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
View: Text | PDF
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

×

Figure 8

Labeled rTEM neutrophils are present in blood and pulmonary vasculature and show an activated phenotype.

Options: View larger image (or click on image) Download as PowerPoint
Labeled rTEM neutrophils are present in blood and pulmonary vasculature ...
(A and B) LysM-EGFP-ki mice were subjected to local cremaster muscle stimulation with IL-1β (2 hours) and an i.v. injection of biotinylated anti-Ly6G (2 μg) at t = 90 minutes. The tissues were then superfused with histamine (30 μM) or vehicle in conjunction with AF647-streptavidin (1 μg/mL) for 2 hours. Peripheral blood was analyzed for the frequency of AF647-streptavidin+ neutrophils. (A) Representative flow cytometry profiles and (B) frequency of AF647-streptavidin+ neutrophils (LysM-GFPhiGr-1hi) (n = 6 mice/group). (C–G) WT mice were subjected to cremaster muscle stimulation with IL-1β or PBS for 2 hours followed by i.v. injection of biotinylated anti-Ly6G (2 μg) at t = 90 minutes. The mice then received an i.s. injection of AF647-streptavidin (400 ng) coadministered with histamine (200 μL of 30 μM solution) or PBS for 2 hours. Peripheral blood and pulmonary vascular washout samples were analyzed by FACS. (C) Expression of indicated markers on AF647-streptavidin+ neutrophils relative to levels on AF647-streptavidin– neutrophils in blood samples collected from mice subjected to IL-1β plus histamine, as measured by geometric MFI (gMFI) (n = 5–8 mice/group). (D) Representative flow cytometry profiles and (E) frequency of pulmonary vascular washout AF647-streptavidin+ neutrophils (Gr-1hiCD115–) (n = 4–8 mice/group). (F) Representative FACS histograms and (G) expression of indicated markers on AF647-streptavidin+ neutrophils relative to levels on AF647-streptavidin– neutrophils in pulmonary vascular washout samples collected from mice stimulated with IL-1β plus histamine, as measured by gMFI (n = 6–9 mice/group). Data are represented as mean ± SEM (each symbol represents 1 mouse/independent experiment). Statistically significant differences from IL-1β (B), gMFI of indicated markers of blood AF647-streptavidin– neutrophils (C), PBS (E), and gMFI of indicated markers on pulmonary vascular washout AF647-streptavidin– neutrophils (G) are shown by *P < 0.05; **P < 0.01 or by indicated comparisons ##P < 0.01, 2-tailed Student’s t test (B, C, and G) or 1-way ANOVA followed by Bonferroni’s post hoc test (E). NS, not significant.