Fig. 2.

Murine macrophages promote epithelial restitution through hepatocyte growth factor (HGF) secretion. (a) Macrophages do not need cell contact to promote wound repair. Schematic representation of the experimental system (a, left panel). Representative images of wounded areas on t = 18 h of CMT-93 cells cultured in serum-deprived medium (no cells) in the presence of bone marrow-derived macrophages (BMM) added directly on top of wounded cells (BMM no insert) or in the presence of BMM added into a cell culture insert (BMM in insert). (b) Recombinant mouse hepatocyte growth factor (rHGF) promotes epithelial repair. The addition of rHGF to wounded epithelial cells promotes repair in a dose-dependent manner. After addition of 400 nM of the c-met inhibitor PHA-665732, the rHGF-induced wound closure is suppressed. (c) HGF protein concentration measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of BMM co-cultured with wounded epithelial cells. HGF concentrations were determined at 18 h of culture in Dulbecco's modified Eagle's medium (DMEM) 0·1% fetal calf serum (FCS). (d) Wound closure ability of BMM in the presence of PHA-665732 (400 nM). (e) HGF mRNA expression measured by quantitative polymerase chain reaction (PCR) and HGF protein concentrations in the culture supernatant of BMM left untreated or electroporated with non-coding siRNA (nc siRNA) or HGF specific siRNA (HGF siRNA). Wound closure and F-actin purse string formation abilities of BMM left unmanipulated (BMM) or transfected with non-coding siRNA (BMM + nc siRNA) or HGF-specific siRNA (BMM + HGF siRNA). The data presented show the cumulated results of at least three independent experiments; bars are mean ± standard error of the mean. P-values were calculated by the Mann–Whitney U-test for (b) and (c); for (d) and (e) comparison between several groups to identify a trend was performed by a one-way analysis of variance (anova) test with Trend test as post-test.