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 2

Induction of microvascular leakage promotes neutrophil reverse transendothelial migration.

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Induction of microvascular leakage promotes neutrophil reverse transendo...
Cremaster muscles of LysM-EGFP-ki mice were subjected to IL-1β–induced inflammation for 120 minutes and analyzed by confocal IVM. AF647-labeled anti-CD31 mAb (i.s.) and 75-kDa TRITC-dextran (i.v.) were used to visualize EC junctions (red) and vascular leakage (blue pseudocolor intensity), respectively. Mice were superfused with histamine (30 μM) or injected i.v. with VEGF (4 μg) 2 hours after IL-1β stimulation. (A) Total neutrophil extravasation (n = 4–5 mice/group). (B) Representative images of a postcapillary venular segment subjected to IL-1β plus histamine stimulation at different time points after application of histamine, illustrating neutrophil TEM and dextran leakage responses (see Supplemental Video 4). Scale bars: 20 μm. (C) Time course of dextran accumulation in the perivascular region of a selected postcapillary venule (n = 3–9 mice/group). (D) Frequency of neutrophil reverse TEM events in relation to total TEM events of 15.2 ± 2.4 (IL-1β), 19.8 ± 2.9 (IL-1β + hist), and 21 ± 3.6 (IL-1β + VEGF) per 300-μm venular segment within 2-hour microscopy periods (mean ± SEM, n = 4–10 mice/group). (E) Temporal association of dextran leakage and cumulative frequency of neutrophil reverse TEM (n = 7–9 mice/group). Data are represented as mean ± SEM (each symbol represents 1 mouse/independent experiment). Statistically significant differences from PBS-treated (A) or IL-1β–treated (D) mice are indicated by *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test.