Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, United Kingdom
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ABSTRACT |
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The epithelium of the gastrointestinal tract
transports ions and water but excludes luminal microorganisms and toxic
molecules. The factors regulating these important functions are not
fully understood. Intestinal myofibroblasts lie subjacent to the
basement membrane, at the basal surface of epithelial cells. We
recently showed that primary cultures of adult human colonic
subepithelial myofibroblasts express cyclooxygenase (COX)-1 and COX-2
enzymes and release bioactive transforming growth factor- (TGF-
).
In this study we have investigated the role of normal human colonic subepithelial myofibroblasts in the regulation of transepithelial resistance and secretory response in HCA-7 and T84 colonic epithelial cell lines. Cocultures of epithelial cells-myofibroblasts and medium
conditioned by myofibroblasts enhanced transepithelial resistance and
delayed mannitol flux. A panspecific antibody to TGF-
(but not
piroxicam) antagonized this effect. In HCA-7 cells, myofibroblasts
downregulated secretagogue-induced change in short-circuit current, and
this effect was reversed by pretreatment of myofibroblasts with
piroxicam. In contrast to HCA-7 cells, myofibroblasts upregulated the
agonist-induced secretory response in T84 cells. This study shows that
intestinal subepithelial myofibroblasts enhance barrier function and
modulate electrogenic chloride secretion in epithelial cells. The
enhancement of barrier function was mediated by TGF-
. In contrast,
the modulation of agonist-induced change in short-circuit current was
mediated by cyclooxygenase products. These findings suggest that
colonic myofibroblasts regulate important functions of epithelial cells
via distinct secretory products.
ion transport; transforming growth factor-; prostaglandins
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INTRODUCTION |
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THE GASTROINTESTINAL TRACT performs the critical functions of extraction of nutrients, minerals, and water but excludes luminal microorganisms, their products, and other toxic molecules (37). These essential functions are mediated by a monolayer of epithelial cells that line the intestinal lumen. The epithelial cells are able to accomplish their functions by their capacity to provide a barrier to luminal contents and to transport ions and solutes into and out of the cells (2, 11, 23, 34). The factors that regulate these two major functions of the epithelium are not fully understood.
It is increasingly recognized that interactions between the intestinal
epithelium and subepithelial components in the lamina propria are
important in the regulation of many epithelial cell functions (42). The
subepithelial components are physically separated from the epithelium
by a basement membrane, which contains numerous discrete pores (26).
These pores can allow mediators secreted by cells in the lamina propria
access to the basal surface of the epithelial cells. Myofibroblasts lie
immediately subjacent to the basement membrane and close to the basal
surface of epithelial cells (24, 33). The coordinated interaction
between epithelial and mesenchymal cells has also been shown to be
important for proliferation and differentiation (7, 8, 19, 39). As constitutive cells situated intraparenchymally between epithelial and
stromal cells, they modulate information between adjacent immune,
neural, and endocrine tissue and may therefore play a crucial role in
inflammation (5, 14). We recently developed an ex vivo model that
allows the isolation and establishment of pure cultures of
subepithelial myofibroblasts from adult human intestinal mucosal
samples (24). Despite prolonged culture and passage, the myofibroblasts
retain a representative and differentiated phenotype. These cells
express transcripts and protein for cyclooxygenase (COX)-1 and COX-2
enzymes, release prostaglandin E2
(PGE2), and express
extracellular matrix proteins that are likely to contribute toward the
formation of the basement membrane (24). Recently, these normal colonic
subepithelial myofibroblasts were shown to release bioactive
transforming growth factor- (TGF-
) (28).
The transepithelial secretion of chloride ions is an important factor in the control mechanism of fluid secretion across the intestinal epithelial surface. A number of inflammatory mediators, including kinins, histamine, serotonin, eicosanoids, and a range of cytokines, are known to mediate this secretory response (4). In situations where the balance of regulatory mechanisms is disturbed, diarrhea is the main feature in the human gut (37, 40).
Although much attention has focused on agents that stimulate electrogenic ion secretion, relatively little is known of endogenous regulatory mechanisms that will attenuate the secretory response and maintain the barrier function of epithelial cells in intestinal inflammation. In this study we have investigated the role of normal human colonic subepithelial myofibroblasts in epithelial barrier function and in the regulation of the response of the colonic epithelium to secretory agonists.
