Effects of growth factors and trefoil peptides on migration and replication in primary oxyntic cultures

Kimitoshi Kato1, Monica C. Chen1, Minh Nguyen1, Frank S. Lehmann1, Daniel K. Podolsky2, and Andrew H. Soll1

1 CURE: Digestive Diseases Research Center, West Los Angeles Veterans Affairs Medical Center, University of California Los Angeles School of Medicine, Los Angeles, California 90073; and 2 Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Restitution, the lateral migration of cells over an intact basement membrane, maintains mucosal integrity. We studied the regulation of migration and proliferation of enzyme-dispersed canine oxyntic mucosa cells in primary culture. Confluent monolayers were wounded and cultured in serum-free medium, and cells migrating into the wound were counted. [3H]thymidine incorporation into DNA was studied using subconfluent cultures. Considerable migration occurred in untreated monolayers; however, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha , basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), two trefoil peptides, and interleukin (IL)-1beta further enhanced migration. The specific EGF receptor (EGFR) monoclonal antibody, MAb-528, inhibited both basal and TGF-alpha - or IL-1beta -stimulated migration, but not the response to trefoil peptide, bFGF, or IGF-I. Exogenous TGF-beta inhibited cell proliferation but did not alter migration. Immunoneutralization with anti-TGF-beta blocked the response to exogenous TGF-beta and produced a small enhancement of basal thymidine incorporation but did not attenuate basal or TGF-alpha -stimulated migration. In conclusion, endogenous EGFR ligands regulate proliferation and migration. TGF-beta inhibits mitogenesis; it did not upregulate migration in these cultures. Although bFGF, IGF-I, and IL-1beta enhance gastric epithelial migration, only IL-1beta acted in a TGF-alpha -dependent fashion.

gastric mucosa; thymidine incorporation; epidermal growth factor receptors; epidermal growth factor receptor antibody; peptic ulcer; cytokines


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTEGRITY OF the gastric mucosa is maintained in the face of continual mechanical, chemical, and inflammatory insult by a summation of a series of defense and repair processes (1, 45): 1) defense mechanisms reducing cell injury (mucous and bicarbonate secretion, intrinsic epithelial cell mechanisms, and mucosal blood flow), 2) epithelial repair mechanisms operative over largely intact basement membrane, and 3) wound healing processes that reconstitute connective tissue and restore a basement membrane allowing epithelial regeneration. The epithelial repair mechanisms operative over intact basement membrane include migration, the lateral movement of cells to fill in gaps created by sloughed cells, and subsequent enhanced cell replication to restore mucosal mass (28, 37, 46).

Restitution in gastrointestinal mucosa has been characterized by studying intact mucosa after ethanol injury (28, 46) and cell lines, such as those derived from rat intestinal mucosa (IEC-6) and human colon carcinoma (T84; see Refs. 10, 30, 37, 51). Elegant studies with IEC-6 cells implicated transforming growth factor (TGF)-beta as a critical modulator of migration. For example, expression of TGF-beta was induced by TGF-alpha , and the action of TGF-alpha was blocked by immunoabsorption with TGF-beta antibody (10, 12). However, there is little information on the actions of potential autocrine and paracrine factors controlling migration in primary gastric epithelial cells. Recently, we have adapted methods to study migration (10, 30, 37, 51) in gastric mucosa using primary cultures of canine cells dispersed with enzymes (26). Studying monolayers formed from these cell preparations, we assessed migration into a wounded zone created by a razor blade scrape. We found a considerable contrast between regulation of migration in this system and findings in the IEC-6 rat intestinal cell line (10, 12). In our primary canine gastric cultures, although TGF-beta potently inhibited thymidine incorporation, it did not enhance migration. Furthermore, immunoneutralization with antibody to TGF-beta did not impair migration in untreated cells or reduce the response to TGF-alpha . Also, in contrast to observations in IEC-6 cells, we found that immunoblockade of epidermal growth factor (EGF) receptors (EGFR) reduced the high rate of spontaneous migration in untreated monolayers, suggesting that endogenous ligands for the EGFR drive migration under these conditions.

