Laboratory of Biophysics of Cell Membranes under Critical State, Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V.A. Negovsky Scientific Research Institute of General Reanimatology, Petrovka Street 25c2, 107031 Moscow, Russia.
Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 119334 Moscow, Russia.
Int J Mol Sci. 2023 Aug 2;24(15):12355. doi: 10.3390/ijms241512355.
Before NETs are released, the neutrophil undergoes structural changes. First, it flattens, accompanied by a change in cell shape and rearrangement of the cytoskeleton. Then, nuclear swelling begins, which ends with the ejection of NETs into the extracellular space. We used widefield and confocal fluorescence microscopy to register morphological and structural changes in neutrophils during activation and NETosis. Different types of activators were used, such as NOX-dependent PMA and calcium ionophore A23187. The measurements were performed in a series of sequential stages. In the first stage (30 s after addition of activators and immediately after stimulation of neutrophils), the response of neutrophils to A23187 and PMA exposure was studied. Subsequently, the characteristics of neutrophils in different phases of activation were examined over a longer period of time (30, 60, 120, 180, and 240 min). The specific features of NETosis development were analyzed separately. During the first 30 s, neutrophils appeared to be heterogeneous in shape and structure of the actin cytoskeleton. Characteristic cell shapes included 30″ type 1 cells, similar in shape to the control, with F-actin concentrated in the center of the cytoplasm, and 30″ type 2 cells, which had flattened (spread) shapes with increased frontal dimensions and F-actin distributed throughout the cell. Later, the development of nuclear swelling, the corresponding changes in neutrophil membranes, and NET release into the extracellular space were evaluated. The conditions determining the initiation of chromatin ejection and two characteristic types of decondensed chromatin ejection were revealed. The results obtained contribute to a better understanding of the biophysical mechanisms of neutrophil activation and NETosis development.
在 NETs 释放之前，中性粒细胞会发生结构变化。首先，它会变平，伴随着细胞形状的变化和细胞骨架的重新排列。然后，核肿胀开始，最终导致 NETs 被喷射到细胞外空间。我们使用宽场和共聚焦荧光显微镜来记录激活和 NETosis 过程中中性粒细胞的形态和结构变化。使用了不同类型的激活剂，如依赖 NOX 的 PMA 和钙离子载体 A23187。测量是在一系列连续的阶段进行的。在第一阶段（加入激活剂后 30 秒，即刺激中性粒细胞后立即），研究了中性粒细胞对 A23187 和 PMA 暴露的反应。随后，在较长时间内（30、60、120、180 和 240 分钟）检查了不同激活阶段中性粒细胞的特征。分别分析了 NETosis 发展的特征。在最初的 30 秒内，中性粒细胞在肌动蛋白细胞骨架的形状和结构上似乎存在异质性。典型的细胞形状包括 30″ 型 1 细胞，其形状与对照相似，细胞质中心的 F-肌动蛋白集中，以及 30″ 型 2 细胞，其形状变平（展开），前向尺寸增加，F-肌动蛋白分布在整个细胞中。后来，评估了核肿胀的发展、中性粒细胞膜的相应变化以及 NET 释放到细胞外空间。揭示了启动染色质喷射的条件和两种特征性的去凝聚染色质喷射类型。所获得的结果有助于更好地理解中性粒细胞激活和 NETosis 发展的生物物理机制。
