will provide a means of evaluating potential Fc epsilon RI antagonists.

Epidermal Langerhans cells from normal human skin bind monomeric IgE via Fc epsilon RI.

Human epidermal Langerhans cells (LC) bearing IgE are found in disease states associated with hyperimmunoglobulinemia E. When studying the mechanism(s) underlying this phenomenon, immunohistology revealed that a majority of epidermal LC from normal skin of healthy individuals can specifically bind monomeric IgE. IgE binding to LC could neither be prevented by preincubation of the tissue with monoclonal antibodies (mAb) against either Fc epsilon RII/CD23 or Fc gamma RII/CD32, nor by the addition of lactose. However, binding could be entirely abrogated by preincubation with the anti-Fc epsilon RI alpha mAb 15-1, which interferes with IgE binding to Fc epsilon RI alpha gamma transfectants. These observations indicated that IgE binding to epidermal LC is mediated by Fc epsilon RI rather than by CD23, CD32, or the D-galactose-specific IgE-binding protein. This assumption gained support from our additional findings that: (a) the majority of LC exhibited distinct surface immunolabeling with the anti-Fc epsilon RI alpha mAbs 15-1 and 19-1, but not with any of eight different anti-Fc epsilon RII/CD23 mAbs; and (b) transcripts for the alpha, beta, and gamma chains of Fc epsilon RI could be amplified by polymerase chain reaction from RNA preparations of LC-enriched, but not of LC-depleted, epidermal cell suspensions. In view of the preeminent role of Fc epsilon RI crosslinking on mast cells and basophils in triggering the synthesis and release of mediators of allergic reactions, the demonstration of this receptor on epidermal LC may have important implications for our understanding of allergic reactions after epicutaneous contact with allergens.

Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI).

It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast cells and basophils. Epidermal LC react with antibodies specific for the alpha subunit of the tetrameric (alpha, beta, 2 gamma) Fc epsilon RI. Specific transcripts for Fc epsilon RI alpha and Fc epsilon RI gamma were detected in LC and correspond to those of human basophils and of the human basophil cell line KU812. Furthermore, human basophils, KU812 cells, and LC express the putative beta subunit. Thus human LC express the complete structure of Fc epsilon RI. This finding opens new perspectives in the putative functional role of this structure on antigen-presenting cells.

Association of the human Fc epsilon RI gamma subunit with novel cell surface polypeptides.

It has recently been demonstrated that the gamma-subunits of the high affinity Fc epsilon RI are required for the cell surface expression of not only the Fc epsilon RI alpha-subunit, but also for the low affinity Fc gamma RIIIA alpha (CD16). In addition, formation of heterodimeric complexes of the gamma-subunit with the zeta- and eta-chains of the TCR have also been reported. The exact role of the gamma-subunit in the function of these receptors is not known. To gain additional insight into the association of the gamma-subunit with these and other cell surface polypeptides, we have generated a mAb, 4D8, directed against the human Fc epsilon RI gamma-subunit. Using this antibody we have been able to demonstrate that Fc epsilon RI alpha and Fc gamma RIIIA alpha are associated with Fc epsilon RI gamma at the cell surface. Furthermore, we have identified the expression of Fc epsilon RI gamma in HL60 and U937 cells, which are negative for the TCR, Fc epsilon RI, and Fc gamma RIII. Analysis of these cells reveals the presence of novel Fc epsilon RI gamma-associated polypeptides. These results suggest that Fc epsilon RI gamma plays a common functional role in a number of different receptor complexes. The availability of the anti-gamma antibody 4D8 will help to define this role, and allow the characterization of cell surface polypeptides that are associated with the Fc epsilon RI gamma-subunit.

Structure-function relationships in the mast cell high affinity receptor for IgE. Role of the cytoplasmic domains and of the beta subunit.

To define functionally critical regions of the high affinity receptor for IgE (Fc epsilon RI), we stably transfected P815 cells with mutated cDNAs coding for subunits with truncated cytoplasmic domains (CD). In addition, to examine further the role of the beta subunit, stable transfectants expressing chimeric Fc epsilon RI without beta subunits were generated. Transfectants were tested for receptor-mediated changes in intracellular Ca2+, for stimulated hydrolysis of phosphoinositides, and for protein tyrosine phosphorylation. In all cases these biochemical signals were affected coordinately, suggesting that they are coupled, possibly in a single pathway. Truncation of the alpha subunit or of the NH2-terminal CD of the beta subunit had no effect, but Fc epsilon RIs with beta subunits missing the COOH-terminal CD were inactive. Interestingly, receptors in cells transfected only with human Fc epsilon RI(alpha) (which utilize the gamma chains endogenously synthesized by the P815 cells but which contain no beta subunits) responded normally. Therefore, the beta subunit influences the functions studied but is not essential. Although structural analysis excluded a straightforward mechanism, truncation of the CD of the gamma chain led to loss of signaling.

