Tomizawa Hiroki, Arima Misaki, Miyabe Yui, Furutani Chikako, Kodama Sahoko, Ito Keisuke, Watanabe Ken, Hasegawa Ryo, Nishiyama Shohei, Hizuka Keinosuke, Yamada Takechiyo, Ueki Shigeharu
Akita University Graduate School of Medicine School of Medicine, Department of General Internal Medicine and Clinical Laboratory Medicine, Akita, Japan.
Akita University Graduate School of Medicine School of Medicine, Department of Otorhinolaryngology, Head & Neck Surgery, Akita, Japan.
Am J Respir Cell Mol Biol. 2024 Aug 1. doi: 10.1165/rcmb.2023-0438OC.
Cytolytic ETosis is a type of programmed cell death distinct from apoptosis and necrosis and plays a major role in the innate immune system and disease progression. Through the process of ETosis, cells release their chromatin with diverse antimicrobial proteins into the extracellular milieu, forming extracellular traps (ETs). Although ETosis has been reported in several leukocyte types, few studies have compared ETosis and the component proteins of ETs in leukocytes. The aim of this study was to better understand the characteristics of eosinophil ETosis (EETosis) compared with other leukocytes. We isolated human blood eosinophils, neutrophils, basophils, monocytes, and lymphocytes and stimulated them with known ETosis inducers, a protein kinase C activator PMA, or a calcium ionophore A23187. Both stimuli induced eosinophil cell death and ET release after 180 minutes of stimulation in a NADPH-oxidase-dependent manner. PMA also induced NADPH-oxidase-dependent ETosis in neutrophils, whereas little or no significant ETosis was observed in basophils, monocytes, or lymphocytes at 180 minutes. Mass spectrometry-based proteomic analysis of eosinophil- and neutrophil-derived ETs identified 997 and 1415 proteins, respectively. Among the physiological stimuli tested, immobilized IgA and IgG induced EETosis. C-C motif chemokine ligand 11 (CCL11) and interleukin 5 (IL-5) were weak inducers of EETosis, but co-stimulation significantly induced rapid EETosis. Under high serum or albumin conditions, co-stimulation with CCL11 and IL-5 paradoxically prolonged cell survival by preventing spontaneous apoptosis. This study provides an in-depth characterization of EETosis and highlights the precise regulation of eosinophil survival and cell death pathways.
细胞溶解性ETosis是一种不同于凋亡和坏死的程序性细胞死亡类型，在先天免疫系统和疾病进展中起主要作用。通过ETosis过程，细胞将其染色质与多种抗菌蛋白释放到细胞外环境中，形成细胞外陷阱（ETs）。尽管在几种白细胞类型中都报道了ETosis，但很少有研究比较白细胞中ETosis和ETs的组成蛋白。本研究的目的是更好地了解嗜酸性粒细胞ETosis（EETosis）与其他白细胞相比的特征。我们分离了人血嗜酸性粒细胞、中性粒细胞、嗜碱性粒细胞、单核细胞和淋巴细胞，并用已知的ETosis诱导剂、蛋白激酶C激活剂PMA或钙离子载体A23187刺激它们。两种刺激在刺激180分钟后均以NADPH氧化酶依赖性方式诱导嗜酸性粒细胞死亡和ET释放。PMA还诱导中性粒细胞中NADPH氧化酶依赖性的ETosis，而在180分钟时，嗜碱性粒细胞、单核细胞或淋巴细胞中几乎没有或没有观察到明显的ETosis。基于质谱的嗜酸性粒细胞和中性粒细胞来源的ETs蛋白质组学分析分别鉴定出997种和1415种蛋白质。在测试的生理刺激中，固定化的IgA和IgG诱导EETosis。C-C基序趋化因子配体11（CCL11）和白细胞介素5（IL-5）是EETosis的弱诱导剂，但共刺激显著诱导快速EETosis。在高血清或白蛋白条件下，CCL11和IL-5的共刺激通过防止自发凋亡反而延长了细胞存活时间。本研究对EETosis进行了深入表征，并突出了嗜酸性粒细胞存活和细胞死亡途径的精确调控。
