| Abstract |
Introduction
Intestinal subepithelial myofibroblasts (SEMFs) are alpha-smooth muscle actin-positive mesenchymal cells located at the interface between the intestinal epithelium and lamina propria. Connective tissue fibrils form a distinct connective tissue barrier called the basal lamina, through which SEMFs and the epithelium can interact through soluble factors or pseudopods.
Early evidence suggested that SEMFs participated in wound healing and subsequent intestinal tissue remodelling, including fibrosis. Mucosa overlying Crohn’s Disease (CD) strictures overexpress profibrotic TGF-β transcripts and tissue inhibitor of metalloproteinases-1 (TIMP-1) proteins, while resident mesenchymal cells overproduce collagen I and III. Early immunohistochemistry studies showed that, beyond lamina propria inflammatory cells, intestinal epithelial cells also exhibit intense immunoreactivity for TGF-β1 during CD-associated inflammation. Growth factors, including TGF-β, accelerated migration of intestinal lamina propria myofibroblasts but its production was not dependent on autocrine loops. Therefore it seems that a crosstalk between SEMFs and other cell types is essential in the wound-healing process and neighbouring epithelial cells are putative candidates.
MMPs have been implicated in tissue remodelling and ulceration in inflammatory bowel disease (IBD) and many matrix metalloproteinases (MMPs) are overexpressed in inflamed
IBD human tissues and animal IBD models. The importance of MMP-9 in inflammatory bowel ulceration was confirmed in studies of dextran sodium sulphate colitis where the extent and severity of intestinal epithelial injury were significantly attenuated in MMP-9-deficient mice. Initial immunohistochemistry studies identified neutrophils as the sole source of MMP-9 in inflamed bowels, despite the localisation of MMP-9 immunoreactivity in the connective tissue adjacent to the crypts, which suggests the possible involvement of SEMFs.
Epithelial cells have been proposed to control the aforementioned processes. Supporting evidence originated from observations in other tissues, such as the skin and the lungs. In the skin, keratinocytes are partially responsible for the induction of α-SMA in fibroblasts via TGF-β and endothelin-1 (ET-1). Interactions between keratinocytes and the underlying fibroblasts also seem to modulate the levels of two key enzymes involved in extracellular matrix remodelling: MMP-2 and MMP-9. In the lungs, under continuous compressive stress, epithelial cells upregulate gene expression and secretion of ET and TGF-β2. Conditioned media from mechanically stressed epithelial cells induce the incorporation of proline into matrix proteins (mostly collagen) by unstressed normal human lung fibroblasts via pathways that involve ET and TGF-β2.
Findings
ELISA on untreated HT-29 and CaCO-2 colonic epithelial cell line supernatants of 24 h showed substantial amounts of TGF-β1. The addition of the pro-inflammatory cytokines IL-1α, TNF-α and IFN-γ into the cell culture media significantly increased the production of TGF-β1 with IL-1α being the most potent stimulus. Combinations of cytokines had an
additive impact, with TGF-β production being highest when colonic epithelial cells were treated with all three cytokines (3C). The other two TGF-β isoforms (TGF-β2 and TGF-β3) were also secreted by non-stimulated colonic epithelial cells in considerable amounts and upregulated pro-inflammatory cytokines in a similar manner. Primary SEMFs and the 18CO cell line secreted very small quantities of TGF-β1 and TGF-β3 after 24 h in culture and were not responsive to IL-1α, TNF-α or IFN-γ, added in isolation or in every possible combination. We were unable to detect any TGF-β2 production in primary SEMF or 18CO cultures, even after stimulation with a cocktail of all three pro-inflammatory cytokines.
