Normal human colonic subepithelial myofibroblasts enhance epithelial migration (restitution) via TGF-beta 3

Brian C. McKaig1, Shavcharn S. Makh1, Christopher J. Hawkey1, Daniel K. Podolsky2, and Yashwant R. Mahida1

1 Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, United Kingdom; and 2 Gastrointestinal Unit, Massachusetts General Hospital, Boston, Massachusetts 02114-2696


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

-After injury and loss of epithelial cells, intestinal barrier function is reestablished by migration of viable epithelial cells from the wound edge (restitution). Myofibroblasts are located close to the basal surface of epithelial cells. This study aimed to investigate the role of human colonic subepithelial myofibroblasts in epithelial restitution. Primary cultures of subepithelial myofibroblasts were established. Monolayers of the epithelial cell lines IEC-6 and T84 were "wounded" in a standard manner to create an in vitro model of restitution. Migration of epithelial cells across the wound edge was assessed following culture in myofibroblast-conditioned medium. Myofibroblast expression of transforming growth factor (TGF)-beta isoforms was examined using RT-PCR, and TGF-beta isoform bioactivity was assessed using Mv 1 Lu bioassay. Myofibroblast-conditioned medium, via a TGF-beta -dependent pathway, significantly enhanced migration of epithelial cells across the wound edge and significantly inhibited cell proliferation in wounded monolayers. Messenger RNA for TGF-beta 1, -beta 2, and -beta 3 was detected in the myofibroblasts, and Mv 1 Lu bioassay showed the presence of predominantly bioactive TGF-beta 3. This study shows that human colonic subepithelial myofibroblasts secrete predominantly bioactive TGF-beta 3 and enhance restitution in wounded epithelial monolayers via a TGF-beta -dependent pathway.

transforming growth factor-beta ; wound repair


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GASTROINTESTINAL TRACT performs the critical functions of extraction of nutrients, minerals, and electrolytes but excludes luminal microorganisms and toxic molecules. These essential functions are mediated by a monolayer of epithelial cells that lines the intestinal lumen. The epithelial monolayer provides a barrier to luminal toxic molecules and microorganisms via intercellular tight junctions. This barrier is disrupted following injury and loss of epithelial cells, directly exposing the lamina propria cells to luminal contents. In the normal intestinal mucosa, epithelial continuity and barrier function are rapidly reestablished following the loss of injured epithelial cells. This occurs via a process designated restitution, in which viable epithelial cells migrate from the wound edge to reestablish epithelial continuity (15, 16, 21, 29, 31, 37, 43, 47). This process can be complete within minutes to hours, depending on the extent of epithelial injury. Cell proliferation over the subsequent 24-48 h allows the replacement of the lost epithelial cells. There is increasing appreciation for the fact that epithelial restitution in vivo involves complex interaction between epithelial cells, the underlying basement membrane, as well as cells within the lamina propria matrix. The cells in the lamina propria may interact with the epithelial cells via pores in the basement membrane (26).

Intestinal subepithelial myofibroblasts are present immediately subjacent to the basement membrane and close to the basal surface of epithelial cells. Ultrastructural studies have shown that these cells share characteristics of both fibroblasts (7, 14, 19) and smooth muscle cells and have therefore been designated myofibroblasts (18, 20, 36, 38). Their location below the basement membrane suggests that these cells may play an important role in the regulation of a number of epithelial cell functions (45). We have recently established an ex vivo model that allows, for the first time, pure populations of subepithelial myofibroblasts from adult human intestinal mucosa to be available (24). The isolated myofibroblasts retain a representative and differentiated phenotype despite prolonged culture and passage (24). Myofibroblasts have been shown to have an important role in wound healing and fibrosis (10), but there have been no studies to date investigating the role of human intestinal myofibroblasts in the initial wound healing process of restitution.

In this study, we have investigated the role of isolated normal primary adult human colonic subepithelial myofibroblasts in the regulation of epithelial restitution. We demonstrate that the myofibroblasts enhance epithelial cell migration via secreted bioactive transforming growth factor (TGF)-beta and that the predominant bioactive isoform is TGF-beta 3.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The nontransformed rat small intestinal epithelial cell line IEC-6 was obtained from the European Collection of Animal Cell Cultures (ECACC; Porton Down, UK) and studied at passages 26-31. The cells were maintained in DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 5% FCS (GIBCO), 2 mM glutamine (Sigma Chemical, St. Louis, MO), 100 U/ml penicillin (Britannia Pharmaceuticals, Surrey, UK), 0.1 mg/ml streptomycin (Evans Medical, Surrey, UK), and insulin (final concentration 4 µg/ml; Sigma).