To gain further understanding of the elements maintaining gastric mucosal integrity, we compared effects on migration and growth of other endogenous growth factors: insulin-like growth factor I (IGF-I; see Ref. 36) and basic fibroblast growth factor (bFGF; see Refs. 13, 38); trefoil peptides (13, 19, 40): human spasmolytic peptide (hSP) and rat intestinal trefoil peptide (rITF); and cytokines (31). We selected these three categories of factors based on their presence in or delivery to gastric mucosa in the normal or inflamed state and their ability to influence epithelial proliferation or restitution (11-13, 38). We utilized canine gastric epithelial cells in short-term primary culture in anticipation that primary gastric cultures will provide a more relevant model for regulatory events in gastric mucosa than will data obtained from cell lines, especially if derived from intestinal or transformed cells. Another advantage of our culture system is that considerable data have already been collected on the control of cell replication in these cultures (6, 7).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Materials for cell preparation and culture were obtained from sources outlined previously (7). Human recombinant EGF and TGF-beta were purchased, respectively, from Amgen (Thousand Oaks, CA) and Bachem (Torrance, CA); porcine TGF-beta 1 was purchased from R&D Systems (Minneapolis, MN); turkey anti-TGF-beta 1 was from Collaborative Research (Bedford, MA); sheep anti-TGF-alpha was from East Acres Biologicals (Southbridge, MA); monoclonal antibody 528 (MAb-528) against EGFR (15, 18) was from Oncogene Science (San Diego, CA), and Matrigel was from Collaborative Research. Human recombinant bFGF was purchased from R&D Systems, and IGF-I was from Bachem. Recombinant hSP and rITF were the generous gifts of Dr. D. Podolsky (40). Human recombinant interferon-gamma (IFN-gamma ) and tumor necrosis factor-alpha (TNF-alpha ) were from Biosource (Camarillo, CA); human recombinant interleukin (IL)-1beta , IL-1alpha , and IL-6 were from Genzyme (Cambridge, MA). Other chemicals were purchased from Sigma (St. Louis, MO).

Tissue dispersion, cell separation, and culture. Canine oxyntic mucosa was dispersed with collagenase and EDTA and filtered through a nylon mesh, and material retained on a fine nylon mesh was incubated in dithiothreitol and deoxyribonuclease, as previously described (7). The resulting cell suspension was then elutriated to enrich glandular cells (parietal and chief cells) and remove endocrine, vascular endothelial, and surface epithelial cells, as well as bacteria (8). For these studies, cells with apparent cell size (estimated from sedimentation velocity) ranging from 14 to 22 µm were collected using a Beckman elutriator (rotor JE-5.0, run in a J-6B centrifuge). This cell fraction was then suspended in DMEM-Ham's F-12 in a 1:1 mixture, supplemented with 20 mM HEPES, 100 µg/ml amikacin, 100 U/ml penicillin, and 100 mg/ml streptomycin; we refer to this medium without added serum or growth factors as R0. Calf serum (2%) was added to R0 for the initial period of culture, but cultures were placed in serum-free conditions before testing.

Proliferation assay. For these studies, cells at low density (0.35 × 106 in 0.5 ml) were plated onto polymerized type I rat tail collagen in 24-well tissue culture plates, and incorporation of [3H]thymidine into DNA was performed, as previously described (7). In brief, cells were washed and incubated for an additional 36 h in fresh R0 medium in the presence of [3H]thymidine (0.5 µCi/ml). For studies with glycosylated hSP, rITF, bFGF with heparin (125 mg/ml), and IGF-I, a 36-h incubation period was used, whereas studies with IL-1alpha , IL-1beta , IL-6, IFN-gamma , and TNF-alpha were done using a 72-h incubation period.

Migration assay. Wound assays were performed using methods adapted from others (12, 30). Higher-density cells (1.4 × 106 in 1 ml) were plated on Matrigel-coated six-well tissue culture plates (diameter 35 mm) or 35-mm petri dishes. Confluent monolayers formed after 40-48 h of culture in R0 with 2% calf serum. Monolayers were then washed in serum-free R0 and scraped with a single-edge razor blade (18 mm length). The scrape, which was started at the center of the dish, was extended 6-8 mm, producing a wounded area that was 18 mm × 6-8 mm. To avoid the plastic culture surface from being scratched, the cutting edge was dragged with gentle pressure at an angle. The medium was immediately replaced with fresh, serum-free R0 to remove cellular debris, and the wounded monolayers were cultured for 24 h in the presence or absence of individual growth factors or antibodies, as noted. Cell migration was quantified by counting in a blinded fashion the number of cells observed across a standardized length of wound border. Random photographs were taken of the migration zone in a field 0.9 mm wide, using 100-fold magnification with an inverted microscope Nikon Diaphot TMS and a Nikon N 6006 Camera (Nikon, Garden City, NY). In addition, the mean distance that cells traveled from the edge of the scraped area was measured.