Furthermore, we found that both colonic epithelial cell lines secreted substantial amounts of TIMP-1- another mediator of fibrosis- into the cell culture supernatant at 24h. Treatment with TNF-α or IFN-γ, but not IL-1α, significantly increased TIMP-1 secretion in both cell lines. Treating with two or all three cytokines together also resulted in a further increase in TIMP-1 production. Primary SEMFs and 18CO cells also exhibited basal TIMP-1 secretion, but, surprisingly, it was unresponsive to stimulation with IL-1α, TNF-α and IFN-γ, in isolation or in combination at 24h, which was similar to TGF-β production.
Moreover, we examined the presence of gelatinases MMP-2 and MMP-9 in the cell culture supernatant of colonic SEMFs. They were detected with gelatin zymography as gelatinolytic bands and discriminated by their molecular weights (MMP-2: 70 kDa, MMP-9: 100 kDa). Primary SEMFs and 18CO cells secreted MMP-2 into the cell culture supernatant without any additional stimulus after 24h in culture but this was not true for MMP-9. Stimulation of primary SEMFs and 18CO cells with TNF-α or IL-1α for 24h induced MMP-9 activity in their supernatants and combination of both had an additive effect. IFN-γ had no effect on its own, but when added to TNF-α, IL-1α or both it suppressed MMP-9 activity in
SEMF supernatants. Therefore, the combination of all three pro-inflammatory cytokines (IL-1α+TNF-α+IFN-γ) did not significantly induce MMP-9 activity. Furthermore, those cytokines in isolation or in every possible combination had no effect on MMP-2 activity. Colonic epithelial cell lines showed no gelatinolytic activity despite stimulation with IL-1α, TNF-α, IFN-γ, in isolation or in combination.
We subsequently explored the effect of epithelial cell-conditioned media on SEMF cultures. Epithelial cell-conditioned (ECC) media from CaCO-2 cultures pretreated for 6h with IL-1α, TNF-α, IFN-γ, alone or in combination, induced substantial amounts of MMP-9 activity in SEMFs and 18CO cells, but at the absence of those cytokines acting directly on myofibroblasts. Similar results were obtained with the HT-29 cell line. Therefore, MMP-9 production by myofibroblasts was triggered by a soluble mediator derived from cytokine-treated epithelial cells. Results were identical when SEMFs from inflammatory non-fibrotic CD colonic tissue segments were utilised.
We next examined if TGF-β was responsible for the induction of MMP-9 activity in SEMFs treated with ECC medium. This hypothesis was based on the observation that exogenous TGF-β1 can induce MMP-9 activity in primary SEMF cultures in a dose-dependent manner. However, neutralisation of all TGF-β isoforms did not significantly affect MMP-9 production by SEMF cultures stimulated with ECC media. Results were similar if CaCO-2 ECC media or CD SEMFs 18CO cells were used.
In the lungs, production of ΕΤ-1 by stimulated bronchial epithelial cells was suspected to induce MMP-9 activity in co-cultured skin fibroblasts. Therefore, we assayed whether MMP-9 upregulation was dependent on endothelin. We demonstrated that specific endothelin
receptor A (ETR-A) inhibition in SEMF cultures blocked the induction of MMP-9 by ECC media. In contrast, specific inhibition of endothelin receptor B (ETR-B) had no effect on induced MMP-9 production. Furthermore, there was no additional effect on MMP-9 inhibition when both ETR-A and ETR-B were blocked. This phenomenon could be reproduced on CD SEMFs.
We further explored possible cooperation between epithelial cells and myofibroblasts in other aspects of connective tissue remodelling, such as collagen production. First, we assessed collagen production by myofibroblasts treated in the absence or presence of pro-inflammatory cytokines. The Sircol assay was used to measure the majority of collagen types cumulatively. SEMFs and 18CO cells secreted minimal amounts of total collagen in cell culture supernatants in preliminary experiments, so we studied total collagen production by measuring total intracellular collagen. IL-1α, TNF-α and IFN-γ treatment alone, in pairs or 3C did not have a significant effect on collagen synthesis at 24 h. Interestingly, stimulation with TGF-β1, a profibrotic mediator associated with intestinal fibrosis produced a minimal, non-significant increase in collagen production at 24h. However, we found that CD SEMFs inherently produced more collagen ex vivo compared to those isolated from control group.
