Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors.

Koichiro Sueyoshi,Kumiko Ishii,Kazuhisa Iwabuchi,Itaru Hamachi,Yoshiaki Inoue,Yasutaka Kurishita,Yoko Kuroda,Hiroshi Tanaka,Hajime Shigemitsu,Hitoshi Nakayama,Yuka Sumi

doi:10.1186/s13613-016-0175-z

Abstract

BackgroundThe 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.MethodsLive 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.ResultsThe 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.ConclusionsThe 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.

Highlights

The activation of polymorphonuclear neutrophils (PMNs) plays an important role in sepsis
We reported that plasma ATP levels were correlated with PMN activation and were significantly higher in mouse models of cecal ligation and puncture (CLP) [7]
The bright green fluorescence emitted by PMAP-1 was predominantly observed on the plasma membrane of PMNs (Fig. 1a)

Summary

Introduction

The activation of polymorphonuclear neutrophils (PMNs) plays an important role in sepsis. PMAP-1 and MitoAP-1, were developed to detect ATP in the plasma membrane and mitochondria of living cells, respectively. We aimed to evaluate ATP localization using these chemosensors in PMNs of sepsis patients. Polymorphonuclear neutrophils (PMNs) are one of the most important phagocytic cell types in defending against invading pathogens and play a pivotal role in inflammation, their activation can be a Sueyoshi et al Ann. Intensive Care (2016) 6:64 through the P2Y2 receptor, one of eight P2Y G proteincoupled receptors, is an essential mechanism for activating PMNs [5, 6]. Two new fluorescent chemosensors were developed (PMAP-1 and MitoAP-1), which can detect ATP and its derivatives in specific regions of living cells [10]. PMAP-1 can localize to the plasma membrane and detect the extracellular release of ATP. MitoAP-1 can spontaneously localize in the mitochondria and detect local ATP

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