Substrate: Collagen vs. Matrigel coating. Studies of migration and thymidine incorporation were performed using Matrigel-coated and collagen I-coated substrate, respectively; in control studies, we found comparable basal and EGF-stimulated thymidine incorporation and migration with these two substrates (n = 3, P > 0.2, data not illustrated). We used collagen coating for the thymidine studies because of lower cost, whereas Matrigel was used in migration studies for better cell adhesion around the wound edge because patches of cells tend to peel from the cut edge more easily with collagen-coated plates.

Immunohistochemistry. For replicating cells, bromodeoxyuridine (BrdU), a thymidine analog incorporated into newly synthesized DNA, was added to cells at the time of wounding, and cells were incubated for 24 h in the absence or presence of EGF (1 nM). Cells were fixed with Bouin's, and avidin-biotin-peroxidase complex staining (22) was performed using a monoclonal antibody for BrdU (Amersham, Arlington Heights IL), as previously described (7). Immunohistochemistry for H+-K+-ATPase and pepsinogen I were performed as previously described (6). Rabbit anti-human MUC5 and chicken anti-human MUC6 antibodies kindly provided by Dr. S. B. Ho (Veterans Affairs Medical Center and University of Minnesota) were used to identify canine gastric mucous cells (20, 21).

Statistical analysis. Data are expressed as means ± SE of at least three independent experiments, with n equal to the number of separate cell preparations. Statistical significance was assessed using the paired Student's t-test. Repeated- measure ANOVA followed by Dunnett's contrast were used for multiple comparisons to a single basal value. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EGF/TGF-alpha enhance migration. In the 24 h after confluent monolayers were wounded with a razor blade, 170 ± 7.0 (mean ± SE, n = 6) cells migrated into the wound per 0.9 mm micrographic field width, traveling a mean distance of 0.31 ± 0.02 mm from the cutting line (Fig. 1A). Treatment with EGF enhanced migration; 320 ± 21 (mean ± SE, n = 6) cells migrated into the wound (Fig. 1B). Data were comparable calculating either the mean traveling distance from the scratch line or the number of cells migrating over the wound edge. Cell number was chosen for subsequent studies.


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Fig. 1.   Photomicrograph of basal and epidermal growth factor (EGF)-stimulated migration. Cells were plated on 35-mm plastic dishes coated with Matrigel. After razor blade wounding, medium was replaced with medium without serum in the presence or absence of growth factor. After a 24-h period, medium was removed and washed by PBS. Control monolayers are illustrated in A, and 1 nM EGF-treated monolayers are in B. Line depicts the initial cut on the culture plate. Original magnification, ×100.

In time course studies, EGF enhancement of migration was evident at 3 h, but this EGF effect was not statistically significant until 12 h after treatment (Fig. 2A). After 24 h, EGF (1 nM) stimulated epithelial cell migration by 92 ± 6% above basal (mean ± SE, n = 3); subsequent studies were performed at this time point. TGF-alpha stimulated cell migration to a similar maximal extent. These two related peptides stimulated migration over a similar concentration range (1 pM-10 nM; Fig. 2, B and C).


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Fig. 2.   Time course and dose response for EGF effects on migration. A: number of cells migrating in the denuded zone was assessed at 3, 6, 12, and 24 h after wounding in the presence () and absence () of 1 nM EGF. Data, the mean ± SE from 3 preparations, are the number of cells migrating in the photographic field. # P < 0.01 compared with untreated controls at the same time point. B and C: EGF (B) and transforming growth factor (TGF)-alpha (C) were added at the time of wounding. Data are means ± SE from 5 preparations. * P < 0.05 and ** P < 0.01 compared with untreated controls (basal).

Controls differentiating migration and proliferation. Hydroxyurea, which inhibits replication but not migration, was used to discriminate these two processes (35). When studied in the subconfluent cultures, a dose of 20 mM hydroxyurea inhibited basal and EGF-stimulated thymidine incorporation by 84.4 ± 2.0% (mean ± SE, n = 5; Fig. 3A). In contrast, 20 mM hydroxyurea did not impair cell viability or reduce basal or EGF-stimulated migration in wounded confluent monolayers (Fig. 3B).


