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J Allergy Clin Immunol. 2013 Aug;132(2):437-45. doi: 10.1016/j.jaci.2013.03.024. Epub 2013 May 16.

Mechanism of Siglec-8-mediated cell death in IL-5-activated eosinophils: role for reactive oxygen species-enhanced MEK/ERK activation.

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Division of Allergy and Immunology, Cincinnati Children's Hospital, and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.



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.


In this report we investigate the mechanism of Siglec-8-mediated cell death in activated eosinophils.


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.


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.


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.


7-Aminoactinomycin D; 7AAD; DPI; DUSP; Diphenyleneiodonium; Dual-specificity phosphatase; EPX; ERK; Eosinophil peroxidase; Eosinophils; Extracellular signal-regulated kinase; H(2)O(2); HRP; Horseradish peroxidase; Hydrogen peroxide; JNK; MAPK; MAPK-ERK kinase; MEK; Mitogen-activated protein kinase; NaN(3); PFA; Paraformaldehyde; ROS; Reactive oxygen species; STAT; Sialic acid–binding immunoglobulin-like lectin; Siglec; Signal transducer and activator of transcription; Sodium azide; c-Jun N-terminal kinase; cell death; signaling

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