Monomeric immunoglobulin E stabilizes FcεRIα from the human basophil cell line KU812 by protecting it from natural turnover |
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Authors: | B M Jensen J B Hansen S Dissing† J Gerwien‡ § P S Skov and L K Poulsen |
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Institution: | Laboratory of Medical Allergology, Allergy Unit FIN. 7542, National University Hospital, Copenhagen, Denmark. bmj@rh.dk |
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Abstract: | BACKGROUND: The high affinity IgE receptor (FcepsilonRI) on mast cells and basophils is up-regulated by its own ligand IgE; however, the mechanism is unknown. OBJECTIVE: To study the IgE-mediated effect on FcepsilonRI on basophils by using the human basophilic cell line KU812. METHODS: 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. RESULTS: 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. CONCLUSION: 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. |
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Keywords: | basophil high affinity IgE receptor IgE |
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