Surface FcepsilonRI Research Articles

Human blood dendritic cells from allergic subjects have impaired capacity to produce interferon‐α via toll‐like receptor 9

High-affinity IgE receptor (Fc epsilon RI) expression on blood dendritic cells reportedly correlates with serum IgE levels. Our studies demonstrate that plasmacytoid dendritic cells (pDCs) secrete pro-inflammatory cytokines (IL-6, TNF-alpha) following Fc epsilon RI stimulation - a mode of activation that simultaneously reduces expression of Toll-like receptor 9 (TLR9). Whether or not TLR9 and/or Fc epsilon RI levels and their function on dendritic cells relate to allergic status is unknown. The aim of this study is to compare the innate (TLR9-mediated) immune response of human pDCs to TLR9 and Fc epsilon RI alpha receptor expression in allergic and non-allergic subjects. Basophil-depleted mononuclear cell fractions containing pDCs were prepared from peripheral blood of allergic and non-allergic subjects. Intracellular TLR9 and surface Fc epsilon RI alpha expression in blood dendritic cell antigen-2-positive cells were determined by flow cytometry. Activating anti-IgE antibody, anti-Fc epsilon RI alpha antibody, and TLR9 agonist were used to stimulate cell suspensions, with cytokine levels determined by ELISA. No difference in the frequency of pDCs was detected among allergic (n=9) vs. non-allergic (n=11) subjects (P=0.261). While there was also no difference in the baseline expression of TLR9, pDCs from allergic subjects produced sixfold less IFN-alpha when stimulated with CpG (P=0.002). Conversely, there was higher Fc epsilon RI alpha expression (P=0.01) on the pDCs of allergic subjects. Impaired TLR9-dependent immune responses in human pDCs are associated with allergic status and inversely correlated with Fc epsilon RI alpha expression. This impaired innate immune response among dendritic cells of allergic subjects may lead to more targeted therapeutic approaches and could provide a better understanding of the mechanisms underlying conventional and CpG-based immunotherapy.

Roles of the transmembrane domain and the cytoplasmic domain of FcɛRIα in immunoglobulin E‐mediated up‐regulation of surface FcɛRI expression1

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

Requirement of Fc Epsilon RI Gamma-subunit for IgE-mediated Up-regulation of Surface Fc Epsilon RI

RATIONALE: Since FcεRIα cannot be expressed at the cell surface without the FcRγ-subunit, it has not been clarified whether the FcRγ and the transmembrane domain (TM) and cytoplasmic domain (CY) of FcεRIα are necessary for IgE-mediated FcεRIα up-regulation.METHODS: To clarify the role of the TM and CY of FcεRI in IgE-mediated cell surface expression of FcεRI, we constructed chimeric receptors and transduced them into a human mast cell line, HMC-1, with a viral vector system. To determine the contribution of the FcRγ to receptor up-regulation, an FcεRI chimera containing the TM and CY of FcγRIα was expressed in cell line NIH3T3 with or without co-transduction of the FcRγ. IgE-mediated FcεRI up-regulation of the chimeric receptors was examined by FACS.RESULTS: An FcεRI chimera containing the TM and CY or just the CY of FcγRIα was expressed on the cell surface, and they mediated efficient IgE-dependent up-regulation. Full-length FcγRI and an FcγRI chimera containing the TM and CY or just the CY of FcεRIα did not mediate IgG1-dependent up-regulation. The FcεRI chimera containing the TM and CY of FcγRIα failed to mediate IgE-dependent up-regulation. Co-transduction of either FcRγ, FcRγ ITAM mutant or a CY deletion mutant for FcRγ to the cells recovered its response to IgE.CONCLUSIONS: IgE-mediated up-regulation of FcγRIα requires FcRγ, but does not require activation signals by FcRγ ITAM. RATIONALE: Since FcεRIα cannot be expressed at the cell surface without the FcRγ-subunit, it has not been clarified whether the FcRγ and the transmembrane domain (TM) and cytoplasmic domain (CY) of FcεRIα are necessary for IgE-mediated FcεRIα up-regulation. METHODS: To clarify the role of the TM and CY of FcεRI in IgE-mediated cell surface expression of FcεRI, we constructed chimeric receptors and transduced them into a human mast cell line, HMC-1, with a viral vector system. To determine the contribution of the FcRγ to receptor up-regulation, an FcεRI chimera containing the TM and CY of FcγRIα was expressed in cell line NIH3T3 with or without co-transduction of the FcRγ. IgE-mediated FcεRI up-regulation of the chimeric receptors was examined by FACS. RESULTS: An FcεRI chimera containing the TM and CY or just the CY of FcγRIα was expressed on the cell surface, and they mediated efficient IgE-dependent up-regulation. Full-length FcγRI and an FcγRI chimera containing the TM and CY or just the CY of FcεRIα did not mediate IgG1-dependent up-regulation. The FcεRI chimera containing the TM and CY of FcγRIα failed to mediate IgE-dependent up-regulation. Co-transduction of either FcRγ, FcRγ ITAM mutant or a CY deletion mutant for FcRγ to the cells recovered its response to IgE. CONCLUSIONS: IgE-mediated up-regulation of FcγRIα requires FcRγ, but does not require activation signals by FcRγ ITAM.

