Leukocyte transendothelial migration at the blood-brain barrier: Molecular mechanisms in vitro to therapeutic potential in vivo

Leukocyte diapedesis (transendothelial migration, TEM) across vascular endothelium and into tissue is a tightly regulated process that is vital to immune surveillance and inflammation. Leukocyte recruitment into sites of inflammation is mediated by a set of adhesive molecular interactions between the circulating leukocytes and the vascular endothelium. PECAM and CD99 are two cellular adhesion molecules that are critical for TEM in vitro and in several inflammatory settings in vivo. In endothelial cells about 1/3 of the total PECAM and CD99 resides in a novel, intracellular compartment in endothelial cells known as the lateral border recycling compartment (LBRC). The LBRC supplies transmigrating leukocytes with unligated adhesion molecules and increased membrane surface area to facilitate their movement across the endothelium.;Most TEM occurs at endothelial cell borders (paracellular). However, there is indirect evidence to suggest that, at the tight junctions of the blood-brain barrier (BBB), leukocytes migrate directly through the endothelial cell body (transcellular). Why leukocytes migrate through the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the tightness of endothelial cell junctions influences the pathway of diapedesis, we developed an in vitro model of the BBB that possessed 10-fold higher electrical resistance than standard culture conditions and strongly expressed the BBB tight junction proteins claudin-5, claudin-3 and occludin. We found that paracellular TEM was still the predominant pathway (≥98%) and TEM was dependent on PECAM-1 and CD99. We show that endothelial tight junctions expressing claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial LBRC is mobilized to the exact site of tight junction remodeling. This preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell border. These findings provide new insights into leukocyte-endothelial interactions at the BBB and suggest that tight junctions are more dynamic than previously appreciated.;At the central nervous system (CNS), a limited number of immune cells cross the BBB to enter the CNS under physiological immune surveillance to detect and eliminate potential mediators of infection or damage. However, unwanted and exacerbated leukocyte trafficking into the CNS is a prominent pathological feature in many CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Remarkably little is known regarding the molecular regulation of the step of diapedesis within the CNS in vivo. Here, we report that CD99 is critical for lymphocyte transmigration, but not adhesion, in a human BBB model. CD99 blockade in vivo ameliorated EAE and decreased the accumulation of infiltrating inflammatory leukocytes into the CNS, including T and B-lymphocytes and inflammatory myeloid-derived dendritic cells. Anti-CD99 therapy was effective administered after onset of disease symptoms and blocked relapse when administered therapeutically after disease symptoms had recurred. These findings underscore an important role for CD99 in the pathogenesis of CNS autoimmunity and suggest it may serve as a novel therapeutic target for controlling neuroinflammation.