CAM‐mediated endocytosis contributes to leukocyte transcellular transmigration

Transcellular transendothelial migration (tTEM) of white blood cells (WBCs) involves formation of endothelial docking structures and endocytic vesicles that coalesce into transmigration pores without disrupting endothelial cell (EC) junctions. Intercellular adhesion molecule 1 (ICAM‐1) is key in tTEM yet the pathways underlying this association are unclear. ICAM‐1 engagement on ECs by anti‐ICAM‐coated beads induces vesiculization via cell adhesion molecule (CAM)‐mediated endocytosis, involving Na + /H + exchanger 1 (NHE1). Here we explored the contribution of CAM‐mediated endocytosis in tTEM using leukocytes, anti‐ICAM beads, microscopy, inhibitors and knockout tools. ICAM‐1 engagement in areas away from the cell border rendered docking structures in cholesterol and sphingomyelin rich‐domains. Acid sphingomyelinase (ASM) was recruited to these areas. These events led to ceramide production, actin stress fiber formation, vesiculization by CAM‐endocytosis, and formation of transcellular pore‐like structures from coalescing vesicles, all in regions away from endothelial cell‐cell border. Disruption of CAM‐mediated endocytosis by inhibiting NHE1 or ASM impaired these events and affected WBC TEM. These data suggest that ICAM‐1/NHE1‐dependent CAM‐mediated signaling supports formation of endothelial pores and WBC transcellular migration.