Deficiency of ROCK1 in BMMs, peritoneal macrophages, and neutrophils results in increased migration and chemotaxis in vitro. (A) WT and ROCK1−/− BMMs (2.5 × 105) were subjected to a migration assay on fibronectin fragment CH-296, which contains the binding sites for both integrins α4β1 and α5β1. Shown is the quantitative assessment of the number of WT and ROCK1−/− BMMs migrated on fibronectin-coated wells. The bar graph represents the average number of WT and ROCK1−/− BMMs migrated per field in an independent experiment performed in replicates of 3. WT versus ROCK1−/−, *P < .05. Similar results were obtained in 3 other independent experiments. (B) Expression of F4/80 on WT and ROCK1−/− BMMs. BMMs were stained with PE-conjugated anti-F4/80 antibody and subjected to flow cytometric analysis. Solid histograms represent the level of F4/80 expression on the surface of WT and ROCK1−/− BMMs, whereas open histograms represent the level of expression using an isotype control antibody. Percentage of F4/80 expression on WT and ROCK1−/− BMMs is shown in the top right corner of each histogram (n = 4, 6 pairs of WT and ROCK1−/− mice). (C-D) BMM migration using 8-μm pore size transwell filters coated with fibronectin. Representative images shown here are from WT and ROCK1−/− BMMs migrated on fibronectin in response to the MCP-1 gradient. The bar graph represents the average number of WT and ROCK1−/− BMMs migrated in response to MCP-1 gradient per field in an independent experiment. Similar results were obtained using 2 other mice of each genotype (total 3 pairs of WT and ROCK1−/− mice, *P < .05). (E) WT and ROCK1−/− peritoneal macrophages (2.5 × 105) were subjected to a migration assay on fibronectin. The bar graph represents average number of migrated peritoneal macrophages derived from WT and ROCK1−/− mice from an independent experiment. WT versus ROCK1−/−, *P < .05. Similar results were obtained using 2 other mice of each genotype (total n = 3). (F) BMN migration assay was performed using the Boyden chamber. Migration was evaluated without fMLP, or in a gradient of 1μM fMLP. The bar graph represents the number of migrated BMNs per field (mean ± SD). Shown is a representative experiment performed in replicates of 3. WT versus ROCK1−/−, *P < .05. Similar results were obtained in 2 additional independent experiments (total n = 3). (G) Expression of integrins on WT and ROCK1−/− BMNs. BMNs were stained with PE-conjugated anti-α4β1 or anti-α5β1 antibody and subjected to flow cytometric analysis. Solid histograms indicate the level of α4β1 and α5β1 expression on the surface of WT and ROCK1−/− BMNs, whereas open histograms in both panels demonstrate the level of expression using an isotype control antibody. Percentage of α4β1 or α5β1 integrin expression on BMNs is shown in the top right quadrant of each histogram. Histograms shown are from a representative experiment. Similar results were obtained in 3 independent experiments (total n = 4). (H) WT and ROCK1−/− BMNs were harvested after 1 week and stained with antibodies that recognize Gr-1 and Mac-1. Stained cells were analyzed by flow cytometry to determine the level of Gr-1 and Mac-1 expression. Percentage of Gr-1 and Mac-1 double-positive cells in either genotype are indicated in the top right quadrant of each dot blot. (I) Increased migration of ROCK1−/− BMMs in a wound-healing assay. WT and ROCK1−/− BMMs were cultured for 8 days in 24-well plates in the presence of 100 ng/mL M-CSF. An artificial wound was created in the macrophage monolayer using a pipette tip. Images were taken immediately and again at indicated time periods after creating the wound. Top panels represent the migration of WT cells; bottom panels represent migration of ROCK1−/− cells. (J) Bar graph shows quantitative analysis of the number of migrated cells in the wounded area from 1 independent experiment. WT versus ROCK1−/−, *P < .05. Similar results were obtained in 2 other independent experiments (total n = 3). (C, I) Images were acquired through a Zeiss Axioskop 2 Plus microscope equipped with a Plan-Neofluar 20×/0.5 objective lens, and were captured with an Axiocam MRC-5 camera and Axiovision 4 software (all from Zeiss).
