Induction of Adhesion Molecules (sE–selectin and sICAM–1) and Interferon–Beta by Human Retinal Vascular Endothelial Cells

Abstract

Abstract: : Purpose: We have recently shown the presence of interferon–beta (IFN–ß), sE–selectin and sICAM–1 in the sera of patients with retinal vasculitis. Since the vascular endothelium of the eye may be of great importance in regulating immune system activity, we evaluated human retinal microvascular endothelial cells to identify the cell source and possible inducers of these molecules. Methods: Human retinal microvascular endothelial cells were propagated in vitro. The cultured cells were incubated with media, a cytokine mix (TNF–α, IFN–γ and IL–1ß), poly I:C (an analog of dsRNA), or poly dI:dC. Supernatant fluids were harvested at varying times after stimulation. EIA was used to determine the presence of IFN–ß, sE–selectin or sICAM–1 proteins. Gene expression was determined by RT–PCR. Results: Supernatant fluids from un–stimulated cells did not contain detectable levels of IFN–α, or sE–selectin but did contain low levels of IFN–ß (2 IU/ml) and sICAM–1 (1.7 ng/ml). Treatment of the cells with the cytokine mix induced the release of sE–selectin (39 ng/ml) and sICAM–1 (42 ng/ml) but not IFN–α or ß. Treatment of cells with poly I:C induced the release of sE–selectin (13 ng/ml), sICAM–1 (35 ng/ml), IFN–ß (44 IU/ml) but not IFN–α. In contrast, treatment of the cells with the control oligonucleotide poly dI:dC did not enhance the secretion of these molecules. RT–PCR analysis revealed that poly I:C treatment increased gene expression of IFN–ß. The induction of sE–selectin, sICAM–1 and IFN–ß by poly I:C was shown to be dose dependent and could be blocked by pretreatment of cells with an antibody to Toll–Like Receptor 3 (TLR3). Conclusions: This study demonstrates that retinal microvascular endothelial cells are a possible source of IFN–ß, sE–selectin and sICAM–1 in patients with retinal vasculitis. Furthermore while the cytokine mix could induce release of sE–selectin and sICAM–1, only poly I:C induced the release of all three molecules (IFN–ß, sE–selectin and sICAM–1) through a mechanism involving TLR3.

Keywords: vascular cells • inflammation • retina