PECAM-1-dependent neutrophil transmigration is independent of monolayer PECAM-1 signaling or localization

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a tyrosine phosphoprotein highly expressed on endothelial cells and leukocytes, is an important component in the regulation of neutrophil transendothelial migration. Engagement of endothelial PECAM-1 activates tyrosine phosphorylation events and evokes prolonged calcium transients, while homophilic engagement of neutrophil PECAM-1 activates leukocyte beta-integrins. Although PECAM-1 modulates polymorphoneutrophil transmigration via homophilic PECAM-1-PECAM-1 interaction, the mechanisms underlying endothelial PECAM-1 function are unknown. Proposed mechanisms include (1) formation of a haptotactic gradient that guides neutrophils to the cell-cell border, (2) service as a passive ligand for neutrophil PECAM-1, ultimately mediating activation of neutrophil beta integrins, (3) regulation of endothelial calcium influx, and (4) mediation of SH2 protein association, and/or (5) catenin and non-SH2 protein interaction. Utilizing PECAM-1-null model endothelial cells (REN cells), we developed a neutrophil transmigration system to study PECAM-1 mutations that specifically disrupt PECAM-1-dependent signaling and/or PECAM-1 cell localization. We report that interleukin-1beta (IL-1beta) elicits PECAM-1-dependent transmigration that requires homophilic PECAM-PECAM-1 engagement, but not heterophilic neutrophil PECAM-1 interactions, and is intercellular adhesion molecule-1 dependent. Conversely, whereas IL-8 and leukotriene-B-4-mediated transmigration is PECAM-1-independent, PECAM-1 and IL-8-dependent transmigration represent separable and additive components of cytokine-induced transmigration. Surprisingly, neither monolayer PECAM-1-regulated calcium signaling, cell border localization, nor the PECAM-1 cytoplasmic domain was required for monolayer PECAM-1 regulation of neutrophil transmigration. We conclude that monolayer (endothelial cell) PECAM-1 functions as a passive homophilic ligand for neutrophil PECAM-1, which after engagement leads to neutrophil signal transduction, integrin activation, and ultimately transmigration in a stimulus-specific manner.