Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2

Y. R. Mahida1, J. Beltinger1, S. Makh1, M. Göke2, T. Gray3, D. K. Podolsky2, and C. J. Hawkey1

Divisions of 1 Gastroenterology and 3 Pathology, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom; and 2 Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Interactions between epithelial cells and subepithelial myofibroblasts are increasingly recognized as important in the regulation of epithelial cell function. We have established primary cultures of subepithelial myofibroblasts from adult human colonic mucosal samples denuded of epithelial cells and maintained in culture. During culture of mucosal tissue, subepithelial myofibroblasts migrated out via basement membrane pores before establishment in culture. Despite prolonged culture and passage, the myofibroblasts maintained their phenotype, as demonstrated by expression of alpha -smooth muscle actin and vimentin. The cells expressed transcripts and protein for cyclooxygenase (COX)-1 and -2 enzymes, and their release of prostaglandin E2 (PGE2) was inhibited by selective COX-1 and -2 inhibitors. The myofibroblasts also expressed the extracellular matrix (ECM) proteins collagen type IV, laminin-beta 1 and -gamma 1, and fibronectin. Adult human colonic subepithelial myofibroblasts may influence epithelial cell function via products of COX-1 and -2 enzymes, such as PGE2 and secreted ECM proteins.

basement membrane; collagen; laminin; prostaglandin

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

INTERACTIONS between epithelial and subepithelial components of the gastrointestinal mucosa are increasingly recognized as being important in the regulation of normal mucosal physiology and pathophysiology (2, 24). Intestinal subepithelial myofibroblasts are present immediately subjacent to the basement membrane and close to the basal surface of the epithelial cells. Early ultrastructural studies showed that these cells have characteristics of fibroblasts (3, 8, 16). In subsequent studies, they were also shown to share ultrastructural and immunochemical characteristics with smooth muscle cells (15, 17, 25, 27) and have therefore been designated myofibroblasts.

Intestinal subepithelial myofibroblasts are likely to be important in the regulation of intestinal epithelial cell proliferation, differentiation, and functions such as electrolyte transport (31). Myofibroblasts may interact with epithelial cells via extracellular matrix (ECM) proteins, especially those making up the basement membrane (28). The latter contains discrete pores (20, 21) that would allow myofibroblast-derived secretory products to reach the basal surface of epithelial cells. Such secreted factors may include prostaglandins, which are important in the regulation of electrolyte transport by epithelial cells (1). Cyclooxygenase (COX) enzymes catalyze the initial step in the formation of prostaglandins from arachidonic acid, and recent studies (5) have demonstrated the existence of two isoforms of the COX enzyme, COX-1 and -2. COX-1 is regarded as constitutively expressed, whereas COX-2 is inducible in a variety of cells.

We describe a model of human intestinal mucosa that allows the establishment of subepithelial myofibroblast cultures. By use of selective inhibitors, myofibroblasts from normal colonic mucosa have been shown to express functional COX-1 and -2 enzymes and also to express the ECM proteins, collagen type IV, laminin-beta 1 and -gamma 1, and fibronectin.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Mucosal tissue. Fresh mucosal samples were obtained from human intestinal specimens resected at operation. Histologically normal colonic mucosal samples, at least 5 cm away from tumor, were obtained from colons resected for carcinoma. Mucosal tissue was also obtained from resected intestine with active inflammatory bowel disease (IBD; 2 ulcerative colitis and 2 Crohn's disease).

Tissue culture. Epithelial cells were detached from mucosal strips as previously described (21). The mucosal samples were incubated with 1 mM dithiothreitol (DTT; Sigma Chemical, St. Louis, MO) for 15 min at room temperature. After washing with calcium- and magnesium-free Hanks' balanced salt solution (HBSS; GIBCO-BRL, Gaithersburg, MD), the mucosal strips were incubated in a shaking water bath (at 37°C) in 1 mM EDTA (Sigma) for three 30-min periods. After each incubation with EDTA, the mucosal strips were extensively washed with HBSS. At the end of EDTA treatment, the mucosal samples were completely denuded of epithelial cells and were subsequently cultured at 37°C in RPMI 1640 (GIBCO-BRL) containing 10% fetal calf serum (FCS; GIBCO-BRL). During culture, numerous cells appeared both in suspension and adherent to the culture dish (21). The cells in suspension were removed after every 24- to 72-h culture period, and the denuded mucosal tissue was maintained in culture for up to 4 wk.

