Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line

• Published in: Journal of leukocyte biology Vol. 75; no. 6; pp. 1045 - 1055
• Main Authors: Al‐Rabia, Mohammed W., Blaylock, Morgan G., Sexton, Darren W., Walsh, Garry M.
• Format: Journal Article
• Language: English
• Published: United States Society for Leukocyte Biology 01.06.2004
• Subjects: Annexin A5 - metabolism; Apoptosis - immunology; asthma; Caspase Inhibitors; Caspases - metabolism; Cell Differentiation; Cell Line; cell‐surface molecules; Enzyme Activation; Enzyme Inhibitors - pharmacology; eosinophils; Eosinophils - physiology; fas Receptor - metabolism; Humans; Leukemia - immunology; Leukemia - metabolism; Leukemia - pathology; Leukocyte Common Antigens - metabolism; Membrane Potentials - drug effects; Mitochondria - drug effects; programmed cell death
• ISSN: 0741-5400; 1938-3673
• DOI: 10.1189/jlb.0803404

Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase‐3, ‐8, and ‐9 activities in receptor ligation‐dependent apoptosis induction in the differentiated human eosinophilic cell line EoL‐1. Differentiated EoL‐1 exhibited bi‐lobed nuclei, eosinophil‐associated membrane receptors, and basic granule proteins. Annexin‐V fluorescein isothiocyanate binding to EoL‐1 revealed significant (P<0.01) apoptosis induction in cells cultured for 20 h with monoclonal antibodies (mAb) specific for CD45 (71%±4.3), CD45RA (58%±2.3), CD45RB (68%±2.4), CD95 (47%±2.6), and CD69 (52%±2.1) compared with control (23%±1.6) or CD45RO mAb (27%±3.9). The pan‐caspase inhibitor Z‐Val‐Ala‐Asp‐fluoromethylketone (fmk) and inhibitors of caspase‐8 (Z‐Ile‐Glu‐Thr‐Asp‐fmk) and caspase‐9 (Z‐Leu‐Glu‐His‐Asp‐fmk) significantly inhibited mAb‐induced apoptosis of EoL‐1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag™ technique and flow cytometry. EoL‐1 treated with pan‐CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase‐3 and ‐9 activation at 12 h post‐treatment, which increased at 16 and 20 h. Activated caspase‐8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase‐3 activation, a modest but significant activation of caspase‐8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase‐9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation‐mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti‐inflammatory therapy for asthma.