A staphylococcal protein A rosetting assay for the demonstration of high affinity IgE receptors on rIL-3-dependent human basophil-like cells grown in mixed cell cultures.
Mature basophils can be differentiated from other blood born cells by their morphology, metachromatically staining granules, histamine content, and the presence on their surfaces of high-affinity IgE receptors. The identification of basophil-like cells arising in mixed human bone marrow cultures, however, is made more difficult because of their immaturity and the morphologic alterations due to in vitro culture. Identification of IgE receptors on individual cells in cell preparations which could simultaneously be examined for their histochemical properties would facilitate the study of basophil growth and differentiation. Because this was difficult using existing techniques, we developed an IgE staphylococcal protein A rosetting assay which allows the identification of cells bearing high-affinity IgE receptors and permits the same cells to be examined by a variety of staining techniques. We then examined the appearance of basophil-like cells bearing IgE receptors in cultures of human bone marrow and correlated this data with measurements of IgE receptor number and affinity. The percentage of total cells grown in the presence of human recombinant IL-3 (rIL-3) that rosetted with Staphylococcus aureus increased from 2 +/- 0.5% at 1 week of culture to 7 +/- 2% at 2 weeks, 14 +/- 5% at 3 weeks, and 18 +/- 5% at 4 weeks. Using 125I-labeled IgEPS, the number of IgE receptors per rosetting basophil-like cell was calculated to be 7.3 x 10(4) at 2 weeks, 6.8 x 10(4) at 3 weeks, and 3.9 x 10(4) receptors per rosette positive basophil-like cell at 4 weeks. Finally, the rate of dissociation of IgE from these cultured cells was found to be 4.0 +/- 1.3 x 10(-5) s-1, indicating that IgE bound to IgE receptors on cultured basophil-like cells with high affinity.
Kirshenbaum
A S
AS
Mast Cell Physiology Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Dreskin
S C
SC
Metcalfe
D D
DD
D000944:Antigens, Differentiation, B-Lymphocyte; D001491:Basophils / Q000276:immunology*; D002478:Cells, Cultured; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D011961:Receptors, Fc / Q000032:analysis*; D017455:Receptors, IgE; D011994:Recombinant Proteins / Q000494:pharmacology; D012397:Rosette Formation; D013205:Staphylococcal Protein A
D000944:Antigens, Differentiation, B-Lymphocyte; D007377:Interleukin-3; D011961:Receptors, Fc; D017455:Receptors, IgE; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A