Cell culture. Established colonies of myofibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% FCS and 1% nonessential amino acids (NEAA; GIBCO-BRL). At confluence, the cells were passaged using 0.1% (wt/vol) trypsin-0.2% (wt/vol) EDTA in a 1:2 to 1:3 split ratio.

Electron microscopy. Untreated mucosal samples and those obtained after culture for varying periods of time were studied by transmission electron microscopy (TEM). The mucosal samples were fixed by immersion in 2.5% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.4) for 2 h, and myofibroblasts adherent to culture dishes were also fixed in the same fixative for 2 h. Subsequent processing was performed as previously described (26). Suitable areas for TEM were selected from 0.5-mm toluidine blue-stained sections. After trimming, 18-nm sections were cut and mounted on copper grids before staining with uranyl acetate and lead citrate. A Jeol 1200 EX transmission electron microscope (Jeol, Welwyn Garden City, UK) was used for TEM.

Immunohistochemistry. Mouse monoclonal antibodies to alpha -smooth muscle actin, vimentin, and desmin (all obtained from Sigma) and to macrophages (anti-CD68; Dako, High Wycombe, UK) were used. Rabbit antibodies to COX-1 and -2 enzymes were obtained from Cayman Chemical (Ann Arbor, MI). Cells were grown on coverslips and fixed with acetone before immunoperoxidase staining using the Vectastain ABC peroxidase kit (Vecta Laboratories, Burlingame, CA). After incubation with the primary antibody, biotinylated goat anti-mouse immunoglobulin (Ig) G or goat anti-rabbit IgG was applied followed by avidin-biotinylated horseradish peroxidase complex. Peroxidase activity was developed with diaminobenzidine, followed by nuclear staining of all the cells present (peroxidase positive and negative) using hematoxylin (from Sigma).

Assessment of ECM proteins by immunoblotting. Cell lysates were harvested, and ECM protein expression was assessed essentially as previously described (11). In brief, ECM proteins were extracted by scraping myofibroblasts into 10 mM EDTA, 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS) containing protease inhibitors (2 mM N-ethylmaleimide, 2 mg/ml aprotinin, 4 mg/ml pepstatin, 10 mg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride). Extracts were cleared by centrifugation at 10,000 g for 15 min, separated into aliquots, and stored at -80°C. Protein concentration in each sample was determined by colorimetric Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Electrophoresis of equal amounts of protein in 7.5-8.5% polyacrylamide gels was performed under reducing conditions (5% vol/vol 2-mercaptoethanol) according to the method of Laemmli (18). Purified human fibronectin (50 ng), purified murine collagen type IV (50 ng), and Engelbreth-Holm-Swarm mouse tumor-derived Matrigel (1.5 ml) (all from Collaborative Biomedical Products/Becton Dickinson, Bedford, MA) served as positive controls for fibronectin, collagen type IV, and laminin immunoblotting, respectively. Proteins were electroeluted onto polyvinylidene difluoride-Immobilon P transfer membranes (Millipore, Bedford, MA) in transfer buffer (50 mM Tris, 0.38 M glycine, 10% vol/vol methanol) for 12 h at 30 mA and then stained (0.1% Ponceau red-1% acetic acid). After washing, membranes were blocked in 1× Tris-buffered saline (TBS), 0.05% Tween 20, and 5% nonfat dry milk (Western blocking buffer) at 4°C overnight. Blots were then incubated with primary antibody diluted in Western blocking buffer for 1 h at room temperature. Rabbit polyclonal anti-rat fibronectin and anti-human laminin antiserum (diluted 1:1,000) were purchased from GIBCO-BRL; rabbit polyclonal IgG anti-mouse collagen type IV (diluted 1:200) was obtained from Collaborative Biomedical Products. After washing in 1× TBS-0.05% Tween 20, hybridization with the secondary antibody (donkey anti-rabbit Ig, horseradish peroxidase-linked antibody diluted 1:20,000, obtained from Amersham Life Science, Arlington Heights, IL) was performed for 30 min at room temperature. After washing in 1× TBS-0.05% Tween 20, detection was performed using Renaissance enhanced chemiluminescence reagents (Du Pont-NEN, Boston, MA). Blots were then subjected to autoradiography.

