Ly6G ligation down-regulates the expression of β2-integrins and neutrophil adhesion and migration via an integrin-dependent mechanism. (A) BM cells were cytospun, fixed, permeabilized, and stained with Abs to CD11a, CD11b, and Ly6G. The images were acquired using confocal microscopy (60× objective). The higher magnification of a representative single-cell image (arrow denotes the cell selected for enlarged viewing) are shown in parallel. The results are representative of at least 3 independent experiments. (B) Coimmunoprecipitation studies were carried out with lysates prepared from BM cells after in situ cross-linking. Ly6G was coimmunoprecipitated with CD18 and CD18 was coimmunoprecipitated with Ly6G. (C) BM cells were cytospun, fixed, permeabilized, and stained with FRET pair as indicated. Shown are representative images from each group displaying interacting τ1 lifetimes in each pixel on a pseudocolor scale (60× objective). (D) Neutrophils were exposed to LTB4 and surface expression of CD11a and CD11b was measured at 60 minutes by flow cytometry. Data are representative of at least 3 independent experiments. (E) Neutrophils were preincubated with anti-Ly6G, anti-CD11a, or the combination at 10 μg/mL for 10 minutes and then with or without LTB4 (100nM) for 30 and 60 minutes. FITC-labeled ICAM-1 binding on neutrophils was detected by flow cytometry. Results are representative of 3 independent experiments. (F) Adhesion of LTB4-activated neutrophils to inflamed endothelium was assessed under high shear stress (10 dyne/cm2) after exposure to anti-Ly6G or control. Data reflect triplicate conditions from 2 independent experiments (CD11a blockade control from 1 experiment). (G) Blockade of transwell migration by anti-Ly6G requires the expression of the β2-integrin CD18. Data are representative of 3 independent experiments. *P < .05; **P < .01 isotype versus anti-Ly6G or anti-CD11a.