Epithelial cell responses were studied in monolayers of HCA-7 (13, 18,
21) and T84 cells (14, 34). We report that human colonic subepithelial
myofibroblasts enhance the barrier function and downregulate the
agonist-induced secretory response in HCA-7 cells. The
myofibroblast-mediated enhancement of barrier function is shown to be
mediated via TGF- and the change in secretory response via
cyclooxygenase products. In contrast to HCA-7 cells, myofibroblasts
upregulated the agonist-induced secretory response in T84 cells. We
also show differences between the two epithelial cell lines in the
expression of COX-1 and COX-2 enzymes, which may explain their
different secretory responses to myofibroblasts.
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METHODS |
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Epithelial cells. HCA-7 colony 29 (18), and T84 cells were grown in DMEM (Sigma Chemical) supplemented with 10% FCS, glutamine (0.29 mg/ml), penicillin (40 µg/ml), streptomycin (368 µg/ml), and nonessential amino acids (NEAA; GIBCO BRL, Gaithersburg, MD) in an atmosphere of 5% CO2 at 37°C. For electrophysiological studies, cells were seeded on Snapwell filters (polycarbonate membrane, pore size 0.45 µm, surface area 1 cm2; Costar) and formed confluent monolayers within 10-12 days, as assessed by an epithelial voltohmmeter (EVOM, World Precision Instruments). HCA-7 and T84 cells were studied between passages 25 and 30 and passages 70 and 75, respectively.
Myofibroblasts. Colonies of myofibroblasts were established and characterized as previously described (24). Established colonies of myofibroblasts from different donor sources were cultured in DMEM supplemented with 10% FCS and 1% NEAA. On confluency, the cells were passaged using 0.1% (wt/vol) trypsin and 0.2% (wt/vol) EDTA in a 1:2-1:3 split ratio.
The interactions between colonic subepithelial myofibroblasts and epithelial cells were investigated in coculture and myofibroblast-conditioned medium. Myofibroblast-epithelial cocultures were established using specially designed filter supports, as described by Madara et al. (22), that allow cell monolayers to be grown on either side of a membrane filter. Plastic rings with the same dimension as the base of the Costar inserts were cleaned by washing in 70% ethanol, dried, and attached to the underside of the insert with silicone glue (no. 732, Dow-Corning), leaving the pores untouched. After they were dried, the inserts were sterilized by submersion in 70% ethanol for 4 h, inverted onto a sterile petri dish in a hood, and allowed to dry. Myofibroblasts were trypsinized and resuspended in 10% FCS-DMEM. The cell suspension (150-200 µl) was seeded, and the myofibroblasts were grown on the inverted filter for 24 h. The membrane filters were subsequently turned and placed into wells containing 10% FCS-DMEM and epithelial cells at a density of 3 × 105/ml seeded on the other, unoccupied side of the membrane. For studies using myofibroblast-conditioned medium, myofibroblasts were grown to confluence in tissue culture flasks in 10% FCS-DMEM. After they were extensively washed (with 0.1% FCS-DMEM), the cells were cultured for 24 h in 0.1% FCS-DMEM. The myofibroblast-conditioned medium was filtered and stored atElectron microscopy. Filters with HCA-7 cells and myofibroblasts were fixed by immersion in 2.5% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.4). Subsequent processing was performed as previously described (36). Areas suitable for transmission electron microscopy were selected from 0.5-µm toluidine blue-stained sections. After they were trimmed, 18-nm sections were cut and mounted on copper grids before they were stained with uranyl acetate and lead citrate. A model 1200 EX transmission electron microscope (Jeol, Welwyn Garden City, UK) was used for transmission electron microscopy.
Electrophysiology.
The filters (filter area = 1 cm2) were placed into an Ussing
chamber (World Precision Instruments), bathed in oxygenated (95% O2-5%
CO2) Krebs-Henseleit solution
(in mM: 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.0 MgSO4, 24.8 NaHCO3, 1.2 KH2PO4
and 11.1 glucose), and maintained at 37°C (3). The epithelial
monolayers, individually or in coculture, were voltage clamped to 0 mV
by continuous application of a short-circuit current (SCC) with a
dual-voltage clamp (model DVC-1000, World Precision Instruments).
Periodic constant-amplitude voltage pulses were used to assess
transepithelial resistance. Basal SCC
(µA/cm2) and resistance
( · cm2)
were measured after the monolayers were allowed to equilibrate for 15 min. Peak change in SCC (
SCC) was recorded in response to
secretagogues administered to the basolateral side of the epithelial monolayers. SCC was digitally recorded and analyzed with the
Acqknowledge III (Biopac Systems) data acquisition system. Values are
means ± SE.
