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Src kinases and neutrophil extravasation
absolute numbers of adherent cells/mm2 in TNFα-stimu- lated post-capillary cremaster muscle venules of SFK-ko and wildtype mice (Figure 2G). We found a similar shear rate dependent decrease in the number of adherent cells/mm2 in SFK-ko mice in vivo compared to wildtype mice. Additionally, we used a pharmacological approach and administered the broad-spectrum tyrosine kinase inhibitor Dasatinib before TNFα-stimulation. Dasatinib has been used in various studies as a SFK inhibitor and led to promising results as an immune modulator.21 Similar to the results in SFK-ko mice, we found a decrease in adher- ent cells with increasing shear rates. Importantly, SKF-ko mice receiving Dasatinib did not show any further decrease in adherent cells at high shear stress levels, indi- cating that Dasatinib exerts its observed effects on adhe- sion via inhibition of SFK (Online Supplementary Figure S1F). Taken together, these in vivo and in vitro findings indi- cate that loss of SFK in neutrophils leads to a shear force dependent inability to firmly adhere to the inflamed endothelium, a prerequisite for subsequent extravasation.
Reduced phosphorylation of cytoskeleton-associated proteins in Src family kinase-deficient neutrophils
To further elucidate the molecular mechanisms of SFK dependent neutrophil extravasation, we analyzed differ- ent steps of integrin outside-in signaling that occur after integrins bind to their receptor. This includes integrin clus- tering and cytoskeletal rearrangements.22 To analyze inte- grin clustering, we performed time-lapse confocal microscopy and studied LFA1 clustering in SFK-ko and wildtype neutrophils in flow chambers coated with E-selectin/ICAM-1/CXCL1 (Figure 3A and Online Supplementary Mov2). We observed spreading and polariz- ing wild-type neutrophils which showed an accumulating LFA1 signal (clustering) over time at the neutrophil uropod (Figure 3B). In contrast, SFK-deficient neutrophils appeared round and unpolarized with no obvious LFA1 clustering. Several cytoskeleton-associated proteins and signaling molecules are known to be tyrosine phosphory- lated upon integrin ligation.23 We investigated the phos- phorylation and activation of the direct SFK target24,25 Syk in SFK-ko and wildtype neutrophils plated on ICAM-1 and then stimulated with CXCL1 or PMA. Western blot analysis of phospho-Syk (Tyr519/520) revealed a strong upregulation of phosphorylation following stimulation of wildtype cells. In contrast, SFK deficiency prevented the upregulation of phosphorylation following stimulation with CXCL1 or PMA (Figure 3C), indicating that Syk acti- vation is defective in the absence of SFK. Moreover, we tested the phosphorylation of the adaptor protein Paxillin, one of the critical proteins for cell adhesion, migration and podosome formation, that is known to be tyrosine phos- phorylated upon β2 integrin activation.26,27 Western blot analysis showed significant upregulation of Paxillin phos- phorylation when stimulated with CXCL1 or PMA in wildtype cells, while no upregulation was detectable in SFK-ko neutrophils (Figure 3D). In addition, we also inves- tigated the tyrosine phosphorylation of Cortactin, which regulates actin branching and therefore cell migration. Likewise, we observed no significant upregulation of Cortactin Tyr421 phosphorylation upon CXCL1 or PMA stimulation in SFK-ko cell, in contrast to wildtype neu- trophils (Figure 3E). Because during neutrophil adhesion and migration SFK do not selectively signal via LFA1, but also via Mac1, we additionally analyzed Paxillin phospho-
rylation after plating the cells on Fibrinogen (Online Supplementary Figure S1G). Similar to ICAM1 we observed a stimulus dependent phosphorylation of Paxillin in wild- type neutrophils, which was absent in SFK-ko neu- trophils. Taken together, these experiments show that β2 -integrin clustering and subsequent outside-in signaling is severely impaired in SFK-ko neutrophils.
Src family kinase are indispensable to pass the vascular basement membrane
For successful extravasation into inflamed tissue, neu- trophils need to cross the endothelial cell layer and the underlying vascular basement membrane. We aimed to investigate whether SFK are required to perform this last step of extravasation. First, we analyzed transmigration capacity of SFK-ko neutrophils in a transwell assay using CXCL1 as chemoattractant (Online Supplementary Figure S2A). In line with previous results,15 we observed no dif- ference in transmigration between SFK-ko neutrophils and wildtype neutrophils. Both groups displayed a proportion- al increase in transmigration with rising CXCL1 concen- trations. As a next step, we coated transwells with laminin-111 (LN1) or a combination of LN1 and PECAM- 1/ICAM-1 and stimulated them with CXCL1 in order to mimic the situation at the vascular BM, as reported previ- ously.6 No difference in transmigration through LN alone was detected between wildtype and SFK-ko neutrophils (Figure 4A). Coating with LN1/PECAM-1/ICAM-1 (LN/P/I) together with CXCL1 stimulation induced a strong increase in transmigration of wildtype cells. However, SFK-ko cells failed to cross the artificial BM. How exactly neutrophils cross the vascular BM is still a matter of debate, especially, whether the BM is perma- nently modified by extravasating neutrophils. To further address this, we conducted transwell assays as described above, coated with a combination of LN1 and PECAM- 1/ICAM-1 and stimulated with CXCL1. We also used the same total cell number (2x105), but this time mixed SFK- ko x Lyz2GFP neutrophils and wildtype neutrophils in a 1:1 ratio (1x105 wildtype and 1x105 SFK-ko x Lyz2GFP neu- trophils). In this manner, transmigrated SFK-ko neu- trophils were identified by their GFP signal. Interestingly, in combination with wildtype neutrophils, SFK-ko neu- trophils were able to overcome the BM to the same extent as wildtype cells, suggesting that wildtype neutrophils facilitate BM penetration by providing exit points for SFK- ko neutrophils (Figure 4A). To confirm these findings in vivo, we performed whole mount staining of TNFα- stimulated cremaster muscles for LN (green) to visualize the BM, and MRP14 (red) to visualize neutrophils, then analyzed the localization of neutrophils by confocal microscopy. In wildtype tissue, we were able to detect local areas of increased extravasation, which is in accor- dance with published literature, describing transmigration hotspots28 (Figure 4B). In contrast, none of these spots could be found in cremaster muscle tissue of SFK-ko mice. MPR14-positive cells remained located within the vascular compartment and only few extravasated neutrophils could be detected, mainly remaining in close vicinity to the vessel (white arrows). Overall, SFK-ko neutrophils covered less distance away from the vessel wall when compared to wildtype neutrophils (8.0 μm vs. 17.3 μm, respectively) (Online Supplementary Figure S2B). These in vivo findings support our earlier in vitro findings that SFK are critical for neutrophils to cross the BM.
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