TY - JOUR
T1 - Mechanism of Siglec-8-mediated cell death in IL-5-activated eosinophils
T2 - Role for reactive oxygen species-enhanced MEK/ERK activation
AU - Kano, Gen
AU - Almanan, Maha
AU - Bochner, Bruce S.
AU - Zimmermann, Nives
N1 - Funding Information:
Disclosure of potential conflict of interest: B. S. Bochner has consultancy arrangements with and owns stock in Allakos, Inc and has one or more patents (planned, pending, or issued) related to Siglec-8 and its ligand with and has received royalties from Johns Hopkins University. N. Zimmermann has been supported by one or more grants from the National Institutes of Health and the Dana foundation and is employed by Cincinnati Children's Hospital. The rest of the authors declare that they have no relevant conflicts of interest.
Funding Information:
Supported in part by National Institutes of Health grants AI088559 (to N.Z.), P30 DK078392 , and AI72265 (to B.S.B.) and a grant from the Dana Foundation (to B.S.B and N.Z.).
PY - 2013/8
Y1 - 2013/8
N2 - 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.
AB - 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.
KW - Eosinophils
KW - cell death
KW - signaling
UR - https://www.scopus.com/pages/publications/84881187383
UR - https://www.scopus.com/pages/publications/84881187383#tab=citedBy
U2 - 10.1016/j.jaci.2013.03.024
DO - 10.1016/j.jaci.2013.03.024
M3 - Article
C2 - 23684072
AN - SCOPUS:84881187383
SN - 0091-6749
VL - 132
SP - 437
EP - 445
JO - Journal of Allergy and Clinical Immunology
JF - Journal of Allergy and Clinical Immunology
IS - 2
ER -