Macrophages promote epithelial repair through hepatocyte growth factor secretion
Version of Record online: 8 SEP 2013
© 2013 British Society for Immunology
Clinical & Experimental Immunology
Volume 174, Issue 1, pages 60–72, October 2013
How to Cite
D'Angelo, F., Bernasconi, E., Schäfer, M., Moyat, M., Michetti, P., Maillard, M. H. and Velin, D. (2013), Macrophages promote epithelial repair through hepatocyte growth factor secretion. Clinical & Experimental Immunology, 174: 60–72. doi: 10.1111/cei.12157
- Issue online: 8 SEP 2013
- Version of Record online: 8 SEP 2013
- Accepted manuscript online: 18 JUN 2013 03:48AM EST
- Manuscript Accepted: 10 JUN 2013
- Swiss National Foundation. Grant Number: 3200B0-120717
- Fondation pour les maladies intestinales et hépatiques
- UCB Pharma AG
Fig. S1. F-actin purse string quantification by Photoshop software. Microphotographs were taken with a fluorescence microscope (Olympus IX81). The fluorescence intensity measured in square sections (arrowhead) defined along the leading edge of the migration front of epithelial cells was measured using Photoshop software. The quantified intensity of phalloidin staining reflects the F-actin purse string formation. At least five wounds per experimental condition were analysed.
Fig. S2. Neutralizing anti-transforming growth factor (TGF)-β antibodies did not reduce the properties of bone marrow-derived macrophages (BMM) significantly to assist epithelial repair. Epithelial repair was performed in the presence of hepatocyte growth factor (HGF) (0·25 ng/ml), TGF-β (1 ng/ml; R&D Systems), BMM (250 000) and/or anti-TGF-β neutralizing monoclonal antibody [20 μg/ml (1D11 clone)]. The data presented show the cumulated results of three independent experiments; bars are means ± standard error of the mean. P-values were calculated by the Mann–Whitney U-test.
Fig. S3. Impact of inflammatory stimuli on macrophage-assisted epithelial repair. One million heat-inactivated Escherichia coli or 10 ng/ml interleukin (IL)-1β were added to 250 000 bone marrow-derived macrophages (BMM) co-incubated with wounded epithelial cell monolayer for 18 h without insert. At the end of the incubation, we measured wound repair and HGF concentrations. The data presented show the cumulated results of three independent experiments; bars are means ± standard error of the mean. P-values were calculated by the Mann–Whitney U-test; n.d.: not done.
Fig. S4. Impact of inflammatory stimuli on human macrophage-assisted epithelial repair. Human monocyte-derived macrophages (MDM) were activated for 24 h with heat-inactivated Escherichia coli at 50 multiplicity of infection (MOI) and 105 were incubated with wounded epithelial cell monolayer. No statistical differences were found between the repair activities of activated or quiescent monocyte-derived macrophages.
Fig. S5. Viability and phenotypical characterization of Crohn's disease (CD) and healthy donor (HD) macrophages. (a) CD14-positive cells were selected by magnetic sorting from peripheral blood mononuclear cells and plated at density of 500 000–1 000 000 cells/ml for 5 days. On days 1 and 4, non-adherent cells were discarded and at day 5 adherent macrophages were recovered by scraping and numerated. The data show the percentage of recovered living macrophages compared to the initial number of plated CD14-positive cells. The data presented show the cumulated results of nine HD and nine CD patients; bars are means ± standard error of the mean. *No statistical difference (P-values were calculated by the Mann–Whitney U-test). (b) Macrophages were surface-stained with monoclonal antibodies to CD14 (clone M5E2; BD Biosciences), CD11b (clone ICRF44; BD Biosciences), CD11c (clone B-ly6; BD Biosciences), human leucocyte antigen D-related (HLA-DR) (clone G46-6; BD Biosciences), CD33 (clone WM53; BD Biosciences), CD68 (clone Y1/82A; BD Biosciences), CD116 (clone hGMCSFR-M1; BD Biosciences), CD163 (clone GHI/61; Biolegend), CD206 (clone 15.2; Biolegend), CX3CR1 (clone 2A9-1, MBL Nagoya, Japan). Data were acquired using a fluorescence activated cell sorter (FACS)scan flow cytometer (BD Biosciences) and analysed with CellQuest software (BD Biosciences). Dead cells were excluded by propidium iodide staining.
Fig. S6. Active transforming growth factor (TGF)-β and epidermal growth factor (EGF) levels in human co-cultures. Active TGF-β and EGF protein concentrations measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems) in the culture supernatant of monocytes derived-macrophages co-cultivated for 72 h with wounded epithelial cells in Dulbecco's modified Eagle's medium (DMEM) 0·1% fetal calf serum (FCS). No differences between TGF-β and EGF levels for healthy donors (HD) (eight individuals), Crohn's disease (CD) (11 individuals) and ulcerative colitis (UC patients) (four individuals) can be observed. 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.
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