Figure 6.

Reducing matrix stiffness promotes macrophage chemotaxis by a MDM4–p53-dependent CX3CL1 paracrine signal derived from lung myofibroblasts. (A) An equal number of IPF lung myofibroblasts were cultured on 0.5–20-kPa PA gels or plastic surface (P). CM was collected and incubated with 2 × 10⁵ THP1 or mBMDMs in 96-Transwell plates. Migrating macrophages were stained by calcein-AM. Chemotaxis index was calculated as a ratio of quantitative fluorescence of cells incubated with the CM to cells incubated with plain DMEM. Mϕ, macrophage. (B) Relative levels of CX3CL1 and CXCL10 in the CM collected from IPF lung myofibroblasts cultured on 1–20-kPa PA gels were determined by ELISA. (C) IPF lung myofibroblasts were cultured on 1-kPa PA gels. CM were collected and incubated with THP1 or mBMDMs in the presence of increasing concentrations (0, 0.1, 1, and 10 µg/ml) of CX3CL1 neutralizing antibody (3CL Ab), nonimmune goat IgG (gIgG), CXCL10 neutralizing Ab (CL10 Ab), or mouse IgG (mIgG). Macrophage chemotaxis index was determined as described above. (D) IPF lung myofibroblasts were cultured on 1- or 20-kPa PA gels in the presence or absence of MDM4-expressing lentiviral vector or 25 µM C646. Levels of CX3CL1 in the CM were determined by ELISA. (E and F) CM collected from cells treated in D were incubated with THP1 or mBMDMs. Effects of MDM4 overexpression (E) and C646 treatment (F) on macrophage chemotaxis were determined as described above. Results are the means ± SD of three separate experiments. *, P < 0.05; **, P < 0.01 (ANOVA).