Interaction of a monoclonal IgE-specific antibody with cell-surface IgE-Fc epsilon RI: characterization of equilibrium binding and secretory response

Aggregation of FcepsilonRI, the high-affinity cell-surface receptor for IgE antibody, is required for degranulation of basophils and mast cells, but not all receptor aggregates elicit this cellular response. The stereochemical constraints on clusters of FcepsilonRI that are able to signal cellular responses, such as degranulation, have yet to be fully defined. To improve our understanding of the properties of FcepsilonRI aggregates that influence receptor signaling, we have studied the interaction of 23G3, a rat IgG(1)(kappa) IgE-specific monoclonal antibody, with IgE-FcepsilonRI complexes on rat mucosal-type mast cells (RBL-2H3 line). We find that 23G3 is a potent secretagogue. This property and the structural features of 23G3 (two symmetrically arrayed IgE-specific binding sites) make 23G3 a potentially valuable reagent for investigating the relationship between FcepsilonRI clustering and FcepsilonRI-mediated signaling events. To develop a mathematical model of 23G3-induced aggregation of FcepsilonRI, we used fluorimetry and flow cytometry to quantitatively monitor equilibrium binding of FITC-labeled 23G3 intact Ab and its Fab' fragment to cell-surface IgE. The results indicate that IgE bound to FcepsilonRI expresses two epitopes for 23G3 binding; that 23G3 binds IgE resident on the cell surface with negative cooperativity; and that 23G3 appears to induce mostly but not exclusively noncyclic dimeric aggregates of FcepsilonRI. There is no simple relationship between receptor aggregation at equilibrium and the degranulation response. Further studies are needed to establish how 23G3-induced aggregation of IgE-FcepsilonRI correlates with cellular responses.     

Critical role of protein kinase C betaII in activation of mast cells by monomeric IgE

Accumulating evidence suggests that IgE-mediated activation of mast cells occurs even in the absence of antigen, which is referred to as "monomeric IgE" responses. Although monomeric IgE was found to induce a wide variety of responses, such as up-regulation of the FcepsilonRI, survival, cytokine production, histamine synthesis, and adhesion to fibronectin, it remains to be clarified how mast cells are activated in the absence of antigen. It has been controversial whether monomeric IgE responses are mediated by a similar signaling mechanism to antigen stimulation, although recent studies suggest that IgE can induce the FcepsilonRI aggregation even in the absence of antigen. In this study, we focused on the role of conventional protein kinase C (cPKC), since this response is suppressed by a specific inhibitor for cPKC. Monomeric IgE-induced Ca(2+) influx was not observed in a mouse mastocytoma cell line, which lacks the expression of PKCbetaII, although Ca(2+) influx induced by cross-linking of the FcepsilonRI was intact. Transfection of PKCbetaII cDNA was found to restore the Ca(2+) influx induced by monomeric IgE in this cell line. Furthermore, the dominant negative form of PKCbetaII (PKCbetaII/T500V) significantly suppressed the Ca(2+) influx, histamine synthesis, and interleukin-6 production in another mouse mast cell line, which is highly sensitive to monomeric IgE. Expression of PKCbetaII/T500V was found not to affect the antigen-induced responses. These results suggest that PKCbetaII plays a critical role in monomeric IgE responses, but not in antigen responses.     

Long term maintenance of IgE-mediated memory in mast cells in the absence of detectable serum IgE

