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

Abstract

Background 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 flow cytometry and morphology. Phosphorylation of MAPK was determined by phospho-luminex, flow cytometry, and Western blotting. ROS accumulation was determined by dihydrorhodamine (DHR) fluorescence. Results Co-stimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cells dying by necrosis accompanied by granule release as compared to stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in co-stimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 co-stimulation. Phosphorylation of ERK1/2 and MEK1 was significantly enhanced and sustained in co42 stimulated cells compared to 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, 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.

Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8

“…S5). None of these mutations affected the overall structure of Siglec-8, as indicated by very similar 2D 1 H, 15 N-HSQC fingerprint spectra compared with the wild-type protein. As expected, substitution of the strictly conserved essential Arg109, which eliminated the salt bridge with the Neu5Ac carboxyl group, completely abrogated binding, confirming its indispensable role in carbohydrate recognition.…”

Eosinophilic Gastroenteritis and Colitis: a Comprehensive Review

“…For example, sialic-acid-binding immunoglobulin-like lectin (siglec-8) is expressed on human eosinophils, where its ligation with ligand-coated polymers or anti-siglec-8 antibodies induces apoptosis [137]. Studies in human tissue from patients with eosinophilic disease demonstrate significant eosinophil cytolysis, which might be due to regulated necrosis.…”

“…Studies in human tissue from patients with eosinophilic disease demonstrate significant eosinophil cytolysis, which might be due to regulated necrosis. The ability to impede necrosis or redirect cell death from necrosis to apoptosis may lead to improved therapies in patients with eosinophilic conditions [137]. Ben Baruch-Morgenstern et al recently demonstrated that IL-5 activity in eosinophils was regulated by paired immunoglobulinlike receptors A and B, which may offer a way of regulating IL-5-induced expansion of the eosinophil [138].…”

Cyclophilin D regulates necrosis, but not apoptosis, of murine eosinophils

“…5D). To specifically assess cytolytic eosinophils, we established morphological criteria to differentiate cytolytic eosinophils from all eosinophils on MBP-stained colonic sections on the basis of previous studies (9,13,18). Notably, after DSS exposure, the percentage of cytolytic eosinophils in the colon was significantly reduced in Ppif Ϫ/Ϫ compared with Ppif ϩ/ϩ mice (Fig.…”

“…Recent in vitro studies demonstrated primary eosinophil lysis after stimulation by a Ca 2ϩ ionophore or Siglec-8 engagement on primed human eosinophils (18,38). In addition, these cell-free eosinophil granules can directly secrete their constituents, including eosinophil cationic protein, EPX, ribonucleases, and cytokines, via cytokines, chemokines, and eicosanoids ligating to a granule's membrane-bound receptors (24,25,31).…”