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

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 9

Accumulation of labeled rTEM neutrophils in lungs is linked with lung injury.

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Accumulation of labeled rTEM neutrophils in lungs is linked with lung in...
The cremaster muscles of LysM-EGFP-ki mice were locally stimulated with IL-1β or PBS (2 hours). Mice were injected i.v. with AF555–anti-CD31 mAb (10 μg), biotin–anti-Ly6G mAb, and fluorescent microspheres (20 nm diameter, 9.1 × 1013 beads) at t = 90 minutes to label the vasculature and neutrophils, and to quantify vascular leakage, respectively. To induce and track rTEM neutrophils stemming from the cremaster muscles, tissues were additionally stimulated with locally applied histamine or vehicle (PBS) in combination with Atto425-streptavidin (AT425-Strept, 400 ng) for 120 minutes. The lungs were then analyzed by confocal microscopy. (A) Representative confocal images of alveolar capillaries in whole-mount imaged lungs of mice in which the cremaster muscles were locally stimulated as indicated (CD31-labeled vessels, red; neutrophils, blue; extravascular beads, green). Scale bars: 20 μm. (B) Lung vascular leakage as quantified by accumulation (MFI) of extravascular beads (n = 4–6 mice/group) and (C) number of AT425-streptavidin+ neutrophils per field of view (n = 4–6 mice per group), in mice subjected to indicated cremaster muscle stimulations. (D) Correlation of the number of AT425-streptavidin+ neutrophils and extravascular beads in lung tissues (n = 4–6 mice/group). Line indicates linear regression and dashed lines 95% confidence band (Spearman’s r = 0.7). (E) High-magnification images of lung sections showing AT425-streptavidin+ neutrophils in close apposition to sites of extravascular fluorescent bead accumulation. Scale bars: 3 μm. (F) Lung vascular leakage in close proximity (<60 μm) of AT425-streptavidin– neutrophils or AT425-streptavidin+ neutrophils (n = 6 mice/group). Data are represented as mean ± SEM (each symbol represents 1 mouse/independent experiment). Statistically significant differences from PBS or indicated group are shown by **P < 0.01; ***P < 0.001, and between indicated groups by ##P < 0.01; ###P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test (B and C), or Spearman’s rank correlation test (D), or paired t test (F). NS, not significant.