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I. Rohwedder et al.
mainly regulated by Rab GTPases and their effector pro- teins with Rab27a being the main Rab molecule involved in the secretory machinery of neutrophils.7 Rab27a func- tion is mediated by the two effector molecules synapto- tagmin-like protein 1 (JFC1, encoded by Sytl1 in mice) and Munc13-4 (Unc13d).8 Although most of these processes rely on integrin signaling, integrins themselves lack enzy- matic activity. Therefore, numerous proteins are recruited to their intracellular tails, among those Src family kinases (SFK). Their early recruitment during integrin activation assigns SFK a critical role in the so-called outside-in signal- ing process and thus regulation of central signaling path- ways downstream of integrin-receptor ligation.9 Neutrophils express three members of this family, namely Hck, Fgr and Lyn.10 Their function has been intensively studied in SFK single, double (Hck-/-Fgr-/-), and triple knock- out (ko) (Hck-/-Fg-/-Lyn-/-) mice,11 demonstrating a role for SFK in integrin activation12 and their downstream signal- ing. Neutrophils isolated from Hck-/-Fgr-/- mice showed poor spreading, indicating that integrin outside-in signal- ing is impaired.13 Additionally, neutrophil migration into the liver of Hck-/-Fgr-/- mice in an in vivo endotoxemia model is severely reduced.14 Furthermore, Kovács et al. recently showed that neutrophils exhibit reduced capabil- ity to create a microinflammatory environment, resulting in diminished neutrophil extravasation in a model of autoantibody-induced arthritis and inflammatory blister- ing skin disease.15
Our study aimed to investigate the effect of SFK on leukocyte recruitment in an in vivo setting of acute inflam- mation, focusing on post-arrest modifications and the molecular mechanism of vascular BM penetration. We show that, in the genetic absence of SFK, these two steps are strongly impaired. Adherent Hck-/-Fgr-/-Lyn-/- neutrophils are unable to withstand shear forces and display dimin- ished LFA1 clustering with reduced phosphorylation levels of Paxillin, Cortactin and Syk, suggesting that SFK deple- tion results in severely impaired adhesion strengthening. In addition, we show that SFK are critical for crossing the vascular BM during neutrophil extravasation by facilitat- ing translocation of VLA3-, VLA6- and NE-containing vesicles to the cell surface. Therefore, SFK are not only important mediators of neutrophil post-arrest modifica- tions, but are also required to breach the vascular BM.
Methods
Animals
Lyz2GFP, SFK-ko (Hck-/-Fgr-/-Lyn-/-) and SFK-ko Lyz2GFP mice were generated as described earlier.16-18 C57BL/6 wildtype mice were purchased from Janvier Labs (Saint Berthevin, France). All animal experiments were approved by the Regierung von Oberbayern, Germany (AZ 55.2-1-54-2531-80-76/12 and 55.2-1-54-2532-102- 2017).
Live cell imaging of in vitro laminin digestion Transmigration of neutrophils was analyzed in μ-Slide mem- brane ibiPore flow chambers (Ibidi, Planegg, Germany) equipped with a 300-nm thick membrane with 5-μm pores, and a subjacent rat-tail collagen gel (1.5 mg/mL) containing 10 μM N-formylme- thionyl-leucyl-phenylalanine (fMLP) as chemoattractant. The upper compartment was coated with Laminin (LN), PECAM-1 and ICAM-1, and, in addition, LN was visualized using an anti-LN antibody conjugated to Alexa Fluor-647 (novusbio, Littleton, CO,
USA). Isolated neutrophils from wildtype and SFK-ko animals were labeled with CellTrackerTM Green CMFDA Dye and CellTrackerTM Red CMTPX Dye (Thermo Fisher, Waltham, MA, USA), respectively, and distributed into the upper chamber com- partment in a 1:1 ratio. Time-lapse microscopy with an interval of 50 seconds was performed using an upright spinning-disk confocal microscope (Examiner; Zeiss) equipped with a confocal scanner unit CSU-X1 (Yokogawa Electric Corporation, Japan), an EMCCD camera (Evolve; Photometrics), and a x20/1.0 NA water immer- sion objective (Plan Apochromat; Zeiss). 3D images (70 z-stacks with a step size of 2μm) were acquired per time point and ana- lyzed by generating maximum z-projections over time using Slidebook 6.0.8 software (3i) and ImageJ. Interaction strength was analyzed with the MosaicIA interaction plugin of Fiji.
Functional in vitro and in vivo experiments, including intravital microscopy,19 peritonitis experiments, flow chamber experiments, vesicle trafficking, flow cytometry, western blot analysis and sta- tistics are described in detail in the Online Supplementary Methods.
Data sharing statement
Original data are available on request.
Results
Src family kinase depletion reduces neutrophil adhesion and extravasation in inflamed postcapillary venules in vivo
We first investigated how loss of neutrophil-expressed SFK influences neutrophil adhesion in TNFα-stimulated cremaster muscle postcapillary venules using intravital microscopy. Interestingly, the absolute number of adher- ent neutrophils was strongly increased compared to wild- type control mice (Online Supplementary Figure S1A). However, after adjusting the number of adherent neu- trophils to the circulating neutrophil count, which was sig- nificantly higher in SFK-ko mice compared to wildtype mice (Online Supplementary Figure S1B and C), the neu- trophil adhesion efficiency calculated as number of adher- ent cells/mm2 divided by the systemic neutrophil count was significantly reduced in SFK-ko mice (0.48±0.04) com- pared to wildtype mice (1.04±0.08) (Figure 1A). This was not due to altered surface expression of rolling and adhe- sion relevant surface proteins including CD18, CD11a, CD11b, CD62L, PSGL1, CXCR2, and CD44 as their sur- face expression was equal between wildtype and SFK-ko neutrophils (Online Supplementary Figure S1D). We there- fore conclude that SFK are critical for neutrophil adhesion in vivo.
Efficient neutrophil extravasation into tissue is required for proper host defense. Recently, Kovács et al. demon- strated in the K/B×N serum-transfer arthritis model that SFK deficiency resulted in lower levels of cytokine produc- tion and release leading to impaired leukocyte extravasa- tion.15 To investigate a cell intrinsic adhesion defect in SFK-ko mice, we injected TNFα into the mouse scrotum. This approach circumvents possible effects of a reduced inflammatory environment and enables us to focus on neutrophil recruitment itself. Performing Giemsa staining of TNFα stimulated cremaster muscle whole mounts revealed strongly reduced numbers of extravasated neu- trophils in SFK-ko mice compared to wildtype mice (306.51±43.7 vs. 578.6±72.9, respectively) (Figure 1B and Online Supplementary Figure S1E). Moreover, we calculated the extravasation efficiency as total number of extravasat-
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haematologica | 2020; 105(7)