The human colon cancer cell line T84 was obtained from the ECACC and studied at passages 70-75. The cells were maintained in DMEM-Ham's F-12 medium (GIBCO) supplemented with 10% FCS (GIBCO), 2 mM glutamine (Sigma), 100 U/ml penicillin (Britannia Pharmaceuticals), and 0.1 mg/ml streptomycin (Evans Medical).

Human colonic subepithelial myofibroblasts were isolated from normal colonic mucosal samples obtained from six colonic resection specimens. The normal colonic mucosal samples were obtained >5 cm from the tumor, and myofibroblasts were isolated as previously described (24). In brief, the mucosal samples were completely denuded of epithelial cells by three 30-min periods of incubation (at 37°C) in 1 mmol/l EDTA (Sigma). The deepithelialized mucosal samples were subsequently cultured (at 37°C in 5% CO2) in RPMI 1640 (GIBCO) containing 10% FCS. The cells in suspension were removed after every 24- to 72-h culture period, and the denuded tissue was maintained in culture for up to 6 wk. Established colonies of myofibroblasts were cultured in DMEM supplemented with 10% FCS and 1% nonessential amino acids (GIBCO), penicillin (100 U/ml), and streptomycin (0.1 mg/ml). The cells were passaged using 0.1% (wt/vol) trypsin-0.2% (vol/wt) EDTA in a 1:2 to 1:3 split ratio. Studies were carried out at passages 2-7 of myofibroblasts isolated from six resection specimens.

For myofibroblast-conditioned medium, subconfluent and confluent monolayers of cells were washed with DMEM and subsequently cultured in 0.1% FCS-DMEM for 24 h. The conditioned medium was stored at -70°C until use in studies on wounded epithelial monolayers and TGF-beta bioassay (see below).

Wounding assays. Wound assays were performed in multiples of six, using a previously described method (27) with modification. Confluent monolayers of IEC-6 and T84 cells in six-well tissue culture plates (Nunc, GIBCO) were wounded under microscopic vision using a razor blade and a Gilson "p2" pipette tip. Cells were washed three times with fresh serum-deprived medium (0.1% FCS-DMEM), and the wounded monolayers were further cultured in fresh serum-deprived medium in the presence or absence of recombinant human TGF-beta 1 (5 ng/ml; R&D Systems, Minneapolis, MN) or myofibroblast-conditioned medium.

Wound assays were also performed in the presence of polyclonal neutralizing antibody to recombinant TGF (rTGF)-beta 1, -beta 2, and -beta 3 (1 µg/ml; R&D Systems).

As previously described, migration of IEC-6 cells was assessed in a blinded fashion by the determination of the mean number of cells found across the wound border in a standardized wound area (12), and migration of T84 cells was assessed in a blinded fashion by determination of the reduction in wound width over 24 h (34). Wound areas were standardized by taking photographs at 100-fold magnification using an Olympus CK-2 inverted microscope and an Olympus OM-1 camera. Experiments were performed in quadruplicate, and data are presented as means ± SD.

Cell proliferation in wounded epithelial monolayers. "Wounded" monolayers of IEC-6 cells in six-well plates were incubated as described in wounding assays. After culture for 20 h, [3H]thymidine (Amersham International, Buckinghamshire, UK) was added to each well (1 µCi/well). After a further incubation of 4 h, the cells were subsequently fixed with methanol-acetic acid (vol/vol, 3:1) at room temperature for 1 h, washed twice with 80% methanol, and lysed with 1 M NaOH. Uptake of [3H]thymidine was determined using an LKB (Wallac, Milton Keynes, UK) beta counter.

Proliferation was also assessed by determination of bromodeoxyuridine (BrdU) uptake, as reflected in the mean number of BrdU-positive cells observed per low-power field as previously described (4). Briefly, BrdU (final concentration of 10 µmol/l; Sigma) was added to the wounded monolayer. BrdU uptake was assessed after culture for 24 h in either serum-deprived medium or myofibroblast-conditioned medium by addition of mouse IgG anti-BrdU antibody (final concentration of 200 mU/ml; Sigma) and detected using a universal Vectastain ABC detection kit (Vector Laboratories, Peterborough, UK).

TGF-beta bioassay. The presence of bioactive TGF-beta in myofibroblast-conditioned medium was determined using a specific bioassay, which is based on the ability of TGF-beta to inhibit proliferation of the mink lung epithelial cell line Mv 1 Lu (ECACC) (25). Latent TGF-beta present in the myofibroblast-conditioned medium was activated by the addition of concentrated HCl to pH 2 and left to stand at room temperature for 60 min, followed by neutralization with NaOH and HEPES (to a final concentration of 16 mmol/l).

