Figure 2
Apically attached neutrophil (PMN) exhibit intercellular adhesion molecule-1 (ICAM-1)- and Mac-1-dependent locomotion. (a, b) Following transepithelial migration (TEM), PMN were allowed to apically adhere to intestinal epithelial cells (IECs) grown in the bottom chamber of transwells. The number of PMN exhibiting locomotion (a) and the traveled distances (b) were visualized and quantified in the presence or absence of the appropriate IgG control, or anti-ICAM-1 and Mac-1 function-blocking antibodies (Abs; 20 μg ml−1), using phase-contrast time-lapse microscopy (Zeiss, Axiovert microscope) and ImageJ software. (c) PMN before (solid black line) and after TEM (dotted line) were analyzed for Mac-1 expression using flow cytometry. Representative flow diagram (n=4) shows robust upregulation of Mac-1 on PMN surface after TEM. The filled area represents control IgG staining. (d) Image sequence depicting representative PMN exhibiting locomotion on T84 IECs in the absence (upper panels) and in the presence of Mac-1-blocking Ab (bottom panels). *Initial PMN position, white arrows track PMN movement. The dashed lines highlight the path traveled by PMN over 40 min. Bar = 20 μm. (e) Movement trajectories (in μm) of six representative PMN from three independent experiments over 40 min time periods on the apical surface of interferon-γ (IFNγ)-stimulated T84 IECs in the absence (upper panels) and in the presence of Mac-1-blocking Ab (bottom panels). n=4 independent experiments in duplicates, **P<0.01, analysis of variance with Newman–Keuls multiple comparison test (a,b).
