Figure 1
Figure 1. Granulocyte blood kinetics and activation status of 111-Indium–labeled eosinophils. After reinjection of 111-Indium–labeled mixed leukocytes, 40-mL blood samples were taken. (A) Neutrophils (NEU) and (B) eosinophils (EO) were isolated from 40-mL blood samples using RoboSep before measurement of cell-associated radioactivity. (A-B) Data represent the mean ± SEM of 7 independent experiments. *P < .05, compared with 2-hour and 6-hour values (1-way ANOVA test). (C) Kinetics of eosinophils isolated after reinjection of purified 111-Indium–labeled eosinophils. Data are mean ± SEM of 6 independent experiments. Non-cell–associated radioactivity was measured by collecting plasma samples (○). Where error bars are not visible, the SEM lies within the symbols. (D) Representative flow cytometry and quantification of CD69, CD44, CD66b, and CD81 expression in freshly isolated whole blood eosinophils (WB), mixed leukocyte eosinophils (ML) ± 111-Indium tropolonate, and CD16 purified eosinophils (EO) ± 111-Indium tropolonate. Histograms represent WB (red line), ML (blue line), or EO (black line). Isotype-matched controls (gray fill) for WB, ML, or EO are shown from left to right. (E) Eosinophil shape change. Freshly isolated WB, and CD16 purified EO ± 111-Indium tropolonate were incubated with 10 ng/mL GM-CSF (GM) before assessment of shape change, as described in supplemental Methods. The means of cell size in forward scatter signal are shown. Data are the mean ± SEM of at least 3 independent experiments. *P < .05, compared with whole blood values (1-way ANOVA test). (F) Representative TEM images of eosinophils in WB, ML ± 111-Indium tropolonate, and EO ± 111-Indium tropolonate incubated with or without IL-5 (10 ng/mL). IL-5–treated eosinophils display signs of vacuolation (black arrows) and loss of granule integrity (white arrows). Sections were examined with a FEI, Tecnai G2 microscope operated at 120 kv using a 20 μm objective aperture. Digital images were captured with an AMT XR60B camera (Deben), saved in TIF format, and trimmed for publication using Microsoft Office Powerpoint. Original magnification ×3500.

Granulocyte blood kinetics and activation status of 111-Indium–labeled eosinophils. After reinjection of 111-Indium–labeled mixed leukocytes, 40-mL blood samples were taken. (A) Neutrophils (NEU) and (B) eosinophils (EO) were isolated from 40-mL blood samples using RoboSep before measurement of cell-associated radioactivity. (A-B) Data represent the mean ± SEM of 7 independent experiments. *P < .05, compared with 2-hour and 6-hour values (1-way ANOVA test). (C) Kinetics of eosinophils isolated after reinjection of purified 111-Indium–labeled eosinophils. Data are mean ± SEM of 6 independent experiments. Non-cell–associated radioactivity was measured by collecting plasma samples (○). Where error bars are not visible, the SEM lies within the symbols. (D) Representative flow cytometry and quantification of CD69, CD44, CD66b, and CD81 expression in freshly isolated whole blood eosinophils (WB), mixed leukocyte eosinophils (ML) ± 111-Indium tropolonate, and CD16 purified eosinophils (EO) ± 111-Indium tropolonate. Histograms represent WB (red line), ML (blue line), or EO (black line). Isotype-matched controls (gray fill) for WB, ML, or EO are shown from left to right. (E) Eosinophil shape change. Freshly isolated WB, and CD16 purified EO ± 111-Indium tropolonate were incubated with 10 ng/mL GM-CSF (GM) before assessment of shape change, as described in supplemental Methods. The means of cell size in forward scatter signal are shown. Data are the mean ± SEM of at least 3 independent experiments. *P < .05, compared with whole blood values (1-way ANOVA test). (F) Representative TEM images of eosinophils in WB, ML ± 111-Indium tropolonate, and EO ± 111-Indium tropolonate incubated with or without IL-5 (10 ng/mL). IL-5–treated eosinophils display signs of vacuolation (black arrows) and loss of granule integrity (white arrows). Sections were examined with a FEI, Tecnai G2 microscope operated at 120 kv using a 20 μm objective aperture. Digital images were captured with an AMT XR60B camera (Deben), saved in TIF format, and trimmed for publication using Microsoft Office Powerpoint. Original magnification ×3500.

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