Transforming Growth Factor-β1 Regulates the Expression of the High-Affinity Receptor for IgE on CD34+ Stem Cell-Derived CD1a+ Dendritic Cells In Vitro

It has been reported that monocytes, Langerhans cells (LC) and other dendritic cells (DC) express the high-affinity receptor for IgE (FcepsilonRI) in patients with atopic diseases. These cells may be instrumental in the control of the immune response and the allergic inflammation. In this context, transforming growth factor beta 1 (TGF-beta1) has been highlighted as a key cytokine involved in the mechanisms aimed to orchestrate tolerance and has been suggested as a candidate gene in atopic diseases. In this report, we investigate the putative role of TGF-beta1 in the regulation of FcepsilonRI on cord blood CD34+ stem cell-derived CD1a+ DC (CD34-derived CD1a+ DC). Kinetic experiments show that FcepsilonRI spontaneously appears on the surface of CD1a+ DC, but decreases when exogenous TGF-beta1 is added at high doses (10 ng per mL) or when endogenous TGF-beta1 is neutralized in the culture conditions. In contrast, low-dose TGF-beta1 (0.5 ng per mL) stabilizes surface FcepsilonRI expression on DC. Increasing TGF-beta1 concentrations leads to the generation of LC-like DC showing an augmentation in stimulatory capacity towards allogeneic T cells. In view of these data, a picture emerges that FcepsilonRI+ on DC is finely modified by the TGF-beta1 concentration in the microenvironment and could be of primary relevance in the context of atopic diseases.

Monomeric immunoglobulin E stabilizes FcepsilonRIalpha from the human basophil cell line KU812 by protecting it from natural turnover.

The high affinity IgE receptor (FcepsilonRI) on mast cells and basophils is up-regulated by its own ligand IgE; however, the mechanism is unknown. To study the IgE-mediated effect on FcepsilonRI on basophils by using the human basophilic cell line KU812. Expression of cell surface FcepsilonRI was assessed by flow cytometry. Western blot technique was used to illustrate tyrosine-phosphorylation and the Ca2+ level in KU812 was measured by fluorescence of Fura-2. Soluble specimens of the alpha-chain from FcepsilonRI (FcepsilonRIalpha) were obtained by lysing 107 KU812 pr. mL. FcepsilonRIalpha was detected by a sandwich immunoradiometric assay employing the IgE-binding capacity of FcepsilonRIalpha in conjunction with a monoclonal antibody. Polyclonal rabbit anti-FcepsilonRIalpha was used for detection of FcepsilonRIalpha by Western blotting. We found that monomeric IgE did not induce tyrosine-phosphorylation in KU812, which was the case when stimulating with IgE cross-linked by anti-IgE binding. Further, only cross-linking of IgE, but not monomeric IgE, increased the Ca2+ level. Using the immunoradiometric assay, we found a temperature dependent reduction in the amount of FcepsilonRIalpha. Samples incubated at 37 degrees C for 5 h displayed a 16-fold decrease in the FcepsilonRIalpha level compared with samples incubated at 4 degrees C. In the presence of IgE the reduction at 37 degrees C was only threefold. These results indicate that IgE does not induce intracellular signals in KU812, i.e., tyrosine-phosphorylation or Ca2+ release. Instead it appears that FcepsilonRIalpha is an unstable protein that IgE stabilizes and thereby protects from a temperature dependent turnover.

Evidence for a differential expression of the FcepsilonRIgamma chain in dendritic cells of atopic and nonatopic donors.

