![]() ![]() LPS-induced acute lung inflammation (ALI) was performed essentially as described ( 22). ARAP3 knockdown in αIIbβ3-expressing Chinese hamster ovary cells Transmigrated neutrophils were labeled for GR1, and eight random fields of view were photographed and counted (×20 magnification EVOS imaging system). Transendothelial migration toward the indicated concentrations of MIP2 (R&D Systems) for 1 h in 6.5-mm transwell inserts with 3-μm pore polycarbonate membranes (Corning) was performed as described ( 17). Adherent neutrophils were fixed with PFA, labeled for GR1 (clone RB6-8C5 BioLegend), and counted in randomly taken frames (EVOS imaging system Advanced Microscopy Group/Thermo Fisher). After 30 min, nonadherent neutrophils were washed away with HBSS (without Ca 2+ and Mg 2+). After washing and careful aspiration, 100 μl of HBSS (with Ca 2+ and Mg 2+) containing 1 × 10 5 neutrophils were added and allowed to bind to the stimulated endothelial cells under gentle rocking. For adhesion to endothelial cells, bEND5 cells were seeded into 24 wells, allowed to form confluent monolayers for 2 d, and stimulated with 5 nM TNF-α for 16 h. Neutrophil adhesion, spreading, and degranulation assays were done as previously described ( 16). Where blocking peptides were employed, neutrophils were plated onto the immobilized stimuli and the competing peptide, such that both were encountered at the same time. Where indicated, neutrophils were preincubated with inhibitors for 10 min at 37☌ at the indicated concentrations. ROS production was measured indirectly using chemiluminescence produced by 5 × 10 5 neutrophils per well at 37☌ with 150 μM luminol and 18.75 U/ml HRP in the presence or absence of TNF-α (20 ng/ml final concentration) in luminescence-grade 96-well plates (Nunc) using a Cytation plate reader (BioTek) essentially as described ( 16). Surfaces were washed three times with PBS, blocked with 10% FBS in PBS, and washed again before addition of prewarmed neutrophils. Tissue culture wells were coated overnight at 4☌ with fibronectin as indicated. All animal work was approved by the University of Edinburgh Animal Welfare Committee and conducted under the control of the U.K. All mice were housed in a specific pathogen–free small animal barrier unit at the University of Edinburgh. For ease of reading, tamoxifen-induced Arap3 fl/fl ERT2Cre + mice (or neutrophils) are referred to in the text as ARAP3-deficient and in figures as −/−, whereas vehicle-induced Arap3 fl/fl ERT2Cre + and tamoxifen-induced Arap3 +/+ ERT2Cre + controls are referred to as controls and +/+, with explanations provided in the figure legends. For in vivo experiments, age- and sex-matched Arap3 fl/fl ERT2Cre + mice and Arap3 +/+ ERT2Cre + controls were subjected to five successive gavages with emulsion containing 1.5 mg of tamoxifen, followed by a rest period of 10 d ( Supplemental Fig. injection with 200 mg/kg tamoxifen or vehicle, with experiments performed 10–12 d after induction as described ( 16). To analyze neutrophils in vitro, 10–12-wk-old sex-matched Arap3 fl/fl ERT2Cre + mice were induced with a single i.p. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation. ![]() In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. ![]() Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. ![]()
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