The Effect of Drugs with α-Glutamyl-Tryptophan for Cytokine Secretion and Level of Surface Molecule ICAM-1 In Vitro.

The study of the molecular mechanisms underlying the action of immunomodulatory drugs is important for substantiating their therapeutic effect. In the present work, spontaneous and TNFα-induced secretion of IL-1α and IL-8 pro-inflammatory cytokines, as well as the level of the ICAM-1 adhesion molecule in EA.hy 926 endothelial cell culture and peripheral blood mononuclear cells of healthy donors, is studied using an in vitro model of inflammation in the presence of α-glutamyl-tryptophan (α-Glu-Trp) and Cytovir-3. The aim was to evaluate cellular mechanisms mediating the immunomodulatory effect of α-Glu-Trp and Cytovir-3 drugs. It was shown that α-Glu-Trp reduced TNFα-induced IL-1α production and increased TNFα-stimulated level of the ICAM-1 surface molecule of endothelial cells. At the same time, the drug reduced secretion of the IL-8 cytokine induced by TNFα and increased the spontaneous level of ICAM-1 in mononuclear cells. Cytovir-3 had an activating effect on EA.hy 926 endothelial cells and human peripheral blood mononuclear leukocytes. In its presence, there was an increase in the spontaneous secretion of IL-8 by endothelial and mononuclear cells. In addition, Cytovir-3 increased the level of TNFα-induced ICAM-1 on endothelial cells and increased the spontaneous level of this surface molecule on mononuclear cells. Suppression of stimulated production of pro-inflammatory cytokines under the action of α-Glu-Trp both separately and as a part of Cytovir-3 may determine its anti-inflammatory properties. However, an increased level of the surface ICAM-1 molecule indicates mechanisms that enhance the functional activity of these cells, which is equally important for the implementation of an effective immune response to infection and repair of damaged tissues during inflammatory response.

[Monocytes of human blood as a target of Streptococcus pyogenes components].

AIM: Study the effect of components of destroyed streptococci on human blood monocyte functions related to processes of trans-endothelial migration in vitro. MATERIALS AND METHODS: Mononuclear leukocytes, isolated from blood of healthy donors, endothelial cells of EA.hy 926 line and supernatant of ultrasound disintegrated Streptococcus pyogenes (DSS) were the objects of the study. Evaluation of adhesion and monocyte migration, level of expression of adhesion molecules and phosphokinases on monocytes was carried out by flow cytometry using monoclonal antibodies. Cytokine concentration was determined by using standard commercial test systems in enzyme immunoassay. RESULTS: Under the effect of DSS, expression of adhesion molecules CD162 and CD11b, as well as phospho-p38 MAPK changed, IL-6 and IL-8 secretion induction took place. DSS caused enhancement of migration and adhesive activity of monocytes, however, inhibited intensity of trans-endothelial migration. CONCLUSION: Products of destroyed streptococci have a multi-directional effect on human blood monocytes, that could be explained by the presence of components with varying biological activity in DSS.

[Regulation of endothelial cells functions by ultrasonic supernatant of Streptococcus pyogenes].

Angiogenesis and vascular remodeling are vital components of inflammation. As an inflammation evolves, vessels expand to supply nutrients and inflammatory mediators, sustaining the accumulation of activated immune cells in the affected tissues. This study demonstrates that ultrasonic supernatant of Streptoccocus pyogenes has anti-angiogenic properties: inhibit EA.hy 926 human endothelial cells metabolism, adhesion, migration, proliferation. At the same time Streptococcal components inhibit signaling pathways that involve FAK and ERK1/2. These effects are not associated with necrosis or apoptosis in cell culture. Taking together, our results suggest that impairing angiogenic function of endothelial cells might contribute to the reduced tissue perfusion, hypoxia, and subsequent regional tissue necrosis caused by Streptococci group A.

In vitro studies of changes in human endothelial cell functions under the effect of imiquimod.

Imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) is an active immunomodulator with antiviral effects. In addition to its stimulatory effect on cell-mediated immunity, in vivo studies have detected its antiviral and antiangiogenic effects. Possible direct effect of imiquimod on endothelial cells remains not studied. We have shown that imiquimod inhibited proliferation and migration of human endothelial cells (EA.hy 926 strain) in vitro and induced apoptosis (but not necrosis) of endothelial cells and production of IL-6 cytokine. These results suggest that imiquimod inhibits angiogenesis via direct modulation of endothelial cell function.