While mast cells and basophils constitutively express the high-affinity IgE receptor (Fc epsilon RI), it is absent or weakly expressed on APCs from normal donors. Fc epsilon RI is strongly upregulated on APCs from atopic donors and involved in the pathophysiology of atopic diseases. Despite its clinical relevance, data about Fc epsilon RI regulation on APCs are scarce. We show that in all donors intracellular alpha chain of the Fc epsilon RI (Fc epsilon RI alpha) accumulates during DC differentiation from monocytes. However, expression of gamma chains of the Fc epsilon RI (Fc epsilon RI gamma), mandatory for surface expression, is downregulated. It is low or negative in DCs from normal donors lacking surface Fc epsilon RI (Fc epsilon RI(neg) DCs). In contrast, DCs from atopics express surface Fc epsilon RI (Fc epsilon RI(pos) DCs) and show significant Fc epsilon RI gamma expression, which can be coprecipitated with Fc epsilon RI alpha. In Fc epsilon RI(neg) DCs lacking Fc epsilon RI gamma, immature and core glycosylated Fc epsilon RI alpha accumulates in the endoplasmic reticulum. In Fc epsilon RI(pos) DCs expressing Fc epsilon RI gamma, an additional mature form of Fc epsilon RI alpha exhibiting complex glycosylation colocalizes with Fc epsilon RI gamma in the Golgi compartment. IgE binding sustains surface-expressed Fc epsilon RI on DCs from atopic donors dependent on baseline protein synthesis and transport and enhances their IgE-dependent APC function. We propose that enhanced Fc epsilon RI on DCs from atopic donors is driven by enhanced expression of otherwise limiting amounts of Fc epsilon RI gamma and is preserved by increased IgE levels.

A Reducing Microenvironment Leads to the Generation of FcεRIhigh Inflammatory Dendritic Epidermal Cells (IDEC)

Inflammatory dendritic epidermal cells present in skin lesions of the atopic eczema/dermatitis syndrome display the highest expression of the high-affinity receptor for IgE (FcepsilonRI), ever detected on human antigen-presenting cells. Owing to the instability of the FcepsilonRI (alphagammagamma) complex and fast cleavage from the cell surface during the interleukin-4/granulocyte-macrophage colony-stimulating factor driven in vitro differentiation of monocytes, a method to generate inflammatory dendritic epidermal cells was not at our disposal in the past and the amount of ex vivo isolated inflammatory dendritic epidermal cells available for functional assays was limited. Therefore, information about the role of inflammatory dendritic epidermal cells and FcepsilonRI on this dendritic cell subtype in atopic and inflammatory skin diseases is completely missing. In this study, we were able to: (i) increase the expression of a functional FcepsilonRI complex on the cell surface of immature monocyte-derived dendritic cells from atopic donors by creating a reducing microenvironment; (ii) enhance significantly the intracellular pool of the FcepsilonRIgamma chains, which is the limiting parameter for the FcepsilonRI surface expression; and (iii) generate monocyte-derived dendritic cells displaying the phenotypical characteristics of inflammatory dendritic epidermal cells, producing high amounts of proinflammatory cytokines and chemokines similar to the cytokines found in lesional skin of the atopic eczema/dermatitis syndrome. Altogether the high expression of functional FcepsilonRI on these cells enables us for the first time to study inflammatory dendritic epidermal cells and FcepsilonRI-mediated mechanisms of inflammatory dendritic epidermal cells in vitro, in order to shed light on the putative role of this important cell type in the atopic eczema/dermatitis syndrome.

Construction of an in vitro allergy reaction evaluation system using human leukemia cell lines.

