Heat inactivation of degranulated contents blocked VE-cadherin degradation (Figure S7C), suggesting that proteases released from neutrophil degranulation were responsible for the degradation process. in Methods. Neutrophils Onjisaponin B were labeled with i.v. injection of 4 g of PE-conjugated anti-mouse Gr-1 antibody and blood vessels were visualized with injection of 50 l of 10 kD FITC-Dextran. Blood vessels in the hind foot phalanges Onjisaponin B were imaged with 2P microscopy between 15-60 min after s.c. injection to assess cell recruitment. Scale bar = 30 m. Time stamp = min:sec. NIHMS1055318-supplement-Video_2.mp4 (1.2M) GUID:?A8968D3A-EB7D-4556-8B7D-7B141E84E691 Video 3: Video 3. Trafficking of WT neutrophils in the accelerated K/BxN arthritis model. K/BxN serum was injected s.c. as described in video 2. Neutrophils were labeled with i.v. injection of 4 g of PE-conjugated Ly6G antibody and vessels were visualized with 50 l of 10 kD FITC-Dextran as described in video 2. Neutrophils labeled with Ly6G antibody were indistinguishable from cells labeled with Gr-1 antibody in terms Onjisaponin B of morphology and cell recruitment behavior, suggesting that Gr-1 antibody used under our experimental conditions specifically labeled neutrophils. Scale bar = 30 m. Time stamp = min:sec. NIHMS1055318-supplement-Video_3.mp4 (1.2M) GUID:?4A0298C6-D658-4D7A-838B-24CE2314A5E1 4. NIHMS1055318-supplement-4.pdf (4.7M) GUID:?CABC2751-7C7C-4175-BA80-8A00753E089B Data Availability StatementData generated in this current study are available from the corresponding authors on reasonable request. Abstract Neutrophils are essential to the pathogenesis of many inflammatory diseases. In the autoantibody-mediated K/BxN model of inflammatory arthritis, the alternative pathway Onjisaponin B (AP) of complement and Fc gamma receptors (FcRs) are required for disease development while the classical pathway is dispensable. The reason for this differential requirement is unknown. We show that within minutes of K/BxN serum injection complement activation (CA) is detected on circulating neutrophils, as evidenced by cell surface C3 fragment deposition. CA requires the AP factor B and FcRs but not C4, implying that engagement of FcRs by autoantibody or immune complexes directly triggers AP C3 convertase assembly. The absence of C5 does not prevent CA on neutrophils but diminishes the upregulation of adhesion molecules. two-photon microscopy reveals that CA on neutrophils is critical for neutrophil extravasation and generation of C5a at the site of inflammation. C5a stimulates the release of neutrophil proteases, which contribute to the degradation of VE-cadherin, an adherens junction protein that regulates endothelial barrier integrity. C5a receptor antagonism Onjisaponin B blocks the extracellular release of neutrophil proteases, suppressing VE-cadherin degradation and neutrophil NOS2A transendothelial migration for 15 min at RT. Red blood cells were lysed, cells were washed with 0.1% BSA in PBS, pelleted and analyzed by FACS analysis using the same antibody panel as analysis (see above). Flow cytometry was performed on the BD FACSCalibur?. Data analysis was performed using BD CellQuest? Pro software. 2.4. C5a ELISA Fresh plasma was prepared from collected blood for C5a ELISA-based quantification. Briefly, plates were coated overnight at 4C with rat anti-mouse C5a (5 g/ml) monoclonal antibody (cat#558027, BD Pharmingen, San Jose, CA). After blocking with reagent diluent (1% BSA in PBS) for 1 h at RT, the plates were washed 3x with ELISA wash buffer [0.05% (v/v) Tween 20 in PBS] and incubated with samples (100 L of same day, freshly obtained plasma diluted 1:20 in reagent diluent) for 1 h at RT. The plates were washed 3x, followed by incubation with biotinylated anti-mouse C5a (500 ng/mL) monoclonal antibody (cat#558028, BD Pharmingen) for 1 h at RT. After washing, the plates were incubated with streptavidin-HRP, 1:200 dilution in reagent diluent (cat# 890803, R&D Systems, Minneapolis, MN) for 30 min, washed and 100 l of peroxidase substrate (cat# DY999, R&D Systems, Minneapolis, MN) was added. The reaction was stopped with 1M H2SO4 and OD of samples measured at 450nm (Molecular devices SpectraMax Plus 384, Molecular Devices, LLC, Sunnyvale, CA). 2.5. Two-photon (2P) microscopy Imaging of mouse paws and neutrophil trafficking was performed as previously described (9). Briefly, neutrophils were labeled with an i.v. injection of 4 g of PE-conjugated anti-Gr-1 antibody (cat#108408, Biolegend) or anti-Ly6G antibody (cat# 127654, Biolegend, San Diego, CA). Administration.