Rat CD16 is defined by a family of class III Fc gamma receptors requiring co-expression of heteroprotein subunits.

Rat CD16 is defined here by a family of highly homologous class III Fc gamma receptor isoforms. Northern blot and polymerase chain reaction analysis indicate that multiple rtFc gamma RIII alpha isoforms are expressed by rat NK and macrophages contrasting the expression of a single class III receptor isoform by human and mouse NK and macrophages. Analysis of genomic Southern blots and splice variants identified by polymerase chain reaction suggests the existence of several homologous rat Fc gamma RIII alpha genes organized similar to human and mouse class III receptor genes. All rat Fc gamma RIII alpha isoform protein sequences have conventional transmembrane insertion sequences containing the unique LFAVDTGL motif conserved in all other class III Fc gamma and Fc epsilon RI alpha receptor sequences. A model is proposed for the protein-protein interactions between this sequence and the transmembrane sequences of two heteroprotein subunits, Fc epsilon RI gamma and CD3 zeta, known to interact with human and mouse class III receptors. Rat NK, monocytes, and neutrophils were all found to express transcripts for both of these subunits, whereas rat macrophages express only Fc epsilon RI gamma subunit transcripts. Furthermore, the Fc epsilon RI gamma subunit was found to promote the cell surface expression of rtFc gamma RIII alpha isoforms in transfected COS cells.

Cytokine mRNA are preferentially increased relative to secretory granule protein mRNA in mouse bone marrow-derived mast cells that have undergone IgE-mediated activation and degranulation.

The levels of mRNA that encode a number of cytokines have been reported by several laboratories to be increased in mouse mast cells after their IgE-bearing receptors have been cross-linked with Ag. In this study, we have compared the mRNA levels for Fc epsilon RI alpha, three cytokines (IL-6, granulocyte-macrophage CSF, and TNF-alpha), actin and three secretory granule-localized proteins (carboxypeptidase A, proteoglycan peptide core, and a generic serine protease) in mouse bone marrow-derived mast cells (BMMC) before and after IgE-mediated activation and degranulation to determine the kinetics and specificity of mRNA induction. An antigen concentration of approximately 10 ng/ml was optimal for the release of histamine from IgE-sensitized BMMC and for the generation and release of a cytokine that was functionally and immunochemically identical to TNF-alpha. In kinetic experiments, the levels of TNF-alpha, IL-6, and granulocyte-macrophage CSF mRNA increased greater than 23-fold 0.5 to 1 h after activation. As assessed by in situ hybridization, virtually all BMMC contained detectable proteoglycan peptide core mRNA before and after exposure to Ag, but only approximately one-half of the Ag-treated cells in the culture contained IL-6 mRNA 1 h after activation. There was a slight transient increase at 4 h in the level of proteoglycan peptide core mRNA, but no increase in the levels of those highly expressed mRNA that encode actin, Fc epsilon RI alpha, carboxypeptidase A, and serine protease. Thus, despite the remarkable increment in the levels of the transcripts that encode cytokines in BMMC after IgE-mediated, Ag-dependent activation, the levels of those transcripts that encode a plasma membrane-localized recognition receptor and several constituents of the secretory granule remain essentially unchanged. The failure to increase substantially the level of protease and proteoglycan peptide core mRNA in mast cells after the activation/secretion response suggests that regranulation of mast cells is a slow process.

CD3 zeta subunit can substitute for the gamma subunit of Fc epsilon receptor type I in assembly and functional expression of the high-affinity IgE receptor: evidence for interreceptor complementation.

