Fig. 3: The vascular endothelium is a double-layered barrier for neutrophil extravasation.

a Transmission electron microscopy revealed a gradual intrusion of neutrophils into the endothelial cell layer (upper panel). The cartoons of the transmission electron micrographs from the upper panel indicate the endothelial cell layer in red and pericytes in blue (lower panel). b Two-photon three-dimensional microscopy revealed the presence of transendothelial migratory neutrophils. See Video 4. c Transmission electron microscopy revealed neutrophil embedment in the endothelial basement between the endothelial cell layer and pericytes (upper panel). The cartoons of the transmission electron micrographs from the upper panel indicate the endothelial cell layer shown in red and pericytes in blue (lower panel). d Two-photon intravital microscopy revealed that neutrophils were embedded in the endothelial basement membrane. See Videos 5 and 6. e Transmission electron microscopy revealed that neutrophils penetrated the pericyte sheath. (1) The leading edge of an embodied neutrophil protruded from the endothelial basement membrane between the endothelial cell layer and the pericyte sheath. (2) Two neutrophils were undergoing penetration at the same site of the pericyte sheath in the panel. (3) Three neutrophils were undergoing penetration at the same site of the pericyte sheath in the panel. (4) The head and tail regions of a neutrophil are localized outside the pericyte sheath in the panel. In all of the corresponding cartoons, the endothelial cells are shown in blue, and the pericytes are in red. Blue and red arrows indicate endothelial cells and pericytes, respectively. N neutrophil, Lu lumen. f Two-photon intravital microscopy revealed three neutrophils that were continually penetrating the vascular endothelium at the same spot within 5 min of each other. The dotted arrows indicate the trajectories of the extravasating neutrophils. See Video 7. For all experiments, the data are representative of five independently repeated experiments with N-formylmethionyl-leucyl-phenylalanine stimulation