Mv 1 Lu cells (in 0.2% FCS-DMEM) were seeded at 5 × 104 cells/well in 24-well cell culture plates (Nunc, GIBCO). Four hours after cells were seeded, acid-treated and untreated myofibroblast-conditioned medium and standardized concentrations of rTGF-beta 1 were added and incubated for 20 h at 37°C (95% O2, 5% CO2). After 20 h, [3H]thymidine (1 µCi/well) was added, and the incubation was continued for an additional 4 h. The cells were then fixed with methanol-acetic acid (3:1), washed twice with 80% methanol, and lysed with 1 M NaOH. Uptake of [3H]thymidine was determined as before. From the standard curve obtained, the concentration of total and biologically active TGF-beta present in the myofibroblast-conditioned medium could be calculated.

The contribution of each TGF-beta isoform to total bioactivity was assessed using Mv 1 Lu bioassay performed in the presence of TGF-beta isoform-specific monoclonal antibodies (1 µg/ml; R&D Systems).

Conditioned medium from confluent and wounded IEC-6 and T84 monolayers was also assessed for TGF-beta bioactivity.

RNA isolation and reverse transcription. RNA was isolated from myofibroblasts using RNAzolB (Biogenesis, Poole, UK). Random hexamer primer (Pharmacia Biotech, Brussels, Belgium) was mixed with 10 µg RNA (final volume of 37.5 µl) and heated to 70°C for 10 min and allowed to cool on ice. Reverse transcription to cDNA was performed by adding the following and incubating at 37°C for 60 min: 5 µl of 10× PCR buffer [0.5 M Tris (pH 8.3), 0.75 M KCl, and 30 mM MgCl2 (Stratagene, La Jolla, CA)], 1.5 µl of 5 mM 2'-deoxyribonucleotide 5'-triphosphate mix (containing dATP, dCTP, dGTP, and dTTP each at 25 mM; Ultrapure dNTP set, Pharmacia), 1 µl of Moloney murine leukemia virus RT (200 U/µl; GIBCO), and 5 µl of 0.1 M DTT. Subsequent enzyme deactivation was performed by heating to 90°C for 5 min, and the cDNA was stored at -20°C.

Polymerase chain reaction. The following reaction mixture was added to 5 µl of the cDNA product: 5 µl of enzyme buffer [0.5 mM KCl, 0.1 M Tris · HCl (pH 9.0), and 1% Triton X-100 (Promega, Madison, WI)], 2 µl of 5 mM dNTPs, 0.5 µl of Taq DNA polymerase (5 U/µl; Promega), and sterile water to make a final solution of 50 µl. The following primer pairs were used (to a final concentration of 5 µM) based on published nucleotide sequences: 5'-CAG AAA TAC AGC AAC AAT TCC TGG-3' (sense) and 5'-TTG CAG TGT GTT ATC CGT GCT GTC-3' (antisense) to amplify 187-bp human TGF-beta 1 product (8); 5'-TCC AAA GAT TTA ACA TCT CCA ACC-3' (sense) and 5'-TCC CAC TGT TTT TTT TCC TAG TGG-3' (antisense) to amplify 310-bp human TGF-beta 2 product (23); 5'-ACA TTT CTT TCT TGC TGG-3' (sense) and 5'-GGG GAA GAA CCC ATA ATG-3' (antisense) to amplify 685-bp human TGF-beta 3 product (9); and 5'-GGT GAA GGT CGG AGT CAA CGG A-3' (sense) and 5'-GAG GGA TCT CGC TCC TGG AAG A-3' (antisense) to amplify 240-bp human glyceraldehyde-6-phosphate dehydrogenase product.

Amplification was performed using a Trio-Thermoblock (Biometra). PCR cycles consisted of denaturation for 45 s at 95°C, annealing at 54°C for 90 s, and extension at 72°C for 90 s. A total of 30 cycles were used and completed by extension for 2 min at 54°C followed by 3 min at 72°C.

PCR products were analyzed by adding 1 µl of ethidium bromide (10 mg/ml) and 5 µl of gel-loading buffer (Sigma Chemical) to 15 µl PCR product and electrophoresis on a 2% agarose gel. The specificity of RT-PCR for TGF-beta 1, -beta 2, and -beta 3 was confirmed by hybridization of the PCR product using specific digoxigenin-labeled internal oligonucleotide probes and subsequent detection using alkaline phosphatase-labeled rabbit anti-digoxigenin antibody (28) and by DNA sequence analysis. The following digoxigenin-labeled probes were used: 5'-GTT GTG CGG CAG TGG-3' (to identify TGF-beta 1 product), 5'-GAG CAG AAG GCG AAT-3' (to identify TGF-beta 2 product), and 5'-GGG AGA AGG AAG GGC-3' (to identify TGF-beta 3 product).

