α4 Integrin/FN-CS1 mediated leukocyte adhesion to brain microvascular endothelial cells under flow conditions

Abstract

Insights into sequential leukocyte–endothelial interactions during leukocyte trafficking have been obtained through experiments using human umbilical vein endothelial cells (HUVEC) under flow conditions. To investigate leukocyte–brain endothelial cell interactions, we developed a dynamic in vitro system, using Transfected Human Brain Microvascular Endothelial Cells (THBMEC) and a parallel plate flow chamber. Human peripheral blood mononuclear cells (PBMC) were perfused across confluent THBMEC cultures at a velocity that approximates the rate found in human brain capillaries. Leukocyte–THBMEC interactions were visualized by phase-contrast microscopy, and images were captured on a CCD camera. To simulate inflammatory conditions, we activated THBMEC with the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), which up-regulated chemokine and adhesion molecule expression in THBMEC without affecting the distribution of immunoreactivity for tight junction-associated proteins. PBMC adhesion was enhanced by cytokine-mediated activation of THBMEC. G protein-coupled receptor (GPCR) activation was essential for leukocyte–THBMEC interaction, as pertussis toxin (PTX) treatment of PBMC abrogated PBMC adhesion to activated THBMEC. The anti-α4 integrin antibody, natalizumab, infused into MS patients, significantly reduced the adhesion of their ex vivo PBMC to activated THBMEC under flow conditions. Further study showed that alternatively spliced fibronectin containing the CS1 region (FN-CS1), but not Vascular Cell Adhesion Molecule type 1 (VCAM-1), was the ligand of α4 integrin on activated THBMEC. Blocking FN-CS1 abrogated PBMC adhesion on activated THBMEC, while anti-VCAM-1 antibodies had no effect. These results established a novel in vitro dynamic BBB model. We also demonstrated the dependence of leukocyte–endothelial interactions in this model on α4 integrins and FN-CS1.

Introduction

Elucidating mechanisms of leukocyte migration across the blood brain barrier (BBB) may be useful for understanding the pathogenesis of disorders including multiple sclerosis (MS), brain and spinal cord trauma, stroke, and HIV infection (Chaudhuri et al., 2008, Danton and Dietrich, 2003, Lossinsky and Shivers, 2004, McCandless et al., 2008, Wang et al., 2008). Components that subserve leukocyte–endothelial cell interactions define the molecular pathways that regulate leukocyte trafficking.

Insights into sequential leukocyte–endothelial interactions have been obtained through experiments using human umbilical vein endothelial cells (HUVEC) under flow conditions (Schreiber et al., 2007, Burns et al., 2000). The step-wise interactions between leukocytes and endothelial cells are governed by adhesion molecules, chemokines, and chemokine receptors. Chemokine engagement of cognate G protein-coupled receptor (GPCRs) activates leukocyte integrins through inside-out signaling, resulting in the leukocyte adhesion to endothelial cells (Doring et al., 2007, Hartmann et al., 2008, Luster et al., 2005, Man et al., 2008, Schreiber et al., 2007, Shulman et al., 2006, Woolf et al., 2007). Unexpectedly, flow conditions promote, rather than antagonize, leukocyte–endothelial interactions (Ransohoff et al., 2007). As one example, T cells encountering apically-presented CXCL12 underwent robust LFA-1-dependent transmigration across HUVEC toward abluminal CCL5 under shear stress, in contrast to negligible transmigration under static conditions (Schreiber et al., 2007).

Leukocytes entering the CNS parenchyma must cross the BBB (Bechmann et al., 2007, Engelhardt and Ransohoff, 2005). The barrier function of the BBB relies on highly specialized brain microvascular endothelial cells, which lack pinocytotic vesicles and fenestrae but possess intercellular tight junctions. These features limit transcellular and paracellular movement of cells and molecules (Man et al., 2007). Mechanisms of leukocyte migration across the BBB are incompletely understood. Transwell cultures of brain endothelial cells have been used to study leukocyte trafficking into the brain (Callahan et al., 2004, Kraus et al., 2004, Luster et al., 2005, Mahad et al., 2006, Man et al., 2008, Ubogu et al., 2006). However, the lack of shear stress limits the interpretation of the results of such studies. The current report describes our progress in developing an in vitro BBB model using human brain microvascular cells and a parallel plate flow chamber to study leukocyte–brain endothelial cell interactions under flow conditions. The current paper focuses in particular on leukocyte adhesion to brain microvascular endothelial cells under flow. Analyses of the earlier stages of leukocyte/endothelial interaction, tethering and rolling, are ongoing.

Anti-α4 integrin antibodies (natalizumab; NTZ) is an FDA approved treatment for MS. Although it was designed to block α4 integrin-mediated leukocyte–endothelial interactions, this effect has not been directly evaluated in MS patients receiving NTZ. Using a static BBB model, we found that NTZ infusion to MS patients inhibited leukocyte migration across BBB, and that this blockade was reversed by removing NTZ with plasmapheresis (Khatri et al., 2009). We extended these results in the present study by examining NTZ effects under flow conditions.