Page 105 - 2020_07-Haematologica-web

Src kinases and neutrophil extravasation
ed leukocytes divided by the total number of adherent leukocytes to differentiate between reduced extravasation due to decreased adhesion versus a specific extravasation defect. Surprisingly, we observed a strongly reduced extravasation efficiency in SFK-ko mice (0.21 in SFK-ko mice vs. 0.77 in wildtype mice) (Figure 1C), indicating that SFK depletion does not only affect neutrophil adhesion, but also interferes with extravasation itself. Finally, we also compared neutrophil cell numbers in the peritoneal cavity 2 hours (h) after TNFα-induced peritonitis (Figure 1D). In SFK-ko animals, peritoneal neutrophil numbers were reduced to 50% compared to wildtype mice (0.42x106 vs. 106, respectively). Taken together, these experiments demonstrate an important role for SFK in neutrophil extravasation.
Adhesion strengthening is severely impaired in Src family kinase-knockout neutrophils in vitro
Our experiments suggest that SFK-deletion in neu- trophils results in an extravasation defect in the presence of the proinflammatory cytokine TNFα. After firm adhe- sion to the inflamed endothelium, neutrophils start to polarize and crawl along the endothelium to find a suit- able spot for extravasation. These changes are highly dependent on β2 integrins LFA1 (αLβ2) and Mac1 (αMβ2)
AB
and integrin outside-in signaling.20 We analyzed neu- trophil crawling using time-lapse microscopy in TNFα- stimulated cremaster muscle venules of SFK-ko and wild- type mice. We observed no differences in crawling direc- tion (Figure 2A), but, interestingly, crawling velocity was significantly increased in SFK-ko mice (12.3 μm/min) com- pared to wildtype mice (10.7 μm/min) (Figure 2B), sug- gesting defective adhesion strengthening. Subsequently, we performed ex vivo flow chamber assays using glass cap- illaries coated with E-Selectin/ICAM-1/CXCL1 to mimic the inflamed endothelium. Plots of single cell tracks dis- played more SFK-ko neutrophils crawling in flow direc- tion compared to wildtype neutrophils (Figure 2C). Crawling velocity was increased in SFK-ko leukocytes (Figure 2D), verifying our in vivo results. Interestingly, only 20% of SFK-ko neutrophils were able to crawl under flow (Figure 2E and Online Supplementary Mov1), compared to 80% of wildtype cells, suggesting that SFK-ko neutrophils are unable to maintain stable adhesion to the substrate. Together, these findings demonstrate a decreased capabil- ity of SFK-ko neutrophils to withstand shear forces, indi- cating that SFK are necessary for adhesion strengthening during post-arrest modifications. To further investigate a shear stress dependent adhesion defect in the absence of neutrophil SFK, we conducted detachment assays in the
CD
Figure 1. Src family kinases (SFK) are required for slow rolling, neutrophil adhesion and extravasation. All data are presented as mean± standard error of the mean (SEM). *P<0.05; ***P<0.001; n.s. : not significant (unpaired Student t-test or two-way ANOVA, Sidak multiple comparison test). (A) Adhesion efficiency from wildtype or SFK-knockout (ko) mice: n= 5 wildtype; n=5 SFK-ko mice. (B) In vivo leukocyte extravasation in TNFα-stimulated venules of mouse cremaster muscle. Differential total cell counts of neutrophils, eosinophils and other cells: n= 4 wildtype; n=5 SFK-ko mice. (C) Extravasation efficiency was calculated by the number of extravasated cells/mm2 divided by number of adherent cells/mm2. (D) Total neutrophil numbers after peritoneal lavage of the unstimulated or TNFα–stimulated peritoneal cavity of wildtype and SFK-ko mice: n=7 wildtype and n=6 SFK-ko mice for unstimulated controls and n=6 wild-type and n=6 SFK-ko mice for TNFα.
haematologica | 2020; 105(7)
1847