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Fig. 3.   Hydroxyurea inhibits replication but not migration. A: [3H]thymidine incorporation was studied using nonconfluent cultures exposed to 1 or 10 nM EGF with or without 20 mM hydroxyurea. Data are means ± SE from 5 preparations. ** and # P < 0.01 vs. control without hydroxyurea. cpm, Counts/min. B: cell migration was studied after wounding confluent monolayers; migration in the denuded area was studied for the same treatment groups as in A. Data are means ± SE from 5 preparations. * P < 0.05 compared with control without hydroxyurea. ** P < 0.05 compared with control with 20 mM hydroxyurea.

Thymidine incorporation was also compared in wounded and unwounded confluent monolayers; no significant changes in thymidine uptake were detected as a function of wounding in the presence or absence of EGF or TGF-alpha (Table 1). However, because cell replication may be localized to the cells in the region of the wound, we used BrdU staining to assess the number of cells that had undergone DNA synthesis during the 24-h postwound incubation period. In both control and EGF-treated monolayers, BrdU-positive cells represented a smaller proportion of the migrating than of the nonmigrating cell population (Table 2).

                              
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Table 1.   Thymidine uptake in wounded and unwounded gastric epithelial confluent monolayer


                              
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Table 2.   Proportion of migrating and nonmigrating cells displaying BrdU, H+-K+-ATPase, and pepsinogen immunoreactivity

We also compared the migrating and nonmigrating cell populations using immunohistochemistry for H+-K+-ATPase and pepsinogen (Table 2) as markers for parietal and pepsinogen-containing cells, respectively. The proportion of parietal and pepsinogen-positive cells was comparable in the migrating and nonmigrating populations under both basal and EGF-stimulated conditions, suggesting that gastric glandular and neck cells readily undergo migration in response to wounding.

Basal migration is mediated by endogenous EGFR ligand. A dose response of the monoclonal antibody to EGFR, MAb-528 in migration, and proliferation was established (data not shown). MAb-528 at 20 nM, but not 1 nM, markedly inhibited migration and thymidine incorporation in response to exogenous TGF-alpha (Fig. 4, A and B). MAb-528 also significantly inhibited basal migration in these cultures by ~40% (Fig. 4B), suggesting that endogenous ligands for the EGFR contribute to the high rate of migration in the basal state. A control antibody, monoclonal antibody against somatostatin (CURE S6; see Refs. 9, 52), at the similar dose did not affect either migration or proliferation.


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Fig. 4.   Effects of anti-EGF receptor (EGFR) monoclonal antibody 528 (MAb-528) on basal and TGF-alpha -stimulated thymidine incorporation and migration. A: effects of 1 nM TGF-alpha on thymidine incorporation were studied in the presence (+) or absence of MAb-528 (20 nM) in nonconfluent cultures. B: cultures were examined for migration as noted above for Fig. 2 with or without treatment with 1 nM TGF-alpha in the presence and absence of the anti-EGFR MAb-528 (20 nM), as indicated. Data are means ± SE from 4 preparations. ** and ## P < 0.01 compared with untreated controls.

We also utilized a sheep anti-TGF-alpha antiserum from East Acres to assess regulation of migration. TGF-alpha antiserum (1:125 dilution) attenuated migration in response to exogenous TGF-alpha in a fashion surmountable by higher doses of TGF-alpha (n = 4, P < 0.05, not illustrated). In contrast, EGF effects on migration were not inhibited by this TGF-alpha antiserum (n = 4, P > 0.2, data not illustrated), consistent with the specificity of this antiserum against TGF-alpha and not EGF (6). However, in contrast to MAb-528 (Fig. 4B), TGF-alpha antiserum did not reduce basal migration (n = 4, P > 0.2, data not illustrated). The failure of TGF-alpha antiserum to reduce basal migration may indicate that TGF-alpha is not the endogenous EGFR ligand enhancing migration. However, because MAb-528 appears highly specific in immunoblockade of EGFR, we suspect that another factor in the unpurified TGF-alpha antisera enhances migration, masking effects of immunoneutralizing TGF-alpha . Serum is likely to contain sufficient quantities of components, such as IGF-I, to obviate interpretation. Also in contrast to MAb-528, this TGF-alpha antiserum blocks stimulation of thymidine incorporation by IGF-I and fibroblast growth factor (FGF), probably indicating inhibition of replication by mechanisms other than or in addition to immunoneutralization of TGF-alpha . Clarifying the role of TGF-alpha requires repeated studies using an affinity- or protein A-purified antibody.

