Oxidant-Mediated Mitochondrial Injury in Eosinophil Apoptosis: Enhancement by Glucocorticoids and Inhibition by Granulocyte-Macrophage Colony-Stimulating Factor

Abstract

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.

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

“…6 In this respect, also a high expression of the mitochondrial anti-oxidant enzyme MnSOD in neutrophils seems to be logical (see Figure 5, lane 4 and Murphy et al 24 ), because this enzyme may have the important physiological role of inactivating excessive ROS in the absence of the functional 'natural' sink of electrons -complex IV. Increased oxidant injury due to loss of the protective function of MnSOD has been proposed to play a role in glucocorticoid-induced apoptosis of eosinophils, 34 which are believed to have a defective mitochondrial respiration like neutrophils. 4 Neutrophil mitochondria lacking cytochrome c and having no complex IV activity do, however, create Dc m (Figure 3), although the underlying mechanism remains unclear.…”

The CDK inhibitor, R‐roscovitine, promotes eosinophil apoptosis by down‐regulation of Mcl‐1

“…Eosinophils are also notoriously difficult to study for a number of reasons: their paucity in the peripheral blood of normal, healthy volunteer blood donors, the laborious isolation techniques required to ensure a pure population and their relative intractability to standard molecular biological techniques such as siRNA. Corticosteroids are known to induce significant eosinophil apoptosis, in vitro, but it is still debatable as to whether this effect is relevant in vivo [6,31,[36][37][38][39][40]. Steroid drugs are extremely effective in the majority of asthmatic patients but some, at the more severe end of the spectrum, are resistant and others experience intolerable and unacceptable side-effects [41][42][43][44][45].…”

c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis

“…[21,22] To further evaluate the possible role of JNK in spontaneous and dexamethasone-induced eosinophil apoptosis, the activation of JNK was studied. The activity of JNK was assessed by calculating the ratio of phosphorylated/total JNK by using antibodies directed against Thr183/Tyr185 and Thr221/Tyr223 phosphorylated (activated) JNK and total JNK, which recognize JNK at molecular weights of 46 and 55kDa.…”

“…[18] Recent evidence suggests that nitric oxide and glucocorticoids may activate MAP kinases in human eosinophils. [21][22][23] Activation of JNK by dexamethasone was associated with induction of eosinophil apoptosis, a process that could be inhibited by GM-CSF and by blocking JNK activation by a JNK inhibitor (SP600125). [21] However, the results concerning the importance of JNK activation in glucocorticoid-induced eosinophil apoptosis remain controversial as JNK1/2 antisense phosphorothioate oligodeoxynucleotides did not exert any significant effect on dexamethasone-induced eosinophil apoptosis.…”

“…[21][22][23] Activation of JNK by dexamethasone was associated with induction of eosinophil apoptosis, a process that could be inhibited by GM-CSF and by blocking JNK activation by a JNK inhibitor (SP600125). [21] However, the results concerning the importance of JNK activation in glucocorticoid-induced eosinophil apoptosis remain controversial as JNK1/2 antisense phosphorothioate oligodeoxynucleotides did not exert any significant effect on dexamethasone-induced eosinophil apoptosis. [22] Given the possible role of JNK in regulating apoptosis of eosinophils and other cell types, the aim of the present study was to elucidate the role of JNK in spontaneous and dexamethasoneenhanced apoptosis of human eosinophils.…”