Mechanisms of allergy/immunology
Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells

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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.

Section snippets

Human eosinophil isolation and culture

Written informed consent for blood donation was obtained using an institutional review board–approved protocol. Eosinophils from blood donors were purified from peripheral blood using density gradient centrifugation, erythrocyte hypotonic lysis, and immunomagnetic negative selection with CD16 beads (Miltenyi Biotec, Bergisch Gladbach, Germany) as described.10 Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and 4′-6-diamidino-2-phenylindole (DAPI)

Siglec-8 is internalized in human eosinophils and mast cell lines

Because Siglec-8 endocytosis has not been studied previously, we first sought to confirm that Siglec-8, like other siglecs, is indeed internalized following its engagement. To measure Siglec-8 endocytosis, cell-surface Siglec-8 was bound by an unlabeled anti–Siglec-8 mAb and, following incubation at 37°C to permit endocytosis, any mAb remaining at the cell surface was detected using a labeled secondary antibody. Upon antibody engagement of the receptor on primary human eosinophils, Siglec-8 was

Discussion

Siglec-8 is considered a potential therapeutic target for allergic disease due to its expression on eosinophils, mast cells, and basophils and its roles in inducing cell death in activated eosinophils and preventing FcεRI signaling-induced mediator release in mast cells.34 However, its potential as a target via which to deliver toxins, chemotherapeutic agents, or drugs had not been explored previously and is especially relevant for mast cells and non–cytokine-primed eosinophils that do not

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    • Cited by (0)

    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).

    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.

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