Panel A shows confocal images of cortactin with neutrophil interaction causing localized endothelial degradation. Panel B shows confocal images indicating extravascular neutrophils do not induce cortactin degradation in vessels. Panel C shows confocal images comparing neutrophil depleted and control conditions under tumor necrosis factor alpha stimulation. Panel D shows bar graph quantifying endothelial cortactin intensity under neutrophil depletion and control conditions. Panel E shows flow cytometry bar graph of endothelial cortactin intensity with neutrophil co incubation conditions. Panel F shows scatter plot correlating endothelial cortactin intensity with ratio of adhered neutrophils per endothelial cell. Panel G shows bar graph comparing endothelial cortactin intensity after co culture with neutrophils and mononuclear cells.
Neutrophils induce endothelial cortactin degradation in vivo and in vitro. (A and B) Representative 3D-confocal images (Nikon A1) of cremaster muscles stimulated with TNFα for 4 h and stained for CD31 (magenta), MRP-14 (cyan), and cortactin (green); scale bars = 100 μm. PCVs (white asterisks) are indicated. Bottom panels show zoomed-in orange, white, and green areas; scale bars = 10 μm. (A) Cortactin degradation in PCV is detected when intravascular neutrophils are observed (white squares). PCV devoid of neutrophil interaction (orange squares) do not show cortactin degradation. (B) Extravascular neutrophils do not induce cortactin degradation in other vessels (green squares). (C and D) Representative Z-stack confocal images (Nikon A1) of cremasteric PCV stimulated with TNFα or PBS for 2 h from neutrophil-depleted mice (α–Gr-1) and mice injected with isotype control antibody (rat IgG2b); scale bars = 20 μm. Right panels show cortactin in 2× zoomed-in orange areas; scale bars = 10 µm. (D) Quantification of cortactin MFI in PCV from the images in C (n = 3–6 mice/group). (E–G) Flow cytometric quantification of cortactin MFI in HUVEC co-cultures. Human peripheral blood neutrophils (1 × 106/ml) or MNC (1 × 106/ml) were co-incubated with confluent untreated or TNFα-treated HUVEC monolayers for 20 min. (E) Cortactin MFI in the EC population with or without neutrophil co-incubation (n = 4–9 independent experiments). (F) Spearman’s correlation analysis of EC cortactin MFI and the ratio of adhered neutrophils per EC was performed using the flow cytometry data in E. (G) Cortactin MFI in EC after co-culture with neutrophils (PMN, 20 min) or MNC (30 min and 1 h) (n = 2–4 independent experiments). Data are represented as means ± SEMs; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.