Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells - 04/05/18
Abstract |
Background |
Sialic 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.
Objectives |
We sought to 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.
Methods |
Siglec-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 the targeting of an agent to these cells through Siglec-8 endocytosis.
Results |
Siglec-8 endocytosis required actin rearrangement, tyrosine kinase and protein kinase C 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.
Conclusions |
Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec-8 endocytic pathway.
Le texte complet de cet article est disponible en PDF.Key words : Siglec-8, endocytosis, eosinophil, mast cell, allergic diseases, chronic eosinophilic leukemia, systemic mastocytosis, immunotoxin, targeting
Abbreviations used : BIM, DAPI, ITIM, ITSM, PI3K, PTP, SFK, Siglec
Plan
This work was supported by the National Heart, Lung, and Blood Institute (grant no. P01HL107151 to B.S.B.), the National Institute of Allergy and Infectious Diseases (grant no. AI072265 to B.S.B. and grant no. T32AI083216 to J.A.O.), and the National Cancer Institute (Cancer Center Support grant no. NCI CA060553 to the Center for Advanced Microscopy at Northwestern University). |
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Disclosure of potential conflict of interest: J. A. O'Sullivan and B. S. Bochner receive grant support from the National Institutes of Health (NIH). B. S. Bochner has current or recent consulting or scientific advisory board arrangements with, or has received honoraria from, Sanofi-Aventis, TEVA, AstraZeneca, and Allakos and owns stock in Allakos and Glycomimetics; receives publication-related royalty payments from Elsevier and UpToDate, and is a coinventor on existing Siglec-8–related patents and thus may be entitled to a share of royalties received by Johns Hopkins University on the potential sales of such products; and is also a cofounder of Allakos, which makes him subject to certain restrictions under university policy. The terms of this arrangement are being managed by the Johns Hopkins University and Northwestern University in accordance with their conflict of interest policies. The rest of the authors declare that they have no relevant conflicts of interest. |
Vol 141 - N° 5
P. 1774 - mai 2018 Retour au numéroBienvenue sur EM-consulte, la référence des professionnels de santé.
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