Statistical analysis. Results are expressed as means ± SD. Statistical analyses were performed using one-way ANOVA and Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myofibroblast-conditioned medium enhances epithelial migration in wounded IEC-6 and T84 monolayers. The results illustrated in Table 1 and Figs. 1 and 2 show that conditioned medium of myofibroblast cultures derived from mucosal samples of six different colons consistently enhanced repair of wounded IEC-6 and T84 monolayers. As expected (4, 12) and shown in Table 1, rTGF-beta 1 also enhanced wound repair.

                              
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Table 1.   Repair of wounded IEC-6 monolayers



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Fig. 1.   Anti-transforming growth factor (TGF)-beta antibody (Ab) significantly inhibits myofibroblast-conditioned medium (MFCM)-mediated enhancement of epithelial restitution in wounded IEC-6 (A) and T84 (B) monolayers. "Wounded" monolayers were cultured in control medium (0.1%), MFCM, or recombinant TGF (rTGF)-beta in the presence or absence of neutralizing antibody to TGF-beta . For both cell lines, in the presence of anti-TGF-beta antibody, there was a significant (* P < 0.01 for IEC-6 and * P = 0.02 for T84 vs. MFCM alone) reduction in wound repair. MFCM and rTGF-beta significantly enhanced cell migration compared with control medium (# P < 0.01 vs. control medium).



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Fig. 2.   Acid-treated (acid Rx) and untreated (UnRx) MFCM enhance epithelial cell migration in wounded IEC-6 monolayers to the same extent (P < 0.01), confirming that in untreated MFCM majority of TGF-beta is in the bioactive form.

Repair of wounded epithelial monolayers may occur as a consequence of cell migration (restitution) or proliferation. To investigate the mechanism by which myofibroblast-conditioned medium enhanced epithelial wound repair, studies were performed using [3H]thymidine incorporation and BrdU uptake as indexes of cell proliferation. As demonstrated in Table 2, myofibroblast-conditioned medium consistently inhibited cell proliferation in the wounded IEC-6 monolayers. This was also the case with rTGF-beta 1 (data not shown). In wounded monolayers cultured in control medium, scattered BrdU-labeled epithelial cells were seen in the remaining monolayer, as previously described (5). In wounded monolayers cultured in myofibroblast-conditioned medium or rTGF-beta 1, there was a significant reduction in the number of BrdU-labeled cells (data not shown).

                              
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Table 2.   [3H]thymidine uptake in wounded IEC-6 monolayers

Myofibroblasts enhance epithelial migration via secreted TGF-beta . The similarity with rTGF-beta 1 in the effect on cell migration and proliferation in wounded epithelial monolayers suggested that the myofibroblast-conditioned medium contained bioactive TGF-beta . This was confirmed by inhibition of myofibroblast-conditioned medium-mediated epithelial migration by polyclonal neutralizing antibody to TGF-beta (Fig. 1A). The myofibroblast-mediated enhanced restitution in wounded T84 cells was also shown to be partially dependent on bioactive TGF-beta (Fig. 1B). Addition of neutralizing antibody alone to wounded IEC-6 monolayers (cultured in control medium) did not significantly inhibit cell migration (data not shown).

The presence of bioactive TGF-beta in myofibroblast-conditioned medium was confirmed using the Mv 1 Lu bioassay (Table 3). After acidification, there was only a small increase in TGF-beta bioactivity detected in the conditioned medium, implying that the majority of TGF-beta secreted by the myofibroblasts is in the biologically active form. The predominance of bioactive TGF-beta in myofibroblast-conditioned medium was confirmed by lack of a difference between acid-treated and untreated samples in the enhancement of restitution in wounded IEC-6 monolayers (Fig. 2). TGF-beta bioactivity present in conditioned medium of IEC-6 and T84 epithelial monolayers was negligible in comparison to that present in myofibroblast-conditioned medium. After acid treatment, the TGF-beta concentration of IEC-6- and T84-conditioned medium was 60 and 6 pg/ml, respectively (TGF-beta concentration range in myofibroblast-conditioned medium was 600-1,500 pg/ml). There was no significant difference in total TGF-beta bioactivity in conditioned medium collected from confluent vs. subconfluent myofibroblast monolayers or from wounded vs. confluent IEC-6 and T84 monolayers (data not shown).

                              
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Table 3.   Assessment of TGF-beta bioactivity in conditioned medium using Mv 1 Lu bioassay

The contribution of each TGF-beta isoform to total bioactivity was assessed by the use of monoclonal isoform-specific neutralizing antibodies. As shown in Fig. 3, TGF-beta 3 was the predominant bioactive isoform secreted by the myofibroblasts. Neutralizing antibodies to TGF-beta 1 and -beta 2 reduced the overall percentage of TGF-beta bioactivity by 16.2 ± 6.7% and 3.2 ± 3.8%, respectively. In contrast, anti-TGF-beta 3 antibody reduced total bioactivity by 81.0 ± 17.6%.


