Department of Emergency and Critical Care Medicine, Juntendo University, Urayasu Hospital, 2-1-1 Tomioka, Urayasu, Chiba, 279-0021, Japan.
Lipid Biology Laboratory, Riken Advanced Science Institute, Wako, Saitama, Japan.
Ann Intensive Care. 2016 Dec;6(1):64. doi: 10.1186/s13613-016-0175-z. Epub 2016 Jul 15.
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.
多形核粒细胞（PMN）的激活在脓毒症中起着重要作用。先前，我们发现 ATP 的释放和通过 ATP 受体的反馈对于 PMN 的激活是必不可少的，但动态过程仍知之甚少。两种新的荧光化学传感器，PMAP-1 和 MitoAP-1，分别被开发用于检测活细胞的质膜和线粒体中的 ATP。在这项研究中，我们旨在使用这些化学传感器评估脓毒症患者 PMN 中的 ATP 定位。
用这些化学传感器对来自 16 名脓毒症患者和健康对照者（HC）的活 PMN 进行染色，并用共聚焦显微镜观察，并通过流式细胞术评估其平均荧光强度（MFI）。还评估了 PMN 中的 CD11b 表达。
脓毒症患者第 0-1 天的 PMAP-1 和 MitoAP-1 的 MFI 和 PMN 中的 CD11b 表达明显高于 HCs。与第 0-1 天相比，第 3-4 天的 PMAP-1 MFI 和 CD11b 表达显著降低，而 MitoAP-1 MFI 保持在高水平。PMAP-1 MFI 与患者的 CD11b 表达、白细胞计数、中性粒细胞计数和 C 反应蛋白水平呈显著正相关。
脓毒症患者中 PMAP-1 和 MitoAP-1 的较高 MFI 表明 ATP 对于 PMN 激活具有重要作用。ATP 水平的时间差异表明，ATP 在粒体和细胞表面发挥不同的作用。这些数据应该有助于了解 PMN 中 ATP 的动态，并有助于开发脓毒症的新疗法。
