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

Jeremy Alastair O'Sullivan, Daniela J. Carroll, Yun Cao, Adriano N. Salicru, Bruce Scott Bochner

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1774-1785.e7
JournalJournal of Allergy and Clinical Immunology
Volume141
Issue number5
DOIs
StatePublished - May 1 2018

Fingerprint

Saliva
Eosinophils
Lectins
Mast Cells
Immunoglobulins
Endocytosis
Mast-Cell Leukemia
Cell Line
Clathrin
Basophils
Neuraminidase
Ribosomes
Confocal Microscopy
Protein-Tyrosine Kinases
Protein Kinase C
Tyrosine
Actins
Flow Cytometry
Membrane Proteins
Cell Death

Keywords

  • Siglec-8
  • allergic diseases
  • chronic eosinophilic leukemia
  • endocytosis
  • eosinophil
  • immunotoxin
  • mast cell
  • systemic mastocytosis
  • targeting

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

@article{2e882b2d237a4693a68223d85aee0e86,
title = "Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells",
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.",
keywords = "Siglec-8, allergic diseases, chronic eosinophilic leukemia, endocytosis, eosinophil, immunotoxin, mast cell, systemic mastocytosis, targeting",
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Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells. / O'Sullivan, Jeremy Alastair; Carroll, Daniela J.; Cao, Yun; Salicru, Adriano N.; Bochner, Bruce Scott.

In: Journal of Allergy and Clinical Immunology, Vol. 141, No. 5, 01.05.2018, p. 1774-1785.e7.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells

AU - O'Sullivan, Jeremy Alastair

AU - Carroll, Daniela J.

AU - Cao, Yun

AU - Salicru, Adriano N.

AU - Bochner, Bruce Scott

PY - 2018/5/1

Y1 - 2018/5/1

N2 - 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.

AB - 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.

KW - Siglec-8

KW - allergic diseases

KW - chronic eosinophilic leukemia

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KW - eosinophil

KW - immunotoxin

KW - mast cell

KW - systemic mastocytosis

KW - targeting

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