Mechanisms of Allergy: Rapid Publication
Regulation of eosinophil apoptosis by nitric oxide: Role of c-Jun-N-terminal kinase and signal transducer and activator of transcription 5,☆☆

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Abstract

Background: Reduced eosinophil apoptosis is considered to be a key mechanism for eosinophilia in allergic diseases such as asthma, rhinitis, and eczema. Objective: The aim of our study was to investigate the possible modulatory effect of nitric oxide (NO) in human eosinophils. Methods: Apoptosis in isolated eosinophils was assessed by relative DNA fragmentation assay, annexin-V binding, and morphologic analysis. The activation of c-Jun-N-terminal kinase (JNK) and signal transducer and activator of transcription 5 (STAT5) was assessed by immunoblot analysis. Results: The NO donor S-nitroso-N -acetylpenicillamine (SNAP) reversed the survival-prolonging effect of IL-5 by inducing apoptosis. This effect was blocked by the NO scavenger (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3oxide.potassium salt), indicating that reversal of IL-5-mediated eosinophil survival was due to NO. The effect of NO on IL-5-afforded cell survival was not mediated by cyclic guanosine 3′: 5′-monophosphate (cGMP), because neither an inhibitor of guanylyl cyclase nor inhibitors of phosphodiesterases had any effect on SNAP-induced eosinophil apoptosis in IL-5-treated cells. SNAP induced a time-dependent increase in the activity of JNK, and an inhibitor peptide specific for JNK, L-JNKI1, completely reversed SNAP-induced apoptosis in IL-5-treated eosinophils. In contrast, SNAP did not inhibit IL-5-induced STAT5 activation. Inhibition of the activity of caspases by Z-Asp-CH2-DCB reversed the effect of SNAP, suggesting that NO promotes apoptosis in IL-5-treated human eosinophils in a caspase-dependent manner. However, this effect of NO was not mediated by means of activation of caspases 3, 8, or 9. Conclusions: Our results suggest that exogenous NO reverses IL-5-mediated eosinophil survival by inducing apoptosis, and this is mediated by means of activation of JNK in a cGMP-independent manner. (J Allergy Clin Immunol 2003;112:93-101.)

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 ).

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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).

    ☆☆

    Reprint requests: Hannu Kankaanranta, MD, PhD, Medical School, FIN-33014, University of Tampere, Tampere, Finland.

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