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.

Staphylococcus aureus Causes the Arrest of Neutrophils in the Bloodstream in a Septicemia Model.

Staphylococcus aureus induces the expression of VCAM-1, P- and E-selectins on the endothelial cells of the EA.hy926 cell line but, at the same time, causes the significant suppression of the force and work of adhesion between these receptors of endotheliocytes and the receptors of neutrophils in an experimental septicemia model. Adhesion contacts between the receptors of neutrophils and endotheliocytes are statistically significantly suppressed under non-opsonized and opsonized S. aureus treatment, which disrupts the initial stage of transendothelial migration of neutrophils-adhesion. Thus, S. aureus causes the arrest of neutrophils in the bloodstream in an experimental septicemia model.

Conditioning adhesive contacts between the neutrophils and the endotheliocytes by Staphylococcus aureus.

We have developed a model for evaluating the integral intercellular interactions in the "endotheliocyte-neutrophil" system and have shown the high variability of adhesion contacts in different donors associated with different expression profiles of neutrophils. Two methods (forсe spectroscopy-spectroscopy and scanning ion-conductance microscopy) showed a decrease in the rigidity of the membrane-cytoskeletal complex of neutrophils under the influence of Staphylococcus aureus 2879 M. Adding this strain to the "endotheliocyte-neutrophil" system caused a statistically significant decrease in the adhesion force and adhesion work, which indicates a change in the expression profile and physicochemical properties of membranes of both types of interacting cells (neutrophils and endotheliocytes).

Dynamics of formation and morphological features of neutrophil extracellular traps formed under the influence of opsonized Staphylococcus aureus.

In the process of performing their protective functions, neutrophils can form neutrophil extracellular traps (NETs), consisting of DNA in combination with enzymes and histones. The aim of the study was to determine the dynamics of the formation of NETs under the influence of opsonized Staphylococcus aureus and to determine the morphological features of their development in real time by atomic force microscopy. It was found that the maximum formation of NETs was observed after 3 hours of co-incubation of neutrophils and opsonized S. aureus. For the first time, the atomic force microscopy method revealed that, at first, large blocks of parallel DNA helices are formed, which then spread in waves, and only then their bifurcation and separation can be observed. Some of the strands formed are covered by a shell, which subsequently completely disappears. Enzymes and histones become clearly visible only after 140 to 150 minutes of observation. The DNA helixes move toward the opsonized S. aureus. After NET formation, the cell remains on the substrate only in the form of traces of focal adhesion. This, and the fact that the maximum amount of NETs is formed after 3 hours of co-incubation with opsonized S. aureus, suggests that the formation of NETs follows the classical mechanism. The study of the dynamics of formation and the microstructure of NETs makes it possible to estimate the time frame for the implementation of this protective mechanism of the human body when performing the compensatory inflammatory reaction.

The interaction between human blood neutrophil granulocytes and quantum dots.

For biomedical applications, it is important to know, which kinds of blood cells can capture quantum dots (QDs). The maximum accumulation of QDs was found for the monocyte fraction of leukocytes, the minimum binding of QDs was observed for lymphocytes. It was found that CdSe/ZnS-MPA QDs are actively absorbed by the cells and have more expressed toxicity. The classical mechanism of the phagocytosis of QDs was revealed for neutrophils, when the QDs are located in phagolysosomes. The capture of QDs by neutrophil granulocytes has resulted in a destruction of certain types of QDs. The interaction of the neutrophils with the QDs has resulted in the death of the cells by one of the following cell death mechanisms: necrosis, apoptosis, autophagy, NETos, or mummification. The aggregation of the QDs manifested as an increase of the hydrodynamic diameter of the QDs was found to occur under the influence of serum and under the influence of blood cells (lymphocytes and neutrophils) in a serum-free medium.

Treatment by serum up-conversion nanoparticles in the fluoride matrix changes the mechanism of cell death and the elasticity of the membrane.

Nanoparticles are increasingly being used for treatment and diagnostic purposes, but their effects on cells is not fully understood. Here, the interaction of fluorescent up-conversion nanoparticles (UpC-NPs) with neutrophils was investigated by imaging and measurement of membrane-cytosceletal elasticity by atomic force microscopy. It was found that UpC-NPs induce the death of neutrophils mainly by necrosis, and to a smaller extent by a novel process called 'mummification'. Necrosis occurs by gradual loss of intracellular contents and nuclei, 45-110min after exposure to UpC-NPs. Mummification is apparent as an increase in the rigidity of the neutrophils' membrane and acquisition of a characteristic bumpy shape with numerous protrusions; this structure does not change during atomic force microscopy scanning. Coating UpC-NPs with protein by incubation with serum leads to (1) formation of nanoparticle aggregates in the nm and µm size range, (2) a reduction in toxicity, (3) reduced mummification of neutrophils, and (4) no significant reduction of the elasticity of the membrane-cytoskeletal complex of neutrophils 30min after exposure to coated UpC-NPs. The study shows that serum proteins greatly curb the toxicity of nanoparticles and reveals mummification as a novel mechanism of UpC-NP-induced cell death.