Mitogenic responses to endogenous EGFR ligands. We sought comparisons of the regulation of migration with proliferation, confirming our previous findings that EGF and TGF-alpha (1 pM and 1 nM, respectively) produced a dose-dependent increase of [3H]thymidine incorporation by nonconfluent cultures (n = 3, data not shown). We now find that the anti-EGFR antibody MAb-528 produced a small (18 ± 2%, n = 4) but significant reduction in [3H]thymidine uptake in basal conditions and completely blocked TGF-alpha (1 nM)-stimulated thymidine uptake (Fig. 4A).

TGF-beta regulates proliferation but also inhibits migration at high doses. TGF-beta 1 effectively inhibited thymidine incorporation by nonconfluent cultures (Fig. 5A). The effects of exogenous TGF-beta 1 were dose dependent over a concentration range from 1 to 100 pM. Inhibition was profound, maximally inhibiting basal thymidine incorporation by ~85 ± 1% (Fig. 5A). Although clearly active on these cells inhibiting thymidine incorporation, TGF-beta 1 did not stimulate migration. To the contrary, TGF-beta 1 (10-100 pM) significantly inhibited cell migration by ~20-40% (Fig. 5B).


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Fig. 5.   TGF-beta 1 effects on thymidine incorporation and migration. A: effects of TGF-beta 1 on thymidine incorporation were tested at the indicated doses. Cells were studied in the absence of exogenous stimuli; data are means ± SE from 4 experiments. B: effects of the indicated concentrations of TGF-beta 1 were tested on migration. Data are means ± SE from 5 preparations. * P < 0.05 and ** P < 0.01 vs. controls.

To test possible effects of endogenous TGF-beta 1 present in these cultures, we utilized an immunoneutralizing monoclonal antibody against TGF-beta 1 that blocks action of endogenous TGF-beta in IEC-6 cells (10, 12). As a positive control, we confirmed that this monoclonal antibody attenuated inhibition of proliferation by exogenous TGF-beta 1 (Fig. 6A). TGF-beta 1 immunoneutralization caused a small but statistically significant enhancement of basal thymidine uptake (Fig. 6A). Although this antibody blocked action of TGF-beta 1 regulating proliferation, it did not reduce migration either in the basal state or in response to TGF-alpha treatment (Fig. 6B). The control antibody (CURE S6) had no effect in these studies (n = 4, data not shown).


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Fig. 6.   Effects of antibody to TGF-beta 1 on replication and migration. A: inhibition of thymidine incorporation by 1 pM TGF-beta 1 was studied in the presence and absence of immunoneutralizing monoclonal antibody (Ab) to TGF-beta 1 (10 µg/ml). Data are means ± SE from 3 preparations. * and ** P < 0.05 vs. untreated controls. B: effects of TGF-alpha on cell migration were studied in the presence and absence of anti-TGF-beta 1 antibody. Data are means ± SE from 3 preparations. * P < 0.05 vs. untreated controls.

IGF-I and bFGF enhance migration and proliferation. IGF-I and bFGF at concentrations from 1 pM to 1 nM dose dependently increased migration (Fig. 7, B and D). Maximal stimulation of migration over basal was 67 and 92% with IGF-I and bFGF, respectively. Studying nonconfluent cultures, bFGF and IGF-I also enhanced thymidine incorporation in a parallel fashion over a similar concentration range (Fig. 7, A and C), with the data for IGF-I confirming our previous findings (7). Although immunoblockade of the EGFR with MAb-528 (20 nM) attenuated both thymidine incorporation and migration in untreated cultures, it did not attenuate stimulation of either thymidine uptake (Fig. 8A) or migration (Fig. 8B) by IGF-I or bFGF.


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Fig. 7.   Effects of insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) on replication and migration. Dose response for IGF-I on thymidine incorporation (A) and migration (B) are illustrated. Dose responses for the effects of bFGF on thymidine incorporation (C) and migration (D) are also illustrated. Data, expressed as means ± SE, are from 5 identical experiments. * P < 0.05 and ** P < 0.01 vs. basal.