RNA isolation and reverse transcription. RNA was isolated from myofibroblasts using RNAzol (Biogenesis, Poole, UK). Random hexamer primer (Pharmacia Biotech) was mixed with 10 µg of RNA (final vol 37.5 µl), heated to 70°C for 10 min, and allowed to cool on ice. Reverse transcription (RT) to cDNA was performed by adding the following and incubating at 37°C for 60 min: 5 µl of 10× RT buffer [0.5 M Tris (pH 8.3), 0.75 M KCl, 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 Biotech), 1 µl of Moloney murine leukemia virus reverse transcriptase (200 U/ml; GIBCO-BRL), 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 -70°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), 1% Triton X-100; Promega, Madison, WI], 6 µl of 2 mM MgCl2, 2 µl of 5 mM dNTPs, and 1 µl of Taq DNA polymerase (5 U/ml; Promega). The following primer pairs were used (to a final concn of 5 mM) based on published nucleotide sequences (6, 14): 5'-GAG TCT TTC TCC AAC GTG AGC-3' (sense) and 5'-ACC TGG TAC TTG AGT TTC CCA-3' (antisense) to amplify 350-bp COX-1 product; 5'-TGA AAC CCA CTC CAA ACA CAG-3' (sense) and 5'-TCA TCA GGC ACA GGA GGA AG-3' (antisense) to amplify 232-bp COX-2 product; 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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) product.

Amplification was performed using a Trio-Thermoblock (Biometra). Polymerase chain reaction (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 was used and then completed by extension for 3 min at 72°C.

PCR products were analyzed by adding 1 µl of ethidium bromide (10 mg/ml) and 3 µl of gel loading buffer (Sigma) to 12 µl of PCR product and electrophoresis on a 2% agarose gel. The specificities of RT-PCR for COX-1 and -2 and GAPDH had previously been confirmed by sequencing of the PCR products and/or hybridization using probes specific to the relevant amplified sequences.

Prostaglandin E2 production. For studies of prostaglandin E2 (PGE2) production, myofibroblasts were grown to confluence in 24-well plates (Nunc; GIBCO-BRL). After three washes in prewarmed (to 37°C) medium, the cells were cultured in quadruplicate in 10% FCS-1% NEAA-DMEM alone or with added indomethacin (10-6 and 10-7 M; from Sigma), SC-58560 (a selective COX-1 inhibitor, 10-6 and 10-7 M; a gift from Searle, Skokie, IL), or NS-398 [a selective COX-2 inhibitor (21), 10-6 and 10-7 M; Cayman Chemical]. After culture for 2 h, cells in all the wells were washed three times, and the myofibroblast monolayers were cultured in fresh medium (10% FCS, 1% NEAA-DMEM; 250 µl/well). After culture for a further 22 h, cell supernatants were obtained and, after centrifugation (at 10,000 g), were stored at -70°C until assayed for PGE2 by a specific enzyme-linked immunosorbent assay (Biotrak; Amersham International, Slough, UK).

Statistical analysis. PGE2 data were analyzed by analysis of variance and paired t-tests.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Studies on mucosal tissue. TEM of untreated normal colonic mucosal tissue showed myofibroblasts lying below the basement membrane, close to the basal surface of overlying epithelial cells (Fig. 1).


