Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study

Abstract

Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.

A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen

“…At higher concentrations (500 nM), neutrophils enlarged and showed signs of plasma membrane permeabilization. This could be explained by the shedding of plasma membrane microvesicles upon neutrophil activation, a process which may be regulated by µ-calpain and ezrin proteins during NETosis [35][36][37]. Moreover, the nuclei were enlarged and the genetic material was decondensed, which could be described as "diffused" or chromatin "cloud" NETs [28,29].…”

Red Blood Cell Storage with Xenon: Safe or Disruption?

“…Scanning areas varied from 100 × 100 to 1 × 1 μm 2 in semi-contact operation mode. The resolution of each image spanned between 512 and 1024 points [ 29 , 30 ]. Image processing was performed using FemtoScan Online software, Version 2.3.239 (5.2) (Advanced Technologies Center, Moscow, Russia, (accessed on 12 January 2023)) [ 31 , 32 , 33 , 34 ].…”

Cell Surface Parameters for Accessing Neutrophil Activation Level with Atomic Force Microscopy

“…During activation, neutrophils can significantly change their morphology and, in particular, their shape, e.g., from spherical to flat (spread) [42,51]. This process may also be accompanied by changes in the pericellular layer (glycocalyx), which are difficult to evaluate using only classical optical methods.…”

“…It is important to note that stimulation of neutrophils, with both physiological and pharmacological stimuli, such as hydrogen peroxide [39][40][41], not only activates effector functions, but also leads to significant changes in the shape of the plasma membrane, including spreading [42]. The mechanisms behind this spreading phenomenon have been described previously [43] and are controlled by a calcium-dependent cysteine proteinase called calpain [43].…”