Local microvascular leakage promotes trafficking of activated neutrophils to remote organs
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
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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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 4

Stimulated microvascular leakage induces movement of small-molecular-weight proteins into the vascular lumen.

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Stimulated microvascular leakage induces movement of small-molecular-wei...
(A–D) Cremaster muscles of WT mice were stimulated with IL-1β or TNF for 2 hours followed by local injection of histamine or i.v. VEGF (or corresponding vehicle), for 30 minutes before collection of tissue and plasma samples for quantification of CXCL1 levels by ELISA. Mice subjected to PBS, histamine, or VEGF alone were used as control (n = 4–7 mice/group). (E) Representative images of selected IL-1β–stimulated postcapillary venules showing movement of i.v. 75-kDa TRITC-dextran (marker of vascular leakage; red) and topically applied 10-kDa AF488-dextran (superfused for 10 minutes; green) after topical superfusion of PBS vehicle (top panels) or histamine (30 μM, lower panels; see Supplemental Video 5). Dashed lines indicate blood vessel borders. Scale bars: 20 μm. (F and G) Time course of 75-kDa TRITC-dextran and 10-kDa AF488-dextran accumulation in the perivascular region of selected IL-1β–stimulated postcapillary venules after superfusion of vehicle (F, n = 3 mice) or histamine (G, n = 3 mice). (H) Quantification of 10-kDa AF488-dextran in plasma samples of mice treated as detailed above (n = 3 mice/group). (I and J) Schematic diagram and mathematical equation of the Péclet number (Pe) (see Supplemental Figure 4). Data are represented as mean ± SEM (each symbol represents 1 mouse/independent experiment). Statistically significant differences from PBS (A–D) or IL1β plus vehicle (H) are indicated by *P < 0.05; **P < 0.01; ***P < 0.001, between groups as indicated by lines or by #P < 0.05, 1-way ANOVA followed by Bonferroni’s post hoc test or 2-tailed Student’s t test. NS, not significant.