View larger version (142K):
[in this window]
[in a new window]
 
Fig. 1.   Representative transmission electron micrograph of a normal colonic mucosal sample showing a myofibroblast (*) lying below basement membrane, close to basal surface of epithelial cells. Longitudinally arranged bundle of microfilaments (arrow) is seen below cell membrane.

To establish long-term cultures of subepithelial myofibroblasts, we adopted our recently described model (21) in which intestinal mucosal samples, denuded of epithelial cells, are maintained in culture. TEM of denuded normal mucosa showed that myofibroblasts migrated out of the lamina propria via the basement membrane pores to lie on the outer surface of the basement membrane (Fig. 2) after 4-8 days of culture. For IBD tissue, such migration was seen over the first 24-h period of culture and became more prominent over the subsequent days. TEM of denuded mucosal samples, after prolonged culture, demonstrated a complete absence of myofibroblasts below the basement membrane.


View larger version (108K):
[in this window]
[in a new window]
 
Fig. 2.   Transmission electron micrograph of a colonic mucosal sample denuded of epithelial cells and cultured for 7 days. A myofibroblast (identified by bundles of microfilament below *) is migrating out of lamina propria via a basement membrane pore.

Studies on myofibroblasts growing in culture dishes. After 3- to 10-day culture of denuded mucosa, myofibroblasts adherent to the culture dishes began to appear. Initially, the myofibroblasts were present with numerous adherent macrophages. Over the next 5-10 days, colonies of myofibroblasts appeared and gradually increased in size until a monolayer of myofibroblasts was formed, with scattered macrophages (CD68 +ve) lying between them (Fig. 3). Subsequently, the macrophages became detached from the culture dishes and were lost during media changes to leave a morphologically homogeneous population of myofibroblasts with no other contaminating cells.


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 3.   Monolayer of normal colonic myofibroblasts with a macrophage (arrow). Cells were derived from cultures of normal colonic mucosal samples denuded of epithelial cells and grown on a coverslip. Subsequent immunohistochemical labeling was performed using a monoclonal antibody to CD68 (specific for macrophages), followed by nuclear staining with hematoxylin. A cytoplasmically immunolabeled macrophage with processes (arrow) is closely associated with a monolayer of cytoplasmically unlabeled (with monoclonal antibody) myofibroblasts (their presence is illustrated by hematoxylin-stained nuclei).

Characterization of myofibroblast cultures. Myofibroblasts from denuded normal colonic mucosal samples were passaged 12 times, and those from IBD tissue were passaged 8 times. The cells proliferate after seeding (cell doubling time of 5 days) until a monolayer covers the culture dish. No overlapping myofibroblasts were seen despite prolonged culture of confluent cells (contact inhibition), and the cells proliferated and were passaged after freezing.

Immunohistochemical studies of normal colonic myofibroblasts at passages 3 and 9 showed that the cells expressed alpha -smooth muscle actin (Fig. 4A) and vimentin (Fig. 4B) and were weakly positive for desmin (not shown). In IBD myofibroblasts, there was heterogeneity in expression of alpha -smooth muscle actin, with some cells demonstrating stronger immunoreactivity than others (Fig. 5). Heterogeneity in the expression of desmin and vimentin was also seen in these cells (data not shown).


View larger version (205K):
[in this window]
[in a new window]
 


View larger version (209K):
[in this window]
[in a new window]
 
Fig. 4.   Expression of alpha -smooth muscle actin (A) and vimentin (B) by normal colonic myofibroblasts. Cells were grown on coverslips and immunohistochemical labeling was performed using relevant monoclonal antibodies, followed by nuclear staining using hematoxylin. Cytoplasmically immunolabeled myofibroblasts are present in both A and B.


