Variation of adhesion molecule expression on human umbilical vein endothelial cells upon multiple cytokine application

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Abstract

Background: A variety of cytokines, mediators, activators, growth factors and other products are simultaneously released into circulation with the activation of the cellular immune system during rejection or infection. The secretion of these biochemical markers potentiates the immunological events associated with these processes. Among other things some cytokines demonstrate regulatory effects on the expression of endothelial cell adhesion molecules. Method: Endothelial cells are detached by trypsinisation and adhesion molecule expression is assessed by means of flow cytometry. Fluorescence-conjugated mouse monoclonal antibodies directed against VCAM-1, ICAM-1, PECAM-1, CD34, E- and P-selectin are used. Results: The combined application of different cytokines synergistically evokes P-selectin expression after a chosen incubation period of 16 h, while under single cytokine treatment P-selectin induction is not observed. Co-stimulation with TNF-α and a second cytokine reduces its influence on E-selectin. IL-1β/IFN-γ lead to E-selectin levels higher than those under treatment with one of the both alone. Concomitant incubation with all cytokines synergistically down-regulates PECAM-1 referred to each cytokine alone. Conclusion: Our investigations in some cases clearly demonstrate that the combination of a second cytokine with TNF-α, IL-1β or IFN-γ can either synergistically or antagonistically modulate the expression of adhesion molecules on HUVECs.

Introduction

An important feature of different phases of the inflammatory process is the localisation of leucocytes. The description of this event documents interactions between leucocytes and endothelial cells lining venules at inflammatory sites. Endothelial cells express a variety of adhesive receptors that regulate leucocyte adhesion and also the adhesion of endothelial cells to the extracellular matrix. These interactions include the first rolling and the consequent stationary adhesion and transmigration of leucocytes and are complex phenomena requiring multiple recognition mechanisms. In acute and chronic inflammation the initial step is the recruitment of leucocytes. Thereby, circulating leucocytes bind loosely to stimulated or injured endothelium via the action of various selectins and their ligands. Leucocytes start to roll along the endothelium and to interact with cytokines presented by the endothelium. This leads to the activation of leucocytes, which increase their integrin adhesiveness, opening up the possibility to firmly interact with endothelial cells. Consequently, the fixed leucocytes migrate across the endothelium through interaction with immunoglobulin-like adhesion molecules [1]. It has been reported that cytokines have regulatory effects on cell adhesion molecule expression [2], [3]. However, not all cytokine combinations released into the blood during inflammatory and immunological processes have been investigated thoroughly so far. Therefore, the present study investigates whether combinations of IL-2, IL-4, IL-6, IL-8 and IL-10 with IL-1β,  TNF-α or  IFN-γ reveal synergistic or antagonistic effects on adhesion molecule expression on human umbilical vein endothelial cells (HUVECs). Additionally, we determine the in vitro effects of a cytokine mixture comprising all these stimulants. As a reference, cells are incubated with every single cytokine mentioned above. It has been shown that TNF-α induces E-selectin leading to peak levels after 6 h of stimulation, which remain up-regulated for 16 h [4]. It has also been found that VCAM-1 has a constant increased expression level under incubation times between 6 and 24 h. Furthermore, it has been demonstrated that ICAM-1 and PECAM-1 reach highest expression levels after stimulation for 24 h. P-selectin expression is known to peak within 10 min after TNF-α stimulation.

In our work, we have chosen a stimulation period of 16 h in order to render it possible to evaluate the modulatory influences of the different cytokine mixtures on the expression of E-selectin, VCAM-1, ICAM-1, PECAM-1 and CD34, which remain at high levels under these conditions. We have also investigated whether P-selectin expression is modulated after this incubation time in the presence of more than one cytokine in the incubation cocktail.

Section snippets

Materials

Medium 199 (Hepes-modification) and heparin were ordered from Sigma-Aldrich, Steinheim, Germany. Penicillin/streptomycin, l-glutamine, trypsin/EDTA (0.05%/0.02% in Hanks balanced salt solution) and PBS (without Ca2+ and Mg2+) were purchased from Gibco-Life Technologies, Merelbeke, Belgium. Cytokines were ordered from R&D Systems, Abington, UK. Fluorescence-labelled (FITC, RPE) mouse monoclonal antibodies directed against PECAM-1, CD34, ICAM-1, VCAM-1, E- and P-selectin were obtained from

Results and discussion

The present study dealt with possible synergistic or antagonistic effects of a number of cytokines (TNF-α, IL-1β, IFN-γ, IL-2, IL-4, IL-6, IL-8 and IL-10)-applied in different dual combinations-on adhesion molecule expression of HUVECs, i. e. PECAM-1, CD34, ICAM-1, VCAM-1, E- and P-selectin. Additionally, the influences of a mixture of all cytokines were investigated. As a reference for the values obtained under concomitant stimulation with two or more cytokines, the cells were also incubated

Acknowledgments

We appreciate the kind help of Mrs Christiane Klein as well as the support of all co-workers at the Department of Gynecology of Kaiser-Franz-Josef Hospital in collecting umbilical cords. Snezana Markovic received a grant from the IFCC attending a professional scientific exchange program.

References (18)

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