Sialic acid–binding immunoglobulin-like lectin 8 (Siglec-8) is an activating receptor mediating β₂-integrin–dependent function in human eosinophils

Background: Siglec-8 is a CD33 subfamily cell-surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec-8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33-related Siglecs function as inhibitory receptors, but the ability of Siglec-8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec-8 might instead function as an activating receptor. Objective: We sought to determine the role of IL-5 priming and identify the signaling molecules involved in Siglec-8 function for human eosinophils. Methods: We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec-8 function for human eosinophils. Results: Cytokine priming was required to elicit the unanticipated finding that Siglec-8 engagement promotes rapid β₂-integrin–dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec-8–mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec-8–mediated eosinophil apoptosis. Finally, engagement of Siglec-8 on IL-5–primed eosinophils resulted in increased phosphorylation of Akt, p38, and c-Jun N-terminal kinase 1 that was also β₂-integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec-8–mediated eosinophil apoptosis. Conclusions: These data demonstrate that Siglec-8 functions uniquely as an activating receptor on IL-5–primed eosinophils through a novel pathway involving regulation of β₂-integrin–dependent adhesion, NADPH oxidase, and a subset of protein kinases.