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.

The LTB4–BLT1 axis regulates actomyosin and β2-integrin dynamics during neutrophil extravasation

“…Overall, we found that NMIIA regulates integrin-based adhesion stability during neutrophil arrest, possibly via different nonexclusive mechanisms, as previously reported (Vicente-Manzanares et al, 2009). We document that Itgb2 redistributes from a dispersed intracellular pool to a defined area of the neutrophil surface that is juxtaposed to the inflamed endothelium (adhesion ring), consistent with previous in vivo and in vitro studies (Hyun et al, 2019;Jones et al, 1988;Shaw et al, 2004). At this stage, we cannot resolve whether Itgb2 clusters observed at the adhesion ring-like structures are formed through lateral diffusion or rapid internalization and recycling back to the PM.…”

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration

“…Interestingly, we observed that once a neutrophil used a JMP to cross the endothelium, other neutrophils followed this path and used the same exit-site ( Figure 2E). This was in line with an increase in endothelial JMP dynamics at exit-sites after the first neutrophil crossed ( Figure 2F), in line with a recent publication by Hyun and colleagues (Hyun et al, 2019). These data suggest that endothelial JMPs can function as exit-recognition sites that are reinforced by transmigration.…”

Characterization of the Dynamic Behavior of Neutrophils Following Influenza Vaccination

“…(Figure 1H). In addition, we observed the presence of multiple hotspots (55) in the wall of high endothelial venules (HEVs), suggesting a relevant role of these areas in the observed recruitment (Figure 1G; Supplementary Movie 1). To confirm this observation, we performed electron microscopy in a PLN HEV, which clearly showed neutrophils associated with the blood vessel endothelium as early as 2 h p.v.…”