Fig. 1: Perivascular MCs promote hotspots of neutrophil migration.

a–c Cremaster muscles or ear skin of WT mice were stimulated with TNF (300 ng) or PBS for 4 h. Tissues were immunostained for neutrophils (MRP14), MCs (avidin) and pericytes/smooth muscle cells (α-SMA). a Mouse cremaster 4 h post TNF-stimulation, representative image of 5 independent experiments, scale bars 100 µm. b, c Correlation between the number of extravascular neutrophils and perivascular MCs (b, n = 63 venules, p value ≤ 0.0001) or ear skin (c, n = 41 venules, p value ≤ 0.001); data pooled from six mice. Line indicated linear regression and dashed lines 95% confidence band. d–j LysM-EGFP-ki; α-SMA-RFPcherry-Tg mice were stimulated with TNF (300 ng) for 2 h. AF532-labelled anti-CD31 and AF647-labelled anti-CD117 mAbs were injected i.s. to visualise EC junctions (dark blue) and MCs (cyan), respectively. d Illustration of the 4-colour confocal IVM methodology to simultaneously visualise the neutrophils, perivascular MCs, pericytes & ECs. Scale bars, 20 µm. e Scheme depicting neutrophil responses quantified. f Frequency of neutrophil TEM and TPM migrating <20 µm from a perivascular MC (n = 5 mice, p value = 0.0191). g Images of TNF-stimulated post-capillary venules showing the last time point of a neutrophil within the sub-EC space. Crawling path (coloured time-coded line) and directionality (white arrow) from site of neutrophil TEM to the site of venular wall exit distant to MC (left images) or adjacent to an MC (right images, see Movie S3); scale bars, 5 µm. h Neutrophil abluminal migration distance (n = 17 neutrophils distant to MC, n = 14 close to MC neutrophils, p value = 0.0277), time (n = 21 neutrophils, p value = 0.0482) and straightness (n = 21/20 neutrophils, p value = 0.0014); data pooled from five mice. i Time lapse IVM images 2 h post TNF-stimulation (see Movie S5) illustrating a hotspot of neutrophil transpericyte migration (TPM) occurring in close apposition to MCs, interstitial view is shown, scale bars 7 µm. j Frequency of neutrophil TPM hotspot <10 µm from a perivascular MC or macrophage (Mac) or not related to perivascular immune cells (n = 5 mice, **p value = 0.006 ^#p value = 0.0379). Mean ± SEM (each mouse represents one independent experiment). b, c Spearman’s rank correlation test; f, h two-tailed Student’s t test; j one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05, **p < 0.01 as compared to TEM, >10 or No Mac or MC or as indicated by ^#p < 0.05. Source data are provided as a Source Data file.