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.

中文翻译：

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利用Siglec-8内吞机制杀死人嗜酸性粒细胞和恶性肥大细胞

背景

结合唾液酸的免疫球蛋白样凝集素（Siglec）-8是一种在人嗜酸性粒细胞，肥大细胞和嗜碱性粒细胞上选择性表达的细胞表面蛋白，使其成为治疗涉及这些细胞类型疾病的理想靶标。然而，将治疗剂有效递送至这些细胞需要了解Siglec-8表面表达的动力学。

目标

我们试图确定Siglec-8是否在人类嗜酸性粒细胞和恶性肥大细胞中被内吞，确定其内吞作用的机制，并证明毒素是否可以靶向含Siglec-8的细胞以杀死这些细胞。

方法

通过使用流式细胞仪，在存在靶向内吞途径成分的抑制剂的情况下，使用外周血嗜酸性粒细胞，肥大细胞系和Siglec-8转导的细胞，对Siglec-8表面动力学进行了检测。共聚焦显微镜观察Siglec-8细胞内运输。将核糖体抑制蛋白沙泊蛋白与Siglec-8特异性抗体偶联，以检查通过Siglec-8内吞作用将药物靶向这些细胞。

结果

Siglec-8内吞作用需要肌动蛋白重排，酪氨酸激酶和蛋白激酶C活性，以及​​网格蛋白和脂质筏。内部化的Siglec-8定位于溶酶体区室。在Siglec-8转导的HEK293T细胞中最大的内吞作用需要完整的基于免疫受体酪氨酸的抑制基序。Siglec-8也通过独特的途径穿梭到地面。嗜酸性粒细胞的唾液酸酶处理表明，Siglec-8被唾液酸化的顺式配体部分掩盖。将saporin靶向Siglec-8一直在嗜酸性粒细胞和人类肥大细胞白血病细胞系HMC-1.2中引起广泛的细胞死亡。

结论

通过利用这种Siglec-8内吞途径，可将治疗有效载荷选择性地靶向嗜酸性粒细胞和恶性肥大细胞。