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).

Pathogenic Escherichia coli change the adhesion between neutrophils and endotheliocytes in the experimental bacteremia model.

Septicemia caused by gram-negative bacteria is characterized by high death rate due to the endotoxin release. Since the septicemia depends not only on biochemical aspects of interactions in the system bloodstream, the study of mechanical interactions is also important. Using a model of experimental septicemia caused by E. coli, a hyperproduction of integrins CD11a and CD11b by neutrophils was shown, but this did not lead to the establishment of strong adhesion contacts between endothelial cells and neutrophils. On the contrary, adhesion force and work, as assessed by FS spectroscopy, were statistically significantly reduced in the presence of bacteria. It has also been shown that exposure to the pathogenic strain E. coli 321 increases the stiffness of the membrane-cytoskeleton complex of endothelial cells and bacteria significantly change their morphology on long-term observation. At the same time, we observed the death of neutrophils by apoptosis. Thus, it was shown that besides lipopolysaccharide release there are other pathogenic factors of E. coli: decrease in the interaction between neutrophil and endothelial cell caused by an increase of the endothelial cell rigidity and apoptotic death of neutrophils probably as a result of adhesins and exotoxin effects. Obtained results should be taken in mind during the therapy of septicemia.

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.