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
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ABSTRACT |
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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 -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-
1 and
-
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
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INTRODUCTION |
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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-1 and
-
1, and fibronectin.
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METHODS |
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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 -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
(106 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.
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RESULTS |
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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).
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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.
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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
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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).
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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 1/
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-
1
and -
1 chains. Using an
anti-human laminin antibody which recognizes the
laminin-
1,
-
1, and
-
1 chains, the
laminin-
1 chain could not be
detected in myofibroblast lysates despite the ability to detect
laminin-
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-
1 and
-
1.
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DISCUSSION |
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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 -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
-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-1 and
-
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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