Mitochondrial respiration is required for activation of ERK1/2 and caspase-3 in human eosinophils stimulated with hydrogen peroxide.
BACKGROUND
Eosinophils are important effector cells in the pathogenesis of allergic diseases such as bronchial asthma. Oxidative stress in the form of cellular reactive oxygen species (ROS) has been implicated in the pathogenesis of several allergic diseases. Recently, it has become evident that mitochondrial-derived ROS are important transducers of apoptosis and intracellular signaling. In this study, we investigated the role of mitochondrial ROS in the activation of extracellular signal-regulated kinases (ERK) 1 and 2-mitogen-activated protein kinase (MAPK) and caspase-3 in human eosinophils stimulated with H2O2.


METHODS
Human eosinophils were purified using immunomagnetic negative selection and then stimulated with H2O. H2O2-induced eosinophil apoptosis was measured by staining cells with annexin V. Activation of ERK1/2 MAPK and caspases was assessed by Western blotting. Eosinophils were pretreated with rotenone, an inhibitor of the mitochondrial electron transport chain, before H2O2 was added.


RESULTS
Treatment with 1 mM H2O2 induced externalization of phosphatidylserine (PS) and activation of caspases in eosinophils. H2O2-triggered PS externalization and cleavage of caspase-3 were markedly inhibited by pretreatment with the mitochondrial ROS scavenger N-acetyl-L-cysteine. In addition, H2O2 strongly induced phosphorylation of ERK1/2, but not ERK5, in eosinophils. Hydrogen peroxide-triggered activation of caspase-3 and ERK1/2 was attenuated by pretreatment with rotenone.


CONCLUSIONS
These results suggest that mitochondrial respiration is essential for activation of ERK1/2 and caspase-3 in human eosinophils stimulated with H2O2.
Lee
Y A
YA
Department of Environmental Medical Biology, Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, Korea.
Shin
M H
MH
D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D053148:Caspase 3 / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D004804:Eosinophils / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000276:immunology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000096:biosynthesis*; D048052:Mitogen-Activated Protein Kinase 3 / Q000096:biosynthesis*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology
D017382:Reactive Oxygen Species; D012402:Rotenone; D006861:Hydrogen Peroxide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D053148:Caspase 3