TGF- bioassay.
The presence of bioactive TGF-
in myofibroblast-conditioned medium
was determined using a specific bioassay (25, 28) that is based on the
ability of TGF-
to inhibit proliferation of the mink lung epithelial
cell line MvLu (ECACC, Porton Down, UK). Latent TGF-
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, then neutralized with NaOH and HEPES (to a final concentration of
16 mmol/l).
Mannitol flux. Penetration of the inert compound mannitol to the basal compartment was studied after addition to the apical compartment. Epithelial cells were seeded onto Transwell filters (polycarbonate membrane, 6.5-mm wells, 0.45 µm pore size; Costar). After incubation of the epithelial cells with control medium or conditioned medium from myofibroblasts for 24 h, medium in the apical and basolateral compartments was replaced with fresh DMEM (0.1% FCS) in the apical compartment supplemented by d-[3H]mannitol (Sigma Chemical; 20 Ci/mmol, 1.0 µCi/µl). The cells were then incubated at 37°C for 2 h. Subsequently, 50 µl were taken from the apical and basal compartments and added to scintillation fluid, and the amount of [3H]mannitol was determined. Inert probe penetration was calculated as the total amount of [3H]mannitol in the basal well divided by that in the apical well at the start of the experiment (17).
Studies to assess influence of cyclooxygenase products on secretory
response.
Myofibroblasts were grown to confluence in tissue culture flasks in
10% FCS-DMEM. After they were extensively washed (with 0.1%
FCS-DMEM), the cells were cultured for 24 h in 0.1% FCS-DMEM, the
first 2 h of this culture in the absence or presence of the cyclooxygenase inhibitor piroxicam
(105 M). The
myofibroblast-conditioned medium was filtered and stored at
70°C until used for electrophysiological studies on HCA-7 cells. To show that piroxicam was indeed able to suppress
PGE2 production under these
conditions, myofibroblasts were grown to confluence in 12-well plates
(Costar). After three washes in prewarmed (to 37°C) medium, the
cells were cultured in quadruplicate in DMEM-0.1% FCS alone or with
added piroxicam at a final concentration of
10
9-10
5
M. After culture for 2 h, cells in the wells were washed and the
myofibroblast monolayers were cultured in fresh medium (0.1% FCS-DMEM,
500 µl/well). After culture for a further 22 h, cell supernatants
were collected and, after centrifugation at 10,000 rpm, stored at
70°C until assayed for
PGE2 by a specific ELISA (Biotrak,
Amersham International, Slough, UK).
RNA isolation and reverse transcription.
RNA was isolated from HCA-7 and T84 cells with use of RNAzolB
(Biogenesis, Poole, UK). Random hexamer primer (Pharmacia Biotech, Brussels, Belgium) was mixed with 10 µg of RNA (final volume 37.5 µl), heated to 70°C for 10 min, and allowed to cool on ice.
Reverse transcription to cDNA was performed by addition of 5 µl of
10× PCR 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's murine leukemia virus RT
(200 U/µl; GIBCO BRL), and 5 µl of 0.1 M dithiothreitol and
incubation at 37°C for 60 min. Subsequent enzyme deactivation was
performed by heating to 90°C for 5 min, and the cDNA was stored at
20°C.
PCR. 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, Madsion, WI), 6 µl of 2 mM MgCl2, 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 mM) on the basis of published nucleotide sequences (10, 16): 5'-GAG TCT TTC TCC AAC GTG ACG-3' (sense) and 5'-ACC TGG TAC TTG AGT TTC CCA-3' (antisense) to amplify the 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 the 232-bp COX-2 product, and 5'-GGT GAA GGT CGG AGT CAA CGG-3' (sense) and 5'-GAG GGA TCT CGC TCC TGG AAG A-3' (antisense) to amplify the 240-bp glyceraldehyde 3-phosphate dehydrogenase product.
The PCR was performed using a Trio-Thermoblock (Biometra, Goettingen, Germany), with the PCR cycle 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 the reaction mixtures were further heated to 54°C for 2 min and 72°C for 3 min to ensure that all the amplified DNA was fully double stranded. The PCR products (12 µl) were added to 3 µl of gel loading buffer (Sigma Chemical) and electrophoresed on a 2% agarose gel containing 0.5 µg/ml ethidium bromide (Sigma Chemical) in Tris-boric acid-EDTA buffer. The specifities of RT-PCR for COX-1, COX-2, and glyceraldehyde 3-phosphate dehydrogenase have previously been confirmed by sequencing of the PCR products and/or hybridization with probes specific to the relevant amplified sequences.Materials.
