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.     

A comparison of mediators released or generated by IFN-gamma-treated human mast cells following aggregation of Fc gamma RI or Fc epsilon RI

The high affinity receptor for IgG (Fc gamma RI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-gamma and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-gamma-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of Fc gamma RI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via Fc epsilon RI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-gamma-treated cultured human mast cells after Fc gamma RI or Fc epsilon RI aggregation. Aggregation of Fc gamma RI resulted in histamine release and PGD(2) and LTC(4) generation. These responses were qualitatively indistinguishable from responses stimulated via Fc epsilon RI. Aggregation of Fc epsilon RI or Fc gamma RI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of Fc gamma RI compared with Fc epsilon RI. TNF-alpha mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-alpha data using IFN-gamma-treated purified human lung mast cells. Thus, aggregation of Fc gamma RI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-alpha, were released and generated to a greater degree after Fc gamma RI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through Fc gamma RI in an IFN-gamma-rich environment.     

Immobilization and internalization of mutated IgE receptors in transfected cells.

Earlier studies have shown that the mast cell receptor IgE (Fc epsilon RI) for is expressed on COS-7 cells transfected with the cDNA for each of the three types of subunits that form the tetrameric, alpha beta gamma 2, receptor. Although such transfected COS cells fail to exhibit some of the early biochemical perturbations initiated by aggregation of the receptor on normal mast cells and related tumor lines, we show here that other characteristics of the endogenous Fc epsilon RI are retained. Thus, the unaggregated transfected wild-type receptors were found to have a restricted translational diffusion similar to that observed for endogenous receptors on mast cells as assessed by fluorescence photobleaching and recovery. Similarly, as with endogenous receptors the mobility of transfected receptors was sharply reduced when the receptors were aggregated by reaction with small oligomers of IgE. In addition, aggregation of the transfected Fc epsilon RI caused them to be internalized by the COS cells by a cytochalasin-sensitive mechanism, albeit at a considerably slower rate than was seen with endogenous receptors on mast cells or with transfected receptors in a line of receptor-deficient mast cells. We also examined the mobility and internalization before and after aggregation, of some 13 different combinations of receptor subunit mutants in which one or more of the five cytoplasmic domains of the receptor had been truncated. Our results show that whatever interactions between the receptor and cellular components may account for the phenomena we studied, such interactions do not critically depend upon the bulk of the cytoplasmic domains of the receptor.     

Transmembrane signaling in P815 mastocytoma cells by transfected IgE receptors

In order to delineate structural-functional relationships of the mast cell receptor for IgE (Fc epsilon RI) by molecular-genetic analysis, a transfectable cell must be identified which resembles mast cells except for being deficient in receptors. We have found that the well known murine mastocytoma P815 is suitable. These cells express no Fc epsilon RI, lack mRNA for the alpha and beta subunits of the receptor, but contain some mRNA for gamma chains. After transfection with the cDNA for each of the subunits, stable clones could be isolated which expressed several hundred thousand normal Fc epsilon RI and synthesized large amounts of mRNA for alpha, beta, and gamma, the last at 3-fold higher levels than in the untransfected cells. Aggregation of the transfected receptors led to opening of presumptive calcium channels and to activation of phospholipase C, phospholipase A2, and protein kinase C. The kinetics and other characteristics of the signals were similar to those observed after stimulation of the rat tumor mast cells from which the receptor genetic material had been derived but were smaller in magnitude. These weaker signals most likely result from an overall reduced reactivity exhibited by the P815 cells since stimulation by other ligands led to weaker or even no responses. The cells failed to degranulate after either receptor aggregation or reaction with ionophores with or without phorbol ester. Both the transfected and untransfected P815 cells express Fc receptors for IgG (Fc gamma RII) which, interestingly, independently triggered similar responses despite their apparently simpler subunit structure.     

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.     

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.     

Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon tyrosine activation motifs in RBL-2H3 cells.     

A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation

Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.     

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.     

Murine recombinant Fc gamma RIII, but not Fc gamma RII, trigger serotonin release in rat basophilic leukemia cells.

