Human colonic subepithelial myofibroblasts modulate transepithelial resistance and secretory response

J. Beltinger, B. C. McKaig, S. Makh, W. A. Stack, C. J. Hawkey, and Y. R. Mahida

Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta (TGF-beta ). 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-beta (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-beta . 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-beta ; prostaglandins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta (TGF-beta ) (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-beta 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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -70°C until used for studies on HCA-7 cells.

Electron 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 (Omega  · cm2) were measured after the monolayers were allowed to equilibrate for 15 min. Peak change in SCC (Delta 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.

Studies were also performed using an ENDOHM chamber connected to an epithelial voltohmmeter (World Precision Instruments). At electrical confluence the monolayers were washed with fresh medium and incubated for 24 h with medium containing 0.1% FCS. The electrical potential difference across the monolayer after passage of a defined current pulse was measured and expressed as Omega  · cm2. Change in electrical resistance across each monolayer was determined serially in a sterile manner, and the value (in Omega  · cm2) just before addition of a potential modulator served as the control value for that monolayer. For calculation of relative monolayer resistance from the electrical resistance, the control value was set at 1.0. Monolayers were incubated with 1) control medium (0.1% FCS-DMEM), 2) conditioned medium of myofibroblasts, 3) conditioned medium of myofibroblasts preincubated for 1 h with a panspecific antibody (rabbit) to TGF-beta (R & D Systems) at a concentration of 5 µg/ml, and 4) recombinant TGF-beta 1 (rTGF-beta 1; R & D Systems) at 5 ng/ml. Monolayer resistance was measured after 24 h.

TGF-beta bioassay. The presence of bioactive TGF-beta in myofibroblast-conditioned medium was determined using a specific bioassay (25, 28) that is based on the ability of TGF-beta to inhibit proliferation of the mink lung epithelial cell line MvLu (ECACC, Porton Down, UK). 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, then neutralized with NaOH and HEPES (to a final concentration of 16 mmol/l).

Mv1Lu cells (in 0.2% FCS-DMEM) were seeded at 5 × 104/well in 24-well cell culture plates (Nunc, GIBCO BRL). Four hours after seeding, 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 a further 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 using a beta counter (LKB, Wallac, Milton Keynes, UK). From the standard curve obtained, the concentration of total and biologically active TGF-beta present in the myofibroblast-conditioned medium could be calculated. Conditioned medium from confluent HCA-7 monolayers and myofibroblast-HCA-7 cocultures was also assessed for TGF-beta bioactivity.

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 (10-5 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).

For electrophysiological studies, monolayers of HCA-7 cells were pretreated (for 24 h) with 1) control medium (0.1% FCS-DMEM), 2) conditioned medium of untreated myofibroblasts, or 3) conditioned medium of piroxicam-pretreated myofibroblasts.

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-beta 1 and panspecific antibody (rabbit) to TGF-beta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Transmission electron micrographs of cocultures of HCA-7 cells and primary adult human colonic subepithelial myofibroblasts. A: each cell type remained confined to either side of membrane filter. Small processes of myofibroblasts are seen in membrane pores but do not reach epithelial cells. B: HCA-7 cells grow as a polarized monolayer of epithelial cells on filter with apical microvilli and tight junctions (arrow). C: myofibroblasts retain their ultrastructural characteristics of abundant rough endoplasmic reticulum and longitudinally arranged bundles of microfilaments below cell membrane.

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 Omega  · 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 Omega  · 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 Omega  · 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 Omega  · cm2 (P < 0.01).


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Fig. 2.   Colonic myofibroblasts upregulate transepithelial resistance in HCA-7 monolayers. A: confluent monolayers of myofibroblasts did not contribute to barrier function, as illustrated by lack of a difference in resistance between membrane filter alone and filter with a confluent monolayer of myofibroblasts. Resistance of HCA-7 monolayers was significantly enhanced in coculture with colonic myofibroblasts. Values are means ± SE; n = 18. B: HCA-7 monolayers preincubated with myofibroblast-conditioned medium (MFCM) for 24 h also showed increased resistance. Values are means ± SE; n = 6.

