Figure 5.

rTEM neutrophils stemming from locally injured aged tissues accumulate in the lungs.

Young and aged mice were subjected to sham or cremasteric IR injury.

(A) Representative confocal images of post-capillary venules (PCVs) immunostained for CD31 and MRP14 (neutrophils) in WT mice (scale bar: 20 μm).

(B) Representative confocal images and quantification of lung vascular leakage in WT mice 4 h post reperfusion (scale bar: 20 μm; n = 4-5 mice/group).

(C) Neutrophil normal TEM events and (D) frequency of neutrophil rTEM in Y→Y or Y→A chimeras (see Figure 1H) as assessed by confocal IVM (n = 6 mice/group).

(E-I) Mice were injected i.v. with a biotinylated anti-Ly6G mAb and AF647-Strept locally applied to the cremaster muscle.

(E) Time-lapse confocal IVM images (Video S4) of a neutrophil rTEM event in an aged Lyz2-EGFP-ki cremaster muscle during IR injury illustrating that the neutrophil exhibiting rTEM is AF647-Strept^hi (Top panel: en face luminal view; bottom panel: isolated neutrophil; scale bar: 4 μm).

(F-I) Representative flow cytometry profiles and frequency of AF647-Strept^hi neutrophils in (F-G) blood and (H-I) pulmonary vascular washouts in WT mice (n = 4-11 mice/group). Numbers indicate the percentage of AF647-Strept^hi neutrophils. Means ± SEM, #p < 0.05, ###p < 0.001, ####p < 0.0001 as compared to age-matched controls, ^∗p < 0.05, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 as indicated.

See also Figure S5.