Background: Siglec-8 is a single transmembrane protein selectively expressed on eosinophils, basophils, and mast cells. It can exist in two alternatively spliced forms, Siglec-8 and Siglec-8L, the latter likely possessing inhibitory signaling activity. We, therefore, explored the expression and function of Siglec-8 and Siglec-8L in these cells. Methods: Flow cytometry was performed using specific mAb. RT-PCR was performed using Siglec-8 and Siglec-8L specific primers. Various viability assays were performed following Siglec-8 ligation with antibodies. Specific inhibitors z-IETD-FMK and z-LEHD-FMK were used to investigate caspase-8 and caspase-9 function, respectively, white the EnzCheck assay system was used to monitor caspase-3 activity. Results: All preparations tested to date of human basophils, cord blood-derived mast cells and HMC-1 cells express both Siglec-8 and Siglec-8L mRNA. Most eosinophil preparations also expressed both isoforms, but some only expressed Siglec-8L. Siglec-8 ligation of eosinophils in vitro with Siglec-8 mAbs mid secondary anti-mouse Ab resulted in rapid induction of apoptosis and caspase-3 activation. In contrast to what is observed with apoptotic responses in eosinophils induced by Fas ligation or glucocorticoids, Siglec-8-induced eosinophil apoptosis was not reversed by IL-5. Using specific inhibitors, we also determined that Siglec-8-induced eosinophil apoptosis was dependent on caspase-8 and caspase-9. Conclusions: These results demonstrate a novel, caspase-dependent apoptosis-inducing effect of Siglec-8 crosslinking on eosinophils that is not rescued by IL-5. Activation via Siglec-8 may provide a new therapeutic approach to reducing eosinophil (and presumably basophil and mast cell) numbers in vivo.
