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Abstract
Purpose
Although CD30 is known to be expressed more on eosinophils undergoing apoptosis, it is still not known how CD30 activation leads to eosinophil apoptosis. In this study, we evaluated whether ligation of CD30 incites apoptosis and investigated whether the mechanisms of CD30 induced eosinophil apoptosis are dependent on caspase activation.
Methods
We drew 90 mL of peripheral blood from healthy donors and then purified eosinophils using a MACS system. Expression of CD30 on eosinophils was measured, and eosinophils were cultured in wells pretreated with anti-CD30 mAb, isotype control immunoglobulin G1, interleukin (IL)-5, and dexamethasone in Roswell Park Memorial Institute 1640 media supplemented with 10% fetal bovine serum. Their rates of apoptosis were then compared using flow cytometry. To evaluate whether caspase-9 is involved in CD30-induced eosinophil apoptosis, the apoptotic rate was evaluated after the addition of caspase-9 inhibitor. The expression of procaspase-9 was also measured using Western blot.
Results
Expression of CD30 molecules on eosinophils increased steadily as the culture time lapse. 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 to 17.1 6.7% and 29.4 9.2% of the ± ± ± ± control at 4 and 24 hours, respectively (bothP<0.05). The apoptotic rates of eosinophils treated with anti-CD30 mAb were even faster than those of eosinophils treated with dexamethasone, and the mAb also suppressed the IL-5-induced enhancing effect of eosinophil survival. Caspase-9 inhibitor suppressed 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 demonstrated that the expression of procaspase-9 with mAb was diminished compared to that of the control and of IL-5.
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Fig. 1.
Expression of CD30 on human blood eosinophils. Isolated eosinophils were incubated in Roswell Park Memorial Institute 1640 media supplemented with 10% fetal bovine serum and then analyzed CD30 expression in a time dependent manner by flow cytometry using anti-CD30 mAb and fluorescein isothiocyanate-conjugated F(ab)' 2 goat anti-mouse immunoglobulin G (∗P< ‡P<0.001 compared with the 0 hour †P<0.01, 0.05, group).
Fig. 2.
Expression of CD30 on human blood eosinophils. Isolated eosinophils were incubated for 24 hours in Roswell Park Memorial Institute 1640 media supplemented with either of 1% fetal bovine serum (FBS), 10% FBS and 10% FBS with 10 ng/ mL of interleukin (IL)-5. The rate was not significantly different each other (P=0.49).
Fig. 3.
Effect of immobilized anti-CD30 mAb on eosinophil apoptosis. Eosinophils were cultured in Roswell Park Memorial Institute 1640 media supple mented with 10% fetal bovine serum of 24 well culture plates previously coated with 20 g/mL of anti µ -CD30 mAb or control immunoglobulin G (IgG)1 mAb for 4 hours (A) and 24 hours (B). 10 ng/mL of interleukin (IL)-5, and 10 M/mL of dexamethasone were added in other µ plates. Values represent the mean SEM of 4 separate experi ± ments (∗P<0.05 compared with isotype matched control or media alone).
Fig. 4.
Effect of caspase-9 inhibitor on immobilized anti-CD30 mAb induced eosinophil apoptosis in Roswell Park Memorial Institute 1640 media supplemented with 10 % fetal bovine serum. Eosinophils were cultured in 24 well culture plates previously coated with 20 g/mL of anti µ -CD30 mAb and 20 g/mL of control immunoglobulin G µ (IgG)1 mAb for 18 hours (A) and 36 hours (B). Caspase-9 inhibitor was added in ∗P <0.05 other plate. Values represent the mean SEM of 4 separate experiments (± compared with isotype matched control, media alone or with inhibitor).
Fig. 5.
Expression of procaspase-9 protein in eosinophils. Eosinophils were cultured in Roswell Park Memorial Institute 1640 media supplemented with 10 fetal bovine serum of 24 well culture plates previously coated with 20 g/mL of µ anti-CD30 mAb, 20 g/mL of control immuno µ globulin G (IgG)1 mAb and with addition of 10 ng/mL of interleukin (IL)-5 for 36 hours. The expression was determined by Western blot (∗P <0.05 compared with eosinophils cultured in the presence of IL-5).
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