Wateridge, David John; (2005) Lymphocyte migration and the regulation of brain endothelial cell junctions. Doctoral thesis , UCL (University College London).

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Abstract

Through bona fide tight junctions and regulated transcytosis, brain endothelial cells (ECs) are able to establish a blood-brain barrier (BBB) that regulates access of leucocytes and solutes to the central nervous systenn (CNS). Occludin was the first transmembrane tight junction protein identified and it has been demonstrated that expression of mutant occludin proteins in epithelial cells dramatically affects neutrophil transmigration and monolayer permeability. To determine if similar strategies could influence transendothelial lymphocyte migration and other endothelial barrier properties, a brain EC line (GPNT) was transfected with a range of occludin proteins and characterised by functional assays. Expression of wild-type occludin reduced T cell migration and, as in epithelial cells, this was shown to be dependent on an unmodified N-terminal domain. The mechanism(s) by which lymphocytes physically cross the endothelial barrier remains a poorly described stage of lymphocyte extravasation. The possibility remains, however, that modulation of endothelial cell-cell junctions is a necessary pre-requisite to physically allow the passage of a leucocyte through the EC wall, i.e the paracellular pathway. The firm adhesion of circulating T cells to BBB ECs is predominantly via ICAM- 1, a cell adhesion molecule of the immunoglobulin superfamily expressed on the EC and previously demonstrated to be capable of signal transduction. A hypothesis was generated that I CAM-1 engagement facilitates diapedesis by activating signalling pathways that regulate cell-cell junctions. Using GPNT EC, the role of ICAM-1 in regulating junctional proteins and integrity was assessed. Following crosslinking of ICAM-1, both VE-cadherin and PECAM showed increased tyrosine phosphorylation with identical experiments showing that ICAM-1 crosslinking correlated with an increase in transmonolayer permeability to molecular tracers. Immunoprecipitated VE-cadherin showed no change in its association with p- or y-catenin following ICAM-1 crosslinking, however, there was increased association of both catenins with PECAM via a rho-dependent/ROCK- independent pathway. The existence of such pathways suggests that pharmacological targeting of ICAM-1-mediated signalling may be advantageous in the therapeutic management of neuroinflammatory diseases.

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