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

Young-Min Hyun,Sang A Park,Minsoo Kim,Young Ho Choe

doi:10.1038/s12276-019-0227-1

Young-Min Hyun, Sang A Park + Show 2 more

Open Access

https://doi.org/10.1038/s12276-019-0227-1

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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.

Highlights

The leukocyte adhesion cascade inside blood vessels includes rolling, adhesion, and crawling[1,2,3,4]
Neutrophils penetrate the endothelium at hotspots during the early stage of inflammation Following crawling along the blood vessels during inflammation, neutrophils adhere to the luminal surface of the blood vessels and subsequently undergo transendothelial migration via the paracellular pathway[5] or the transcellular pathway[6,7]
Using two-photon intravital imaging of neutrophil extravasation in the stimulated blood vessels, we defined a specific site on a blood vessel as a hotspot when at least three continuous neutrophils infiltrated at that same site (Fig. 1a and Video 1)

Summary

Introduction

The leukocyte adhesion cascade inside blood vessels includes rolling, adhesion, and crawling[1,2,3,4]. Upon completion of transendothelial migration, leukocytes encounter the endothelial basement membrane and pericyte sheath. Pericytes and other perivascular resident cells are actively involved in leukocyte migration[8]. Penetrating through the endothelial cell layer is a rapid stage of leukocyte migration in the migration cascade through the vascular endothelium, but penetration of the endothelial basement membrane takes much longer[9]. In contrast to the well-studied early stages of leukocyte migration, the mechanisms underlying the termination of transendothelial migration and subsequent migration through the basement membrane remain elusive. Using two-photon intravital imaging, we previously revealed that, after the completion of transendothelial migration and prior to proceeding toward the interstitial area, leukocytes detach their trailing edge from the basolateral side of the endothelial layer and/or basement

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