| Leukocyte transendothelial migration is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. Platelet/endothelial cell adhesion molecule (PECAM) is well established as a molecular mediator of transmigration, and CD99 was recently defined as a second mediator in an in vitro study of monocyte transmigration. Homophilic interaction between CD99 on the monocyte and CD99 at the endothelial cell border is critical for transendothelial migration to proceed. We developed an in vitro assay for studying neutrophil-endothelial cell interactions during transmigration. Using this assay, we found that antibodies against CD99 block upwards of 75% of neutrophils from transmigrating across endothelial cell monolayers. This profound effect was observed despite an apparent scarcity of CD99 molecules expressed on the surface of circulating neutrophils. We found that upon activation and engagement with the endothelial cells, CD99 was mobilized to the neutrophil surface from an intracellular source. Live imaging revealed that CD99-blocked PMN did not transmigrate, nor were they firmly arrested on the apical surface of the endothelial cells: instead, these blocked neutrophils exhibited dynamic lateral movement within endothelial cell junctions demonstrating that only the diapedesis step was blocked by interfering with CD99. Confocal microscopy confirmed the location of CD99-blocked neutrophils lodged within junctions, and demonstrated that anti-CD99 antibodies blocked neutrophils at a site downstream of neutrophils blocked by anti-PECAM antibodies. Considered together, the evidence in this thesis supports our model that the diapedesis step of leukocyte emigration is in itself a multistep process. PECAM-PECAM and CD99-CD99 homophilic interactions between leukocytes and endothelial cells regulate distinct, sequential steps in the transendothelial migration of both neutrophils and monocytes during inflammation. |
