Abstract
BackgroundEosinophils are critically involved in the pathogenesis of asthma. Nitric oxide (NO) is produced in high amounts in asthmatic lungs and has an important role as a regulator of lung inflammation. NO was previously shown to induce eosinophil apoptosis mediated via c-jun N-terminal kinase (JNK) and caspases. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS) and JNK.MethodsApoptosis was determined by flow cytometric analysis of relative DNA content, by Annexin-V labelling and/or morphological analysis. Immunoblotting was used to study phospho-JNK (pJNK) expression. Mitochondrial membrane potential was assessed by JC-1-staining and mitochondrial permeability transition (mPT) by loading cells with calcein acetoxymethyl ester (AM) and CoCl2 after which flow cytometric analysis was conducted. Statistical significance was calculated by repeated measures analysis of variance (ANOVA) or paired t-test.ResultsNO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) induced late apoptosis in GM-CSF-treated eosinophils. SNAP-induced apoptosis was suppressed by inhibitor of mPT bongkrekic acid (BA), inhibitor of JNK SP600125 and superoxide dismutase-mimetic AEOL 10150. Treatment with SNAP led to late loss of mitochondrial membrane potential. Additionally, we found that SNAP induces early partial mPT (1 h) that was followed by a strong increase in pJNK levels (2 h). Both events were prevented by BA. However, these events were not related to apoptosis because SNAP-induced apoptosis was prevented as efficiently when BA was added 16 h after SNAP. In addition to the early and strong rise, pJNK levels were less prominently increased at 20–30 h.ConclusionsHere we demonstrated that NO-induced eosinophil apoptosis is mediated via ROS, JNK and late mPT. Additionally, our results suggest that NO induces early transient mPT (flickerings) that leads to JNK activation but is not significant for apoptosis. Thereby, we showed some interesting early events in NO-stimulated eosinophils that may take place even if the threshold for irreversible mPT and apoptosis is not crossed. This study also revealed a previously unknown physiological function for transient mPT by showing that it may function as initiator of non-apoptotic JNK signalling.
Highlights
Eosinophils play a crucial role in the pathogenesis of asthma
SNAP-induced apoptosis is dependent on late mitochondrial permeability transition (mPT) but preceded by early partial mPT Mitochondrial membrane permeabilization including mPT and loss of ΔΨm are critical steps in mitochondrial apoptotic pathway [16]
We found that SNAP reduced calcein fluorescence by 34.6 ± 7.7% when compared to penicillamine-treated cells at 1 h indicating that mPT occurs and CoCl2 enters mitochondria to quench calcein fluorescence (Figures 2A-D)
Summary
Eosinophils play a crucial role in the pathogenesis of asthma. By releasing toxic granule proteins, lipid mediators and other proinflammatory components, eosinophils contribute especially to exacerbations of asthma [1]. Eosinophils typically exist in low numbers in human peripheral blood complicating studies on their functions. They undergo spontaneous apoptosis in few days in the absence of any survivalprolonging cytokines. Blood eosinophils obtained from patients with asthma show delayed apoptosis when compared to eosinophils from healthy controls [4] and elevated levels of eosinophil survival-prolonging cytokines interleukin (IL)-5 and granulocyte macrophage-colony stimulating factor (GM-CSF) have been found from the bronchoalveolar lavage fluid of asthmatics [5]. Apoptosis is an efficient way to discard eosinophils from the airways by avoiding inflammation. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS) and JNK
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