To determine whether the enhanced transepithelial resistance in cocultures was mediated by factors secreted by myofibroblasts, HCA-7 monolayers were studied after incubation in myofibroblast-conditioned medium or control medium (0.1% FCS-DMEM) for 24 h. In paired experiments, monolayers incubated in control medium (0.1% FCS-DMEM) showed a resistance of 138 ± 11.4 Omega  · cm2, whereas in monolayers treated with myofibroblast-conditioned medium (0.1% FCS-DMEM), the resistance increased significantly to 180.7 ± 7.1 Omega  · cm2 (P = 0.01; Fig. 2B).

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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Fig. 3.   [3H]mannitol flux studies. Compared with control medium, confluent monolayers of HCA-7 cells precultured (for 24 h) with conditioned medium of myofibroblasts showed a time-dependent lag in penetration of inert marker [3H]mannitol from apical to basal compartment. Penetration of mannitol to basal compartment is expressed as a percentage of that originally applied to apical compartment. Values are means ± SE; n = 6. # P < 0.01; * P < 0.03.

Role of myofibroblast-derived TGF-beta in transepithelial resistance. Human colonic subepithelial myofibroblasts have recently been shown to release bioactive TGF-beta (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-beta significantly antagonized the effect of myofibroblast-conditioned medium on transepithelial resistance in HCA-7 cells (expressed as relative resistance; Fig. 4A). Moreover, rTGF-beta also significantly enhanced transepithelial resistance in HCA-7 monolayers.



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Fig. 4.   A: role of TGF-beta in upregulation of transepithelial resistance. Change in electrical resistance across each monolayer was determined, and value (in Omega  · cm2) just before addition of a potential modulator served as control value. For calculation of relative monolayer resistance, control value was set at 1.0. Monolayers were incubated with control medium (0.1% FCS-DMEM), conditioned medium of myofibroblasts, conditioned medium of myofibroblasts preincubated for 1 h with a panspecific antibody to transforming growth factor-beta (TGF-beta , at 5 µg/ml), and recombinant TGF-beta 1 (at 5 ng/ml). Values are means ± SE; n = 11. # P < 0.05, control vs. TGF-beta 1; * P < 0.01, myofibroblast-conditioned medium vs. myofibroblast-conditioned medium + panspecific antibody to TGF-beta . B: assessment of TGF-beta bioactivity in conditioned medium by use of Mv1Lu bioassay. Conditioned medium was obtained after HCA-7 cells or myofibroblasts were cultured for 24 h with and without panspecific antibody to TGF-beta . IgG was used as a nonspecific control antibody. Acidification showed a small increase in TGF-beta bioactivity in myofibroblast-conditioned medium, implying that majority of secreted TGF-beta is in biologically active form. TGF-beta bioactivity was reduced after preincubation with a panspecific (rabbit) antibody to TGF-beta (* P < 0.05), but control rabbit IgG had no effect.

The presence of bioactive TGF-beta in myofibroblast-conditioned medium was confirmed using the Mv1Lu bioassay (Fig. 4B). Compared with myofibroblast-conditioned medium, there was very little TGF-beta bioactivity in untreated conditioned medium of HCA-7 cells alone. After acidification, only a small increase in TGF-beta bioactivity was detected in the myofibroblast-conditioned medium, implying that the majority of TGF-beta secreted by the myofibroblasts is in the biologically active form. This TGF-beta bioactivity was significantly abrogated by panspecific antibody to TGF-beta (rabbit), whereas control rabbit IgG had no effect.

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 (10-6 M) and carbachol (10-4 M). As shown in Fig. 5A, bradykinin- and carbachol-induced Delta 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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Fig. 5.   A: colonic myofibroblasts downregulate agonist-induced electrogenic chloride secretion in HCA-7 cells. Agonist-induced maximal change in short-circuit current (Delta SCC) in HCA-7 cells is expressed as percentage of control monolayers. There was a significant reduction in bradykinin- or carbachol-induced chloride secretory response when HCA-7 monolayers were grown with myofibroblasts (MF) in coculture (n = 20, P < 0.04 and 0.03, respectively) or preexposed to myofibroblast-conditioned medium (n = 6, P < 0.04 and 0.03, respectively). B: colonic myofibroblasts upregulate agonist-induced electrogenic chloride secretion in T84 cells. Compared with control monolayers, there was a significant increase in bradykinin- or carbachol-induced chloride secretory response when T84 cells were grown with myofibroblasts in coculture. Values are means ± SE of 6 cultures. * P < 0.01.

