Proceedings of The Physiological Society

University of York (2002) J Physiol 539P, S018


Activated T cells modulate P-glycoprotein expression in an in vitro model of the blood-brain barrier

K.H. Tan, W.M. Purcell, S.J.R. Heales*, J.D. McLeod and R.D. Hurst

Centre for Research in Biomedicine, Faculty of Applied Sciences, University of West of England, Bristol and *Department of Neurochemistry, Institute of Neurology, University College London, London, UK

The blood-brain barrier (BBB) acts as a boundary between the central nervous system (CNS) and the blood and is actively involved in both the regulation of CNS immune-surveillance and in the limitation of cytotoxic agents by the expression of the multi-drug transporter P-glycoprotein (Pgp). During inflammatory disease, reactive leucocytes (mostly lymphocytes) readily gain access to the brain and mediate cerebral cell damage/death via the production and action of pro-inflammatory cytokines and/or oxidative stress (Conlon et al. 1999). During CNS inflammation there is also evidence of a breakdown in BBB function. Hence, the aim of this study was to evaluate whether similar processes could also affect endothelial cell function (monolayer integrity and Pgp expression).

This work was performed using the human umbilical endothelial-like cell line (ECV304), which when co-cultured with rat C6 glioma cells comprises a useful in vitro BBB model (Hurst & Fritz, 1996). Human Jurkat T lymphocytes pre-activated with mitogen (phyto-haemogglutinin), were co-incubated with confluent endothelial monolayers. Endothelial cell viability (vital stain exclusion), barrier integrity (transendothelial electrical resistance, TEER), and Pgp expression (flow cytometric analysis with anti-Pgp antibody MRK16) were assessed. Activated lymphocytes dose-dependently mediated endothelial cell dysfunction and damage which was reflected by visible breaks in the monolayers, a decline in TEER, and an increase in endothelial cell mortality. The process of this dysfunction may well be mediated by oxidative stress (De Vries et al. 2001) and indeed, recent studies have indicated that Pgp expression changes following free radical exposure (Felix & Barrand, 2001). In agreement with these reports, exposure of our BBB model to activated T cells resulted in an increase in the expression level of Pgp on the endothelial cells (332 ± 134 % of the control, n = 6). Pgp expression has been suggested to regulate apoptosis and oxidative stress (Johnstone et al. 2000) and may therefore be a mechanism for protection against further T cell-induced damage.

In summary, our findings suggest that in inflammatory CNS disease, activated lymphocytes may play a role in endothelial cell/barrier dysfunction (cell death and monolayer integrity). Furthermore, T cell-mediated modulation of endothelial cell Pgp expression may represent a protective mechanism.

Where applicable, experiments conform with Society ethical requirements