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 pathol ogy of allergic inflammatory disease, especially bronchial asthma. We recen tly investigated the activation of eosinophils by applying whole blood to a flow cytometer. We measured here beta(1) and beta(2) integrin on eosinophi ls stimulated by phorbol-12-myristate-13-acetate (PMA)/ionomycin to evaluat e eosinophil activation in vitro using whole blood. Methods: Heparinized wh ole blood was diluted with the same volume of RPMI 1640, then cells were in cubated in the presence or absence of PMA and ionomycin for 45 min at 37 de grees C. After hemolyzation with lysing solution, flow-cytometric findings for CR3, LFAl-alpha, LFA1-beta and VLA-4 expression on eosinophils were exa mined. Results: Mean fluorescent intensity (MFI) of CR3 and LFA1-beta stimu lated by PMA and ionomycin was significantly higher than that of the unstim ulated control. MFI of LFA1-alpha 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 an d may allow intensive investigation of eosinophils in clinical laboratories as well as in research laboratories. We are currently investigating the in fluence of different kinds of stimulations, regulation factors or agents on eosinophils using this method.