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Fig. 8.   Effects of MAb-528 on proliferative and migration responses to bFGF and IGF-I. Thymidine incorporation (A) and migration (B) are illustrated. For both migration and thymidine incorporation, cultures were studied untreated and in response to 10-9 M bFGF and IGF-I in the absence (open bars) and presence (filled bars) of 20 nM MAb-528. Data are means ± SE from 3 preparations. ** P < 0.01 vs. basal without MAb-528.

Trefoil peptides enhance migration. Glycosylated hSP stimulated cell migration over a concentration range from 0.1 to 1.0 mg/ml (Fig. 9B), producing a maximal 77% increase over basal migration. The nonglycosylated hSP had a similar effect (data not shown) on migration. We only used the glycosylated hSP for detailed dose response and further studies due to the availability. rITF also stimulated migration (Fig. 9D). hSP produced a modest, but statistically significant, increase in thymidine incorporation at a concentration of 0.1 but not 0.5 mg/ml (Fig. 9A). rITF did not enhance thymidine incorporation at the tested doses (Fig. 9C). MAb-528 reduced the overall magnitude of migration in response to hSP (0.5 mg/ml). This decrease was accounted for by the reduced basal migration in MAb-528-treated cells; the hSP-induced increment was comparable in the presence and absence of MAb-528 (Fig. 9E).


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Fig. 9.   Effects of trefoil peptide human spasmolytic peptide (hSP) and rat intestinal trefoil peptide (rITF) on thymidine incorporation and migration. Cultures were studied in the absence or presence of the indicated doses of hSP for thymidine incorporation (A) and migration (B). Effects of rITF on thymidine incorporation (C) and migration (D) are also illustrated. Data are means ± SE from 5 separate cell preparations. * P < 0.05 and ** P < 0.01 vs. untreated controls. E: effect of anti-EGFR antibody on migration response to hSP. Migration was tested as noted in the legend to Fig. 8 in the absence (open bars) and presence (filled bars) of 20 nM MAb-528. Data are means ± SE from 5 separate cell preparations. ** P < 0.01 vs. untreated controls. # P < 0.05 vs. hSP only without MAb-528.

Variable effects on migration and proliferation by cytokines. Effects on migration and proliferation of IL-1alpha , IL-1beta , TNF-alpha , IFN-gamma , and IL-6 were tested over dose ranges from 5 to 100 U/ml. Only IL-1beta , at concentrations from 1 to 50 U/ml, significantly enhanced migration (Fig. 10B). TNF-alpha (5 U/ml) produced a small (20%) inhibitory effect on migration, whereas the other cytokines appeared to be without effect (Fig. 11B). However, all five of these cytokines enhanced thymidine incorporation during a 72-h incubation (Figs. 10A and 11). In contrast to the other factors we studied, trends but not significant effects were observed after a 36-h incubation period.


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Fig. 10.   Effects of interleukin (IL)-1beta on thymidine incorporation and migration. A and B: thymidine incorporation was studied over a 72-h incubation period for these studies with IL-1beta . Data are means ± SE, with n = 5 for A and n = 6 for B. * P < 0.05 and ** P < 0.01 vs. basal. C: effects of EGFR immunoblockade on migration response to IL-1beta . Cultures were studied untreated and in response to 5 U/ml IL-1beta in the absence (open bars) and presence (filled bars) of 20 nM MAb-528. Data are means ± SE from 4 preparations. * P < 0.05, # P < 0.05, and ** P < 0.01 vs. untreated cells. + P < 0.05 vs. IL-1beta in the absence of MAb-528.



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Fig. 11.   Effects of IL-1alpha , IL-6, interferon-gamma (IFN-gamma ), and tumor necrosis factor-alpha (TNF-alpha ) on thymidine incorporation and cell migration. A: thymidine incorporation was studied over a 72-h incubation period with the indicated cytokines over a dose range from 5 to 50 U/ml. Data are depicted for only the 5 U/ml concentration. Data are means ± SE, n = 5. * P < 0.05 and ** P < 0.01 vs. basal. B: migration was studied for the indicated cytokines; the only significant change was the inhibition produced by TNF-alpha . Data are expressed as means ± SE, n = 6. * P < 0.05 vs. basal.

