The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation.

Spinning Disk Confocal Intravital Microscopy of the Murine Liver after Focal Hepatic Necrosis The Animal Care and Use Committees of North Rhine Westphalia (Germany) approved all animal experiments. Mice were anesthetized intraperitoneally with a mixture of ketamine hydrochloride (125 mg/kg, Sanofi Winthrop Pharmaceuticals) and xylazine (12.5 mg/kg, TranquiVed, Phonix Scientific). After anesthesia, mice were placed on a heating pad set to 37°C to maintain the body temperature for the duration of all performed experiments. The left carotid artery was cannulated for administration of additional anesthetics. For intravital microscopy of the liver, mice were prepared as previously described ( McDonald et al., 2010 ). In brief, a midline laparotomy was performed followed by removal of the skin and abdominal muscle to expose the liver lobe. A thin platinic wire was heated and briefly pressed on the liver to induce a focal injury on the surface of the liver. With a single application of 2 μl of a propidium iodide solution (1.0 mg/ml) diluted 1:25 in PBS to the surface of the liver, necrotic cells were labeled immediately. To avoid dehydration, all exposed tissues were moistened with PBS-soaked tissues. Directly after preparation, the exposed liver was visualized with an upright spinning disc confocal microscope (CellObserver SD, Zeiss) equipped with a 5×/0.25 FLUAR objective and time-lapse Z-stacks were recorded for up to 4 hr after focal injury. The number of adherent neutrophils was determined for different time points per field of view, or after 4 hr after injury within specific regions (within injury, proximal 150 μm around injury, and beyond 150 μm from injury border). Duration of adhesion and neutrophil crawling was determined within the necrotaxis zone 2.5 hr after injury. 30 neutrophils per field of view were randomly selected for tracking, and chemotaxis parameters (migration plots, forward migration index, crawling velocities) were set. All investigated parameters were analyzed with FIJI or Chemotaxis and Migration tool (Ibidi). The determination of recruited neutrophils to the liver 4 hr after focal injury or sham procedure via flow cytometry was performed as previously described ( Rossaint et al., 2014 ). The levels of the transaminases GOT and GPT before and 4 hr after focal hepatic necrosis in serum of blood in WT and Btk −/− mice was determined with a high-volume hematology analyzer (ADVIA, Siemens). Intravital Microscopy of the Murine Cremaster Muscle Mice were anesthetized with injection of 125 mg/kg ketamine hydrochloride (Sanofi Winthrop Pharmaceuticals) and 12.5 mg/kg xylazine (Tranqui Ved, Phonix Scientific) i.p., and the cremaster muscle was prepared for intravital imaging as previously described ( Mueller et al., 2010 ). Postcapillary venules with a diameter between 20 and 40 μm were investigated. To determine adhesion and transmigration in vivo, mice were injected intrascrotally with 16 μg fMLF (Sigma-Aldrich) 4 hr before preparation of the cremaster muscle. Intravital microscopy was performed on an upright microscope (Axioskop, Carl Zeiss) with a 40×0.75 NA saline immersion objective. Neutrophil adhesion was determined by transillumination intravital microscopy, whereas neutrophil extravasation was investigated by reflected light oblique transillumination microscopy as described previously ( Mueller et al., 2010 ). Recorded images were analyzed off-line with ImageJ (NIH) and AxioVision (Carl Zeiss) software. Emigrated cells were determined in an area 75 × 100 μm to each side of a vessel (representing 1.5 × 10 4 μm 2 tissue area). The microcirculation was recorded with a digital camera (Sensicam QE, Cooke). Blood flow centerline velocity was measured with a dual photodiode sensor system (Circusoft Instrumentation). Centerline velocities were converted to mean blood flow velocities as previously described ( Mueller et al., 2010 ). For GPCR-induced arrest, the carotid artery was cannulated for injection of fMLF (16 μg), CXCL-1 (500 ng), or blocking antibodies. In a representative vessel, the initial number of adherent neutrophils was determined, fMLF was injected, and the vessel was recorded for 15 min. Movies were analyzed with Slidebook software (Intelligent Imaging Innovations). For blocking experiments, monoclonal antibodies against Mac-1 (30 μg/mouse; eBioscience), LFA-1 (30 μg/mouse; eBioscience), or both were injected i.v. immediately before the experiments.

Soluble ICAM-1- and Fibrinogen-Binding Assay

The soluble ICAM-1- and fibrinogen-binding assays were performed as previously described ( Lefort et al., 2012 ). In brief, to assess LFA-1-specific ICAM-1 binding, isolated murine neutrophils were preincubated with a functional blocking anti-CD11b (clone M1/70, 10 μg/ml) antibody to prevent Mac-1-dependent ICAM-1 binding or a blocking anti-CD11a antibody (clone TIB217, 10 μg/ml) to prevent LFA-1-dependent ICAM-1 binding. Afterward, neutrophils were stimulated with fMLF (10 μM) or CXCL-1 (100 ng/ml, 3 min, 37°C) or left unstimulated in the presence of ICAM-1/Fc (20 μg/ml, R&D Systems) or IgG control and APC-conjugated anti-human IgG1 (Fc-specific, Southern Biotechnology). Neutrophils were fixed on ice and stained with FITC-conjugated anti-Ly6G (Biolegend). LFA-1-specific binding to ICAM-1/Fc was measured by flow cytometry. To investigate Mac-1 affinity to fibrinogen, isolated murine neutrophils were incubated for 10 min at 37°C with 150 μg/ml Alexa 647-conjugated fibrinogen (Invitrogen) and stimulated with fMLF or CXCL-1 or were left unstimulated. Neutrophils were treated with EDTA (2 mM) as negative controls. LFA-1 crosslinking to induce Mac-1 activation was performed by incubating murine neutrophils with anti-CD11a (Biolegend, clone M17/4) and an anti-rat secondary antibody (Santa Cruz) for 10 min at 37°C. Fluorescence intensity was measured by flow cytometry. The percentage of neutrophil positive for fibrinogen binding was calculated by defining a threshold of the fluorescence intensity where 95% of neutrophils in the EDTA control were considered as negative.

Supplementary Material Supplemental figures movie S1 movie S2 movie S3 movie S4