Characterisation of apoptosis, caspase pathways and phagocytosis in human cultured eosinophils and an eosinophilic cell line
The present research was carried out to investigate the differential activation of caspases-3, -8 and -9 using the novel CaspTagTM technique and flow cytometry together with examination of changes in mitochondrial transmembrane potential (DYm) in human cultured eosinophils and an eosinophilic cell line, EoL-1 following mAb-dependent ligation of CD45, CD45 isoforms, CD95, and CD69. The membrane receptor expression and the storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) in these cells were also studied. This study also established that airway epithelial cells could recognise and ingest apoptotic and not freshly differentiated EoL-1. Peripheral blood eosinophils (PBE), cultured eosinophils (CE), and differentiated EoL-1 express pan-CD45 and the isoforms RA, RB, and RO, CD95, CD69, cytokine and chemokine receptors. They also express and store the eosinophil granule proteins MBP and ECP. My findings suggest that although PBE, CE, differentiated EoL-1 have many phenotypic properties in common there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in cultured eosinophils or that most likely reflect the leukaemic nature of the EoL-1 cell line. Annexin-V fluroescein isothiocyanate binding to cultured eosinophils and differentiated EoL-1 revealed significant apoptosis induction in cells cultured with monoclonal antibodies (mAb) specific for CD45, CD45RA, CD45RB, CD95 and CD69 compared with isotope controls. In contrast, dexamethasone failed to induce significant apoptosis at all concentrations or time points examined in this study. The pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (fmk) or caspase-8 (Z-IIE-Glu-Thr-Asp-fimk) and caspase-9 (Z-Leu-Glu-His-Asp-fmk) inhibitors demonstrated a role for these caspases in membrane receptor ligation-reduced apoptosis in differentiated EoL-1 cells. The study also demonstrated that apoptosis induction in CE or differentiated EoL-1 by mAb-dependent receptor ligation involved differential caspase activation.