Cell Surface FcepsilonRI Research Articles

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.

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.

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.

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.

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.