Human leukemia cell lines, KU812 and KU812F, are immature prebasophillic cell lines and have a potential to differentiate into basophils. Hydrocortisone (HC) and sodium nitroprusside (SNP) can enhance the cell surface FcepsilonRI expression of KU812 cells. However, the differentiated phenotypes of KU812 cells were unstable, hindering the application of KU812 cells to construct a practical invitro allergy reaction evaluation system. Here, we attempted to enhance the cell surface expression of FcepsilonRI on hydrocortisone (HC)- or sodium nitroprusside (SNP)-treated KU812 cells by IgE. The cell surface FcepsilonRI expression was observed in about 20, 20 and 26% of 1 muM HC-, 1 nM SNP- and 450 ng ml(-1) IgE-treated KU812 cells, respectively. Whereas, the cell surface FcepsilonRI expression was observed in about 54% of KU812 cells treated with both 450 ng ml(-1) IgE and 1 muM HC for 8 days, and in about 33% of KU812 cells treated with both 450 ng ml(-1) IgE and 1 nM SNP for 4 days. Ninety five% of the IgE/HC- or IgE/SNP-treated KU812 cells expressed CD 13 antigen, a cell surface marker of basophils. An electrophoretic mobility shift assay revealed that AP-1, NF-AT and NF-kappaB transcription factors were all activated in IgE/HC- and IgE/SNP-treated KU812 cells. Since the differentiated KU812F cells were more sensitive than KU812 cells for histamine release by sensitization with human IgE and anti-IgE antibody, a practical in vitro allergy reaction evaluation system for general use was constructed using IgE/HC-treated KU812F cells. The differentiated KU812F cells sensitized with an allergicpatient's IgE and mite allergen exhibited histamine release. The constructed in vitro allergy reaction evaluation system using differentiated human leukemia KU812F cells will be useful to study allergic reaction and to analyze physiologically functional substances in allergic disease.

Regulation of mouse mast cell surface Fc epsilon RI expression by dexamethasone.

It is now clear that the mast cell's functional response to IgE-dependent stimulation can be influenced significantly by the level of expression of the high-affinity IgE receptor (Fc epsilon RI) on the cell's surface. Thus, modulation of Fc epsilon RI surface expression represents a potentially important mechanism for regulating mast cell activity in allergic reactions. In this study, we examined whether a glucocorticoid, dexamethasone (DEX), can influence levels of mast cell Fc epsilon RI expression either in the presence or absence of IgE, an up-regulator of the mast cell surface Fc epsilon RI level. In the absence of IgE, DEX decreased the surface Fc epsilon RI levels in mouse peritoneal mast cells, mouse bone marrow-derived cultured mast cells and a mouse mast cell line, Cl.MC/C57.1. Moreover, DEX also partially suppressed the ability of IgE to enhance surface expression of Fc epsilon RI in these cells. Three different glucocorticoids, DEX, methylprednisolone and hydrocortisone, suppressed Fc epsilon RI expression in mast cells, whereas sex steroids, i.e. estradiol, progesterone and testosterone, did not, indicating that the Fc epsilon RI-suppressing effect is glucocorticoid specific. On the other hand, DEX did not affect levels of Fc epsilon RI alpha, beta or gamma mRNA, suggesting that its ability to decrease surface Fc epsilon RI reflects a post-transcriptional mechanism. Finally, DEX-treated mast cells showed a reduced degranulation response to antigenic stimulation through down-regulation of surface Fc epsilon RI expression in addition to DEX-induced changes in downstream signals. These results show that mast cell surface Fc epsilon RI expression is suppressed by glucocorticoids in both the presence and absence of IgE, and suggest that reduction of mast cell surface Fc epsilon RI levels may be one of the favorable anti-allergic actions of glucocorticoids.

Induction of basophilic and eosinophilic differentiation in the human leukemic cell line KU812.

We have demonstrated that an immature prebasophilic cell line,KU812 cells can be induced to differentiate into basophil-like cells when cultured with hydrocortisone (HC) with enhanced cell surface expression of FcepsilonRI, a high affinity IgE receptor. In this study, we report that sodium nitroprusside (SNP), an intracellular NO donor, also induces cell surface expression of FcepsilonRI on KU812 cells. Cell surface FcepsilonRI expression was detected in about 20% of KU812 cells treated with SNP for 14 days as well as the cells treated with HC for 7 days, while non-treated KU812 cells did not express FcepsilonRI on their cell surface. However, Wright-Giemsa staining and flowcytometry analysis of CD13 and CD15 antigens on HC and SNP treated KU812 cells demonstrated that SNP induced eosinophilic differentiation in KU812 cells differently from HC which induced basophilic differentiation. To further confirm this result, we performed RT-PCR against mRNAs specific for eosinophils, such as eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase(EPO). SNP treated KU812 cells but not HC treated cells expressed EDN and EPO mRNA depending upon the induction of differentiation,clearly demonstrating that SNP induces eosinophilic differentiation in KU812 cells. To clarify that different signaling cascades were activated in HC and SNP treated KU812 cells, we analyzed activities of AP-1, NF-AT and NF-kappaB transcription factors by EMSA, which are known to be involved in signal transduction pathways downstream from the FcepsilonRI molecule of basophils. All these three transcription factors were activated in HC treated KU812 cells,but not in non-treated and SNP treated KU812 cells. These results indicate that KU812 cells are multi-potent precursor cells which can be induced to differentiate into basophils and eosinophils upon exogenous signals, and that NO is an important factor to decide the eosinophilic differentiation in KU812 cells with enhanced surface expression of FcepsilonRI, and further suggest that different signaling cascades can be activated between basophilic and eosinophilic differentiation in KU812 cells.

