Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells

Publication date: Available online 20 July 2017
Source:Journal of Allergy and Clinical Immunology
Author(s): Jeremy A. O'Sullivan, Daniela J. Carroll, Yun Cao, Adriano N. Salicru, Bruce S. Bochner
BackgroundSialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell-surface protein expressed selectively on human eosinophils, mast cells, and basophils, making it an ideal target for the treatment of diseases involving these cell types. However, the effective delivery of therapeutic agents to these cells requires an understanding of the dynamics of Siglec-8 surface expression.ObjectiveTo determine whether Siglec-8 is endocytosed in human eosinophils and malignant mast cells, identify mechanisms underlying its endocytosis, and demonstrate whether a toxin can be targeted to Siglec-8–bearing cells to kill these cells.MethodsSiglec-8 surface dynamics were examined by flow cytometry using peripheral blood eosinophils, mast cell lines, and Siglec-8–transduced cells in the presence of inhibitors targeting components of endocytic pathways. Siglec-8 intracellular trafficking was followed by confocal microscopy. The ribosome-inhibiting protein saporin was conjugated to a Siglec-8–specific antibody to examine targeting of an agent to these cells through Siglec-8 endocytosis.ResultsSiglec-8 endocytosis required actin rearrangement, tyrosine kinase and PKC activities, and both clathrin and lipid rafts. Internalized Siglec-8 localized to the lysosomal compartment. Maximal endocytosis in Siglec-8–transduced HEK293T cells required an intact immunoreceptor tyrosine-based inhibitory motif. Siglec-8 was also shuttled to the surface via a distinct pathway. Sialidase treatment of eosinophils revealed that Siglec-8 is partially masked by sialylated cis ligands. Targeting saporin to Siglec-8 consistently caused extensive cell death in eosinophils and the human mast cell leukemia cell line HMC-1.2.ConclusionTherapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this endocytic pathway.

Teaser

Our findings demonstrate that Siglec-8 can be exploited as an endocytic receptor to deliver drugs or toxins to kill human eosinophils and mast cells. This approach holds promise to treat diseases involving these cell types.


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