View larger version (144K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of alpha -smooth muscle actin by myofibroblasts derived from cultures of active ulcerative colitis colonic mucosal samples denuded of epithelial cells. Passage 5 cells were grown on coverslips, and cells were labeled immunohistochemically using specific antibody. There is heterogeneity in expression of alpha -smooth muscle actin as illustrated by presence of strongly (arrows) and weakly immunoreactive cells.

Both normal and IBD myofibroblasts expressed alpha -smooth muscle actin despite prolonged culture in medium containing 0.1% FCS.

Ultrastructural studies showed that both normal colonic and IBD myofibroblasts expressed abundant rough endoplasmic reticulum and longitudinally arranged bundles of microfilaments below the cell membrane (Fig. 6). In some cultures, ECM was also seen outside the cells (Fig. 6).


View larger version (141K):
[in this window]
[in a new window]
 
Fig. 6.   Transmission electron micrograph of a confluent monolayer of colonic myofibroblasts. Mitochondria, rough endoplasmic reticula (small arrows), and longitudinally arranged bundles of microfilament (large arrows) are present within cells. Secreted matrix (*) is also present.

Expression of COX-1 and -2 and PGE2 production. mRNA transcripts (Fig. 7) and immunoreactive COX-1 and -2 enzymes were expressed by the normal colonic and IBD myofibroblasts. In contrast to COX-1 (Fig. 8A), COX-2 expression was prominent in the perinuclear region in many cells (Fig. 8B).


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 7.   Expression of mRNA transcripts for cyclooxygenase (COX)-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in colonic myofibroblasts. RNA was isolated from a confluent monolayer of cells and reverse transcribed, and relevant transcripts were amplified by polymerase chain reaction using specific primers. DNA size markers are indicated on left.


View larger version (148K):
[in this window]
[in a new window]
 


View larger version (155K):
[in this window]
[in a new window]
 
Fig. 8.   Expression of immunoreactive COX-1 (A) and COX-2 (B) in colonic myofibroblasts. Immunohistochemical labeling was performed on a confluent monolayer of cells grown on coverslips. Strong COX-2 immunoreactivity is seen in perinuclear region of a number of cells (arrows).

Functional activity of the two COX enzymes was investigated in normal colonic myofibroblasts by determining PGE2 production after pretreatment with selective COX-1 and -2 inhibitors. The selective COX-1 inhibitor SC-58560 caused a marked dose-dependent reduction of released PGE2 (Fig. 9). The COX-2 inhibitor NS-398 also significantly reduced the amount of PGE2 in the medium, but this appeared to be less marked.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 9.   Inhibition of prostaglandin E2 (PGE2) production in colonic myofibroblasts by selective COX-1 and -2 inhibitors. Cells were grown to confluence in 24-well plates (in quadruplicate), and monolayers were cultured in medium alone or in presence of 2 different concentrations (10-6 and 10-7 M) of indomethacin (Indo), SC-58560 (a selective COX-1 inhibitor), or NS-398 (a selective COX-2 inhibitor) for 2 h. After cells were washed, all monolayers were cultured in fresh medium (250 µl/well) for 22 h. Cell supernatants were subsequently assayed for PGE2 using a specific enzyme-linked immunosorbent assay. * P < 0.03 (vs. control).

Expression of ECM proteins. Western blot analysis showed that normal colonic myofibroblasts expressed several ECM proteins. The size of detected proteins matched those of the corresponding controls under reducing conditions including fibronectin at 240 kDa (Fig. 10A), two collagen type IV alpha 1/alpha 2 signals at 185 and 195 kDa, with the 185-kDa signal intensity being more pronounced (Fig. 10B), and two laminin signals at 220 kDa (Fig. 10C). These two signals represent the laminin-beta 1 and -gamma 1 chains. Using an anti-human laminin antibody which recognizes the laminin-alpha 1, -beta 1, and -gamma 1 chains, the laminin-alpha 1 chain could not be detected in myofibroblast lysates despite the ability to detect laminin-alpha 1 (400 kDa) in Matrigel used as a positive control. Collectively, these data clearly suggest that myofibroblasts isolated from human adult colonic mucosa express the ECM proteins fibronectin, collagen type IV, and laminin-beta 1 and -gamma 1.


