Figure 6

rmOPN-mediated neutrophil migration in vivo and in vitro. C57BL/6 mice were injected with rmOPN at a dose of 2.5 μg/mice, intratracheally. After 20 h, cells from the lung tissues were isolated and stained with APC-anti-Gr-1 Ab and then subjected to flow cytometry. (A) Representative dot blots indicating the percentages of Gr-1-positive cells are shown. (B) The mean percentages of Gr-1-positive cells obtained from PBS- and rmOPN-injected mice are shown. Data are expressed as means ± SEM (n = 4 mice/group) and compared by Student’s t test (^* P <0.05 vs. PBS). (C) A total of 5 × 10⁵ primary neutrophil cells isolated from mouse bone marrow were placed into the insert of a Boyden chamber. The bottom compartment contained the RPMI medium with PBS or rmOPN at a dose of 10 μg/ml as a chemotactic stimulus. After 2 h, the migrated primary neutrophil cells were counted. A representative image of the migrated primary neutrophil cells labeled with PI (red fluorescence) on the bottom of the transwell membrane is shown. Cells were observed at × 200 original magnification. (B) Migrated primary neutrophils were counted in five random microscopic fields per well and averaged in each group. Data are expressed as means ± SEM (n = 4/group) and compared by Student’s t test (^* P <0.05 vs. PBS). Ab, antibody; OPN, osteopontin; PBS, phosphate-buffered saline; PI, propidium iodide; rmOPN, recombinant mouse OPN; SEM, standard error of the mean.