Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors.
Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors.
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DOI：
10.1186/s13613-016-0175-z
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发表时间：
2016-12
影响因子：
8.1
通讯作者：
Tanaka H
中科院分区：
文献类型：
--
作者：
Sueyoshi K;Sumi Y;Inoue Y;Kuroda Y;Ishii K;Nakayama H;Iwabuchi K;Kurishita Y;Shigemitsu H;Hamachi I;Tanaka H
多形核中性粒细胞（PMNs）的激活在脓毒症中起重要作用。之前，我们发现ATP的释放和通过ATP受体的反馈对PMN的激活至关重要；然而，人们对其中的动态仍然知之甚少。两种新型荧光化学传感器PMAP-1和MitoAP-1分别用于检测活细胞质膜和线粒体中的ATP。在这项研究中，我们旨在利用这些化学传感器评估脓毒症患者pmn中ATP的定位。用这些化学传感器对16例脓毒症患者和健康对照（hc）分离的活PMNs进行染色，并用共聚焦显微镜观察，并用流式细胞术评估其平均荧光强度（mfi）。CD11b在PMNs中的表达也被评估。脓毒症患者PMNs中PMAP-1、MitoAP-1的mfi及CD11b的表达在0-1 d显著高于hc。与第0-1天相比，第3-4天PMAP-1和CD11b的MFI表达明显降低，而MitoAP-1的MFI维持在较高水平。PMAP-1 MFI与患者CD11b表达、白细胞计数、中性粒细胞计数、c反应蛋白水平呈显著正相关。脓毒症患者PMAP-1和MitoAP-1较高的mfi提示ATP在PMN激活中的关键作用。ATP水平的时间差异表明ATP在线粒体和细胞表面起着不同的作用。这些数据将有助于了解pmn中ATP的动态，并有助于开发一种新的败血症治疗方法。
The activation of polymorphonuclear neutrophils (PMNs) plays an important role in sepsis. Previously, we showed that ATP release and feedback via ATP receptors are essential for PMN activation; however, the dynamics remain poorly understood. Two new fluorescent chemosensors, PMAP-1 and MitoAP-1, were developed to detect ATP in the plasma membrane and mitochondria of living cells, respectively. In this study, we aimed to evaluate ATP localization using these chemosensors in PMNs of sepsis patients. Live PMNs isolated from 16 sepsis patients and healthy controls (HCs) were stained with these chemosensors and observed by confocal microscopy, and their mean fluorescence intensities (MFIs) were evaluated using flow cytometry. CD11b expression in PMNs was also evaluated. The MFIs of PMAP-1 and MitoAP-1 and CD11b expression in PMNs from sepsis patients on days 0–1 were significantly higher than those of HCs. The MFI of PMAP-1 and CD11b expression on days 3–4 decreased significantly compared to those observed at days 0–1, whereas MitoAP-1 MFI was maintained at a high level. The PMAP-1 MFI was significantly positively correlated with CD11b expression, white blood cell counts, neutrophil counts, and C-reactive protein levels in patients. The higher MFIs of PMAP-1 and MitoAP-1 in sepsis patients suggest a pivotal role of ATP for PMN activation. The temporal difference in ATP levels suggests that ATP plays different roles in the mitochondria and on the cell surface. These data should contribute to the understanding of the dynamics of ATP in PMNs and help to develop a novel therapy for sepsis.