To determine whether secreted factor(s) from myofibroblasts contributed to the reduction in bradykinin- and carbachol-induced Delta SCC, HCA-7 monolayers were precultured for 24 h in myofibroblast-conditioned medium before electrophysiological studies. Compared with control medium, there was a significant reduction in bradykinin- and carbachol-induced Delta SCC in HCA-7 monolayers pretreated with myofibroblast-conditioned medium: 68 ± 5% of control values (P < 0.04) for bradykinin and 61 ± 14% of control values (P < 0.03) for carbachol (Fig. 5A).

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 (10-6 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 10-5 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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Fig. 6.   A: piroxicam pretreatment of colonic myofibroblasts reverses myofibroblast-mediated downregulation of electrogenic chloride secretion in HCA-7 cells. Myofibroblasts were cultured with 10-5 M piroxicam for 2 h; after they were washed extensively, myofibroblasts were cultured in fresh medium (0.1% FCS-DMEM) for 22 h. In matched experiments (n = 4), HCA-7 monolayers were cultured for 24 h with control medium (0.1% FCS-DMEM), conditioned medium of untreated myofibroblasts (MFCM), or conditioned medium of prioxicam-pretreated myofibroblasts (MFCM + Pirox). Delta SCC is expressed relative to that in control monolayers. * P < 0.05, MFCM vs. MFCM + Pirox. B: inhibition of PGE2 production in human colonic subepithelial myofibroblasts pretreated with piroxicam. Confluent monolayers of myofibroblasts were incubated for 2 h with control medium only (0.1% FCS-DMEM) or with different concentrations of piroxicam. After they were washed, cells were incubated in fresh medium (0.1% FCS-DMEM) for further 22 h. Prostaglandin E2 (PGE2) levels in supernatants were assayed by ELISA.

Piroxicam pretreatment did not affect the myofibroblast-mediated enhancement of transepithelial resistance in HCA-7 monolayers. The resistance was 221 ± 56 Omega  · cm2 in HCA-7 monolayers precultured (for 24 h) in control medium, 393 ± 24 Omega  · cm2 for conditioned medium of untreated myofibroblasts, and 431 ± 36 Omega  · cm2 for conditioned medium of myofibroblasts pretreated with 10-5M piroxicam [n = 5, control medium vs. myofibroblast (untreated or piroxicam pretreated)-conditioned medium, P < 0.01].

However, piroxicam pretreatment reversed myofibroblast-mediated downregulation of agonist-stimulated Delta SCC in HCA-7 cells. Thus bradykinin- and carbachol-induced Delta SCC in HCA-7 monolayers precultured with conditioned medium of piroxicam-pretreated myofibroblasts was not significantly different from that with control medium (0.1% FCS-DMEM; Fig. 6A).

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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Fig. 7.   Expression of mRNA transcripts for cyclooxygenase-1 and -2 (COX-1 and COX-2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HCA-7 and T84 cells. RNA was isolated from confluent monolayers of cells and reverse transcribed; relevant transcripts were amplified by PCR with use of specific primers. DNA size markers are indicated on left. In contrast to HCA-7 cells, neither COX-1 nor COX-2 transcripts were present in T84 cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta , 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-beta abolished the myofibroblast-mediated enhancement of transepithelial resistance. The secretion of bioactive TGF-beta by the myofibroblasts was demonstrated using the Mv1Lu bioassay. Recent studies have shown that the predominant isoform of TGF-beta secreted by normal human colonic subepithelial myofibroblasts is TGF-beta 3 (28). In the present study we also showed that rTGF-beta 1 enhances transepithelial resistance in HCA-7 monolayers. In studies by other investigators, TGF-beta has also been reported to maintain and/or enhance epithelial barrier function (29, 31).

The mechanism by which TGF-beta enhances epithelial barrier function remains to be determined. TGF-beta 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-beta , 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-beta mRNA, with the highest concentration of transcript localized to cells closest to the luminal surface (1). Therefore, TGF-beta 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 Delta 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 Delta 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 Delta 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 Delta SCC in T84 cells. Thus we have found opposing effects of the myofibroblasts on agonist-induced Delta 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 Delta 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 Delta 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-beta . By contrast, the modulation of agonist-induced Delta 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).


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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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