The high-affinity receptor for IgE (Fc epsilon RI) is a four-subunit structure consisting of three distinct polypeptides: the IgE-binding alpha chain, the four-fold membrane-spanning beta chain, and the disulfide-linked gamma-gamma homodimer. cDNAs encoding each subunit have previously been isolated. Here we show that microinjection of Xenopus oocytes with a mixture of in vitro transcribed RNAs encoding each subunit results in expression of IgE receptors at the oocyte surface as detected by binding of IgE or anti-Fc epsilon RI alpha subunit monoclonal antibody to intact oocytes. Surface expression of Fc epsilon RI requires injection of all three subunits (alpha, beta, and gamma) RNAs. In particular, omission of Fc epsilon RI gamma RNA from the mixtures abolishes surface binding of either IgE or anti-Fc epsilon RI alpha monoclonal antibody to microinjected oocytes. However, addition of CD3 zeta RNA to Fc epsilon RI alpha and Fc epsilon RI beta RNAs restores IgE receptor surface expression when this combination is microinjected into oocytes. Metabolic labeling and immunoprecipitation of oocyte microinjected with a mixture of CD3 zeta plus Fc epsilon RI alpha and Fc epsilon RI beta RNAs reveals a noncovalent association between the CD3 zeta-zeta disulfide-linked homodimer and Fc epsilon RI alpha-beta. These results provide direct evidence for the functional relatedness of CD3 zeta and Fc epsilon RI gamma.

Characterization of rat tissue cultured mast cells

Twelve continuous rat tissue cultured mast cell (MC) lines were established by prolonged culture of rat peritoneal MC in the absence of added factors or feeder layers. Two of these lines, RCMC1 and RCMC2, have been briefly described previously, seven others are now also described. Both RCMC1 and RCMC2 lack a marker chromosomes present on RBL-CA10.7 cells. All lines were found to express the phenotype of mucosal MC as defined by alcian blue-positive and safranin O-negative staining, the presence of rat MC protease II and a low histamine content. When analyzed for high-(Fc epsilon RI) and low-affinity (Fc epsilon RL) receptors for IgE, the various lines yielded a variety of receptor patterns. Northern blot analysis of the RNA of RCMC1, RCMC2 and RBL-CA10.7 revealed that all three cell lines contained the same mRNA species for the alpha, beta and gamma subunits for Fc epsilon RI previously found in another rat basophilic leukemia cell line. Quantitation of the relative amounts of alpha, beta and gamma mRNA did not correlate with the expression of the relative amounts of Fc epsilon RI(alpha) in these cells. The relative amounts of mRNA for all these subunits of RCMC2 were equal or higher than those of RCMC1, suggesting that the low expression of Fc epsilon RI(alpha) on the former was a consequence of post-transcriptional events. Analysis of a RCMC1 clone over a 6-month period revealed changes in the expression of both Fc epsilon RI(alpha) and Fc epsilon RL.

Gene mapping of the three subunits of the high affinity FcR for IgE to mouse chromosomes 1 and 19.

The high affinity receptor for IgE (Fc epsilon RI) is a tetrameric structure consisting of a single IgE-binding alpha subunit, a single beta subunit, and two disulfide-linked gamma subunits. The alpha subunit of Fc epsilon RI and most Fc receptors are homologous members of the Ig superfamily. By contrast, the beta and gamma subunits from Fc epsilon RI are not homologous to the Ig superfamily. The gamma-chains do share a region of high homology with the zeta-chain of the TCR. No homology has been found to date for beta with any published sequence. Here, we report that a single copy gene encodes Fc epsilon RI beta and that the locus for Fc epsilon RI beta is found on mouse chromosome 19, genetically linked to the Ly-1 (Ly-12) locus and in a region that also contains Ly-10 and Ly-44 (CD20). Homology comparisons among these molecules reveal limited regions of homology between Fc epsilon RI beta and Ly-44 (CD20) as well as other striking similarities: both molecules have four putative transmembrane segments and a probably topology where both amino- and carboxytermini protrude into the cytoplasm. In addition, we show that a single gene for FC epsilon RI gamma is found at the distal end of mouse chromosome 1, clustered in a region where Fc epsilon RI alpha has also been linked to Fc gamma RII. At least one of the two forms of Fc gamma RII has recently been shown to contain gamma subunits identical to the gamma subunits of Fc epsilon RI. The close association of the genes for Fc epsilon RI alpha, FC gamma RII, and their shared gamma subunits raises interesting implications regarding coordinate regulation of gene expression.

Receptors for Fc epsilon and Fc gamma are linked on mouse chromosome 1.

Recently isolated cDNA clones for the high affinity Fc epsilon receptors on mast cells and basophils (Fc epsilon RI alpha) and Fc gamma receptors on macrophages and lymphocytes (Fc gamma 2b/gamma 1R) are homologous members of the Ig supergene family. Analysis of the segregation of restriction fragment length polymorphism in crosses of inbred mice now establish that the structural genes encoding both Fc epsilon RI alpha and Fc gamma 2b/gamma 1R are indeed discrete genes and are linked at the distal end of mouse chromosome 1. This finding raises the possibility that a family of Fc receptors could be found in a region that is known to contain immunologically important markers of lymphocyte surface Ag and autoimmune defects.