Figure 4. Neutrophils extravasate adjacent to perivascular macrophages in inflamed dermal vessels.

(a) Time-lapse intravital multi-photon imaging of ear dermis of DPE-GFP mice infected with ΔHla S. aureus depicting the adherence and extravasation of adoptively transferred neutrophils isolated from mT/mG mice (red, numbered 1–5) in close proximity to perivascular macrophages (green). Lines (purple) represent migration tracks of selected neutrophils (red) and the arrow (yellow) represents the direction of blood flow in the vessel (cyan). 00:00:00, hr:min:sec. Scale bar 30μm (b) Statistical analysis of neutrophil extravasation site with respect to perivascular macrophages. Numbers of neutrophils extravasating at theoretical/random (white bars) or observed (black bars) distances from GFP⁺ perivascular macrophages within the same vessels. Data represents 45 total extravasation events from 5 mice pooled from 2 independent experiments. Bars represent mean±SEM. Statistical significance was determined by two-way ANOVA with a Bonferroni’s multiple comparisons test. (c) Relative gene expression by the indicated cell populations (GFP⁺ or GFP⁻ macrophages, dendritic cells (DC), γδ T cells and keratinocytes) isolated from ears of ΔHla S. aureus infected or control (PBS) treated mice at 6h p.i. Data shown are mean±SEM of 3 independent experiments. P-values were calculated using two-way ANOVA with a Bonferroni’s multiple comparison test (between treatment comparisons) and one-way ANOVA with Dunnett’s multiple comparison test (determination of differences between GFP⁺ macrophages and other cell types from infected animals), *P<0.05; **P<0.01. n.s., not significant.