Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.
Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.
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DOI：
10.3389/fimmu.2021.737988
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发表时间：
2021
影响因子：
7.3
通讯作者：
O'Sullivan JA
中科院分区：
文献类型：
--
作者：
Carroll DJ;Cao Y;Bochner BS;O'Sullivan JA
唾液酸结合免疫球蛋白样凝集素（Siglec）-8是一种聚糖结合受体，具有基于免疫受体酪氨酸的抑制和转换基序（分别为ITIM和ITSM），选择性表达于嗜酸性粒细胞、肥大细胞和嗜碱性粒细胞（程度较轻）。先前的工作表明，Siglec-8与IL-5致敏的嗜酸性粒细胞的结合通过CD 11b/CD 18整联蛋白介导的粘附和NADPH氧化酶活性引起细胞死亡，并鉴定了连接粘附、活性氧（ROS）产生和细胞死亡的信号分子。然而，由Siglec-8接合直接激活的近端信号级联仍然难以捉摸。Siglec家族的大多数成员具有相似的细胞质信号传导基序，并募集与ITIM介导的信号传导一致的蛋白酪氨酸磷酸酶SHP-1/2，以抑制细胞活化。然而，尚未研究嗜酸性粒细胞中Siglec-8功能对这些磷酸酶的依赖性。使用Siglec-8抗体结合和药理学抑制结合测定CD 11b整联蛋白的细胞表面上调和构象活化、ROS产生和细胞死亡，我们试图鉴定参与Siglec-8信号传导的分子，并确定每个分子发挥作用的过程阶段。我们在此证明Src家族激酶（SFK）、Syk、SHIP 1、PAK 1、MEK 1、ERK 1/2、PLC、PKC、酸性鞘磷脂酶/神经酰胺酶和Btk的酶活性都是Siglec-8诱导的嗜酸性粒细胞死亡所必需的，而SHP-1/2、SHIP 2或c-Raf没有明显作用。虽然大多数这些信号传导分子是Siglec-8诱导的嗜酸性粒细胞表面CD 11b整联蛋白上调所必需的，但Btk在信号传导级联中被磷酸化并在稍后被激活，而是CD 11b激活所必需的。相比之下，SFKs和ERK 1/2在此过程中更早地磷酸化，这与它们在增加细胞表面CD 11b水平中的作用一致。此外，用latrunculin B或jasplakinastatin预处理嗜酸性粒细胞显示，肌动蛋白丝的解体是必要的，足以使表面CD 11 B整合素上调，肌动蛋白聚合是下游ROS产生所必需的。这些结果表明，Siglec-8通过IL-5致敏的嗜酸性粒细胞中的一组未预料到的信号传导分子进行信号传导以诱导细胞死亡，并挑战了携带ITIM的Siglec通过涉及蛋白酪氨酸磷酸酶的抑制性途径进行信号传导以实现其下游功能的预期。
Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a glycan-binding receptor bearing immunoreceptor tyrosine-based inhibitory and switch motifs (ITIM and ITSM, respectively) that is selectively expressed on eosinophils, mast cells, and, to a lesser extent, basophils. Previous work has shown that engagement of Siglec-8 on IL-5–primed eosinophils causes cell death via CD11b/CD18 integrin–mediated adhesion and NADPH oxidase activity and identified signaling molecules linking adhesion, reactive oxygen species (ROS) production, and cell death. However, the proximal signaling cascade activated directly by Siglec-8 engagement has remained elusive. Most members of the Siglec family possess similar cytoplasmic signaling motifs and recruit the protein tyrosine phosphatases SHP-1/2, consistent with ITIM-mediated signaling, to dampen cellular activation. However, the dependence of Siglec-8 function in eosinophils on these phosphatases has not been studied. Using Siglec-8 antibody engagement and pharmacological inhibition in conjunction with assays to measure cell-surface upregulation and conformational activation of CD11b integrin, ROS production, and cell death, we sought to identify molecules involved in Siglec-8 signaling and determine the stage of the process in which each molecule plays a role. We demonstrate here that the enzymatic activities of Src family kinases (SFKs), Syk, SHIP1, PAK1, MEK1, ERK1/2, PLC, PKC, acid sphingomyelinase/ceramidase, and Btk are all necessary for Siglec-8–induced eosinophil cell death, with no apparent role for SHP-1/2, SHIP2, or c-Raf. While most of these signaling molecules are necessary for Siglec-8–induced upregulation of CD11b integrin at the eosinophil cell surface, Btk is phosphorylated and activated later in the signaling cascade and is instead necessary for CD11b activation. In contrast, SFKs and ERK1/2 are phosphorylated far earlier in the process, consistent with their role in augmenting cell-surface levels of CD11b. In addition, pretreatment of eosinophils with latrunculin B or jasplakinolide revealed that actin filament disassembly is necessary and sufficient for surface CD11b integrin upregulation and that actin polymerization is necessary for downstream ROS production. These results show that Siglec-8 signals through an unanticipated set of signaling molecules in IL-5–primed eosinophils to induce cell death and challenges the expectation that ITIM-bearing Siglecs signal through inhibitory pathways involving protein tyrosine phosphatases to achieve their downstream functions.
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影响因子：
8.8
作者：
Chu JY;Dransfield I;Rossi AG;Vermeren S
通讯作者：
Vermeren S
影响因子：
7.4
作者：
Dubey, Megha;Singh, Abhishek K.;Dikshit, Madhu
通讯作者：
Dikshit, Madhu
DOI：
10.4049/jimmunol.1500871
发表时间：
2015-09-15
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Esnault S;Kelly EA;Shen ZJ;Johansson MW;Malter JS;Jarjour NN
通讯作者：
Jarjour NN
影响因子：
4.3
作者：
Angata, Takashi;Tabuchi, Yukako;Nakamura, Mitsuru
通讯作者：
Nakamura, Mitsuru
影响因子：
4.4
作者：
Brooks, Robert;Fuhler, Gwenny M.;Kerr, William G.
通讯作者：
Kerr, William G.