We next proceeded to study the effect of ECC media on collagen production in myofibroblasts. ECC media significantly increased collagen production in primary SEMFs. The observed effect was moderate when we used conditioned medium of untreated epithelial cells. In contrast, when we used ECC media of epithelial cells pretreated with 3C, we observed a pronounced increase of collagen production. Similar but less pronounced effects were found when 18CO cells were used as the responding myofibroblasts.
We attempted to identify the epithelial cell-derived factors responsible for the increase in collagen synthesis by SEMFs. TGF-β was produced by epithelial cells and upregulated by 3C; therefore, it was a probable candidate. However, neutralisation of all three forms of TGF-β in the ECC media did not affect the induction of collagen production by control or CD SEMFs. Therefore, TGF-β was not a prerequisite messenger for the induction of collagen synthesis induction by ECC media in treated SEMFs. Previous studies demonstrated that an interaction between TF and activated FVII could induce pro-fibrotic CTGF in human keratinocytes. In addition, SEMFs have been shown to upregulate collagen production in an autocrine manner via the secretion of CTGF and TF. Here, we demonstrated the production of CTGF by primary SEMF cultures following treatment with ECC media, especially if colonic epithelial cells were treated with pro-inflammatory cytokines. CTGF expression is dependent on the presence of thrombin and TF, and is abolished by anti-thrombin-III treatment. TF production and subsequent collagen production is dependent on ET-1 receptor signalling. Thus, we explored the hypothesis that CTGF, TF or thrombin regulate pro-fibrotic signalling in our two-cell system of intestinal fibrosis. However, neutralisation of CTGF or adding antithrombin-III did not block of collagen production by ECC stimulated control or CD SEMFs. The same stood true for neutralization TF in similarly treated control SEMFs. In addition endothelin receptor antagonism had no effect on the induction of collagen production on control or CD SEMFs.
SEMF mobility/ migration was assessed with the wound-healing scratch assay and quantified as the percentage of wound repair in vitro. Addition of exogenous TGF-β1 in primary SEMF cultures was found to promote SEMF mobility/migration in a dose-dependent manner. In contrast TNF-α or IFN-γ reduced wound healing. In combination, these cytokines
exerted an additive effect. IL-1α did not alter migration either when added alone or when used in combination with TNF-α or IFN-γ.
To study the potential impact of epithelial cells on this process, we used ECC media to stimulate primary SEMFs. ECC media derived from non-stimulated CaCO-2 cells had no effect on wound healing. Alternatively, SEMF mobility/migration was reduced if CaCO-2 were pretreated for 6 h with IL-1α, TNF-α or IFN-γ, and subsequently wound closure was significantly slowed. Stimulating the epithelial cell cultures with all three cytokines resulted in stronger inhibition of wound closure despite the absence of the initial pro-inflammatory stimuli. Similar results were obtained if HT-29 ECC was used for SEMF treatment. Thus, it appears that pro-inflammatory stimuli could inhibit the migratory potential of SEMFs both directly and indirectly through epithelial cell-derived soluble factors. Moreover, the effect of intestinal epithelial cells on wound healing by adjacent SEMFs differed from that predicted for TGF-β induction; i.e., the net effect of crosstalk might involve other factors.
Conclusions
This study provides indications of potential crosstalk between intestinal epithelial cells and SEMFs. In vitro stimulation of epithelial cells with pro-inflammatory cytokines, which partially mimics epithelial cell injury during intestinal inflammation, provoked changes in SEMFs similar to those observed during intestinal fibrosis. These changes included the upregulation of collagen, endothelin receptor A-dependent induction of MMP-9 and inhibition of migration. Our results support the necessity for further investigation and in vivo validation of cooperation between epithelial cells and adjacent myofibroblasts in the regulation of extracellular matrix remodelling and inflammation-induced intestinal fibrosis.
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