Interactions between Siglec-8 and endogenous sialylated cis ligands restrain cell death induction in human eosinophils and mast cells

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a sialoside-binding receptor expressed on the surface of eosinophils and mast cells that bears an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in its cytoplasmic domain. On eosinophils that have been primed with IL-5, GM-CSF, or IL-33, antibody ligation of Siglec-8 leads to cell death through a pathway involving the β2 integrin-dependent generation of reactive oxygen species (ROS) via NADPH oxidase. In contrast, engagement of Siglec-8 on mast cells has been described to inhibit mast cell activation and mediator release but not impact cell viability. The differences in responses between cytokine-primed and unprimed eosinophils, and between eosinophils and mast cells, to Siglec-8 ligation are not understood. In addition, we have previously found that Siglec-8 binds to sialylated ligands on the surface of the same cell (so-called cis ligands) on eosinophils, preventing interaction with a Siglec-8 ligand in trans. However, the functional relevance of these cis ligands have not been elucidated. We therefore explored the potential influence of cis ligands of Siglec-8 on the surfaces of both eosinophils and mast cells. Removal of sialic acid using exogenous sialidase had profound effects on the consequences of Siglec-8 antibody engagement on both cell types, eliminating the need for cytokine priming of eosinophils to facilitate cell death and enabling Siglec-8-dependent mast cell death without impacting anti-Siglec-8 antibody binding. The cell death process licensed by incubation with sialidase resembled that characterized in IL-5-primed eosinophils, including the upregulation of CD11b, production of ROS, and requirement for the signaling activities of Syk, PI3K, and PLC. These results implicate cis ligands in restraining Siglec-8 function on eosinophils and mast cells and reveal a promising approach to the selective depletion of mast cells in patients with mast cell-mediated diseases.