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Fig. 3.   Relative TGF-beta isoform bioactivity in MFCM. Neutralizing antibodies (Ab) to TGF-beta 1 or -beta 2 did not significantly reduce overall TGF-beta bioactivity. In contrast, neutralizing antibody to TGF-beta 3 significantly reduces bioactivity by 81.0 ± 17.6% (n = 5, * P < 0.05).

Studies using RT-PCR demonstrated that mRNA transcripts for TGF-beta 1, -beta 2, and -beta 3 are expressed by myofibroblasts (Fig. 4). These PCR products were confirmed to be those of the individual isoforms of TGF-beta by hybridization using digoxigenin-labeled internal oligonucleotide probes and by DNA sequencing as described in MATERIALS AND METHODS.


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Fig. 4.   Expression of mRNA transcripts for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TGF-beta 1, -beta 2, and -beta 3 (lanes 1-4, respectively) in human colonic myofibroblasts. RNA was isolated from a confluent monolayer of cells and reverse transcribed, and relevant transcripts were amplified by PCR using designed primers and electrophoresed on a 2% agarose gel. DNA size markers are indicated on left. TGF-beta PCR products were isolated and gel purified. Specificity of the TGF-beta PCR products was determined as described in MATERIALS AND METHODS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the intestine, the surface monolayer of epithelial cells plays a critical role in the maintenance of mucosal integrity, providing an essential barrier to penetration by luminal microorganisms and their products. Injury to the epithelial cells results in a loss of monolayer continuity and barrier function and allows penetration by luminal contents into the lamina propria. It is therefore of importance to the host to repair any such breach in the barrier to avoid or limit an inflammatory response. Epithelial continuity and barrier function is normally rapidly restored by migration of viable cells adjacent to the wound edge to cover the mucosal defect, a process termed restitution (15, 16, 21, 29, 31, 37, 43, 47). This process is independent of proliferation and begins within minutes of the initial injury. Proliferation of epithelial cells, required to make up for the lost cells, occurs over 24-48 h after the injury (16). Previous studies of epithelial cell restitution have shown that a large number of peptide growth factors (interleukin-1beta , interferon-gamma , epidermal growth factor, TGF-alpha , hepatocyte growth factor, basic fibroblast growth factor, intestinal trefoil factor) can influence this process (11-13, 32). However, TGF-beta plays a central role in mediating the effects of many of these peptide growth factors (12).

It is recognized that the process of restitution in vivo involves complex interactions between epithelial cells, cells within the lamina propria, and components of the extracellular matrix. Previous studies have shown that components of the extracellular matrix can regulate epithelial restitution (2, 3, 17); however, there is little information on the role of individual cell types within the lamina propria in the regulation of epithelial restitution. In this study, we have examined the role of primary adult human colonic subepithelial myofibroblasts in epithelial cell migration. Cultures of subepithelial myofibroblasts were established as recently described (24). During culture of mucosal samples denuded of epithelial cells, myofibroblasts migrate out of the lamina propria via pores in the basement membrane. These cells subsequently become adherent to the culture dish and proliferate to form monolayers of myofibroblasts. We have previously shown that, despite prolonged culture and passage, the myofibroblasts retain a representative and differentiated phenotype (24). Myofibroblast cultures used in this study were established as previously reported, and their phenotypes were confirmed by demonstration of the expression of alpha -smooth muscle actin and vimentin (data not shown). The use of the IEC-6 and T84 cell lines to study restitution in vitro is well recognized. Extensive studies have shown the similarity between the untransformed small intestinal rat epithelial IEC-6 cell line and the normal rat crypt intestinal epithelial cell (35). These cell lines allow the study of epithelial cell responses without the ambiguity of contamination inherent to studies with preparations of primary epithelial cells.

The studies in this report show that myofibroblasts secrete a factor or factors that enhance cell migration in wounded epithelial monolayers while they paradoxically inhibit epithelial cell proliferation, suggesting that this effect was mediated by TGF-beta . This was confirmed by the addition of neutralizing antibody to TGF-beta , which abrogated the enhanced epithelial cell migration seen in response to the myofibroblast-conditioned medium. This response was more pronounced in the nontransformed IEC-6 cell line compared with the transformed T84 cell line. This could be explained by the relative resistance of transformed cell lines to the effect of TGF-beta , one of the postulated mechanisms for the development of colonic carcinoma.