Suppression of Microphthalmia Transcriptional Activity by Its Association with Protein Kinase C-interacting Protein 1 in Mast Cells

Microphthalmia (mi) is a transcription factor that plays a major role in the regulation of growth and function in mast cells and melanocytes. Association of mi with other proteins is a critical step in the regulation of mi-mediated transcriptional activation. We found protein kinase C-interacting protein 1 (PKCI) specifically associated with mi in yeast two-hybrid screening. Immunoprecipitation of mi from quiescent rat basophilic leukemic cells or mouse melanocytes resulted in the specific co-immunoprecipitation of PKCI. This association was significantly reduced on engagement of the surface FcepsilonRI of mast cells or engagement of the Kit receptor on melanocytes. Hence, cell activation caused disengagement of mi from PKCI. Microphthalmia was previously shown to activate the mouse mast cell protease 6 (mMCP-6) promoter. Cotransfection of mi with PKCI in NIH 3T3 fibroblasts containing an mMCP-6 promoter-luciferase reporter demonstrated an up to 94% inhibition of mi-mediated transcriptional activation. PKCI by itself, although localized in the cytosol and nucleus of the cells, has no known physiological function and did not demonstrate transcriptional activity. Its ability to suppres mi transcriptional activity in the transient transfected fibroblast system suggests that it can function in vivo as a negative regulator of mi-induced transcriptional activation.

Effect of recombinant human IL-4 on tryptase, chymase, and Fc epsilon receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells.

The effect of recombinant human IL-4 (rhIL-4) on the development of recombinant human stem cell factor-dependent fetal liver-derived mast cells was examined. RhIL-4 attenuates the number of mast cells that develop, preferentially affecting the MC(T) type of mast cell. Cellular levels of tryptase and chymase mRNA normalized to that of glyceraldehyde-3-phosphate dehydrogenase were not appreciably affected. Tryptase mRNA levels peaked at least 2 wk before tryptase protein and before chymase mRNA and protein, indicating that tryptase mRNA expression is an early marker of commitment to a mast cell lineage. In contrast, alpha-tryptase and beta-tryptase mRNA levels increased and decreased in parallel. The most dramatic effect of rhIL-4 was to induce expression of functional surface Fc epsilonRI. Expression was maximal by 21 days with 20 ng/ml of rhIL-4 and reached a plateau by 2 ng/ml of rhIL-4 at 4 wk. Fc epsilonRI+ cells increased modestly when myeloma IgE was added to the developing mast cells, but increased synergistically when both myeloma IgE and rhIL-4 were present together. Delayed addition of rhIL-4 progressively diminished Fc epsilonRI expression, as did withdrawal of rhIL-4 during the first 2 wk of culture. RhIL-4 selectively increased Fc epsilonRI alpha mRNA levels at least 10-fold. Mast cells developed in the presence of rhIL-4 released tryptase when exposed to anti-Fc epsilonRI alpha. In conclusion, induction of functional Fc epislonRI on recombinant human stem cell factor-dependent human fetal liver-derived mast cells by rhIL-4 harmonizes with the well-accepted ability of this cytokine to enhance IgE production by B cells.

Expression and functions of the high-affinity IgE receptor on human platelets and megakaryocyte precursors.

