5 years ago

Siglec-8 is an activating receptor mediating β2 integrin-dependent function in human eosinophils

Siglec-8 is a CD33 subfamily cell surface receptor that is selectively expressed on human eosinophils. Following 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 may instead function as an activating receptor. Objective To determine the role of IL-5 priming and to identify the signaling molecules involved in Siglec-8 function for human eosinophils. Methods We used a mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8, in combination with integrin blocking antibodies, pharmacological 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 via NADPH oxidase activation, because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC23766 (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 JNK1 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 uniquely functions as an activating receptor on IL-5 primed eosinophils via a novel pathway involving regulation of β2-integrin-dependent adhesion, NADPH oxidase and a subset of protein kinases.


Siglec-8 can unexpectedly function as an activating receptor, not an inhibitory receptor, because its engagement on IL-5-primed eosinophils promotes β2-integrin-mediated adhesion and activates NADPH oxidase and protein kinases, all of which are necessary for causing human eosinophil apoptosis.

Publisher URL: www.sciencedirect.com/science

DOI: S0091674917314239

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