Macrophages promote epithelial repair through hepatocyte growth factor secretion - PubMed (original) (raw)
Macrophages promote epithelial repair through hepatocyte growth factor secretion
F D'Angelo et al. Clin Exp Immunol. 2013 Oct.
Abstract
Macrophages play a critical role in intestinal wound repair. However, the mechanisms of macrophage-assisted wound repair remain poorly understood. We aimed to characterize more clearly the repair activities of murine and human macrophages. Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of healthy donors (HD) or Crohn's disease (CD) patients or isolated from the intestinal mucosa of HD. In-vitro models were used to study the repair activities of macrophages. We found that murine and human macrophages were both able to promote epithelial repair in vitro. This function was mainly cell contact-independent and relied upon the production of soluble factors such as the hepatocyte growth factor (HGF). Indeed, HGF-silenced macrophages were less capable of promoting epithelial repair than control macrophages. Remarkably, macrophages from CD patients produced less HGF than their HD counterparts (HGF level: 84 ± 27 pg/mg of protein and 45 ± 34 pg/mg of protein, respectively, for HD and CD macrophages, P < 0·009) and were deficient in promoting epithelial repair (repairing activity: 90·1 ± 4·6 and 75·8 ± 8·3, respectively, for HD and CD macrophages, P < 0·0005). In conclusion, we provide evidence that macrophages act on wounded epithelial cells to promote epithelial repair through the secretion of HGF. The deficiency of CD macrophages to secrete HGF and to promote epithelial repair might contribute to the impaired intestinal mucosal healing in CD patients.
Keywords: Crohn's disease; inflammatory bowel diseases; intestinal ulcers; mucosal healing; restitution.
© 2013 British Society for Immunology.
Figures
Fig. 1
Murine macrophages promote in-vitro wound repair. (a) Wounded CMT-93 epithelial cells were cultivated for 18 h in the absence (without cells) or presence of bone marrow-derived macrophages (BMM) or splenic CD11b+ cells. Six independent experiments were performed; results are represented as mean ± standard error of the mean. _P_-values were calculated by the Mann–Whitney _U_-test. (b) Representative images of CMT-93 epithelial cell monolayers stained with Alexa Fluor 488 Phalloidin to detect F-actin (1, 2, 3) or with Syto 60 red fluorescent nucleic acid stain and Alexa Fluor 488 anti-Ki67 antibodies (4, 5, 6) (bars: 100 μm). 1 and 4: epithelial monolayer before wounding, actin microfilaments (green fluorescence) can be detected close to the cell membrane (1) and Ki-67+ proliferating epithelial cells (with light blue nuclei, *, 4) are scarce; 2 and 5: wounded epithelial monolayers after 8 h incubation with BMM placed in cell culture insert, actin microfilaments reorganized and accumulated at the edge of wounded epithelial cell monolayers (2, **), while there is no evident epithelial cell proliferation (*, 5) at the wound edges; 3 and 6: fully repaired wounded epithelial monolayers after 18 h incubation with BMM placed in cell culture insert. Accumulation of actin microfilaments at the edge of wounded epithelial cells disappeared (3) and no increased cell proliferation was detected in the epithelial cells covering the wounded area (6). White bars show the initial wounded area. (c) Representative image of wounded CMT-93 epithelial cells cultivated for 10 h in presence of PKH26-labelled BMM (red cells). Arrow shows BMM making direct contact with epithelial cells at the leading edge of the migration front; arrowhead shows that once epithelial cells covered the wounded area, some adherent BMM can be detected under the epithelial cell monolayer.
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.
Fig. 3
Human macrophages promote in-vitro wound repair through hepatocyte growth factor (HGF) secretion. (a) Representative images of Caco2 epithelial cell monolayers after wounding (day 0) or after 3 days of incubation. Caco2 cells were seeded with either Dulbecco's modified Eagle's medium (DMEM) 0·5% fetal calf serum (FCS) (–), or DMEM 10% FCS (+) or with 105 human macrophages (macrophages). (b) Wounded Caco2 cells were seeded in DMEM 0·5% FCS either directly with increasing numbers of macrophages, with 105 macrophages into a cell culture insert or with 105 peripheral blood mononuclear cells (PBMC). In negative (–) and positive (+) controls wounded Caco2 cells were seeded with DMEM 0·5% FCS or DMEM 10% FCS, respectively. Comparison between different numbers of macrophages was performed by a one-way analysis of variance (
anova
) test with Trend test as post-test. (c) c-Met inhibitor inhibits macrophage-assisted repair. Wounded Caco2 cells were seeded in DMEM 0·5% FCS with 300 pg/ml rHGF or with 105 macrophages in the presence or not of PHA-665732 (600 nM). (d) HGF protein concentrations measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatant of monocytes derived-macrophages or intestinal macrophages (IMACs). HGF concentrations were determined at 72 h of co-culture with wounded epithelial cells in DMEM 0·1% FCS. (e) Wounded Caco2 cells were seeded in DMEM 0·5% FCS with 105 macrophages isolated from human intestinal mucosa (IMACs). In negative (–) and positive (+) controls wounded Caco2 cells were seeded with DMEM 0·5% FCS or DMEM 10% FCS, respectively. (f) c-Met inhibitor inhibits IMACs-assisted repair. Wounded Caco2 cells were seeded in DMEM 0·5% FCS with 105 IMACs in the presence or not of PHA-665732 (600 nM). In negative (–) and positive (+) controls wounded Caco2 cells were seeded with DMEM 0·5% FCS or DMEM 10% FCS, respectively. Each dot represents a healthy donor. Comparison between several groups to identify a trend was performed by a one-way
anova
test with Trend test as post-test.
Fig. 4
Macrophages from Crohn's disease (CD) patients are unable to promote wound repair. (a) Wounded Caco2 cells were seeded with Dulbecco's modified Eagle's medium (DMEM) 0·5% fetal calf serum (FCS)with 105 macrophages from healthy donors (HD, n = 14), Crohn's disease patients (CD, n = 22, 11 in remission and 11 in active phase of the disease) or ulcerative colitis patients (UC, n = 10, seven in remission and three in the active phase of the disease). In negative (–) and positive (+) controls wounded Caco2 cells were seeded with DMEM 0·5% FCS or DMEM 10% FCS, respectively. (b) Wounded Caco2 cells were seeded with DMEM 0·5% FCS with 105 macrophages of healthy donors (HD, n = 14), of Crohn's disease patients with disease in remission (RCD, n = 11) or with active disease (ACD, n = 11). (c) hepatocyte growth factor (HGF) protein concentrations measured by enzyme-linked immunosorbent assay (ELISA) and expressed as pg/mg of total protein in the culture supernatant of wounded Caco2 cells seeded with 105 macrophages from healthy donors (HD, n = 9), Crohn's disease (CD, n = 13) or ulcerative colitis (UC, n = 4) patients. HGF concentrations were determined at 72 h of culture. 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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