title: The role of caspase activation and mitochondrial depolarisation in cultured human apoptotic eosinophils.
abstract: Caspases are key intracellular molecules in the control of apoptosis, but little is known concerning their relative contribution to the cascade of events leading to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation dependent apoptosis induction in the cultured eosinophils (CE). CE cultured alone for 48 hours exhibited constitutive apoptosis (12% ± 1.2). Significant (P < 0.05) enhancement of eosinophil apoptosis was observed following monoclonal antibody (Mab) treatment with CD45 (40% ± 0.7), CD95 (36% ± 1.6), or CD69 (34% ± 0.2). Caspase activity was analysed using the novel CaspaTagTM technique and flow cytometry. CE ligated with CD45 (Bra55), CD95 (Fas) and CD69 Mab resulted in caspase-3 and -9 activation after 16 hours post-ligation. This trend in caspase-3 and -9 activation continued to increase significantly through to the 20 and 24 hours time points when compared to isotype control. Activated up-stream caspase-8 was detected 16 and 20 hours after treatment with CD45, CD95 and CD69 Mab followed by a trend toward basal levels at 24 hours. Ligation of CD95 was followed by mitochondrial permeabilization, as demonstrated by marked increase in mitochondrial transmembrane potential ([Formula: see text]) at all time points. However, ligation with CD45 and CD69 failed to induce a change in [Formula: see text] at 16 hours post-treatment compared to isotype control even though there was an alteration in mitochondrial downstream-caspase activity following ligation with these Mab(s) at this time point. At 20 and 24 hours post-ligation, CD45 or CD69 induce significantly altered levels of [Formula: see text]. 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.
authors: Department of Med. Lab. Sci., College of Appl. Med. Sci., Al-Kharaj University, PO Box 422, Al-Kharj 11942, Saudi Arabia.
authors: Faris Q
authors: FQ
authors: Alenzi
authors: Badi Q
authors: BQ
authors: Alenazi
authors: Fatma H
authors: FH
authors: Al-Anazy
authors: Abdulla M
authors: AM
authors: Mubaraki
authors: Mohamed L
authors: ML
authors: Salem
authors: Ali A
authors: AA
authors: Al-Jabri
authors: Mahmoud
authors: M
authors: Lotfy
authors: Mohammad S
authors: MS
authors: Bamaga
authors: Mohammed W
authors: MW
authors: Alrabia
authors: Richard K H
authors: RK
authors: Wyse
keywords: Apoptosis; Asthma; Caspases; Eosinophils; Mitochondria
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