Comparison of Immortalized bEnd5 and Primary Mouse Brain Microvascular Endothelial Cells as in vitro Blood–Brain Barrier Models for the Study of T Cell Extravasation

Abstract

Important insights into the molecular mechanism of T cell extravasation across the blood-brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs.

In Vivo Analysis of Uropod Function during Physiological T Cell Trafficking

“…Although this suggests that lymphocytes use the paracellular, rather than the transcellular, TEM pathway in vitro, we cannot exclude that factors in addition to the increased tight junction functionality of primary endothelial cells compared with immortalized cell lines affect the transcellular TEM efficiency. This is particularly relevant because pMBMECs display different F-actin networks, which may influence transcellular TEM (35). Irrespective of the TEM pathway, we hypothesize that the pore size is the decisive factor for the requirement of ROCK activity.…”

“…The brain-derived endothelial cell line bEnd5 is characterized by its .10-fold greater permeability rate and its less elaborate tight junction organization compared with pMBMECs (35). Superfused control T cells moved efficiently on and through CCL21-coated bEnd5 cells (Fig.…”

“…Based on the above findings, we set out to examine dynamic T cell crawling and transmigration through endothelial beds showing a complete, or incomplete, tight junction organization and overall permeability. First, we superfused control or Y27632-pretreated T cells over TNF-a-activated and CCL21-coated pMBMECs, which possess a low permeability rate (35). Under these conditions, control T cells exhibited dynamic motility and transmigration ( Fig.…”

β2 Integrin–Mediated Crawling on Endothelial ICAM-1 and ICAM-2 Is a Prerequisite for Transcellular Neutrophil Diapedesis across the Inflamed Blood–Brain Barrier

“…Paracellular permeability of pMBMECs seeded on Matrigel-coated filter inserts (0.4 mm Polycarbonate Membrane, Transwell Permeable Support; Costar, Corning, NY) was determined using Alexa Fluor 680-labeled 3-kDa dextran (10 mg/ml) (LuBioScience, Luzerne, Switzerland) as published (27). Fluorescence of diffused dextran was measured using the Odyssey Imaging System (LI-COR Biosciences).…”

“…To this end, we employed an in vitro model of the BBB, in which primary mouse brain microvascular endothelial cells (pMBMECs) that maintain physiological barrier characteristics (27), are placed in a flow chamber. Combined with high-magnification live-cell imaging, this model has proven to reliably mimic the dynamic behavior and the molecular mechanisms involved in the interaction of T cells or neutrophils with the BBB in neuroinflammatory disorders (12,28,29).…”