Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Biomedical Research, Danube University Krems, Krems, Austria.
Core Facility Flow Cytometry & Surgical Research Laboratories, Medical University of Vienna, Vienna, Austria.
Front Immunol. 2018 Dec 11;9:2797. doi: 10.3389/fimmu.2018.02797. eCollection 2018.
Secretion and exchange of biomolecules by extracellular vesicles (EVs) are crucial in intercellular communication and enable cells to adapt to alterations in their microenvironment. EVs are involved in a variety of cellular processes under physiological conditions as well as in pathological settings. In particular, they exert profound effects on the innate immune system, and thereby are also capable of modulating adaptive immunity. The mechanisms underlying their interaction with their recipient cells, particularly their preferential association with monocytes and granulocytes in the circulation, however, remain to be further clarified. Surface molecules exposed on EVs are likely to mediate immune recognition and EV uptake by their recipient cells. Here, we investigated the involvement of Tyro3, Axl, and Mer (TAM) tyrosine kinase receptors and of integrin CD11b in the binding of platelet-derived EVs, constituting the large majority of circulating EVs, to immune cells in the circulation. Flow cytometry and Western Blotting demonstrated a differential expression of TAM receptors and CD11b on monocytes, granulocytes, and lymphocytes, as well as on monocyte subsets. Of the TAM receptors, only Axl and Mer were detected at low levels on monocytes and granulocytes, but not on lymphocytes. Likewise, CD11b was present on circulating monocytes and granulocytes, but remained undetectable on lymphocytes. Differentiation of monocytes into classical, intermediate, and non-classical monocyte subsets revealed distinct expression patterns of Mer and activated CD11b. Co-incubation of isolated monocytes and granulocytes with platelet-derived EVs showed that the binding of EVs to immune cells was dependent on Ca. Our data do not support a particular role for TAM receptors or for activated CD11b in the association of platelet-derived EVs with monocytes and granulocytes in the circulation, as anti-TAM antibodies did not interfere with EV binding to isolated immune cells, as binding was not dependent on the presence of TIM4 acting synergistically with TAM receptors, and as neither low levels of Gas6, required as a linker between phosphatidylserine (PS) on the EV surface and TAM receptors on immune cells, nor masking of PS on the EV surface did interfere with EV binding.
细胞外囊泡（EVs）通过分泌和交换生物分子在细胞间通讯中起着至关重要的作用，并使细胞能够适应其微环境的变化。EVs 在生理条件下以及在病理条件下参与各种细胞过程。特别是，它们对先天免疫系统产生深远影响，因此也能够调节适应性免疫系统。然而，它们与受体细胞相互作用的机制，特别是它们在循环中优先与单核细胞和粒细胞结合的机制，仍有待进一步阐明。暴露在 EV 表面的分子可能介导免疫识别和 EV 被受体细胞摄取。在这里，我们研究了 Tyro3、Axl 和 Mer（TAM）酪氨酸激酶受体以及整合素 CD11b 在血小板衍生的 EV 与循环中的免疫细胞结合中的作用，这些 EV 构成了循环中大多数 EV。流式细胞术和 Western Blotting 表明 TAM 受体和 CD11b 在单核细胞、粒细胞和淋巴细胞以及单核细胞亚群上的表达存在差异。在单核细胞和粒细胞上，只有 Axl 和 Mer 以低水平表达，但在淋巴细胞上则检测不到。同样，CD11b 存在于循环中的单核细胞和粒细胞上，但在淋巴细胞上则检测不到。单核细胞分化为经典、中间和非经典单核细胞亚群，显示出 Mer 和激活的 CD11b 的不同表达模式。将分离的单核细胞和粒细胞与血小板衍生的 EV 共孵育表明，EV 与免疫细胞的结合依赖于 Ca2+。我们的数据不支持 TAM 受体或激活的 CD11b 在血小板衍生的 EV 与循环中的单核细胞和粒细胞结合中的特定作用，因为抗 TAM 抗体不干扰 EV 与分离的免疫细胞的结合，因为结合不依赖于与 TAM 受体协同作用的 TIM4 的存在，并且 PS 表面上的低水平 Gas6（作为 EV 表面上的 PS 与免疫细胞上的 TAM 受体之间的连接物）和 EV 表面上 PS 的掩蔽都不干扰 EV 结合。
