Devine, Lesley; (1996) Cellular interaction between lymphocytes and retinal pigment epithelium. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Posterior uveitis is an ocular inflammatory condition which can cause extensive retinal damage. Evidence indicates that this is a T cell mediated disorder and it is likely that induction of the disease is brought about by the migration of lymphocytes from the circulation into the neuroretina. For this to occur lymphocytes must first cross the blood- retinal barrier (BRB) which consists of the retinal vascular endothelium in the anterior portion of the retina and the retinal pigment epithelium (RPE) at the posterior aspect. Lymphocyte migration into the retina can therefore occur via these two separate routes; a direct route by crossing the vascular endothelium and an indirect route via the RPE. For migration to occur by this latter path lymphocytes must first be captured from the circulation by the choroidal endothelium. From here they must them migrate into the extracellular space and penetrate Bruch's membrane, before interacting with adhesion molecules expressed on the RPE which are likely to control migration into the retina. The role of the vascular endothelium in controlling lymphocyte migration has been extensively studied but the role of other cellular barriers in modulating lymphocyte passage is poorly understood. This project was therefore undertaken to investigate the factors involved in lymphocyte adhesion to and migration across RPE monolayers in vitro. PVG rat RPE cells were first isolated and characterised by immunostaining techniques for the expression of cytokeratins and the rat RPE specific monoclonal antibody PE2. In addition, these cells were shown to constitutively express ICAM-1 and MHC class I which was upregulated upon activation with IFN-γ. MHC class II expression, however, was only found on IFN-γ activated cells. Due to the difficulty associated with primary cell culture a rat RPE cell line was developed by immortalising primary cultures of rat RPE using SV40 large T. These cells were characterised and shown to express similar levels of RET-PE2 and cytokeratins as primary cultures. Rat lymphocytes used in this study were characterised using flow cytometric analysis to determine the phenotype and state and mode of activation of each group studied. The ability of peripheral lymph node (PLN) cells (activated and non-activated) and antigen specific CD4+ T cell line lymphocytes to adhere to RPE cells in vitro was evaluated. Results demonstrate that the state and mode of lymphocyte activation is crucial in determining their ability to adhere to and migrate across the monolayers. Antigen specific T cell lines were more adhesive and migratory than untreated or activated PLN cells. The effect of activating the RPE with the cytokines interferon-γ (IFN-γ) and interleukin-1 (IL-1) on adhesion and migration were also studied. It was found that adhesion was not increased by cytokine activation whereas migration was increased by activation of cells with IFN-γ but not IL-1. The adhesion molecules involved in the migration of antigen specific T cell lines across RPE monolayers was also studied. Migration was found to be predominantly ICAM-l/LFA-1 mediated, with VCAM-l/VLA-4 interactions playing a role only when monolayers had been activated for 72 hours with IFN-γ. In addition, the ability of the immortalised RPE cells to support lymphocyte migration was determined. The migration of PLN cells was similar to that observed with primary cultures, although the migration of antigen specific T cell line lymphocytes was found to be lower. To compare results form RPE-lymphocyte interactions with that of retinal endothelial cell-lymphocyte interactions, it was necessary to study lymphocyte migration across PVG rat derived retinal endothelial cells as retinal endothelial studies in our department had employed endothelium isolated from Lewis rats. During the course of this study it was noted that lymphocyte migration was significantly lower across PVG retinal endothelium compared with Lewis rat derived retinal endothelium. Lewis rats are more susceptible to the animal model for uveitis, therefore this may be one of the factors responsible for this. To study this further, ICAM-1 and VCAM-1 expression were compared on retinal endothelium isolated from the two rat strains and it was found that PVG rats expressed lower constitutive levels of ICAM-1.

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