Blood eosinophils from atopic donors express messenger RNA for the α, β, and γ subunits of the high-affinity IgE receptor (FcϵRI) and intracellular, but.

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Presentation transcript:

Blood eosinophils from atopic donors express messenger RNA for the α, β, and γ subunits of the high-affinity IgE receptor (FcϵRI) and intracellular, but not cell surface, α subunit protein Susan J. Smith, PhDa, Sun Ying, MDa, Qui Meng, MDa, Mark H.F. Sullivan, PhDa, Julia Barkans, BSca, Onn Min Kon, MDa, Bhupinder Sihra, MRCPa, Mark Larché, PhDa, Francesca Levi-Schaffer, PhDb, A.Barry Kay, MDa Journal of Allergy and Clinical Immunology Volume 105, Issue 2, Pages 309-317 (February 2000) DOI: 10.1016/S0091-6749(00)90081-2 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 ISH of purified eosinophils. mRNA for the 3 subunits of FcϵRI were detected with antisense cDNA riboprobes for the α, β, and γ chains (A, C, and D, respectively). B, Corresponding sense control for α chain. Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 mRNA expression for 3 FcϵRI subunits, α, β, and γ, on eosinophils cultured for 20 hours under different conditions (see Methods for details). Combination of reagents included coculture of eosinophils with human foreskin fibroblasts (HS27) in medium supplemented with both IgE (10 μg mL–1) and IL-4 (2.5 ng mL–1). Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Example of flow cytometry of permeabilized and nonpermeabilized peripheral blood eosinophils stained with anti-FcϵRI-α. Purified, freshly isolated eosinophils were stained with 22E7 or IgG1 isotype control and detected with phycoerythrin-labeled goat antimouse IgG. Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Flow cytometry of permeabilized and nonpermeabilized peripheral blood eosinophils. Results are expressed as smf of 22E7, which is calculated as the difference between mean fluorescence intensity (mfi) of IgG1 control and mfi of 22E7. Mean smf of nonpermeabilized eosinophils was 0.123 compared with 0.771 for permeabilized cells (P = .005). Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 Immunocytochemistry of permeabilized and nonpermeabilized eosinophils. Peripheral blood eosinophil cytospins stained with IgG1 control or 22E7 without (A and B ) and with (C and D ) saponin permeabilization. Staining of a single cell having morphologic appearances of a basophil is observed in nonpermeabilized staining with 22E7 preparation (B). In contrast, after permeabilization with saponin, eosinophils are clearly staining with 22E7 (D) but not with IgG1 control (C). Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 6 Expression of α subunit of FcϵRI on eosinophils under differing culture conditions (see Methods for details) compared with freshly isolated basophils. Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 7 Effect of IL-3, IL-5, and GM-CSF on surface expression of eosinophils that were cultured for 20 hours in presence of 10 ng mL–1 of either IL-3, IL-5, or GM-CSF compared with culture in medium alone (RPMI containing 10% FCS) and freshly isolated. Cells were stained with FITC-labeled 22E7 or FITC-labeled IgG1 isotype control. Results are expressed as smf of 22E7, which is calculated as difference between mfi of IgG1 control and mfi of 22E7. Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 8 Histamine release from basophils and EPO release from eosinophils. Eosinophils were incubated in microtiter plates coated with either human IgA (open circles) or F(ab ́)2 fragments of 22E7 (closed circles), n = 6. Histamine release from PBMCs containing basophils incubated with F(ab ́)2 fragments of 22E7 (open squares) or IgG1 (open triangles), n = 5. Journal of Allergy and Clinical Immunology 2000 105, 309-317DOI: (10.1016/S0091-6749(00)90081-2) Copyright © 2000 Mosby, Inc. Terms and Conditions