View larger version (21K):
[in this window]
[in a new window]
 


View larger version (37K):
[in this window]
[in a new window]
 


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 10.   Expression of fibronectin (A), collagen type IV (B), and laminin (C) in lysates of colonic myofibroblasts. Lysates of confluent monolayers of myofibroblasts and purified fibronectin (FN), collagen type IV (Col IV) or Matrigel were electrophoresed on polyacrylamide gels and electroeluted onto polyvinylidene difluoride-Immobilon P transfer membranes. Immunostaining was performed using polyclonal antibodies. Myofibroblasts express fibronectin, collagen type IV, and laminin-beta 1 and -gamma 1 (LN).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Pericryptal cells of the large intestine were first described in the 1960s (3) with ultrastructural evidence that these cells were fibroblasts (3, 8, 16). Subsequent studies (15, 17, 25, 27) provided ultrastructural and immunocytochemical evidence that the pericryptal cells are indistinguishable from myofibroblasts. These cells were shown to contain microfilaments with dense bodies and intercellular junctions and also the intermediate filament desmin, myosin, and the actin-binding protein filamin. They were also labeled by antibody PR2D3, in a manner similar to myofibroblasts in other sites in the body (25). Studies with tritiated thymidine (16, 22, 23) have also shown that the pericryptal myofibroblasts replicate and are in a state of flux, migrating to the subepithelial tissues of the villus tips and the surface of the colon.

Interactions between subepithelial myofibroblasts and epithelial cells have increasingly been recognized to play an important role in regulating epithelial proliferation, differentiation, and electrolyte transport (24, 31). Their role in the regulation of epithelial cell proliferation is suggested by prominence at the base of crypts, an increase in number in synchrony with epithelial proliferation, and their presence close to the proliferative epithelium of villous adenomas (27). In vitro studies suggest that subepithelial myofibroblasts may induce epithelial differentiation (19, 30), probably via synthesis of ECM proteins (28).

Our ex vivo model allows the direct investigation of human intestinal myofibroblasts in culture. Denuding mucosal samples of epithelial cells allows the subepithelial myofibroblasts to migrate out via pores in the basement membrane. After initial residence on the outer surface of the basement membrane, they migrated to the culture dish to become resident with adherent macrophages. After myofibroblast proliferation, the macrophages were lost, leaving a morphologically homogeneous population of myofibroblasts. TEM after prolonged culture of mucosal samples demonstrated a complete absence of myofibroblasts in the subepithelial region. We therefore believe that the myofibroblasts migrating out via basement membrane pores and subsequently becoming established in culture are derived from the subepithelial region. It is possible that myofibroblasts present in the lamina propria also migrate via basement membrane pores and become established in culture.

The migration of subepithelial myofibroblasts out of denuded mucosa via the basement membrane pores is likely to represent a response of the intestinal mucosa to injury and loss of surface epithelial cells. We found that the myofibroblasts were initially resident on the outer surface of the basement membrane, in a position where, in vivo, they could provide protection against luminal products. It is likely that these cells may also play an important role in enhancing wound repair by synthesizing ECM and factors such as fibroblast growth factor that would allow restitution by remaining epithelial cells (7). Myofibroblasts may also reduce the surface area of the ulcerated intestinal mucosa by their ability to mediate wound contraction (4, 12).

Previous studies (9, 17) have shown that the endoderm in the developing intestine induces the expression of alpha -smooth muscle actin in fibroblasts, suggesting a requirement of epithelial cells for maintenance of the myofibroblast phenotype. Our studies show that the subepithelial myofibroblasts derived from adult human intestine retain their expression of alpha -smooth muscle actin and other ultrastructural and immunocytochemical characteristics despite prolonged culture and passage. Continued contact with epithelial cells may therefore not be necessary for expression of their phenotypic (and possibly also functional) characteristics, and our studies suggest that paracrine-type interactions may be important.

