Methods: We purified eosinophils using MACS system. Expression of CD30 on eosinophils were measured and eosinophils were cultured in the wells pretreated with anti-CD30 mAb and isotype control IgG1, IL-5 and dexamethasone in RPMI 1640 media supplemented with 10% FBS, and the apoptotic rate were measured using flow cytometry. To evaluate whether caspase-9 is involved in CD30- induced eosinophil apoptosis, the apoptotic rate was evaluated with addition of caspase-9 inhibitor and the expression of procaspase-9 was also measured using Western blot.
Results: The apoptotic rates of eosinophils cultured in the presence of anti-CD30 mAb were significantly increased to 29.1±6.1% and 47.3%±4.7% compared with 17.1±6.7% and 29.4±9.2% of the control at 4 and 24 hours, respectively. (both P<0.05) Caspase-9 inhibitor suppressed the mAb induced eosinophil apoptosis from 54.8±6.9% and 71.5±11.6% to 24.5±6.0% and 47.8±11.4% at 18 and 36 hours, respectively. (both P<0.001) We also showed the expression of procaspase-9 with the mAb was diminished compared with that of the control and of IL-5.
Conclusions: This study showed CD30 activation enhances the eosinophil apoptosis and the effect is mediated by caspase-9 activation