Previous reports have shown that cells can produce latent TGF-beta as well as plasminlike protease activity required to effect bioactivation through liberation of the mature TGF-beta dimer (39, 40, 44). We have shown that myofibroblasts themselves express mRNA transcripts for all three human TGF-beta isoforms (TGF-beta 1, -beta 2, and -beta 3). The secreted TGF-beta is biologically active, suggesting that the myofibroblasts also produce plasminlike protease activity. This finding differs from that of TGF-beta secretion by platelets (33) or other cell types (6, 22, 46). In previous studies, TGF-beta mRNA in the normal colon has been shown to be expressed predominantly in the lamina propria closest to the surface epithelium (1). From our current studies, we postulate that TGF-beta derived from myofibroblasts may play an important role in epithelial restitution in vivo. The myofibroblasts lie close to the epithelium, and the secreted bioactive TGF-beta could reach the epithelial cells both by passive diffusion and via pores in the basement membrane (26). It is also possible that there are other myofibroblast-derived factors that enhance epithelial restitution by inducing secretion of TGF-beta by the epithelial cells (4, 5). In addition, the subepithelial network of myofibroblasts may facilitate repair of the injured intestinal epithelium by contraction, thereby reducing the denuded surface area to be reepithelialized (30).

It is recognized that myofibroblasts also participate in the formation of the extracellular matrix and granulation tissue leading to fibrosis during repair (10), processes that appear to be influenced to a large extent by the TGF-beta superfamily. This superfamily is one of the most complex groups of cytokines, with widespread effects on many aspects of growth, development, and wound healing. The three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, and -beta 3) have been localized in healing wounds, and there is evidence that manipulation of the ratios of the TGF-beta isoforms may influence the latter stages of the repair process, namely scarring and fibrosis (41). Thus recent studies maintain that either neutralization of TGF-beta 1 and -beta 2 or exogenous addition of TGF-beta 3 reduces scarring in cutaneous rat wounds (42). In our study, we have shown for the first time that primary cultures of normal human colonic subepithelial myofibroblasts release predominantly bioactive TGF-beta 3. Such predominant expression of bioactive TGF-beta 3 in vivo would imply a capacity for wound healing without fibrosis. Myofibroblasts may therefore play a pivotal role in regulating several stages of wound repair from restitution to end-stage fibrosis.


    ACKNOWLEDGEMENTS

This work was supported by the Digestive Disorders Foundation (UK) and The National Association for Colitis and Crohn's Disease (UK).


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Y. R. Mahida, Division of Gastroenterology, Univ. Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK.

Received 25 September 1998; accepted in final form 4 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Babyatsky, M. W., G. Rossiter, and D. K. Podolsky. Expression of transforming growth factors alpha  and beta  in colonic mucosa in inflammatory bowel disease. Gastroenterology 110: 975-984, 1996[Medline].

2.   Basson, M. D., I. M. Modlin, S. D. Flynn, B. P. Jena, and J. A. Madri. Independent modulation of enterocyte migration and proliferation by growth factors, matrix proteins, and pharmacological agents in an in vitro model of mucosal healing. Surgery 112: 299-308, 1992[Medline].

3.   Basson, M. D., I. M. Modlin, and J. A. Madri. Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J. Clin. Invest. 90: 15-23, 1992[Medline].

4.   Ciacci, C., S. E. Lind, and D. K. Podolsky. Transforming growth factor beta  regulation of migration in wounded intestinal epithelial monolayers. Gastroenterology 105: 93-101, 1993[Medline].

5.   Ciacci, C., Y. R. Mahida, A. U. Dignass, M. Koizumi, and D. K. Podolsky. Functional interleukin-2 receptors on intestinal epithelial cells. J. Clin. Invest. 92: 527-532, 1993[Medline].

6.   Connor, T. B., Jr., A. B. Roberts, M. B. Sporn, D. Danielpour, L. L. Dart, R. G. Michels, S. de Bustros, C. Enger, H. Kato, M. Lansing, H. Hayashi, and B. M. Glaser. Correlation of fibrosis and TGF-beta type 2 levels in the eye. J. Clin. Invest. 83: 1661-1666, 1989[Medline].

7.   Dean, H. W. Some electron microscopic observations on the lamina propria of the gut, with comments on the close association of macrophages, plasma cells and eopsinophils. Anat. Rec. 149: 453-473, 1964.

8.   Derynck, R., J. A. Jarrett, E. Y. Chen, D. H. Eaton, J. R. Bell, R. K. Assoian, A. B. Roberts, M. B. Sporn, and D. V. Goeddel. Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells. Nature 316: 701-705, 1985[Medline].

