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Local microvascular leakage promotes trafficking of activated neutrophils to remote organs
Charlotte Owen-Woods, … , Mathieu-Benoit Voisin, Sussan Nourshargh
Charlotte Owen-Woods, … , Mathieu-Benoit Voisin, Sussan Nourshargh
Published January 23, 2020
Citation Information: J Clin Invest. 2020;130(5):2301-2318. https://doi.org/10.1172/JCI133661.
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Research Article Inflammation Vascular biology

Local microvascular leakage promotes trafficking of activated neutrophils to remote organs

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Abstract

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability–enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

Authors

Charlotte Owen-Woods, Régis Joulia, Anna Barkaway, Loïc Rolas, Bin Ma, Astrid Fee Nottebaum, Kenton P. Arkill, Monja Stein, Tamara Girbl, Matthew Golding, David O. Bates, Dietmar Vestweber, Mathieu-Benoit Voisin, Sussan Nourshargh

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Figure 7

Development of an in vivo labeling strategy for tracking rTEM neutrophils.

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Development of an in vivo labeling strategy for tracking rTEM neutrophil...
(A) Diagram detailing the labeling method. Cremaster muscles of LysM-EGFP-ki mice were stimulated with IL-1β (50 ng for 2 hours) followed by an i.v. injection of biotinylated anti-Ly6G (2 μg) to label blood neutrophils at t = 90 minutes. The tissues were superfused with histamine (30 μM) or vehicle in conjunction with AF647-streptavidin (1 μg/mL) for 2 hours. (B) Representative confocal image of an IL-1β–stimulated postcapillary venule illustrating the extent of AF647-streptavidin labeling of neutrophils at different stages of trafficking (left panel). Right panels shown enlarged images of the boxed regions and illustrate examples of AF647-streptavidin– luminal, AF647-streptavidin+ sub-EC, and AF647-streptavidin+ interstitial neutrophils. Scale bars: 5 μm. (C) Representative confocal IVM images of a tissue stimulated with IL-1β plus histamine (see Supplemental Video 6) illustrating the effective labeling of an rTEM event. The exemplified neutrophil shows that once the cell has breached an EC junction, the leading body part in the sub-EC space rapidly becomes AF647-streptavidin+ while the luminal body segment remains AF647-streptavidin–. The AF647-streptavidin+ neutrophil can be easily tracked as it migrates in a reverse manner toward the vascular lumen and reenters the bloodstream. Luminal and cross-sectional views are shown with the arrows indicating the direction of motility of the indicated neutrophil. Scale bars: 3 μm. (D) Fluorescence intensity of AF647-streptavidin on neutrophils in the venular lumen, tissue, and cells exhibiting rTEM (n = 4 mice/group) during an IL-1β plus histamine reaction. Data are represented as mean ± SEM (each symbol represents 1 mouse/independent experiment). Statistically significant differences from luminal neutrophils are shown by **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test.

Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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