Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line.
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 trade mark 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.
Al-Rabia
Mohammed W
MW
Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
Blaylock
Morgan G
MG
Sexton
Darren W
DW
Walsh
Garry M
GM
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000276:immunology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004804:Eosinophils / Q000502:physiology*; D006801:Humans; D007938:Leukemia / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D019014:fas Receptor / Q000378:metabolism*
D017304:Annexin A5; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D019014:fas Receptor; D017493:Leukocyte Common Antigens; D020169:Caspases