Murine Fc gamma RII and Fc gamma RIII have highly homologous extracellular domains, but unrelated transmembrane and intracytoplasmic (IC) domains. Murine Fc gamma RIIb1 and b2 are two isoforms of single-chain receptors which differ only by 47 aa in their IC domain. Murine Fc gamma RIII are composed of an IgG-binding alpha-chain, the intracellular portion of which is unrelated to that of Fc gamma RII, and of a homodimeric gamma-chain which also associates with Fc epsilon RI. Murine mast cells express Fc gamma RII, Fc gamma RIII, and Fc epsilon RI. They can be induced to degranulate by murine IgG immune complexes or by F(ab')2 fragments of the rat anti-murine Fc gamma RII/III mAb 2.4G2, complexed to mouse anti-rat (MAR) F(ab')2. In order to determine which murine Fc gamma R can activate mast cells, cDNA encoding murine Fc gamma RIIb1, Fc gamma RIIb2 or Fc gamma RIII alpha were stably transfected into RBL-2H3 cells. Murine Fc gamma RIII but not Fc gamma RIIb1 or Fc gamma RIIb2 induced serotonin release when aggregated by (2.4G2-MAR) F(ab')2 complexes. The respective roles of the IC domains of murine Fc gamma RIII subunits in signal transduction were investigated by stably transfecting cDNA encoding IC-deleted or chimeric murine Fc gamma R into RBL-2H3 cells. The substitution of the IC domain of murine Fc gamma RII for that of murine Fc gamma RIII gamma, but not that of murine Fc gamma RIII alpha, conferred the ability to trigger serotonin release. The deletion of IC sequences of the alpha subunit did not alter the ability of murine Fc gamma RIII to trigger serotonin release. It follows that 1) murine Fc gamma RIII, but not Fc gamma RII, can induce RBL cells to release serotonin, 2) the aggregation of the IC domain of the murine Fc gamma RIII gamma subunit is sufficient, but 3) the IC domain of the murine Fc gamma RIII alpha subunit is neither sufficient nor necessary for triggering serotonin release.     

IgE-mediated activation of NK cells through Fc gamma RIII

NK cells express Fc gamma RIII (CD16), which is responsible for IgG-dependent cell cytotoxicity and for production of several cytokines and chemokines. Whereas Fc gamma RIII on NK cells is composed of both Fc gamma RIII alpha and FcR gamma chains, that on mast cells is distinct from NK cells and made of Fc gamma RIII alpha, FcR beta, and FcR gamma. Mast cells show degranulation and release several mediators, which cause anaphylactic responses upon cross-linking of Fc gamma RIII as well as Fc epsilon RI with aggregated IgE. In this paper, we examined whether IgE activates NK cells through Fc gamma RIII on their cell surface. We found that NK cells produce several cytokines and chemokines related to an allergic reaction upon IgE stimulation. Furthermore, NK cells exhibited cytotoxicity against IgE-coated target cells in an Fc gamma RIII-dependent manner. These effects of IgE through Fc gamma RIII were not observed in NK cells from FcR gamma-deficient mice lacking Fc gamma RIII expression. Collectively, these results demonstrate that NK cells can be activated with IgE through Fc gamma RIII and exhibit both cytokine/chemokine production and Ab-dependent cell cytotoxicity. These data imply that not only mast cells but also NK cells may contribute to IgE-mediated allergic responses.     

Peroxisome proliferator-activated receptor ligands negatively regulate the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells

The high-affinity IgE receptor Fc epsilon RI is expressed on the cell surface of mast cells and basophils, and plays a central role in IgE-mediated inflammatory reactions. Recently, peroxisome proliferator-activated receptors (PPARs) have been implicated in the anti-inflammatory response. To investigate a possible role for PPAR in human basophils, the effect of PPAR ligands on Fc epsilon RI expression in human basophilic KU812 cells was studied. The PPARalpha ligand, leukotriene B(4), did not affect the cell surface expression of Fc epsilon RI. However, prostaglandin (PG) A(1) and 15-deoxy-Delta(12,14) PGJ(2) (15d-PGJ(2)), which are PPARbeta and gamma ligands, respectively, were both able to decrease Fc epsilon RI expression. Treatment with PGA(1) or 15d-PGJ(2) separately also reduced histamine release from KU812 cells in response to cross-linkage of Fc epsilon RI. In addition, RT-PCR analysis showed that KU812 cells expressed the mRNA for PPARalpha, beta, and gamma, indicating that PPARbeta or gamma may negatively regulate the cell activation via Fc epsilon RI. Cells treated with 15d-PGJ(2) expressed lower levels of Fc epsilon RI alpha and gamma mRNA, and PGA(1) treatment decreased the level of Fc epsilon RI gamma mRNA. These results suggest that the suppression of Fc epsilon RI expression by PPARs may be due to the down-regulation of Fc epsilon RI alpha or gamma mRNA.     

The phospholipase C gamma 1-dependent pathway of Fc epsilon RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.     