It seems likely that myofibroblasts play a major role in regulating epithelial cell proliferation, differentiation, and functions such as ion transport (31). Myofibroblasts may mediate their effects via secreted products of COX enzymes, which catalyze the initial step in the formation of prostaglandins from arachidonic acid. Our studies using selective inhibitors have shown that myofibroblasts isolated from normal human colonic mucosa express functional COX-1 and -2 enzymes and synthesize prostaglandins in amounts likely to influence epithelial cell function. Our recent immunohistochemical studies on sections of human gastrointestinal mucosa also suggest that subepithelial myofibroblasts express COX-1 and -2 enzymes in vivo (unpublished observations).

In myofibroblast cell lines derived from neonatal tissue, Hinterleitner et al. (13) have demonstrated constitutive expression of COX-1 but very little COX-2 mRNA. Our studies demonstrating constitutive expression of both COX-1 and -2 mRNA and protein therefore underscore the importance of using primary myofibroblasts derived from adult intestinal mucosa in studies to investigate their interactions with epithelial cells.

Studies in the developing intestine suggest that both epithelial cells and myofibroblasts contribute to the formation of the basement membrane (29). In intestinal tissue, collagen type IV and laminin have been shown to be produced mainly by myofibroblasts (28, 29). We have shown that subepithelial myofibroblasts isolated from adult normal human colonic mucosa also produce collagen type IV, laminin-beta 1 and -gamma 1, and fibronectin. Thus, with respect to ECM protein production, the isolated colonic myofibroblasts behave in a fashion similar to that observed in vivo.

In conclusion, our model therefore allows pure populations of isolated subepithelial myofibroblasts from healthy and diseased adult human intestinal mucosa to be available. The myofibroblasts that we have described retain a representative and differentiated phenotype despite prolonged culture. They are thus likely to yield informative data on the in vivo interactions between myofibroblasts and epithelial cells in processes such as secretion, barrier function, and repair in both health and disease.

    ACKNOWLEDGEMENTS

These studies were supported by the Medical Research Council (UK) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41557 and DK-43351. J. Beltinger is supported by the Swiss National Science Foundation.

    FOOTNOTES

Present address of M. Göke: Abteilung Gastroenterologie und Hepatologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany.

Address for reprint requests: Y. R. Mahida, Div. of Gastroenterology, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK.

Received 17 March 1997; accepted in final form 8 September 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bern, M. J., C. W. Sturbaum, S. S. Karayalcin, H. M. Berschneider, J. T. Wachsman, and D. W. Powell. Immune system control of rat and rabbit colonic electrolyte transport. Role of prostaglandins and enteric nervous system. J. Clin. Invest. 83: 1810-1820, 1989[Medline].

2.   Castro, G. A., and C. J. Arntzen. Immunophysiology of the gut: a research frontier for integrative studies of the common mucosal immune system. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G599-G610, 1993[Abstract/Free Full Text].

3.   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 eosinophils. Anat. Rec. 149: 453-473, 1964.

4.   Desmoulière, A., and G. Gabbiani. The role of the myofibroblast in wound healing and fibrocontractive diseases. In: The Molecular and Cellular Biology of Wound Repair (2nd ed.), edited by R. A. F. Clark. New York: Plenum, 1996, chapt. 13, p. 391-423.

5.   De Witt, D., and W. L. Smith. Yes, but do they still get headaches? Cell 83: 345-348, 1995[Medline].

6.   Diaz, A., A. M. Reginato, and S. A. Jimenez. Alternative splicing of human prostaglandin G/H synthase mRNA and evidence of differential regulation of the resulting transcripts by transforming growth factor beta 1, interleukin 1beta and tumor necrosis factor alpha . J. Biol. Chem. 267: 10816-10822, 1992[Abstract/Free Full Text].

7.   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].