9.   Derynck, R., P. B. Lindquist, A. Lee, D. Wen, J. Tamm, J. L. Graycar, L. Rhee, A. J. Mason, D. A. Miller, R. J. Coffey, H. L. Moses, and E. Y. Chen. A new type of transforming growth factor-beta , TGF-beta 3. EMBO J. 7: 3737-3743, 1988[Abstract].

10.   Desmouliere, A., and G. Gabbiani. The role of the myofibroblast in wound healing and fibrocontractive disease. In: The Molecular and Cellular Biology of Wound Repair (2nd ed.), edited by R. A. F. Clark. New York: Plenum, 1996, p. 391-423.

11.   Dignass, A. U., K. Lynch-Devaney, H. Kinden, L. Thim, and D. K. Podolsky. Trefoil peptides promote epithelial migration through a transforming growth factor beta -independent pathway. J. Clin. Invest. 94: 376-383, 1994[Medline].

12.   Dignass, A. U., and D. K. Podolsky. Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor beta . Gastroenterology 105: 1323-1332, 1993[Medline].

13.   Dignass, A. U., S. Tsunekawa, and D. K. Podolsky. Fibroblast growth factors modulate intestinal epithelial cell growth and migration. Gastroenterology 106: 1254-1262, 1994[Medline].

14.   Donnellan, W. L. The structure of the colonic mucosa. The epithelium and subepithelial reticulohistiocytic complex. Gastroenterology 49: 496-514, 1965[Medline].

15.   Feil, W., E. Wentzl, P. Vattay, M. Starlinger, R. Sogukoglu, and R. Schiessel. Repair of rabbit duodenal mucosa after acid injury in vivo and in vitro. Gastroenterology 92: 1973-1986, 1987[Medline].

16.   Göke, M., and D. K. Podolsky. Regulation of the mucosal epithelial barrier. Baillieres Clin. Gastroenterol. 10 (3): 393-407, 1996[Medline].

17.   Göke, M., A. Zuk, and D. K. Podolsky. Regulation and function of extracellular matrix in intestinal epithelial restitution in vitro. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G729-G740, 1996[Abstract/Free Full Text].

18.   Joyce, N. C., M. F. Haire, and G. E. Palade. Morphologic and biochemical evidence for a contractile network within the rat intestinal mucosa. Gastroenterology 92: 68-81, 1987[Medline].

19.   Kaye, G. I., N. Lane, and R. R. Pascal. Colonic pericryptal fibroblast sheath: replication, migration, and cytodifferentiation of a mesenchhymal cell system in adult tissue. II. Fine structural aspects of normal rabbit and human colon. Gastroenterology 54: 852-865, 1968[Medline].

20.   Kedinger, M., P. Simon-Assmann, F. Bouziges, C. Arnold, E. Alexandre, and K. Haffen. Smooth muscle actin expression during rat development and induction in fetal skin fibroblastic cells associated with intestinal embryonic epithelium. Differentiation 43: 87-97, 1990[Medline].

21.   Lacy, E. R. Epithelial restitution in the gastointestinal tract. J. Clin. Gastroenterol. 10, Suppl. 1: 72-77, 1988.

22.   Lawrence, D. A., R. Pircher, C. Kryceve-Martinerie, and P. Jullien. Normal embryo fibroblasts release transforming growth factor-beta in a latent form. J. Cell. Physiol. 121: 184-188, 1984[Medline].

23.   Madisen, L., N. R. Webb, T. M. Rose, H. Marquardt, T. Ideda, D. Twardzik, S. Seyedin, and A. F. Purchio. Transforming growth factor-beta 2: cDNA cloning and sequence analysis. DNA (NY) 7: 1-8, 1988[Medline].

24.   Mahida, Y. R., J. Beltinger, S. Makh, M. Göke, T. Gray, D. K. Podolsky, and C. J. Hawkey. Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G1341-G1348, 1997[Abstract/Free Full Text].

25.   Mahida, Y. R., S. Djelloul, C. Ciacci, M. de Beaumont, S. Chevalier, C. Gespach, and D. K. Podolsky. Resistance to TGF-beta in SV40 large T-immortalized rat intestinal epithelial cells is associated with down-regulation of TGF-beta type 1 receptor. Int. J. Oncol. 9: 365-374, 1996.

26.   Mahida, Y. R., A. Galvin, T. Gray, M. E. McAlindon, S. Makh, H. Sewell, and D. K. Podolsky. Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells. Clin. Exp. Immunol. 109: 377-386, 1997[Medline].

27.   McCormack, S. A., M. J. Viar, and L. R. Johnson. Migration of IEC-6 cells: a model for mucosal healing. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G426-G435, 1992[Abstract/Free Full Text].