Expression of a functional high-affinity IgG receptor, Fc gamma RI, on human mast cells: Up-regulation by IFN-gamma

Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor Fc epsilon RI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor Fc gamma RI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for Fc gamma RI. We next found that IFN-gamma up-regulated the expression of Fc gamma RI. This was confirmed by flow cytometry, where Fc gamma RI expression on human mast cells was increased from approximately 2 to 44% by IFN-gamma exposure. Fc epsilon RI, Fc gamma RII, and Fc gamma RIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4-5 x 108 M-1) increased from approximately 2,400 to 12,100-17,300 per cell. Aggregation of Fc gamma RI on human mast cells, and only after IFN-gamma exposure, led to significant degranulation as evidenced by histamine release (24.5 +/- 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-alpha, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases.     

Signal transduction by the cytoplasmic domains of Fc epsilon RI-gamma and TCR-zeta in rat basophilic leukemia cells.

The gamma subunit of the high affinity IgE receptor, Fc epsilon RI, is a member of a family of proteins which form disulfide-linked dimers. This family also includes the zeta- and eta-chains of the T cell receptor. Engagement of Fc epsilon RI activates src-related protein tyrosine kinases in basophils and mast cells. However, the role of individual subunits of Fc epsilon RI in this activation is still not known. In an effort to determine the function of Fc epsilon RI-gamma, we used chimeric proteins containing the extracellular and transmembrane domains of the alpha chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domains of either T cell receptor-zeta or Fc epsilon RI-gamma. We show that while cross-linking of the Tac chimeras in the rat basophilic leukemia cell line RBL-2H3 resulted in the tyrosine phosphorylation of a subset of proteins and a portion of the degranulation normally observed after Fc epsilon RI-mediated stimulation, no detectable activation of p56lyn or pp60c-src was observed. In contrast, an apparent transient deactivation of these two kinases was observed after Tac chimera cross-linking. These observations suggest that Fc epsilon RI-gamma is responsible for some, but not all, of the signaling that occurs after engagement of its receptor, and that other receptor subunits may also play important roles in this signaling process.     

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.     

Intracellular expression and release of Fc epsilon RI alpha by human eosinophils.

Although Fc epsilon R have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, Fc epsilon RI, and Fc epsilon RII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance Fc epsilon R on other cells) failed to induce any detectable surface Fc epsilon R. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for Fc epsilon RI alpha showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR gamma-chain, but no Fc epsilon RI beta. Surface biotinylation followed by immunoprecipitation again failed to detect surface Fc epsilon RI alpha, although surface FcR gamma was easily detected. Since we were able to detect intracellular Fc epsilon RI alpha, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of Fc epsilon RI alpha into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of Fc epsilon RI alpha that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of Fc epsilon RI alpha remains to be determined.     

Functional comparison of Fc epsilon RI, Fc gamma RII, and Fc gamma RIII in mast cells.

The cellular responses initiated by cross-linking rodent Fc gamma RII-b1, Fc gamma RII-b2, Fc gamma RIII, and Fc epsilon RI in mast cells were compared. Individual murine Fc gamma R isoforms were transfected into rat basophilic leukemia cells and after cross-linking the FcR, changes in the phosphorylation of protein tyrosines, in the level of intracellular Ca2+, in the hydrolysis of phosphoinositides, and in the release of arachidonic acid metabolites and hexosaminidase were monitored. Cross-linking of Fc gamma RIII initiated all of these early and late biochemical functions, and although they were quantitatively somewhat smaller, the responses were qualitatively indistinguishable from those stimulated by the endogenous Fc epsilon RI. However, despite ample expression, neither Fc gamma RII-b1 nor Fc gamma RII-b2 stimulated these functions when cross-linked. The functional differences between Fc gamma RII and Fc gamma RIII were studied further by assessing the responses to cross-linking of the endogenous Fc gamma R (Fc gamma RII-b1, Fc gamma RII-b2, and Fc gamma RIII) on P815 mouse mastocytoma cells that had been transfected with normal or functionally defective Fc epsilon RI. Two types of mutant subunits had previously been observed to impair the activity of Fc epsilon RI: gamma-chains missing the cytoplasmic domain, and beta-chains missing the COOH-terminal cytoplasmic domain. In both types of transfectants the functional inhibition of the endogenous Fc gamma R paralleled that of the transfected Fc epsilon RI. These results are consistent with the gamma subunit being associated with the functions of Fc gamma RIII as well as of Fc epsilon RI. The functional results also complement the recently reported evidence that Fc gamma RIII can interact with Fc epsilon RI beta-subunits (J. Exp. Med. 175:447, 1992).