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I. Rohwedder et al.
of observation (Online Supplementary Figure S3C and D), indicating that this accumulation is not random, but high- ly dependent. These experiments strongly support the concept of SFK-dependent BM degradation by extravasat- ing wildtype neutrophils. In addition, our results indicate that LN degradation fragments might exert chemotactic activity.
To finally show that these in vitro findings are also rele- vant under in vivo conditions, we injected labeled (CellTrackerTM Deep Red Dye) SFK-ko or wildtype neu- trophils into Lyz2GFP or SFK-ko x Lyz2GFP mice, respectively, and observed neutrophil behavior using multi-photon microscopy of the mouse cremaster muscle vasculature. Compared to SFK-ko x Lyz2GFP mice, where neutrophils accumulated in the vessel or were stuck to the abluminal side, we observed strongly elevated numbers of extravasated neutrophils in the periphery, when wildtype neutrophils were present (Figure 5H and Online Supplementary Mov5 and 6). Additionally, we were able to visualize SFK-ko x Lyz2GFP neutrophils accumulating around adherent wildtype cells, confirming the observed behavior of SFK-ko neutrophils in the in vitro setting (Figure 5I). Also, in the experiment in which we inverted the procedure, by injecting fluorescently labeled SFK-ko neutrophils into Lyz2GFP mice, we could observe extravasated SFK-ko neutrophils (Online Supplementary Figure S3E and Online Supplementary Mov7 and 8). These experiments strengthen the concept of wildtype neu- trophils creating a path through the BM which enables SFK-ko neutrophils to extravasate into inflamed tissue in vivo.
Discussion
Integrin outside-in signaling is critical for neutrophil firm adhesion to and extravasation through the vessel wall into inflamed tissue. The short intracellular integrin tail has no enzymatic activity and acts exclusively as a binding site for recruited proteins, which, in turn, activate a broad range of pathways, spanning from cytoskeletal rearrange- ments and integrin clustering to vesicle trafficking. It has been widely accepted that tyrosine phosphorylation of ITAM motives by members of the Src family kinases is one way of integrin outside-in signaling.35 Therefore, SFK triple knockout mice (Hck-/-Fgr-/-Lyn-/-, here named SFK-ko) have been used to study the function of these kinases in neutrophils in an in vivo model of acute inflammation. Interestingly, Hck-/-Fgr-/- neutrophils displayed no adhesion defect in earlier static adhesion experiments on ICAM- 1,13,36 while our findings show a dramatic decrease in neu- trophil adhesion efficiency in inflamed cremaster muscle venules in vivo implying that SFK-dependent neutrophil adhesion is sensitive to shear stress. We also found that neutrophil extravasation in SFK-deficient mice was dra- matically decreased when compared to wildtype mice, which was caused by an SFK-dependent intrinsic extrava- sation defect. This observation is in line with various reports in different inflammation models with SFK-ko mice14,15,21 where decreased numbers of neutrophils were observed. Lowell et al. linked low PMN numbers in the liver during endotoxemia to a failure of Hck-/-Fgr-/- neu- trophils to rearrange their actin cytoskeleton, while cytokine production was unchanged. We clearly demon- strate that in the absence of SFK, neutrophils are unable to
strongly adhere to the substrate under flow and detach in vitro and in vivo, due to defective LFA1 clustering and poor adhesion strengthening. The application of the tyro- sine inhibitor Dasatinib led to a comparable shear rate dependent decrease in adherent neutrophils in inflamed venules in vivo. This strengthens previous findings of an improved outcome during sepsis after Dasatinib applica- tion, where leukocyte infiltration into the inflamed peri- toneum was reduced in a dose-dependent manner.21 Interestingly, most in vitro studies were performed under steady state conditions, where integrin clustering and crawling were unaffected in SFK deficient leukocytes,15,37 neglecting the impact of shear stress on neutrophil adhe- sion. This phenomenon was previously observed in WASP- and mAbp1-deficient mice, where integrin cluster- ing and migration defects only arose when neutrophils were analyzed under shear stress.38,39 Our study demon- strates that neutrophil adhesion strengthening under flow conditions is dependent on SFK which mediate clustering of integrins, the rearrangement of the actin cytoskeleton along with polarization of the cell.40 Several cytoskeleton associated proteins required for adhesion are reported phosphorylation targets of SFK (directly or indirectly via Syk) like Paxillin and Cortactin.41-43 Here we clearly show reduced phosphorylation of these adhesion-relevant pro- teins.
We also aimed to answer the question as to whether SFK are required for the extravasation process beyond the endothelial layer. It was suggested that leukocytes pre- dominantly cross the vascular BM at LN low expression regions (LER),44 and recent publications, including those from our lab, showed that neutrophils need to translocate vesicles containing VLA3, VLA6 and NE to their surface in a MST1-Rab27a dependent manner in order to overcome the BM.3-6,45,46 Our results identify for the first time SFK to be an additional critical component of this process. We show that SFK-ko neutrophils fail to pass an artificial LN barrier, and even when stimulated with PECAM-1 and ICAM-1, SFK-ko neutrophils are unable to release azurophilic granules and translocate integrin containing secretory vesicles. Exocytosis and vesicle transfer in neu- trophils is strongly regulated by the GTPase Rab27a and its two effectors JFC1 and Munc13-4.8,47 We demonstrate that PECAM-1/ICAM-1 and CXCL1 stimulation facilitates the translocation of all three proteins to the membrane of wildtype neutrophils, suggesting that SFK regulate vesicle transport in a Rab27a-dependent manner. How MST1 and SFK co-operate in this process is still unclear and needs further investigation. Of note, MPO release was not defec- tive in MST1 deficient neutrophils (M Sperandio, 2019, unpublished data/personal communication) compared to SFK- ko neutrophils, where a marked impairment in MPO release could be observed, suggesting that SFK are involved in neutrophil vesicle trafficking independently of MST1.
The role of NE during neutrophil extravasation is still controversial, as is the role of BM degradation by elastases or MMP.5,48-52 However, recent findings suggest that this might, at least in part, be related to the experimental model used. Reichel et al. could clearly demonstrate decreased extravasation into inflamed cremaster muscle tissue after blockage of gelatinases by the specific inhibitor MMP-2/-9 inhibitor III.53 Furthermore, Young et al. were able to show NE-dependent extravasation using the same model.29 It was also suggested that NE/MMP-
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haematologica | 2020; 105(7)