LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) distinctly regulate neutrophil extravasation through hotspots I and II

Abstract

Precise spatiotemporal regulation of leukocyte extravasation is key for generating an efficient immune response to injury or infection. The integrins LFA-1(CD11a/CD18) and Mac-1(CD11b/CD18) play overlapping roles in neutrophil migration because they bind the same as well as different ligands in response to extracellular signaling. Using two-photon intravital imaging and transmission electron microscopy, we observed the existence of preferred sites for neutrophil entrance into the endothelial cell monolayer and exit from the basement membrane and pericyte sheath during neutrophil extravasation, namely, hotspots I and II, by elucidating distinctive roles of LFA-1 and Mac-1. To penetrate the vascular endothelium, neutrophils must first penetrate the endothelial cell layer through hotspot I (i.e., the point of entry into the endothelium). Neutrophils frequently remain in the space between the endothelial cell layer and the basement membrane for a prolonged period (>20 min). Subsequently, neutrophils penetrate the basement membrane and pericyte sheath at hotspot II, which is the final stage of exiting the vascular endothelium. To further investigate the roles of LFA-1 and Mac-1, we newly generated LFA-1 FRET (CD11a-YFP/CD18-CFP) mice and Mac-1 FRET (CD11b-YFP/CD18-CFP) mice. Using both FRET mice, we were able to determine that LFA-1 and Mac-1 distinctly regulate the neutrophil extravasation cascade. Our data suggest that the vascular endothelium functions as a double-layered barrier in the steps of neutrophil extravasation. We propose that the harmonized regulation of neutrophil penetration through the endothelium via hotspots I and II may be critical for vascular homeostasis during inflammation. In response to infection or tissue damage, white blood cells known as leukocytes exit blood vessels at specific sites, “hotspots.” Many leukocytes follow each other out through the same hotspot. As part of the body’s initial response to infection or tissue damage, leukocytes are carried to infected or damaged tissues by blood vessels. To fight infection, they must exit the vessels, but how they exited was poorly understood. Young-Min Hyun at the Yonsei University College of Medicine in Seoul, South Korea and co-workers used advanced imaging techniques to visualize leukocyte delivery. They found that many leukocytes use a single hotspot to enter the blood vessel wall, travel through the wall’s interior, and then exit the wall at another hotspot. Using hotspots is thought to minimize the number of perforations in the vessel wall, maintaining vessel integrity. These results illuminate a key aspect of how efficiently the body fights infection from the viewpoint of leukocyte migration.