Mast cells and basophils involved in allergic responses do not have clonotypic Ag receptors. However, they can acquire Ag specificity through binding of Ag-specific IgE to FcepsilonRI expressed on their surface. Previous studies demonstrated that IgE binding induced the stabilization and accumulation of FcepsilonRI on the cell surface and resulted in up-regulation of FcepsilonRI. In this study we have further analyzed the maintenance of IgE-mediated memory in mast cells and basophils in vivo by comparing kinetics of serum IgE levels, FcepsilonRI expression, and ability to induce systemic anaphylaxis. A single i.v. injection of trinitrophenyl-specific IgE induced 8-fold up-regulation of FcepsilonRI expression on peritoneal mast cells in B cell-deficient (micro m(-/-)) mice. Serum IgE levels became undetectable by day 6, but the treatment of mice with anti-IgE mAb induced a significant drop in body temperature on days 14, 28, and 42. The administration of trinitrophenyl -BSA, but not BSA, in place of anti-IgE mAb gave similar results, indicating the Ag specificity of the allergic response. This long term maintenance of Ag-specific reactivity in the allergic response was also observed in normal mice passively sensitized with IgE even though the duration was shorter than that in B cell-deficient mice. The appearance of IgE with a different specificity did not interfere with the maintenance of IgE-mediated memory of mast cells and basophils. These results suggest that IgE-mediated stabilization and up-regulation of FcepsilonRI enables mast cells and basophils not only to acquire Ag specificity, but also to maintain memory in vivo for lengthy periods of time.     

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.     

Human eosinophils and human high affinity IgE receptor transgenic mouse eosinophils express low levels of high affinity IgE receptor, but release IL-10 upon receptor activation.

FcepsilonRI expressed by human eosinophils is involved in IgE-mediated cytotoxicity reactions toward the parasite Schistosoma mansoni in vitro. However, because receptor expression is low on these cells, its functional role is still controversial. In this study, we have measured surface and intracellular expression of FcepsilonRI by blood eosinophils from hypereosinophilic patients and normal donors. The number of unoccupied receptors corresponded to approximately 4,500 Ab binding sites per cell, whereas 50,000 Ab binding sites per cell were detected intracellularly. Eosinophils from patients displayed significantly more unoccupied receptors than cells from normal donors. This number correlated to both serum IgE concentrations and to membrane-bound IgE. The lack of FcepsilonRI expression by mouse eosinophils has hampered further studies. To overcome this fact and experimentally confirm our findings on human eosinophils, we engineered IL-5 x hFcepsilonRIalpha double-transgenic mice, whose bone marrow, blood, spleen, and peritoneal eosinophils expressed FcepsilonRI levels similar to levels of human eosinophils, after 4 days culture with IgE in the presence of IL-5. Both human and mouse eosinophils were able to secrete IL-10 upon FcepsilonRI engagement. Thus, comparative analysis of cells from patients and from a relevant animal model allowed us to clearly demonstrate that FcepsilonRI-mediated eosinophil activation leads to IL-10 secretion. Through FcepsilonRI expression, these cells are able to contribute to both the regulation of the immune response and to its effector mechanisms.     

Nuclear import of N-terminal FAK by activation of the FcepsilonRI receptor in RBL-2H3 cells

As FAK integrates membrane receptor signalling, yet is also found in the nucleus, we investigated whether nuclear FAK is regulated by membrane receptor activation. Activation of the mast cell FcepsilonRI receptor leads to the release and synthesis of inflammatory mediators as well as increased proliferation and survival. Using RBL-2H3 basophilic leukaemia cells, FAK and the FcepsilonRI receptor were co-localised following cross-linking of IgE with antigen. This also resulted in a significant increase in the nucleus of several N-terminal FAK fragments, the largest of which included the kinase domain but not the focal adhesion targeting domain. This was confirmed using cells that stably expressed recombinant EGFP-FAK. Furthermore, treatment of EGFP-FAK expressing cells with Leptomycin B, an inhibitor of nuclear export, resulted in increased nuclear localisation of EGFP-FAK. Therefore, FAK can shuttle between the nuclear and cytoplasmic compartments and the cellular distribution of N-terminal FAK is regulated by membrane receptor activation.     

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody

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

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.     

A difference between epigallocatechin-3-gallate and epicatechin-3-gallate on anti-allergic effect is dependent on their distinct distribution to lipid rafts

The high-affinity IgE receptor FcepsilonRI expresses on the cell surface of mast cells and basophils, which is the key molecule in allergic reactions. We previously found that the major green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has the suppressive effect of the FcepsilonRI expression in the human basophilic KU812 cells, whereas (-)-epicatechin-3-O-gallate (ECG) has not. For understanding the mechanism of catechins, interactions of catechins with cellular membranes were investigated. Both catechins were shown to bind the cell surface of KU812 cells by surface plasmon resonance assay. EGCG highly associated with cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. On the other hand, the level of ECG in rafts was lower than that of EGCG, suggesting that the association with lipid rafts may have an important role in the FcepsilonRI-suppressive effect of catechins.     

