Mechanism of Leukocyte Transendothelial Migration at the Blood Brain Barrier in vivo

Leukocyte recruitment during inflammation involves specific adhesive molecular interactions that occur between circulating leukocytes and the vascular endothelium. Transendothelial migration (TEM; diapedesis) is the process by which leukocytes migrate across the endothelium to reach inflamed tissue and is arguably the most critical stage in the inflammatory response. PECAM and CD99, adhesion molecules concentrated at endothelial cell (EC) borders and expressed diffusely on the surface of leukocytes, are crucial to diapedesis. Importantly, an interconnected vesicular endothelial cell network called the lateral border recycling compartment (LBRC), contains PECAM and CD99. During TEM in a process known as targeted recycling (TR), LBRC membrane is recruited to the leukocyte where it exteriorizes unligated PECAM and CD99 to promote diapedesis. Blocking targeted trafficking of the LBRC blocks TEM. Most TEM occurs across endothelial cell borders, but leukocytes are also capable of TEM through the EC body. Both routes require PECAM, CD99 and TR of the LBRC. Why leukocytes migrate through the cell body as opposed to the cell borders is unknown. The relative tightness of the EC junctions and the ability of the leukocyte to breach them may dictate the pathway of diapedesis. Therefore, transcellular migration may predominate in vasculature with exceptionally tight junctions, such as the blood brain barrier (BBB). The BBB is composed of highly specialized EC that are interconnected by complex tight junctions, which regulate central nervous system (CNS) homeostasis by restricting the passage of ions, macromolecules, and noxious blood borne agents. Aberrant leukocyte recruitment into the CNS is a key pathologic feature in many CNS diseases, including stroke and infection. Using high resistance in vitro human BBB models (~250 ohm-cm2), we determined that tight junctions undergo significant remodeling to accommodate paracellular TEM and TEM was dependent on PECAM and CD99. However, brain endothelial cell junctions in vivo are significantly tighter (2000 ohm-cm2). How leukocytes cross the BBB and the molecules governing this process in vivo is unknown. Recent advances in microscopy allow for direct visualization of TEM in vivo with ultra-high resolution, through a technique called intravital microscopy. Using intravital microscopy, I will identify the pathway and the molecular mechanisms governing TEM across the BBB in response to acute neuroinflammation.