Figure 8.

Inhibition of BMDN migration by MFG-E8 is mediated through regulation of CXCR2 and GRK2. (A and B) BMDN were isolated from WT and Mfge8^−/− mice. (A) BMDN were placed in the upper well of a modified Boyden chamber for migration assays. The media containing rmMIP-2 (1 ng/ml) was placed in the lower wells as chemotactic stimulus. After 2 h, migrated cells were fixed and stained with PI. Representative images of migrated BMDN are shown. Five random microscopic fields per well were counted. (B) CXCR2 expression in BMDN was analyzed by flow cytometry. Granulocytes were gated according to forward and side scatter dot plots, followed by identification of neutrophils with staining of FITC-CD11b and APC-Ly6G antibodies. Representative histograms of expression intensity of CXCR2 in CD11b⁺Ly6G⁺ cells stained with PerCP/Cy5.5-CXCR2 antibody are shown. The average of mean fluorescent intensities (MFI) for CXCR2 in BMDN is plotted. Data are expressed as means ± SE (n=5 mice/group). *P < 0.05 vs. WT. (C-E) BMDN isolated from WT mice were treated with isotype antibody (Ab) (1 µg/ml) + rmMFG-E8 (500 ng/ml) or anti-MFG-E8 Ab (1 µg/ml) + rmMFG-E8 (500 ng/ml) for 2 h before analysis. (C) BMDN with various treatments were subjected to migration assay. Representative images of migrated BMDN are shown. Five random microscopic fields per well were counted. (D) CXCR2 expression in BMDN with various treatments was analyzed by flow cytometry. (E) Intracellular GRK2 expression in BMDN with various treatments was analyzed by flow cytometry. Representative histograms of expression intensity of GRK2 in CD11b⁺Ly6G⁺ cells stained with PE-GRK2 antibody are shown. The average of MFI for GRK2 in BMDN is plotted. Data are expressed as means ± SE (n=5 independent experiments). *P < 0.05 vs. PBS control; ^#P < 0.05 vs. isotype Ab + rmMFG-E8.