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

9.   Duluc, I., J.-N. Freund, C. Leberquier, and M. Kedinger. Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development. J. Cell Biol. 126: 211-221, 1994[Abstract].

10.   Futaki, N., S. Takahashi, I. Yokoyama, I. Arai, S. Higuchi, and S. Otomo. NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro. Prostaglandins 47: 55-59, 1994[Medline].

11.   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].

12.   Grinnell, F. Mini-review on the cellular mechanisms of disease. Fibroblasts, myofibroblasts and wound contraction. J. Cell Biol. 124: 401-404, 1994[Medline].

13.   Hinterleitner, T. A., J. I. Saada, H. M. Berschneider, D. W. Powell, and J. D. Valentich. IL-1 stimulates intestinal myofibroblast COX gene expression and augments activation of Cl- secretion in T84 cells. Am. J. Physiol. 271 (Cell Physiol. 40): C1262-C1268, 1996[Abstract/Free Full Text].

14.   Hla, T., and K. Neilson. Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA 89: 7384-7388, 1992[Abstract].

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

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

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

18.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680-684, 1970.

19.   Louvard, D., M. Kedinger, and H. P. Hauri. The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures. Annu. Rev. Cell Biol. 8: 157-195, 1992.

20.   Low, F. N., and S. G. McClugage. Microdissection by ultrasonication: scanning electron microscopy of the epithelial basal lamina of the alimentary canal in the rat. Am. J. Anat. 169: 137-147, 1984[Medline].

21.  Mahida, Y. R., A. Galvin, T. Gray, S. Makh, M. E. McAlindon, H. F. 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. In press.

22.   Parker, F. G., E. N. Barnes, and G. I. Kaye. The pericryptal fibroblast sheath. IV. Replication, migration, and differentiation of the subepithelial fibroblasts of the crypt and villus of the rabbit jejunum. Gastroenterology 67: 607-621, 1974[Medline].

23.   Pascal, R. R., G. I. Kaye, and N. Lane. Colonic pericryptal fibroblast sheath: replication, migration, and cytodifferentiation of a mesenchymal cell system in adult tissue. I. Autoradiographic studies of normal rabbit colon. Gastroenterology 54: 835-851, 1968[Medline].

24.   Perdue, M. H., and D. M. McKay. Integrative immunophysiology in the intestinal mucosa. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G151-G165, 1994[Abstract/Free Full Text].

25.   Richman, P. I., R. 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].

26.   Robinson, G., and T. Gray. Electron microscopy. 2. Tissue preparation, sectioning and staining. In: Theory and Practice of Histological Techniques (3rd ed.), edited by J. D. Bancroft, and A. Stevens. London: Churchill Livingstone, 1990, p. 525-562.

27.   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.

28.   Simon-Assmann, P., F. Bouziges, J.-N. Freund, F. Perrin-Schmitt, and M. Kedinger. Type IV collagen mRNA accumulates in the mesenchymal compartment at early stages of murine developing intestine. J. Cell Biol. 110: 849-857, 1990[Abstract].

29.   Simon-Assman, P., F. Bouziges, J.-C. Lissitzky, L. Sorokin, M. Kedinger, and P. Simon-Assman. Dual and asynchronous deposition of laminin chains at the epithelial-mesenchymal interface in the gut. Gastroenterology 102: 1835-1845, 1992[Medline].

30.   Stallmach, A., U. Hahn, H. J. Merker, E. G. Hahn, and E. O. Riecken. Differentiation of rat intestinal epithelial cells is induced by organotypic mesenchymal cells in vitro. Gut 30: 959-970, 1989[Abstract].

31.   Valentich, J. D., and D. W. Powell. Intestinal subepithelial myofibroblasts and mucosal immunophysiology. Curr. Opin. Gastroenterol. 10: 645-651, 1994.


AJP Gastroint Liver Physiol 273(6):G1341-G1348
0193-1857/97 $5.00 Copyright © 1997 the American Physiological Society