Chloride transport in functionally active phagosomes isolated from Human neutrophils.
Chloride transport in functionally active phagosomes isolated from Human neutrophils.
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从人类嗜中性粒细胞分离的功能活性吞噬体中的氯化物转运。
DOI：
10.1016/j.freeradbiomed.2012.10.542
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发表时间：
2012-12-15
影响因子：
7.4
通讯作者：
Wang, Guoshun
中科院分区：
文献类型：
--
作者：
Aiken, Martha L.;Painter, Richard G.;Zhou, Yun;Wang, Guoshun
氯离子对中性粒细胞吞噬体内次氯酸的产生和微生物的杀灭起着至关重要的作用。然而，这种负离子被输送到细胞器的分子机制却知之甚少。本研究采用优化的顺磁性乳胶微球和快速磁分离方法，从人中性粒细胞中分离出具有膜包裹和功能活性的吞噬小体。回收的吞噬小体高度富含与该细胞器相关的特定蛋白标记物，如溶酶体相关膜蛋白-1、髓过氧化物酶(MPO)、乳铁蛋白和NADPH氧化酶。当FITC-葡聚糖加入吞噬培养基时，大部分分离的吞噬小体在分离后仍保留有荧光标记，表明膜结构完整。对纯化的吞噬小体中的荧光pH指示剂吖啶橙进行的流式细胞仪检测表明，处于隔离状态的细胞器能够通过液泡型ATPase质子泵(V-ATPase)将质子输送到吞噬体腔内。当供应NADPH时，分离的吞噬小体结构性地氧化二氢罗丹明123，表明它们有能力产生过氧化氢。当用高氯酸盐特异的荧光探针R19-S用流式细胞仪检测时，这些制剂也显示出在吞噬体腔内有强劲的高氯酸盐产生。定量测定了MPO介导的与吞噬小球共价结合的蛋白质的碘化程度。氯离子通道抑制剂CFTRinh-172和NPPB可显著抑制吞噬体对碘的摄取和蛋白质的碘化。进一步的实验确定，V-ATPase驱动的质子通量进入分离的吞噬小体需要氯的共运输，而cAMP激活的CFTR氯通道是氯运输的主要贡献者。综上所述，这些数据表明，本文描述的吞噬体制剂保留了离子传输特性，包括CFTR在内的多个氯离子通道负责将氯供应给中性粒细胞吞噬体。
Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes.
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影响因子：
3.9
作者：
Behe, P.;Segal, A. W.
通讯作者：
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影响因子：
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