Exploring the Process of Neutrophil Transendothelial Migration Using Scanning Ion-Conductance Microscopy.

The dynamics of neutrophil transendothelial migration was investigated in a model of experimental septicopyemia. Scanning ion-conductance microscopy allowed us to determine changes in morphometric characteristics of endothelial cells during this process. In the presence of a pyogenic lesion simulated by Staphylococcus aureus, such migration was accompanied by both compensatory reactions and alteration of both neutrophils and endothelial cells. Neutrophils demonstrated crawling along the contact sites between endothelial cells, swarming phenomenon, as well as anergy and formation of neutrophil extracellular traps (NETs) as a normergic state. Neutrophil swarming was accompanied by an increase in the intercellular spaces between endothelial cells. Endothelial cells decreased the area of adhesion to the substrate, which was determined by a decrease in the cell projection area, and the cell membrane was smoothed. However, endothelial cell rigidity was paradoxically unchanged compared to the control. Over time, neutrophil migration led to a more significant alteration of endothelial cells: first, shallow perforations in the membrane were formed, which were repaired rather quickly, then stress fibrils were formed, and finally, endothelial cells died and multiple perforations were formed on their membrane.

ROS Production by a Single Neutrophil Cell and Neutrophil Population upon Bacterial Stimulation.

The reactive oxygen species (ROS) production by a single neutrophil after stimulation with S. aureus and E. coli was estimated by an electrochemical amperometric method with a high time resolution. This showed significant variability in the response of a single neutrophil to bacterial stimulation, from a "silent cell" to a pronounced response manifested by a series of chronoamperometric spikes. The amount of ROS produced by a single neutrophil under the influence of S. aureus was 5.5-fold greater than that produced under the influence of E. coli. The response of a neutrophil granulocyte population to bacterial stimulation was analyzed using luminol-dependent biochemiluminescence (BCL). The stimulation of neutrophils with S. aureus, as compared to stimulation with E. coli, caused a total response in terms of ROS production that was seven-fold greater in terms of the integral value of the light sum and 13-fold greater in terms of the maximum peak value. The method of ROS detection at the level of a single cell indicated the functional heterogeneity of the neutrophil population, but the specificity of the cellular response to different pathogens was the same at the cellular and population levels.