28.   McLaughlin, J. M., R. Seth, G. Vautier, R. A. Robins, B. B. Scott, C. J. Hawkey, and D. Jenkins. Interleukin-8 and inducible nitric oxide synthase mRNA levels in inflammatory bowel disease at first presentation. J. Pathol. 181: 87-92, 1997[Medline].

29.   Moore, R., S. Carlson, and J. L. Madara. Rapid barrier restitution in an in vitro model of intestinal epithelial injury. Lab. Invest. 60: 237-244, 1989[Medline].

30.   Moore, R., S. Carlson, and J. L. Madara. Villus contraction aids repair of intestinal epithelium after injury. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G274-G283, 1989[Abstract/Free Full Text].

31.   Nusrat, A., C. Delp, and J. Madara. Intestinal epithelial restitution. J. Clin. Invest. 89: 1501-1511, 1992[Medline].

32.   Nusrat, A., C. A. Parkos, A. E. Bacarra, P. J. Godowski, C. Delp-Archer, E. M. Rosen, and J. L. Madara. Hepatocyte growth factor/scatter factor effects on epithelia. J. Clin. Invest. 93: 2056-2065, 1994[Medline].

33.   Pircher, R., P. Jullien, and D. A. Lawrence. beta -Transforming growth factor is stored in human blood platelets as a latent high molecular weight complex. Biochem. Biophys. Res. Commun. 136: 30-37, 1986[Medline].

34.   Playford, R. J., T. Marchbank, D. P. Calman, J. Calam, P. Royston, J. J. Batten, and H. F. Hansen. Human spasmolyitc polypeptide is a cyotprotective agent that stimulates cell migration. Gastroenterology 108: 108-116, 1995[Medline].

35.   Quaroni, A., J. Wands, T. L. Trelstad, and T. L. Isselbacher. Epithelial cell cultures from rat small intestine. J. Cell Biol. 80: 245-265, 1979.

36.   Richman, P. I., R. J. Tilly, J. R. Jass, and W. F. Bodmer. Colonic pericrypt sheath cells: characterisation of cell type with new monoclonal antibody. J. Clin. Pathol. 40: 593-600, 1987[Abstract].

37.   Rutten, M. J., and S. Ito. Morphology and electrophysiology of guinea pig gastric mucosal repair in vitro. Am. J. Physiol. 244 (Gastrointest. Liver Physiol. 7): G171-G182, 1983[Abstract/Free Full Text].

38.   Sappino, A.-P., P.-Y. Dietrich, O. Skalli, S. Widgren, and G. Gabbiani. Differentiation pattern in embryogenesis and phenotypic modulation in epithelial proliferative lesions. Virchows Arch. 415: 551-557, 1989.

39.   Sato, Y., F. Okada, M. Abe, T. Seguchi, M. Kuwano, S. Sato, A. Furuya, N. Hanai, and T. Tamaoki. The mechanism for the activation of latent TGF-beta during co-culture of endothelial cells and smooth muscle cells: cell-type specific targeting of latent TGF-beta to smooth muscle cells. J. Cell Biol. 123: 1249-1254, 1993[Abstract].

40.   Sato, Y., and D. B. Rifkin. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor beta -1 like molecule by plasmin during co-culture. J. Cell Biol. 109: 309-315, 1989[Abstract].

41.   Shah, M., D. M. Foreman, and M. W. J. Ferguson. Neutralising antibody to TGF-beta 1, 2 reduces cutaneous scarring in adult rodents. J. Cell Sci. 107: 1137-1157, 1994[Abstract/Free Full Text].

42.   Shah, M., D. M. Foreman, and M. W. J. Ferguson. Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds decreases scarring. J. Cell Sci. 108: 985-1002, 1995[Abstract/Free Full Text].

43.   Silen, W. Gastric mucosal defence and repair. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 1044-1069.

44.   Suemori, S., C. Ciacci, and D. K. Podolsky. Regulation of transforming growth factor expression in rat intestinal epithelial cell lines. J. Clin. Invest. 87: 2216-2221, 1991[Medline].

45.   Valentich, J. D., and D. W. Powell. Intestinal subepithelial myofibroblasts and mucosal immunophysiology. Current Opin. Gastro. 10: 645-651, 1994.

46.   Wakefield, L. M., D. M. Smith, T. Matsui, C. C. Harris, and M. B. Sporn. Distribution and modulation of the cellular receptor for transforming growth factor beta . J. Cell Biol. 105: 965-975, 1987[Abstract].

47.   Waller, D. A., N. W. Thomas, and T. J. Self. Epithelial restitution in the large intestine of the rat following insult with bile salts. Virchows Arch. A. Pathol. Anat. Histopathol. 414: 77-81, 1988[Medline].


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