Regulation of eosinophil apoptosis by nitric oxide: Role of c-Jun-N-terminal kinase and signal transducer and activator of transcription 5

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.