Bradykinin, carbachol, piroxicam,
[3H]mannitol, DMEM,
and FCS were purchased from Sigma Chemical, NEAA from GIBCO BRL, and rTGF-1 and panspecific antibody
(rabbit) to TGF-
from R & D Systems.
Statistical analysis. Values are means ± SE. ANOVA and two-tailed Student's t-test were used to determine the significance of differences between means. P < 0.05 was accepted as statistically significant.
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RESULTS |
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Morphology of myofibroblast-epithelial cocultures.
Transmission electron microscopy of cocultures of HCA-7 cells and
colonic myofibroblasts showed that each cell type remained confined to
either side of the membrane filter. Small processes of myofibroblasts
were seen in membrane pores but did not reach the epithelial cells on
the other side of the membrane (Fig.
1A). In
cocultures, myofibroblasts retained their ultrastructural
characteristics of abundant rough endoplasmic reticulum and
longitudinally arranged bundles of microfilaments below the cell
membrane (Fig. 1C). HCA-7 cells grew
as a polarized monolayer of epithelial cells on the filter with apical
microvilli and tight junctions (Fig.
1B).
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Effect of myofibroblasts on transepithelial resistance in epithelial
cells.
Transepithelial resistance was measured in the Ussing chamber by
application of a voltage pulse (2 mV) after equilibration of the
epithelium. The filter membrane alone showed a resistance of 12 ± 0.5 · cm2,
which was not significantly different from that of a confluent monolayer of colonic myofibroblasts grown on one side of the membrane (membrane and myofibroblasts: 10.5 ± 0.4
· cm2).
These studies demonstrate that subepithelial colonic myofibroblasts did
not contribute to transepithelial resistance (Fig.
2A).
HCA-7 cells alone exhibited a mean resistance of 140 ± 12.4
· cm2 when
grown to confluence on the membrane filter. In cocultures of HCA-7
cells and colonic myofibroblasts, transepithelial resistance was
significantly increased to 246.7 ± 20.4
· cm2
(P < 0.01).
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Effect of myofibroblast-conditioned medium on mannitol flux.
The modulatory effect of secreted factors of colonic myofibroblasts on
epithelial barrier function was further assessed by using the inert
marker [3H]mannitol.
In preliminary studies, full equilibration was achieved at 1 h after
application of
[3H]mannitol to the
membrane filter alone and on subconfluent monolayers of HCA-7 cells
(data not shown). Compared with control, confluent monolayers of HCA-7
cells precultured (for 24 h) with conditioned medium of myofibroblasts
showed a lag in penetration of the inert marker mannitol from the
apical to the basal compartment (Fig. 3).
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Role of myofibroblast-derived TGF- in
transepithelial resistance.
Human colonic subepithelial myofibroblasts have recently been shown to
release bioactive TGF-
(28). Its role in myofibroblast-mediated enhancement of transepithelial resistance in HCA-7 monolayers was
therefore investigated. The presence of a panspecific antibody to
TGF-
significantly antagonized the effect of
myofibroblast-conditioned medium on transepithelial resistance in HCA-7
cells (expressed as relative resistance; Fig.
4A).
Moreover, rTGF-
also significantly enhanced transepithelial
resistance in HCA-7 monolayers.
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Effect of myofibroblasts on agonist-stimulated ion tranport in HCA-7
cells.
To investigate the effect of colonic myofibroblasts on
secretagogue-induced ion transport, monolayers of HCA-7 cells, alone and in culture with the myofibroblasts, were stimulated with bradykinin (106 M) and carbachol
(10
4 M). As shown in Fig.
5A,
bradykinin- and carbachol-induced
SCC in HCA-7 monolayers was
markedly reduced when the epithelial cells were grown in coculture with
myofibroblasts: 44 ± 4% of control values
(P < 0.04) for bradykinin and 66 ± 5% of control values (P < 0.03) for carbachol.
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Effect of myofibroblasts on agonist-stimulated ion transport in T84
cells.
With the same experimental design used for HCA-7 cells, we investigated
the effect of myofibroblasts on T84 cells. T84 cells were grown on
filters to confluence, alone or in culture with human colonic
myofibroblasts, and were subsequently transferred into an Ussing
chamber. In contrast to HCA-7 cells, the secretory responses to
bradykinin (106 M) and
carbachol (10
4 M) were
significantly (P < 0.01) upregulated
in T84-myofibroblast cocultures compared with T84 monolayers alone
(Fig. 5B).
