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

Mohammed Al-Rabia, Morgan Graeme Blaylock, Darren William Sexton, Garry Michael Walsh

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 CaspaTag(TM) 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.

Original languageEnglish
Pages (from-to)1045-1055
Number of pages10
JournalJournal of Leukocyte Biology
Volume75
Issue number6
DOIs
Publication statusPublished - Jun 2004

Keywords

  • asthma
  • cell-surface molecules
  • eosinophils
  • programmed cell death
  • COLONY-STIMULATING FACTOR
  • PROTEIN-KINASE
  • IN-VITRO
  • CONSTITUTIVE APOPTOSIS
  • FAS LIGATION
  • CYCLIC-AMP
  • EXPRESSION
  • ASTHMA
  • DEATH
  • MECHANISMS

Cite this

Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line. / Al-Rabia, Mohammed; Blaylock, Morgan Graeme; Sexton, Darren William; Walsh, Garry Michael.

In: Journal of Leukocyte Biology, Vol. 75, No. 6, 06.2004, p. 1045-1055.

Research output: Contribution to journalArticle

Al-Rabia, Mohammed ; Blaylock, Morgan Graeme ; Sexton, Darren William ; Walsh, Garry Michael. / Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line. In: Journal of Leukocyte Biology. 2004 ; Vol. 75, No. 6. pp. 1045-1055.
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AU - Blaylock, Morgan Graeme

AU - Sexton, Darren William

AU - Walsh, Garry Michael

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N2 - 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(TM) 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.

AB - 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(TM) 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.

KW - asthma

KW - cell-surface molecules

KW - eosinophils

KW - programmed cell death

KW - COLONY-STIMULATING FACTOR

KW - PROTEIN-KINASE

KW - IN-VITRO

KW - CONSTITUTIVE APOPTOSIS

KW - FAS LIGATION

KW - CYCLIC-AMP

KW - EXPRESSION

KW - ASTHMA

KW - DEATH

KW - MECHANISMS

U2 - 10.1189/jlb.0803404

DO - 10.1189/jlb.0803404

M3 - Article

VL - 75

SP - 1045

EP - 1055

JO - Journal of Leukocyte Biology

JF - Journal of Leukocyte Biology

SN - 0741-5400

IS - 6

ER -