The team found that approximately one-third of leukocytes made their way across an in vitro vascular endothelial monolayer by transcellular migration. Carman suggests that, in vivo, at least in some settings, the proportion might be higher. Transcellular migration might even be favored over the paracellular route, as the latter could potentially weaken cell–cell junctions.
To investigate the mechanism of transcellular migration, the team used a combination of live fluorescence and electron microscopy. Shortly after leukocytes were added to the monolayer, invaginations in the endothelial membranes appeared coincident with the formation of podosomes by the leukocytes.
The leukocytes used these podosomes to prod and poke the endothelial cell until they find a route of least resistance. Organelles other than the nucleus were then pushed out of the way as one large, invasive podosome created a transcellular pore. Leukocytes that were deficient for an actin regulatory protein called WASP formed podosomes poorly and were thus inefficient in forming transcellular pores. Paracellular migration continued as normal, however.
Endothelial cells survived despite this continuous puncturing by migrating leukoctyes. Indeed, multiple leukocytes were able to pass through a single endothelial cell—Carman has spotted up to eight at a time—lending further evidence that this could be the predominant route for transendothelial leukocyte migration in vivo.