1. Mechanism of Siglec-8-mediated cell death in IL-5-activated eosinophils: role for reactive oxygen species-enhanced MEK/ERK activation.
- Author
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Kano G, Almanan M, Bochner BS, and Zimmermann N
- Subjects
- Antigens, CD genetics, Antigens, Differentiation, B-Lymphocyte genetics, Apoptosis drug effects, Cells, Cultured, Enzyme Activation drug effects, Eosinophils physiology, Humans, Lectins genetics, Mitogen-Activated Protein Kinase 3 genetics, Mitogen-Activated Protein Kinases drug effects, Mitogen-Activated Protein Kinases genetics, Phosphorylation, Reactive Oxygen Species metabolism, Antigens, CD metabolism, Antigens, Differentiation, B-Lymphocyte metabolism, Cell Death drug effects, Eosinophils immunology, Interleukin-5 immunology, Lectins metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Mitogen-Activated Protein Kinases metabolism, Reactive Oxygen Species pharmacology
- Abstract
Background: Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is expressed on human eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-mediated cell death is markedly enhanced by the presence of the activation and survival factor IL-5 and becomes independent of caspase activity., Objective: In this report we investigate the mechanism of Siglec-8-mediated cell death in activated eosinophils., Methods: Human peripheral blood eosinophils were treated with agonistic anti-Siglec-8 antibody and IL-5, and cell death was determined by using flow cytometry and morphology. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by using phosphoLuminex, flow cytometry, and Western blotting. Reactive oxygen species (ROS) accumulation was determined by using dihydrorhodamine fluorescence., Results: Costimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cell death by means of necrosis accompanied by granule release compared with that seen after stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in costimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 costimulation. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and MAPK-ERK kinase (MEK) 1 was significantly enhanced and sustained in costimulated cells compared with that seen in cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors blocked anti-Siglec-8/IL-5-induced cell death. ROS accumulation was induced by Siglec-8 ligation in a MEK-independent manner. In contrast, an ROS inhibitor prevented the anti-Siglec-8/IL-5-induced enhancement of ERK phosphorylation and cell death. Exogenous ROS mimicked stimulation by anti-Siglec-8 and was sufficient to induce enhanced cell death in IL-5-treated cells. Collectively, these data suggest that the enhancement of ERK phosphorylation is downstream of ROS generation., Conclusions: In activated eosinophils ligation of Siglec-8 leads to ROS-dependent enhancement of IL-5-induced ERK phosphorylation, which results in a novel mode of biochemically regulated eosinophil cell death., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)
- Published
- 2013
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