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Src kinases and neutrophil extravasation
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Figure 4. Rab27a dependent vesicle transfer is impaired in Src family kinases (SFK)-knockout (ko) neutrophils, leading to an impaired basement membrane pen- etration. (A) Neutrophil transmigration in a transwell assay with or without CXCL1 through filters coated with laminin (LN), or LN, PECAM-1, and ICAM-1 (LN/P/I). All data are presented as mean± standard error of the mean (SEM). *P<0.05; ***P<0.001; n.s. : not significant (two-way ANOVA, Sidak multiple comparison test). N=3 wildtype and 3 SFK-ko x Lyz2GFP mice, transwell assays were performed in duplicates. (B) Maximum projections of confocal microscopic images of venules in TNFα–stimulated cremaster muscle whole mounts from wildtype and SFK-ko mice. Basement membrane was visualized by anti-LN5 (green), neutrophils by anti- MRP14 (red). Surrounding muscle tissue was visualized by bright field microscopy. White arrows point at extravasated, but still attached neutrophils in SFK-ko tissue. Scale bar: 20 μm. (C) and (E) immunostaining of representative wildtype and SFK-ko neutrophils on BSA- or PECAM-1/ICAM-1/CXCL1–coated (P/I/CXCL1) coverslips for (C) VLA6 and (E) VLA3 analyzed by confocal microscopy. Scale bar: 10 μm. (D) and (F) Quantification of ring formation for VLA6 (D) and VLA3 (F). At least 80 cells from 3 wildtype and 3 SFK-ko mice were analyzed for each condition. (G) Immunostaining of representative wildtype and SFK-ko neutrophils on BSA- or PECAM- 1/ICAM-1/CXCL1–coated coverslips for Rab27a (upper panel), Munc13-4 (middle panel), and JFC1 (lower panel). Scale bar is 5μm. (H) Quantification of ring forma- tion for Rab27a, Munc13-4 and JFC1. At least 80 cells from 3 wildtype and 3 SFK-ko mice were analyzed for each condition. All data are presented as mean±SEM. *P<0.05; P<0.005; ***P<0.001; n.s. : not significant (two-way ANOVA, Sidak multiple comparison test).
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