Whole-Blood Flow-Cytometric Analysis of Induction of Adhesion Molecules Expression on Eosinophils Stimulated by Phorbol-12-Myristate-13-Acetate/Ionomycin

Background: Activation of eosinophils is closely associated with the pathology of allergic inflammatory disease, especially bronchial asthma. We recently investigated the activation of eosinophils by applying whole blood to a flow cytometer. We measured here β 1 and β 2 integrin on eosinophils stimulated by phorbol-12-myristate-13-acetate (PMA)/ionomycin to evaluate eosinophil activation in vitro using whole blood. Methods: Heparinized whole blood was diluted with the same volume of RPMI 1640, then cells were incubated in the presence or absence of PMA and ionomycin for 45 min at 37°C. After hemolyzation with lysing solution, flow-cytometric findings for CR3, LFA1-α, LFA1-β and VLA-4 expression on eosinophils were examined. Results: Mean fluorescent intensity (MFI) of CR3 and LFA1-β stimulated by PMA and ionomycin was significantly higher than that of the unstimulated control. MFI of LFA1-α showed no significant difference from the unstimulated control. On the other hand, MFI of VLA-4 tended to decrease. Conclusions: Our method to distinguish eosinophils from various cell groups in whole blood is simple and time-saving, similar to conditions in vivo and may allow intensive investigation of eosinophils in clinical laboratories as well as in research laboratories. We are currently investigating the influence of different kinds of stimulations, regulation factors or agents on eosinophils using this method.