Effect of cytokines on adhesion molecules expression in cell culture of blood vessel endothelium

Abstract

The interaction between leukocytes and endothelial cells plays the essential role in inflammation. Endothelial cells express a variety of adhesive receptors that regulate their adhesion to leukocytes and also to the extracellular matrix. These interactions are complex phenomena that require multiple recognition mechanisms, and include the first rolling and later the stationary adhesion and transmigration of leukocytes. It is known that cytokines have regulatory effects on cell adhesion molecules expression. In the present study we investigated the influence of cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8 IL-10, TNF-a, IFN-g) and the combined use of IL-2, IL-4, IL-6, IL-8 and IL-10 with IL-1b, TNF-b or IFN-g on the expression of adhesion molecules of cultured human umbilical vein endothelial cells (HUVECs) after stimulation for 16 hours. Likewise, in vitro model described herein is designed to mimic the activation of endothelial cells by cytokines as seen during inflammatory processes. This process is mediated by specific cell adhesion molecules being crucial for the generation of immune and inflammatory responses. Therefore, HUVECs are treated with two different cytokine combinations consisting of either IL-2, IL-6, IL-8 IFN-g and TNF-a or IL-1b, IL-2, IL-4, IL-6, IL-10, IFN-g and TNF-a. Endothelial cells were collected from hunam umbilical vain using collagense type II, and cell cultures in complete medium were kept in the incubator (37.4°C, 5% CO2). After stimulation cells were prepared for analysis using tripsinisation procedure. The surface expression of the following adhesion molecules was determined in cultured human umbilical vein endothelial cells (HUVECs) by means of flow-cytometric analysis: CD 62P (P-selectin), CD 62E (E-selectin, ELAM-1) CD 106CD 34 (L-selectin ligand). The highest CD 62E expression on the surface of HUVECs was found when endothelial cells were stimulated with TNF-a alone. Also they were increased after stimulation with IL-1b, while IL-4 led to down-regulation of CD 62E. Incubation of HUVEC monolayers with IL-1b, IL-4 as well as TNF-a and IFN-g, statistically significant, reduced the surface expression of CD 34 while other cytokines did not affect CD 34 expression. Incubation of HUVECs with a single cytokine caused no statistically significant changes in CD 62P expression compared to controls. The most potent effect on CD 54 expression was found under TNF-a stimulation; IL-1b and IFN-g had also amplifying effects, while all other tested cytokines caused no significant changes in surface molecule expression. Surface expression of CD 106 was amplified during incubation with IL-1b, IL-4, TNF-a and IFN-g. Single stimulation of tested cytokines did not significantly alter the cell surface expression of CD 31. Concomitant stimulation with IL-2, IL-4, IL-6, IL-8 or IL-10 with IL-1b, TNF-a or IFN-g led to different effects compared with effects of single cytokine stimulation: CD 62E were up-regulated under co-stimulation with combination of IL-1b and IFN-g, IL-6 and IL-1b, and also in all combinations with TNF-a. Statistically significant differences were found in CD 62P surface expression after concomitant stimulation with IL-1b and IFN-g, and in combinations with TNF-a. Co-stimulation with IL-10 and IL- 1b, TNF-a or IFN-g, or IL-8 with IL-1b or IFNg, IL-6 with IFN-g, IL-4 with TNF-a or IFN-g and IL-2 with IFN-g significantly decreased the level of CD 34 surface expression. (VCAM-1), CD 54 (ICAM-1), CD 31 (PECAM-1) and CD 54 expression was up-regulated after stimulation with IL-1b and IFN-g, and under concomitant stumulation with TNF-a. Surface expression of molecule CD 106 was higher after co-stimulation of cytokines with TNF-a, and IL-4 or IL-10 with IL-1b. These effects indicate modulation of single cytokine effects. Intracellular mechanisms included in those effects need to be investigated. Also there were found modulatory effects of cytokine combinations. Some effects of cytokine combinations were different in comparison to single cytokine effect. This finding indicates that intracellular mechanisms are present and responsible for signal modulation of single cytokine. The application of these two cytokine combinations mimicing inflammation reactions results in effects of comparable dimensions significantly increasing the mean fluorescence intensity of E-selectin, VCAM-1 and ICAM-1 surface expression accompanied by the induction of P-selectin expression. The experiments reveal a strong up-regulation of these cell surface antigens under conditions mimicing inflammation. This is an essential finding stressing the importance of endothelial cells during inflammatory processes.