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I. Rohwedder et al.
Impaired vesicle transport in the absence of Src family kinase in neutrophils
We and others have previously shown that mobilization of vesicles containing VLA3 and VLA6 to the plasma membrane is an important process during vascular BM penetration.3,4,6 To investigate whether SFK have a role in this process, we analyzed the SFK-dependent transloca- tion of VLA6 and VLA3 to the plasma membrane of SFK- ko and wildtype neutrophils. Bone marrow derived neu- trophils of SFK-ko and wildtype mice were plated on BSA or, again, on a combination of PECAM-1/ICAM-1 and CXCL1 and subsequently stained for VLA6 or VLA3. Plasma membrane translocation was visualized by confo- cal microscopy as “ring formation” (Figure 4C and E). We observed fewer SFK-ko cells (37%) displaying a ring of VLA6, when plated on PECAM-1/ICAM-1/CXCL1 com- pared to wildtype (55%) (Figure 4D). This observation was also true for VLA3, where only 52% of SFK-deficient neutrophils showed vesicle translocation, compared to 82% of wildtype cells (Figure 4F). To test whether ring for- mation can also be observed in vivo, whole mount cremas- ter muscle stainings were conducted for VLA6, which revealed a similar trend as in vitro. In wildtype whole mounts, extravasated neutrophils displayed a ring-like staining for VLA6, while intravascular neutrophils showed only a diffuse signal. In SFK-ko whole mounts, intravascu- lar and extravasated cells showed no clear peripheral staining (Online Supplementary Figure S2C). Next, we plat- ed wildtype neutrophils on BSA or PECAM-1/ICAM- 1/CXCL1 and stained for SFK. Confocal micrographs revealed diffuse intracellular SFK staining when cells were plated on BSA (Online Supplementary Figure S2D). Upon stimulation with PECAM-1/ICAM-1/CXCL1, we observed a clear SFK signal at the cell border. Again, we quantified SFK distribution and observed a pronounced SFK translocation to the cell periphery when neutrophils were plated on PECAM-1/ICAM-1/CXCL1 (Online Supplementary Figure S2E). These experiments support a critical role for SFK by strongly interfering in vesicle traf- ficking of neutrophils in vitro and in vivo.
Src family kinase regulate Rab27a-dependent vesicle translocation
Vesicle trafficking in neutrophils is mainly regulated by Rab27a GTPases and their corresponding effector pro- teins. In neutrophils, Rab27a works in collaboration with two of its known effectors, JFC1 and Munc13-4, on vesicle trafficking and exocytosis of secretory vesicles and azurophilic granules.8 We analyzed the translocation of Rab27a, JFC1 and Munc13-4 to the cell periphery upon PECAM-1/ICAM-1/CXCL1 stimulation as described above. In wildtype neutrophils, stimulation resulted in translocation of Rab27a and its effectors (Figure 4G). While 60% of wildtype neutrophils displayed a ring-like structure, only 40.1% showed Rab27a translocation in SFK-ko neutrophils (Figure 4H). Similarly, staining for JFC1 (50.1% vs. 29.2%) and Munc13-4 (60.9% vs. 31.3%) revealed an equal trend for wildtype vs. SFK-ko neu- trophils, respectively. Taken together, these experiments show a clear SFK-dependent activation of the secretory machinery in neutrophils.
NE-dependent laminin degradation is defective in Src family kinase-ko neutrophils
Neutrophil extravasation is not only dependent on the
translocation of LN binding β1 integrins, but also on the serine proteinase neutrophil elastase (NE).5,29 We first ana- lyzed the localization of NE in neutrophils plated on either BSA or PECAM-1/ICAM-1/CXCL1 as described above (Figure 5A). Similar to VLA3 and VLA6, we observed an increase in ring formation from 21.9% in BSA stimulated wildtype neutrophils to 59.1% of wildtype neutrophils stimulated on PECAM-1/ICAM-1/CXCL1 (Figure 5B). This upregulation of vesicle translocation was absent in SFK-ko neutrophils (20.0% for BSA vs. 31.2% for PECAM- 1/ICAM-1/CXCL1 coating). In neutrophils, NE is stored in azurophilic granules together with a variety of other pro- teinases and antimicrobial proteins such as myeloperoxi- dase (MPO). Neutrophils release MPO after stimulation with TNFα.30,31 Thus, we additionally quantified MPO release in the serum of SFK-ko and wildtype mice by ELISA 2 h after TNFα stimulation. While we did not observe upregulation of MPO in the serum of SFK-ko mice 2 h after TNFα stimulation (158.4 ng/mL before vs. 184.0 ng/mL after TNFα stimulation) (Figure 5C), MPO levels were markedly increased in the serum of TNFα stimulated wildtype mice (113.0 ng/mL before vs. 473.5 ng/mL after TNFα stimulation). This indicates that SFK are required for the release of azurophilic granule content including NE and MPO. Earlier studies revealed that NE is able to pro- teolytically cleave laminins. In addition, NE activates dif- ferent matrix metalloproteinases (MMP) that are involved in matrix degradation.32-34 However, the relevance of these NE functions for neutrophil transmigration are still not completely understood. By applying a NE-fluorescent acti- vatable substrate (NE680FAST) in postcapillary venules with or without TNFα stimulation we were able to inves- tigate NE activity in wildtype and SFK-ko cremaster whole mounts. Compared to unstimulated controls, TNFα stimulation led to a robust NE activity signal in wildtype mice (Figure 5D). As expected, this activity was reduced in SFK-ko venules, indicating that there is reduced NE dependent substrate cleavage in the absence of SFK (Figure 5D).
Next, we investigated whether wildtype neutrophils are able to degrade BM constituents under in vitro conditions. To this end, we performed transwell experiments in a sys- tem that allows us to image neutrophil crawling and extravasation by spinning disc confocal microscopy. Neutrophils from wildtype and SFK-ko mice were isolated and stained with different cell trackers (CellTrackerTM Red CMTPX Dye and CellTrackerTM Green CMFDA Dye,) to image both neutrophil populations in one transmigration chamber. In addition, laminin was visualized with an Alexa-647 coupled antibody. We observed intensive degradation of LN by wildtype neutrophils only, which could be identified by the disappearance of fluorescently labeled LN over time (Figure 5E, Online Supplementary Figure S3A and Online Supplementary Mov3). When we used SFK-ko neutrophils only, no degradation of LN was observed (Online Supplementary Figure S3B). Interestingly we found that SFK-ko neutrophils, which were unable to digest the LN layer, migrated strongly towards wildtype neutrophils during the observation period (Figure 5F and G and Online Supplementary Mov4). We quantified this association of SFK-ko neutrophils around wildtype cells in a randomization approach. Here, the interaction strength is a measure of the degree of dependence of spatial distri- bution between wildtype and SFK-ko neutrophils. We observed an interaction strength of 5.1 after 1,000 seconds
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haematologica | 2020; 105(7)