Attenuation of IgE affinity for FcepsilonRI radically reduces the allergic response in vitro and in vivo

The high affinity of IgE for its receptor, FcepsilonRI (K(a) approximately 10(10) M(-1)), is responsible for the persistence of mast cell sensitization. Cross-linking of FcepsilonRI-bound IgE by multivalent allergen leads to cellular activation and release of pro-inflammatory mediators responsible for the symptoms of allergic disease. We previously demonstrated that limiting the IgE-FcepsilonRI interaction to just one of the two Cepsilon3 domains in IgE-Fc, which together constitute the high affinity binding site, results in 1000-fold reduced affinity. Such attenuation, effected by a small molecule binding to part of the IgE:FcepsilonRI interface or a distant allosteric site, rather than complete blocking of the interaction, may represent a viable approach to the treatment of allergic disease. However, the degree to which the interaction would need to be disrupted is unclear, because the importance of high affinity for immediate hypersensitivity has never been investigated. We have incorporated into human IgE a mutation, R334S, previously characterized in IgE-Fc, which reduces its affinity for FcepsilonRI approximately 50-fold. We have compared the ability of wild type and R334S IgE to stimulate allergen-induced mast cell activation in vitro and in vivo. We confirmed the expected difference in affinity between wild type and mutant IgE for FcepsilonRI (approximately 50-fold) and found that, in vitro, mast cell degranulation was reduced proportionately. The effect in vivo was also marked, with a 75% reduction in the passive cutaneous anaphylaxis response. We have therefore demonstrated that the high affinity of IgE for FcepsilonRI is critical to the allergic response, and that even moderate attenuation of this affinity has a substantial effect in vivo.     

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.     

Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of Fcepsilon RI and Syk in RBL cells

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.     

Does IgE bind to and activate eosinophils from patients with allergy?

Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcepsilonRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcepsilonRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcgammaRII, CD32), but no expression of FcepsilonRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcepsilonRI mAb, suggesting that eosinophils do express minimal amounts of FcepsilonRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express approximately 0.5% of the FcepsilonRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.     

gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization

We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis.     

IgE-enhancing activity directly and selectively affects activated B cells: evidence for a human IgE differentiation factor

T cells from highly atopic individuals spontaneously secrete in vitro a factor that specifically induces IgE synthesis from normal human B cells. We investigated the effects of such T cell supernatants derived from atopic individuals (TCSN-A) on functionally distinct B cell subsets to determine at what developmental stage B cells become responsive to this IgE-enhancing activity. B cells from normal and allergic donors were separated into subsets of small resting and large activated cells by density centrifugation or unit gravity sedimentation. When stimulated by TCSN-A, large activated B cells made more IgE than small resting B cells. The difference was as much as 3300% in comparing these subsets from allergic donors. Similarly, resting B cells stimulated by Staphylococcus aureus Cowan I (SAC) made 52 to 125% more IgE in response to TCSN-A than unstimulated small resting B cells. However, IgE production from large B cells, already activated in vivo, was not enhanced by the addition of SAC. Notably, the IgE level synthesized by in vivo large activated B cells from allergic persons was markedly greater than that seen with similar cells from normal donors, whereas resting B cells purified from allergic and normal donors produced comparable levels of IgE in response to TCSN-A. These results suggest that this enhancing activity functions as an IgE differentiation factor for activated B cells. This was further confirmed by the effects of TCSN-A on the IgM- and IgE-secreting EBV-transformed human B cell line K1D5. TCSN-A specifically enhanced IgE synthesis from these cells; TCSN from normal donors, IL 2, IFN-gamma, and BCGF did not. These results confirm that this activity functions as an IgE-specific differentiation factor, directly influencing activated B cells to synthesize IgE.     