Platelets can be activated by IgE and are therefore involved in IgE-mediated effector mechanisms against parasites and in allergic disorders. Here we show that, besides the low-affinity IgE receptor (Fc epsilon RII/CD23), platelets express the high-affinity receptor for IgE (Fc epsilon RI). Flow cytometry analysis revealed the existence of surface Fc epsilon RI on platelets with a large heterogeneity among individual donors, and a low proportion of platelets co-expressing Fc epsilon RI and FC epsilon RII/CD23. Northern hybridization and reverse transcription polymerase chain reaction confirmed the presence of mRNA encoding the alpha, beta and gamma chains of Fc epsilon RI in platelets and in their megakaryocytic precursors. Cross-linking of Fc epsilon RI with monoclonal antibody (mAb) to alpha chain using either the whole molecule or F(ab')2 triggered platelet cytotoxicity for Schistosoma mansoni larvae. Anti-Fc epsilon RII/CD23 mAb significantly inhibited IgE- or Fc epsilon RI-mediated cytotoxicity, indicating down-regulatory effects of Fc epsilon RII/CD23 on Fc epsilon RI-dependent functions. These results demonstrate functional properties for Fc epsilon RI on platelets and indicate unsuspected interactions between the low- and the high-affinity IgE receptors.

Basis of the 1:1 stoichiometry of the high affinity receptor Fc epsilon RI-IgE complex.

A soluble fragment of the high-affinity IgE receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the C epsilon 2, C epsilon 3 and C epsilon 4 domains of the epsilon-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG1, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the C epsilon 2 and C epsilon 3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging C epsilon 2 domains. To test this hypothesis we have expressed a recombinant epsilon-chain fragment containing C epsilon 3 and C epsilon 4. This product, Fc epsilon 3-4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface Fc epsilon RI. Titration experiments, together with molecular mass measurements of the Fc epsilon 3-4/sFc epsilon RI alpha complex, reveal that Fc epsilon 3-4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by C epsilon 2 accounts for the unexpected stoichiometry.

Characterization of Fc epsilonRI expressing human monocytic cell lines. 1. The role of CD45 on signal transduction in primary monocytes and cell lines.

Recently, the high affinity receptor for IgE (Fc epsilonRI), which plays a major role in allergies, has been identified on a number of different antigen-presenting cell types, including human monocytes from atopic and nonatopic donors. In this report human monocytic cell lines were used to test for the expression of Fc epsilonRI, reasoning that a monocytic cell line expressing Fc epsilonRI constitutively would be a useful tool for large scale studies on the regulation of IgE binding and signal transduction. Reverse transduction polymerase chain reaction was applied to identify Fc epsilonRI alpha-chain message, flow cytometry to detect Fc epsilonRI surface expression and signal transduction on the cell lines generated by transfection. We report the establishment of monocytic cell lines constitutively expressing Fc epsilonRI (THP1-alpha01 to THP1-alpha40) generated by transfection of the cell line THP1 with a plasmid encoding the Fc epsilonRI alpha-chain only. Fc epsilonRI on the THP1-alpha lines specifically binds IgE and is functional with regard to ligand binding and signal transduction. Comparative studies between the transfectants and primary human monocytes from nonatopic donors demonstrated the regulatory role of the tyrosine phosphatase CD45 on Fc epsilonRI-mediated cell activation. Monocytic cell lines carry Fc epsilonRI alpha-chain RNA and enhancement by transfection results in surface Fc epsilonRI expression on THP1. Triggering the receptor on the THP1-alpha lines or on human monocytes, which express native Fc epsilonRI, elicits a rapid and transient calcium mobilization, prevented by co-cross-linking of Fc epsilonRI and CD45.

Thy‐1‐mediated activation of rat basophilic leukemia cells does not require co‐expression of the high‐affinity IgE receptor

The glycosylphosphatidylinositol (GPI)-anchored protein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia cells, RBL-2H3. Antibody-mediated aggregation of Thy-1 induces in these cells release of secretory components; so does aggregation of the receptor with high affinity for IgE (Fc epsilon RI). To examine whether there is any relationship between Thy-1- and Fc epsilon RI-mediated activation, we have isolated from mutagenized RBL-2H3 cells a variant cell line deficient in the expression of surface Fc epsilon RI, and analyzed its ability to be activated by an antibody to Thy-1. Northern and immuno-blot analyses revealed that the variant cells were deficient in the expression of a structural or a regulatory gene for Fc epsilon RI gamma subunit. The cells did not respond by release of secretagogues and protein-tyrosine phosphorylation to IgE and antigen and anti-Fc epsilon RI monoclonal antibody (mAb) but their response to anti-Thy-1.1 mAb and calcium ionophore A23187 was retained. Transfection of the cloned Fc epsilon RI gamma subunit into the variant cells restored the surface expression of Fc epsilon RI and responsiveness to both the antigen and anti-Fc epsilon RI mAb but had no effect on responsiveness to anti-Thy-1 mAb. The combined data indicate that aggregation of surface Thy-1 glycoproteins activates a metabolic pathway which is independent of the presence of Fc epsilon RI gamma subunit and surface expression of Fc epsilon RI.

