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A more recent version of this article appeared on June 1, 2004

Published online before print April 1, 2004
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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0803404

Received for publication August 28, 2003.
Revised February 11, 2004.
Accepted for publication February 19, 2004.

Article

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

Mohammed W. Al-Rabia , Morgan G. Blaylock , Darren W. Sexton , and Garry M. Walsh @

Department of Medicine & Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, United Kingdom

@ To whom correspondence should be addressed. E-mail: g.m.walsh{at}abdn.ac.uk.


  Abstract

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 CaspaTagTM 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.

Key Words: asthma • cell-surface molecules






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