Mechanisms of Allergy: Rapid PublicationRegulation of eosinophil apoptosis by nitric oxide: Role of c-Jun-N-terminal kinase and signal transducer and activator of transcription 5☆,☆☆
Section snippets
Eosinophil purification
Blood (50-100 mL) was obtained from normal individuals. Eosinophils were isolated to >99% purity under sterile conditions as previously reported.6, 18 The cells were resuspended at 106 cells/mL and cultured in RPMI 1640 or L -arginine, nitrate, and nitrite-free Dulbecco modified Eagle medium supplemented with 100 μmol/L L -arginine (for nitrite production and NOS inhibitor cultures), 10% fetal calf serum, and antibiotics.
Determination of eosinophil apoptosis
Unless otherwise stated, eosinophil apoptosis was determined by propidium
Effects of NO on IL-5-mediated eosinophil survival
When eosinophils were cultured in cytokine-deprived conditions for 40 hours, the apoptotic index was 0.43 ± 0.06 (n = 6). IL-5 increased cell survival in a concentration-dependent manner during 40-hour incubation by delaying apoptosis. The maximal antiapoptotic effect of IL-5 was obtained at 10 pmol/L concentration (apoptotic index 0.15 ± 0.05, n = 6). NO-donor SNAP (1-1000 μmol/L) reversed IL-5-mediated cell survival in a concentration-dependent manner by inducing apoptosis (Fig 1, A -C ).
Discussion
In this study, we found that exogenous NO reversed IL-5-mediated eosinophil survival by inducing apoptosis. The NO donors SNAP and GEA3175 increased the number of apoptotic cells in the presence of IL-5 in a concentration-dependent manner, and this effect was abolished by a NO scavenger. Our results are in agreement with the study of Beauvais and Joly,30 in which it was suggested that other NO donors reduced survival and induced apoptosis of IL-5-primed eosinophils. The main physiologic
Acknowledgements
We appreciate the skillful technical assistance of Mrs Tanja Kuusela.
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Supported by Jalmari and Rauha Ahokas Foundation (Finland), Tampere Tuberculosis Foundation (Finland), the Finnish Anti-tuberculosis Association Foundation (Finland), the Academy of Finland, and the Medical Research Fund of Tampere University Hospital (Finland).
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Reprint requests: Hannu Kankaanranta, MD, PhD, Medical School, FIN-33014, University of Tampere, Tampere, Finland.