Adhesion and recruitment of K3KI neutrophils are suppressed in vitro and in vivo. (A) BM neutrophils isolated from WT and K3KI mice were stained with an anti-Gr1 antibody and analyzed by flow cytometry. (B) Western blotting to measure kindlin-3 expression in BM neutrophils. (C) Pull-down assay to measure interaction of glutathione S-transferase (GST)-β₂CT protein with kindlin-3 in neutrophil lysates. GST alone was used as a control. Bound kindlin-3 protein in the precipitates was evaluated by western blotting; GST and GST-β₂CT proteins were evaluated by Coomassie blue staining. (D) Surface expression of the β₂-integrin subunits on BM neutrophils of WT and K3KI mice were measured by flow cytometry. (E-G) BM neutrophils were incubated with immobilized Fgn for 30 minutes in the presence of PMA (20 nM). The adherent cells were fixed and counted by microscopy (E); the representative images were exhibited (F); and percentage of spread cells were analyzed (G). Scale bar = 10 µm. (H) PMA-stimulated BM neutrophils were allowed to adhere to tumor necrosis factor-α primed EC monolayer, and the adherent neutrophils were fixed, stained with an fluorescein isothiocyanate-conjugated anti-Gr1 antibody and quantified under a fluorescence microscope. (I) Representative histologic images (×20 objective) of liver tissues isolated from the endotoxemic mice with IHC staining of Gr1. (J) Gr1-positive neutrophils recruited in the livers of endotoxemic mice. (K) MPO was extracted from the fresh liver tissues of endotoxemic mice and MPO activity was measured as described before.²²  (L) Representative histologic images (×4 objective) of the ligated mouse IVC tissues with staining of hematoxylin and eosin. (M) Thrombi formed in the ligated IVC tissues were isolated and quantified by weighing. (N) Neutrophil recruitment was visualized in histologic sections (×20 objective) of the ligated IVC tissues with staining of hematoxylin and eosin. IHC staining for Gr1 was shown in the insertion (×40 objective). (O) The accumulated neutrophils along the vessel walls (arrowhead) in the ligated IVC tissues were quantified. Data represent mean ± SEM of 3 or more independent experiments; *P < .05; **P < .01 (paired Student t test). EC, endothelial cell; Fgn, fibrinogen.