Role of myofibroblast-derived cyclooxygenase products.
Human colonic subepithelial myofibroblasts have previously been shown
to express functional COX-1 and COX-2 enzymes (24). Studies were
performed to determine whether cyclooxygenase products could be
responsible for the myofibroblast-mediated regulation of resistance and
secretory response. Colonic myofibroblasts were precultured (for 2 h)
with 105 M piroxicam, and
after they were washed extensively the cells were cultured for a
further 22 h in fresh medium only (0.1% FCS-DMEM). Cyclooxygenase
inhibition in the myofibroblasts, by piroxicam pretreatment, was
confirmed by a dose-dependent reduction in
PGE2 release (Fig.
6B).
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Expression of COX-1 and COX-2 in epithelial cells.
In view of the opposite effects of colonic myofibroblasts on
agonist-induced secretory responses in HCA-7 and T84 monolayers, the
expression of COX-1 and COX-2 enzymes in these epithelial cells was
investigated. Studies by RT-PCR showed that although HCA-7 cells
expressed transcripts for COX-1 and COX-2, T84 cells used in our
studies did not express transcripts for either isoform of the
cyclooxygenase enzyme (Fig. 7). The absence
of functional cyclooxygenase enzymes in our T84 cells was confirmed by
the lack of detectable PGE2 in
their supernatants. As previously shown (3, 6),
PGE2 was present in supernatants
of HCA-7 monolayers (data not shown).
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DISCUSSION |
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In this study we have investigated the role of normal primary adult
human colonic subepithelial myofibroblasts in the regulation of barrier
function and electrogenic chloride secretion in a model of the human
colonic epithelium. Pure cultures of normal colonic subepithelial
myofibroblasts were established using a recently described technique in
which mucosal samples denuded of epithelial cells are maintained in
culture (24). During culture, subepithelial myofibroblasts migrate out
of the lamina propria via basement membrane pores and are subsequently
established in the culture dish. They express functional COX-1 and
COX-2 enzymes, bioactive TGF-, and extracellular matrix proteins
(24, 28). Despite prolonged culture and passage, they retain their
phenotype and functional properties, such as expression of COX-1 and
COX-2 enzymes (24). Therefore, they are suitable for functional studies
of their interactions with epithelial cells.
In studies using HCA-7 epithelial cells, we have shown that the colonic
myofibroblasts enhance epithelial barrier function and downregulate
agonist-induced secretory responses. In studies performed after
coculture of cells on permeable supports, myofibroblasts were shown to
enhance transepithelial resistance in HCA-7 monolayers. That this
effect was due to secreted products was confirmed by studies in which
myofibroblast-conditioned medium was used; this medium also delayed
penetration of
[3H]mannitol to the
basolateral compartment. The enhancement of transepithelial resistance
by the myofibroblast-conditioned medium was not affected by
pretreatment of the cells with piroxicam, a cyclooxygenase inhibitor.
In contrast, preincubation of the conditioned medium with panspecific
antibody to TGF- abolished the myofibroblast-mediated enhancement of
transepithelial resistance. The secretion of bioactive TGF-
by the
myofibroblasts was demonstrated using the Mv1Lu bioassay. Recent
studies have shown that the predominant isoform of TGF-
secreted by
normal human colonic subepithelial myofibroblasts is
TGF-
3 (28). In the present
study we also showed that
rTGF-
1 enhances transepithelial
resistance in HCA-7 monolayers. In studies by other investigators,
TGF-
has also been reported to maintain and/or enhance epithelial
barrier function (29, 31).
The mechanism by which TGF- enhances epithelial barrier function
remains to be determined. TGF-
also has other potent biological effects on epithelial cells. Thus it has been shown to play a central
role in intestinal epithelial restitution (10), to inhibit cell
proliferation, and to regulate the synthesis and deposition of
extracellular matrix (16, 27, 35). It can be postulated that, after
exposure to luminal contents, because of increased epithelial
permeability, subepithelial myofibroblasts would be stimulated to
release bioactive TGF-
, which would act as a paracrine factor in the
recovery of epithelial barrier function. This hypothesis is supported
by studies in which colonic mucosa affected by ulcerative colitis and
Crohn's disease showed increased expression of TGF-
mRNA, with the
highest concentration of transcript localized to cells closest to the
luminal surface (1). Therefore, TGF-
appears to be a key cytokine
that facilitates recovery of epithelial function during periods of
active inflammation.
