Dexamethasone inhibits cytokine-induced intercellular adhesion molecule-1 up-regulation on endothelial cell lines

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Abstract

Intercellular adhesion molecule-1 (ICAM-1) expression on three endothelial cell lines was differently modulated by pro-inflammatory cytokines, such as interleukin-1β and tumour necrosis factor-α (TNF-α) and the glucocorticoid hormone dexamethasone. Incubation of EA.hy926 cells with 1 μM dexamethasone prior to addition of TNF-α consistently reduced ICAM-1 induction by approximately 40%. EA.hy926 cell responsiveness to the steroid was validated by detecting specific dexamethasone binding, with a calculated affinity constant of 1.3 nM and a maximal number of sites of 35×103 per cell. To establish the generality of dexamethasone inhibition upon ICAM-1 up-regulation, two other endothelial cell lines were assessed. Incubation of LT4 and ECV304 cells with interleukin-1β or TNF-α produced a significant increase in ICAM-1 expression on their cell surface (ranging from a 2-fold increase for interleukin-1β to a 5-fold increase for TNF-α). Addition of dexamethasone was again able to significantly reduced this induction. Finally, the effect of the steroid on cytokine-induced ICAM-1 up-regulation was functionally related to its ability to suppress in vitro neutrophil trans-endothelial passage. Overall these data indicate that ICAM-1 is a likely molecular target for the anti-inflammatory action exerted by dexamethasone. Inhibition of ICAM-1 up-regulation may, at least in part, mediate the potent anti-migratory action displayed by this class of anti-inflammatory drugs.

Introduction

Intercellular adhesion molecule-1 (ICAM-1), a member of the immunoglobulin superfamily of adhesion molecules, is basally expressed on the cell surface of endothelial cells, where its levels can be increased during inflammatory conditions (Duperray et al., 1995; Malik and Lo, 1996). In the sequela of events regulating the interaction between leukocytes and the endothelium of post-capillary venules, endothelial ICAM-1 promotes leucocyte firm adhesion and the subsequent emigration through the endothelial gaps into the sub-endothelial space (Luscinskas et al., 1991; Sligh et al., 1993).

The importance of ICAM-1 and other adhesion molecules in mediating the cellular response characteristic of inflammatory and infectious pathologies makes them ideal candidates to be targeted for anti-inflammatory therapy. However, the efficacy of the most potent anti-inflammatory drugs available, glucocorticoid hormones, in inhibiting ICAM-1 induction in human umbilical vein endothelial cells, as well as on endothelial cells from other sources, has been conflicting (Cronstein et al., 1992; Forsyth and Talbot, 1992). Similar conflicting results have been found using cells from different lineages. The synthetic glucocorticoid, dexamethasone, reduced interleukin-1β-dependent ICAM-1 induction on a bronchial epithelial cell line (van de Stolpe et al., 1993) and on primary murine macrophages (Perretti et al., 1996), whereas the steroid was ineffective when a renal tubular epithelial cell line was used (Hogan and Foster, 1996). The reported discrepancies may also be related to the stimulus used to achieve cell activation, as indicated in a recent study in which dexamethasone inhibited ICAM-1 induction following cell activation by lipolysaccharide but not by a combination of tumour necrosis factor-α (TNF-α) and γ-interferon (Burke-Gaffney and Hellewell, 1996).

In recent years the availability of endothelial cell lines to many laboratories has been particularly useful to investigate the responsiveness of the endothelium to pro- and anti-inflammatory agents. EA.hy926 cells were the first human umbilical vein endothelial cell-derived cell line to be available and maintained most of the characteristics of primary human umbilical vein endothelial cells (Edgell et al., 1983), including increased adhesion molecule expression on stimulation with cytokines (Thornill et al., 1993).

Using three distinct human umbilical vein endothelial cell-derived cell lines, the present study was undertaken to test the effect of dexamethasone on ICAM-1 levels on the endothelial cell surface following activation with pro-inflammatory cytokines. We found that all three cell lines displayed specific glucocorticoid receptors, and consequently responded to the steroid in terms of inhibition of ICAM-1 up-regulation. Finally, the effect of dexamethasone was evident at longer (24 h) but not shorter (4 h) time-points of cell stimulation with TNF-α.

Section snippets

Cell cultures

EA.hy926 cells were kindly provided by Dr. C.-J. Edgell (Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA). These cells are an hybridoma between human umbilical vein endothelial cells and the epithelioma A549, and retain most of the features of human umbilical vein endothelial cells, including the human factor VIII-related antigen (Edgell et al., 1983). EA.hy926 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10%

Dexamethasone effect on EA.hy926 cell activation

In basal conditions, EA.hy926 cells expressed negligible levels of ICAM-1 on their cell surface whereas PECAM-1 (monitored as an internal control, since not inducible by cytokines) was highly present (Fig. 1). Cell incubation with TNF-α produced a time-dependent increase in ICAM-1 levels (up to 100-fold increase by 24 h post-stimulation), associated with a modest (between 10 and 20%) but consistent reduction in PECAM-1 expression. Addition of dexamethasone prevented both phenomena, with the

Discussion

The data obtained in this study contribute to clarify the apparent disparity in published reports which have investigated the ability of dexamethasone to inhibit ICAM-1 induction on the plasma membrane of endothelial cells. Using three human umbilical vein endothelial cell-derived cell lines we show that dexamethasone significantly reduced TNF-α- or interleukin-1β-induced endothelial cell activation (measured as ICAM-1 up-regulation and, for EA.hy926 cells, by the modest PECAM-1 shedding). A

Acknowledgements

This work was supported by an endowment made to The William Harvey Research Institute by The Ono Pharmaceutical Co. (Osaka, Japan). We thank Ms. Semal Butt for help in the glucocorticoid receptor binding experiments.

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