[CD14++CD16- and CD14+CD16+ human monocytes adhesion to endothelial cells].

Two subsets of monocytes were identified in humans and other mammals blood based on different levels of CD14 and CD16 expression. These subsets have different patterns of adhesion molecules and chemokine receptors, which suggest different modes of interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14+CD16+ and CD14++CD16- monocytes to adhesion to endothelial cells monolayer in presence and in the absence of pro- and anti-inflammatory cytokines. We demonstrated that CD14+CD16+ monocytes had higher level of adhesion to intact endothelial cells monolayer than CD14++CD16- monocytes. Significant increase in adhesion of CD14++CD16- and CD14+CD16+ monocytes subpopulations was observed in the presence of both TNF alpha and TNF alpha combinations with other cytokines. IFN gamma and IL-4 showed no independent effects on adhesion of monocytes. These results have demonstrated that both CD14++CD16- and CD14+CD16+ monocytes can be recruited to inflamed endothelium, but, in the absence of inflammation, CD14+CD16+ monocytes adhere to endothelial cells two times stronger than CD14++CD16- monocytes.

[Effect of sutent and celecoxib on the properties of endothelial cells in vitro].

We studied the anti-angiogenic properties of sutent (SU11248) and celecoxib in human endothelial cell line EA. hy 926 in vitro. Sutent 0.05-0.5 microg/ml suppressed their proliferation and migration depending on dose while celecoxib did the same at 5.0 microg/ml. We were the first to demonstrate that endothelial cell incubation was followed by increase in 5'-nucleotidase activity in the presence of sutent while celecoxib did not produce such effect. It may be suggested that elevated 5'-nucleotidase concentration at the membranes of endothelial cells might in turn contribute to the pool of extracellular adenosine to stimulate antiinflammatory effect. Our data also contribute to the knowledge about the anti-angiogenic properties of sutent and celecoxib.

[Influence of products of bacterial origin on the expression of surface molecules in monocyte-derived and endothelial cells].

AIM: To study the influence of lypopolysaccharide (LPS) of Gram-negative bacterium (Escherichia coli O55:B5) and lysate of Gram-positive bacteria (Streptococcus pyogenes - group A, type M1, strain 40/58) on the level of expression of important surface molecules of monocyte-derived cells from continuous cell line THP-1 and endothelial cells from continuous cell line EA.hy 926. MATERIALS AND METHODS: Expression of surface molecules HLA-DR, CD11b, CD14, CD16, CD32, and CD54 was assessed using FITC- or PE-labeled monoclonal antibodies (Beckman Coulter, USA). Intensity of fluorescence was measured by flow cytometer Epics Altra manufactured by Beckman Coulter (USA). RESULTS: Studied components of Gram-positive and Gram-negative bacteria stimulated expression of CD14, CD16, CD32, and CD54 molecules on cells from THP-1 line; incubation of cells from EA.hy 926 line in the presence of the same bacterial components increased expression levels of CD54 and HLA-DR molecules. CONCLUSION: Endothelial cells of EA.hy 926 line was less sensitive to LPS of E. coli and lysate of S. pyogenes compared to monocyte-derived cells of THP-1 line. Usage of THP-1 cells allowed to reveal differences between effects of components of Gram-positive and Gram-negative bacteria. The stimulating effect of LPS was more pronounced compared to effect of S. pyogenes lysate in relation to expression of HLA-DR, CD11b, and CD54 molecules, whereas lysate of S. pyogenes better stimulated expression of CD14, CD16, and CD32 molecules.

Changes in phenotype of monocyte-like THP-1 cells associated with transendothelial migration.

The level of expression of CD11b and HLA-DR surface molecules on monocyte-like THP-1 cells increased significantly as a result of transmigration of these cells through a monolayer of endothelial cells. The expression of all studied markers (CD11b, HLA-DR, and CD-14) increased significantly after transendothelial migration in the presence of TNF-alpha and IFN-gamma. The changes in surface phenotype of THP-1 cells after transendothelial migration in the presence of TNF-alpha were more pronounced than in the presence of IFN-gamma. Transendothelial migration of THP-1 cells in the presence of IL-4 caused less pronounced changes in the surface phenotype, which did not differ from changes in transmigration without cytokines.