Soluble form of the human high-affinity receptor for IgE inhibits recurrent allergic reaction in a novel mouse model of type I allergy.

The recombinant soluble form of the human high-affinity receptor for IgE alpha subunit (sFc epsilon RI alpha) is able to block the binding of IgE to the cell surface Fc epsilon RI and prevent the activation of mast cells and basophils. To evaluate its anti-allergic effects in vivo, we established a novel model for type I allergy by transplanting antigen-specific IgE-secreting hybridomas into syngeneic mice. The hybridomas continuously produced anti-2,4,6-trinitrophenyl IgE in vivo, and ear swelling responses could be elicited upon applying picryl chloride to the ears of these mice, which reached their maximum 1-2 h after the antigen challenge. The second swelling response, in extent comparable to the first response, was induced by the second antigen challenge several days later. When the sFC epsilon RI alpha was intravenously administered before the first challenge and periodically between the first and the second challenges, the ear swelling response to the second challenge, but not to the first challenge, was suppressed almost completely. Immunohistochemical examination revealed that treatment with the sFc epsilon RI alpha blocked the binding of IgE to the cell surface IgE receptors after the first challenge. Thus, our results indicate that the sFc epsilon RI alpha suppresses recurrent allergic reactions by preventing IgE binding to the cell surface Fc epsilon RI.

Intracellular degradation of Fc gamma RIII in mouse bone marrow culture-derived progenitor mast cells prevents its surface expression and associated function.

Although mouse interleukin-3-dependent, bone marrow culture-derived progenitor mast cells (BMMC) and a Kirsten sarcoma virus (KiSV)-immortalized mouse mast cell line (MC4w) both express on their surfaces receptors for the Fc portion of IgG (Fc gamma R), only MC4w degranulate upon Fc gamma R perturbation. As shown by surface iodination and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated proteins immunoprecipitated with the Fc gamma R-specific monoclonal antibody 2.4G2, a 26-kDa protein, identified as Fc gamma RIII by immunoblotting with antibody to Fc gamma RIII, was predominantly expressed on the surface of MC4w but minimally on BMMC. However, both BMMC and MC4w expressed mRNA for Fc gamma RIII as determined by RNA blot analysis, and both translated Fc gamma RIII as assessed by intrinsic radiolabeling and SDS-polyacrylamide gel electrophoresis analysis of deglycosylated monoclonal antibody 2.4G2 immunoprecipitates. Pulse-chase analysis showed that intrinsically radiolabeled Fc gamma RIII was stable in MC4w cells but was degraded rapidly in BMMC and that newly synthesized Fc gamma RIII remained sensitive to digestion by endoglycosidase H in BMMC but rapidly became resistant in MC4w. These data suggest that the deficiency in surface Fc gamma RIII expression on BMMC is due to the degradation of Fc gamma RIII in the endoplasmic reticulum. Immunoprecipitation of surface Fc gamma R and Fc receptors for IgE (Fc epsilon RI) from digitonin-extracted cells followed by immunoblotting with antibody to Fc epsilon RI gamma-chain showed that gamma-chain is associated with surface Fc epsilon RI and Fc gamma R in MC4w, but only with Fc epsilon RI in BMMC, which lack surface Fc gamma RIII. Inasmuch as BMMC are progenitors of serosal mast cells, which, like MC4w, express surface Fc gamma RIII and undergo Fc gamma R-mediated activation, the data suggest that maturation of BMMC enables Fc gamma RIII to bypass degradation in the endoplasmic reticulum, resulting in the acquisition of functional Fc gamma RIII/gamma-chain complexes on the cell surface.

Protein-tyrosine phosphorylation: an essential component of FcεRI signaling

Mast cell and basophil activation can be achieved by antigen-mediated aggregation of cell surface Fc epsilon RI molecules. At least two signaling pathways are triggered by this activation. Both involve tyrosine phosphorylation. This aspect of Fc epsilon RI signaling is examined here in detail and its position in a complex network of post-binding events assessed.