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 1

Hyperpermeability inflammatory reactions are associated with neutrophil reverse transendothelial migration.

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Hyperpermeability inflammatory reactions are associated with neutrophil ...
Cremaster muscles of LysM-EGFP-ki mice were subjected to IL-1β– or LTB4-induced inflammation (120 and 30 minutes, respectively), or to IR injury (40 minutes ischemia + 1–2 hours reperfusion) and analyzed by confocal IVM. PBS-treated or sham-operated animals were used as control. AF647-labeled anti-CD31 mAb was injected i.s. to visualize EC junctions (red) and i.v. fluorescent (75 kDa) TRITC-dextran was used to visualize vascular leakage (blue pseudocolor intensity). (A) Representative images of IR-stimulated cremasteric venules (see Supplemental Video 1), showing the development of an inflammatory response in terms of neutrophil migration (green GFPbright neutrophils; top panels) and dextran leakage (blue; bottom panels) at the indicated times after reperfusion. Scale bars: 20 μm. (B) Total neutrophil extravasation (n = 5–12 mice/group). (C) Time course of dextran accumulation in the perivascular region of a selected postcapillary venule (n = 3–10 mice/group). (D) Representative images of an IR-stimulated cremasteric postcapillary venule at different times after reperfusion (see Supplemental Videos 2 and 3) illustrating a normal neutrophil TEM (top) and a reverse TEM (bottom) event. Luminal and cross-sectional views with arrows indicating the direction of motility of the indicated neutrophil. Scale bars: 5 μm. (E) Frequency of neutrophil reverse TEM events in relation to total TEM events of 20.7 ± 2.1 (IL-1β), 31.3 ± 5.9 (LTB4), and 15 ± 2.4 (IR injury) per 300-μm venular segment within 2-hour microscopy periods (mean ± SEM, n = 6–9 mice/group). (F) Temporal association of dextran leakage and cumulative frequency of neutrophil reverse TEM (n = 4 mice). Data are mean ± SEM (each symbol represents 1 mouse/independent experiment). Statistically significant differences from PBS-treated (B) or IL-1β–treated (E) mice are indicated by **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test.