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

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

A lipid raft-associated 67kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcepsilonRI expression

(-)-Epigallocatechin-3-O-gallate (EGCG), a major green tea polyphenol, has previously exhibited a suppressive effect on the expression of the high-affinity IgE receptor (FcepsilonRI). This effect has been shown to be elicited by interaction with the plasma membrane microdomain lipid rafts. Recently, we have identified the 67 kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates an anti-cancer action. Here we show that the 67LR is highly associated with lipid rafts on human basophilic KU812 cells. Experiments using 67LR-enhanced and -reduced cells revealed that the EGCG's ability to downregulate FcepsilonRI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the FcepsilonRI-suppressive action of EGCG.     

Fc epsilon RI gamma-ITAM is differentially required for mast cell function in vivo

The cross-linking of IgE-bound FcepsilonRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcepsilonRIgamma signaling to IgE/FcepsilonRI-mediated mast cell responses has not yet been elucidated. In this study FcepsilonRIgamma(-/-) mast cells were reconstituted with either wild-type or mutant FcepsilonRIgamma in transgenic mice and transfected mast cells in vitro. We demonstrate that FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcepsilonRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcepsilonRIgamma-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcepsilonRI is independent of FcepsilonRIgamma signaling. These results indicate that FcepsilonRIgamma-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.     

Mast cells, Fc epsilon RI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant

In certain models of allergic airway disease, mast cells facilitate the development of inflammation and airway hyper-responsiveness (AHR). To define the role of the high affinity IgE receptor (FcepsilonRI) in the development of AHR, mice with a disruption of the alpha subunit of the high affinity IgE receptor (FcepsilonRI(-/-)) were exposed on 10 consecutive days to nebulized OVA. Forty-eight hours after the last nebulization, airway responsiveness was monitored by the contractile response of tracheal smooth muscle to electrical field stimulation (EFS). After the 10-day OVA challenge protocol, wild-type mice demonstrated increased responsiveness to EFS, whereas similarly challenged FcepsilonRI(-/-) mice showed a low response to EFS, similar to nonexposed animals. Further, allergen-challenged FcepsilonRI(-/-) mice showed less airway inflammation, goblet cell hyperplasia, and lower levels of IL-13 in lung homogenates compared with the controls. IL-13-deficient mice failed to develop an increased response to EFS or goblet cell hyperplasia after the 10-day OVA challenge. We transferred bone marrow-derived mast cells from wild-type mice to FcepsilonRI(-/-) mice 1 day before initiating the challenge protocol. After the 10-day OVA challenge, recipient FcepsilonRI(-/-) mice demonstrated EFS-induced responses similar to those of challenged wild-type mice. Transferred mast cells could be detected in tracheal preparations. These results show that FcepsilonRI is important for the development of AHR after an aerosolized allergen sensitization protocol and that this effect is mediated through FcepsilonRI on mast cells and production of IL-13 in the lung.     

Activated N-formyl peptide receptor and high-affinity IgE receptor occupy common domains for signaling and internalization

Immune cells display multiple cell surface receptors that integrate signals for survival, proliferation, migration, and degranulation. Here, immunogold labeling is used to map the plasma membrane distributions of two separate receptors, the N-formyl peptide receptor (FPR) and the high-affinity IgE receptor (FepsilonRI). We show that the FPR forms signaling clusters in response to monovalent ligand. These domains recruit Gi, followed by the negative regulatory molecule arrestin2. There are low levels of colocalization of FPR with FcepsilonRI in unstimulated cells, shown by computer simulation to be a consequence of receptor density. Remarkably, there is a large increase in receptor coclustering when cells are simultaneously treated with N-formyl-methionyl-leucyl-phenylalanine and IgE plus polyvalent antigen. The proximity of two active receptors may promote localized cross-talk, leading to enhanced inositol-(3,4,5)-trisphosphate production and secretion. Some cointernalization and trafficking of the two receptors can be detected by live cell imaging, but the bulk of FPR and FcepsilonRI segregates over time. This segregation is associated with more efficient internalization of cross-linked FcepsilonRI than of arrestin-desensitized FPR. The observation of receptors in lightly coated membrane invaginations suggests that, despite the lack of caveolin, hematopoietic cells harbor caveolae-like structures that are candidates for nonclathrin-mediated endocytosis.