Spontaneous and cytokine-mediated survival of basophils in comparison to eosinophils and neutrophils. (A) Time course evaluation of spontaneous apoptosis and effects of GM-CSF, IL-5, and IL-3 on granulocyte survival. Granulocytes were cultured for 0 to 72 hours in the absence or presence of the cytokine indicated (all at 50 ng/mL), and apoptosis was assessed by double staining of cells with annexin V–FITC and propidium iodide (PI). Live cells are shown as percentage of annexin V/PI-negative cells. Data are from 4 independent experiments and shown as the mean plus or minus SEM. The P values show the levels of probability for differences between the indicated treatments (two-way ANOVA). Significantly different from the mean value of the control: *P < .05, **P < .01, ***P < .001, cytokine versus medium. (B) A comparative Western blot analysis of the expression of proteins regulating apoptosis in granulocytes. Granulocytes were freshly isolated (Fr.is.) or cultured for 24 hours in medium alone without or with the indicated cytokines. Equal amounts (20 μg) of protein extracts derived from different granulocyte subtypes were resolved by SDS-PAGE and immunoblotted using specific antibodies recognizing cIAP2, XIAP, Mcl-1, Bcl-X_L, Bcl-2, PARP, and caspase 3. β-Actin is shown as loading control. (C) Spontaneous apoptosis of basophils is regulated through a caspase-dependent mechanism. Basophils were cultured in the absence or presence of the pan-caspase inhibitor zVAD-fmk (30 μM) or IL-3 (50 ng/mL). (Left panel) Apoptosis was evaluated by flow cytometry using annexin V–FITC staining after 24 hours or 48 hours of culture. Apoptotic cells are shown as percentage of annexin V-positive cells. Results of 5 independent experiments are reported as the mean plus SEM. *P < .05 for comparison of the treatment with medium control, by 1-way ANOVA. (Right panel) Western blot analysis of caspase 3 activation and caspase substrate cleavage (BCL-2, PARP) in protein extracts derived from basophils cultured for 24 hours.