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


CONCLUSIONS
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.
Jensen
B M
BM
Laboratory of Medical Allergology, Allergy Unit FIN. 7542, National University Hospital, Copenhagen, Denmark. bmj@rh.dk
Hansen
J B
JB
Dissing
S
S
Gerwien
J
J
Skov
P S
PS
Poulsen
L K
LK
D001491:Basophils / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D010766:Phosphorylation; D017455:Receptors, IgE / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D012995:Solubility; D013696:Temperature; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D017455:Receptors, IgE; D007073:Immunoglobulin E; D014443:Tyrosine; D002118:Calcium