title: IL-33 enhances Siglec-8 mediated apoptosis of human eosinophils.
abstract: IL-33 activates eosinophils directly via the ST2 receptor. Like IL-5, IL-33 induces eosinophilia and eosinophilic airway inflammation in mouse models and primes human eosinophil responses. Previously, we reported that IL-5 priming enhances Siglec-8 mediated mitochondrial and reactive oxygen species (ROS)-dependent eosinophilic apoptosis and eliminates caspase dependence of this cell death process. Whether IL-33, like IL-5, augments pro-apoptotic pathways involving receptors such as Siglec-8 and in a similar manner has not been explored. Annexin-V labeling was performed to detect apoptosis in human eosinophils pre-incubated with or without a range of concentrations of IL-33 and/or IL-5 in the presence or absence of Siglec-8 monoclonal antibody (mAb) 2C4 and inhibitors of caspases. Tetramethyl-rhodamine staining was used as a marker of mitochondrial membrane potential loss and injury. ROS production was determined by measuring the superoxide dismutase-inhibitable reduction of cytochrome c. Cleavage of poly(ADP-ribose) polymerase (PARP) was assessed using Western blotting. Eosinophils cultured alone or with mAb 2C4 underwent low levels of apoptosis at 24h. 2C4-induced eosinophil apoptosis was markedly and equally enhanced after culture for 24h with either IL-33 or IL-5, although IL-5 was more potent. Effects on apoptosis with IL-33 and IL-5 were synergistic. In contrast, percentages of cells exhibiting reduced mitochondrial membrane potential were greater with IL-33 than IL-5 and effects of these cytokines were also synergistic. Antimycin, an inhibitor of mitochondrial electron transport, almost completely inhibited 2C4-induced apoptosis with either IL-33 or IL-5. Surprisingly, 2C4-induced eosinophil ROS production was significantly enhanced with IL-5 but not IL-33. Siglec-8-mediated apoptosis in the presence of IL-33 was more sensitive in magnitude than IL-5 to inhibition by the pan-caspase inhibitor Z-VAD-FMK, yet both cytokine conditions were associated with PARP cleavage. These data demonstrate that IL-33 is as effective but less potent than IL-5 in enhancing Siglec-8-mediated eosinophil apoptosis, and can synergize with IL-5. Eosinophils primed by IL-33 and/or IL-5 in vivo would be expected to display enhanced susceptibility to undergoing Siglec-8-induced apoptosis.
authors.affiliation: Department of Medicine, Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
authors.forename: Ho Jeong
authors.initials: HJ
authors.lastname: Na
authors.forename: Sherry A
authors.initials: SA
authors.lastname: Hudson
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Bochner
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D004804:Eosinophils / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D000067596:Interleukin-33; D015848:Interleukin-5 / Q000494:pharmacology; D007378:Interleukins / Q000494:pharmacology*; D037102:Lectins / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C506536:IL33 protein, human; D000067596:Interleukin-33; D015848:Interleukin-5; D007378:Interleukins; D037102:Lectins; D017382:Reactive Oxygen Species; C403967:SIGLEC8 protein, human; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases
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