Bradykinin and carbachol induce a defined secretory response in HCA-7
cells, as demonstrated by SCC, which has been shown to reflect
epithelial chloride secretion (21). In this study, primary normal
colonic myofibroblasts, in coculture and via conditioned medium,
reduced bradykinin- and carbachol-induced
SCC in HCA-7 monolayers.
This effect of the myofibroblasts was abolished after their
pretreatment with piroxicam, which led to reduced
PGE2 release. These studies
suggest that, in contrast to its effects on barrier function,
myofibroblast modulation of chloride secretion in HCA-7 monolayers is
mediated via cyclooxygenase products.
In previous studies, fibroblasts have been shown to enhance
agonist-induced SCC in T84 epithelial monolayers (5), and this
effect was abolished by the cyclooxygenase inhibitor indomethacin. Similar effects by myofibroblast cell lines (derived from neonatal intestine) on T84 cells have also been reported (14). Our study also
showed that primary adult human colonic myofibroblasts enhance agonist-induced
SCC in T84 cells. Thus we have found opposing effects of the myofibroblasts on agonist-induced
SCC in T84 and HCA-7 monolayers. To investigate this further, expression of COX-1 and
COX-2 by the epithelial cells was studied. In contrast to HCA-7 cells,
T84 cells used in our studies did not express COX-1 or COX-2
transcripts, nor did they release
PGE2. In other studies on T84
cells, expression of COX-1 (but not COX-2) mRNA transcripts, with very
limited prostaglandin production, has been reported (41). In addition,
bradykinin has been reported to induce a slow increase in SCC in T84
cells, with limited prostaglandin production (14). HCA-7 cell
expression of COX-1 and COX-2 enzymes and of high levels of
cyclooxygenase products has also been reported (3, 6). In HCA-7 cells,
bradykinin stimulates chloride secretion mediated intracellularly by
calcium and cAMP, and induction of the latter appears to be mediated
via eicosanoid production by the epithelial cells (21).
It is possible that the myofibroblast-derived
PGE2 suppresses agonist-induced
SCC in HCA-7 cells by suppressing eicosanoid production by the
epithelial cells. Support for such an explanation is provided by recent
studies in J774 macrophages in which exogenous PGE2 dose dependently suppressed
COX-2 expression (30). We postulate that myofibroblast-derived
PGE2 may similarly suppress
chloride secretion in HCA-7 cells by suppressing expression of COX-2.
The myofibroblast-mediated enhancement of agonist-induced
SCC in T84
cells could be explained by the lack of expression of COX-2, with
absent or limited expression of COX-1 in these epithelial cells.
In contrast to COX-1, COX-2 is the inducible form of the enzyme in many cells. COX-2 protein is not expressed by normal colonic epithelial cells in vivo, but its expression is induced in colonic epithelial cells in active inflammatory bowel disease (38). Thus T84 cells may reflect responses of epithelial cells of the normal colonic mucosa, whereas HCA-7 monolayers may reflect those of epithelial cells in the inflamed mucosa, where chloride secretion likely plays an important role in fluid secretion and diarrhea (32, 37).
In conclusion, our studies have shown that primary adult human colonic
subepithelial myofibroblasts enhance barrier function and modulate
electrogenic chloride secretion in intestinal epithelial monolayers.
The myofibroblast-induced enhancement of barrier function was mediated
by TGF-. By contrast, the modulation of agonist-induced
SCC was
mediated by myofibroblast-derived cyclooxygenase products. These
studies show that colonic myofibroblasts regulate two important functions of epithelial cells via distinct secretory products that
would interact with the basal surface of epithelial cells in vivo via
pores in the basement membrane (24).
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ACKNOWLEDGEMENTS |
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We thank Trevor Gray for assistance with the transmission electron microscopy. HCA-7 colony 29 cells were a kind gift from Dr. Susan Kirkland (London, UK).
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FOOTNOTES |
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J. Beltinger was supported by a grant of the Swiss National Science Foundation and the Novartis Stiftung (formerly Ciba-Geigy-Jubilaeums-Stiftung), Switzerland. B. C. McKaig was supported by The Digestive Disorders Foundation (United Kingdom) and The National Association for Colitis and Crohn's Disease (United Kingdom). Equipment funded by a grant from the Wellcome Trust was used for the electron microscopy studies.
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, Div. of Gastroenterology, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Received 4 December 1998; accepted in final form 5 April 1999.
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