The mainstay of asthma therapy, glucocorticosteroids (GCs) have among their therapeutic effects the inhibition of inflammatory cytokine production and induction of eosinophil apoptosis. In the absence of prosurvival cytokines (e.g., GM-CSF), eosinophils appear to be short-lived, undergoing apoptosis over 96 h in vitro. In a dose-dependent manner, GC further enhances apoptosis, while prosurvival cytokines inhibit apoptosis and antagonize the effect of GC. The mechanisms of eosinophil apoptosis, its enhancement by GC, and antagonism of GC by GM-CSF are not well-understood. As demonstrated in this study, baseline apoptosis of eosinophils resulted from oxidant-mediated mitochondrial injury that was significantly enhanced by GC. Mitochondrial injury was detected by early and progressive loss of mitochondrial membrane potential and the antioxidant protein, Mn superoxide dismutase (SOD). Also observed was the activation/translocation of the proapoptotic protein, Bax, to mitochondria. Underscoring the role of oxidants was the inhibition of mitochondrial changes and apoptosis with culture in hypoxia, or pretreatment with a flavoprotein inhibitor or a SOD mimic. GCs demonstrated early (40 min) and late (16 h) activation of proapoptotic c-Jun NH2-terminal kinase (JNK) and decreased the antiapoptotic protein X-linked inhibitor of apoptosis, a recently demonstrated inhibitor of JNK activation. Similarly, inhibition of JNK prevented GC-enhanced mitochondrial injury and apoptosis. Importantly, GM-CSF prevented GC-induced loss of X-linked inhibitor of apoptosis protein, late activation of JNK, and mitochondrial injury even in the face of unchanged oxidant production, loss of MnSOD, and early JNK activation. These data demonstrate that oxidant-induced mitochondrial injury is pivotal in eosinophil apoptosis, and is enhanced by GC-induced prolonged JNK activation that is in turn inhibited by GM-CSF.

Prolonged eosinophil survival, i.e., reduced apoptosis, is implicated in the pathogenesis of chronic allergic inflammation. Here we demonstrate that interleukin (IL)-15, in the presence or absence of tumor necrosis factor (TNF)-alpha, reduces spontaneous apoptosis in freshly isolated human eosinophils. The prosurvival effect of IL-15 was abrogated by neutralizing antibody to granulocyte macrophage-colony stimulating factor (GM-CSF), although GM-CSF was not detected in conditioned media by ELISA. Additionally, the effect of IL-15 on spontaneous eosinophil apoptosis appeared to require nuclear factor-kappaB (NF-kappaB) activation based on evidence for NF-kappaB nuclear translocation and abrogation of the effect by the NF-kappaB inhibitor, Bay 11- 7082. Finally, the data demonstrate that IL-15 expression is higher in the submucosa of endobronchial tissues from subjects with moderate to severe asthma when compared with control subjects. Thus, our results suggest that IL-15, either alone or in combination with TNF-alpha, may perpetuate allergic inflammation by reduction of spontaneous eosinophil apoptosis through autocrine production of GM-CSF and NF-kappaB activation.

Percutaneous application of 6 mg/mL glycerinated histamine solution, 50% glycerosaline solution and 0.9% buffered saline produced similar positive and negative control wheals. These observations warrant further studies of percutaneous allergen testing in cats with hypersensitivity dermatitis.

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