MAb-528 EGFR antibody (20 nM) attenuated stimulation of thymidine incorporation by cytokines IL-1beta , IL-1alpha , TNF-alpha , and IFN-gamma , each studied at 5 U/ml (data not shown). Furthermore, IL-1beta stimulation of migration was completely blocked by MAb-528 (Fig. 10C). In contrast, the control antibody (CURE S6) did not block IL-1beta -enhanced migration (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our data with short-term primary culture of canine gastric cells support the conclusion that endogenous EGFR ligands play an important role regulating cell migration. Not only do EGF and TGF-alpha strongly stimulate migration but blockade of EGFR by the monoclonal antibody MAb-528 reduced cell migration in the basal state by 40%. Blockade of EGFR by MAb-528 in these cultures produced definite but modest inhibition of thymidine incorporation, supporting our previous conclusion that endogenous EGFR ligands regulate cell replication in the basal state (6). We found that EGF and TGF-alpha act with equal potency stimulating migration (present study) and thymidine incorporation (7), contrasting with previous findings with guinea pig gastric mucous cells of Rutten and co-workers (41), which indicated that TGF-alpha was more potent than EGF at gastric mucosal EGFR.

Our conclusions regarding the physiological role of endogenous EGFR ligands in our cultures rest upon the specificity of the EGFR antibody MAb-528, which we carefully assessed. At a concentration of 20 nM, MAb-528 completely blocked stimulation of cell migration and thymidine incorporation by exogenous EGF and TGF-alpha . In contrast, this concentration did not block stimulation of migration or thymidine incorporation by IGF-I, bFGF, or hSP. Based on this specificity, we are confident that the inhibition of basal migration by MAb-528 implicates endogenous EGFR ligands as regulators of migration and proliferation.

We suspect that the endogenous EGFR ligand driving basal migration in these cultures is TGF-alpha . TGF-alpha was found in parietal cells in these cultures, and radioimmunoassay indicated concentrations in culture supernatants of ~100 pM (6), which is sufficient to stimulate both replication and migration. However, the unpurified TGF-alpha antiserum that we used probably has actions independent of TGF-alpha immunoneutralization. Therefore, additional studies will be necessary to confirm the identity of the endogenous EGFR ligand driving migration and replication. Consideration must also be given to other members of the EGF family in gastrointestinal mucosa, which include amphiregulin and heparin-binding EGF (4, 34).

TGF-beta potently and effectively inhibited cell proliferation in our system, confirming well-recognized actions in other epithelial cells (3, 33). Although exogenous TGF-beta was clearly active in these preparations downregulating proliferation, it inhibited cell migration only at high concentrations. Additionally, immunoneutralization with TGF-beta antibody blocked the response to exogenous TGF-beta but did not alter the high rate of migration in control cells or attenuate the response to TGF-alpha . These findings are in direct contrast to studies with IEC-6 cells in which TGF-beta stimulated migration and TGF-beta immunoneutralization reduced the response to exogenous TGF-alpha (12).

Several factors might explain the failure to detect TGF-beta -positive actions on migration in our canine gastric cultures. The antibody we utilized, generated against human TGF-beta 1, may not fully cross-react with canine peptide. However, this antibody does cross-react with porcine TGF-beta and did produce a trend suggesting enhanced basal thymidine incorporation, suggesting effective immunoneutralization of canine peptide. This latter finding also indicates that TGF-beta is probably activated under these conditions. We conclude that TGF-beta receptors present on canine gastric cells downregulate thymidine incorporation, and we speculate that endogenous TGF-beta exerts negative control on cell replication. However, we do not know whether the contrast to TGF-beta upregulation of migration in the IEC-6 rat intestinal cell line reflects differences between species (rat vs. dog), tissue (intestine vs. stomach), or experimental model (immortalized cell lines vs. primary cultures).

IGF-I, also known as somatomedin, enhances both cell migration (present study) and replication (7) in our system. IGF-I is produced by liver and is a potent mitogen for many cell types. IGF-I has been reported to independently promote migration in several different cell types (2, 25, 36). In our previous studies we found that IGF-I was more potent than insulin in regulating replication and therefore concluded involvement of an IGF-I receptor (7). We did not test this point in the present studies of migration. However, previous studies of IGF-I action on migration of arterial smooth muscle indicated mediation via an IGF-I receptor (24). Furthermore, the EGFR antibody MAb-528 did not attenuate IGF-I effects on migration or replication, indicating that IGF-I acts directly on gastric cells, rather than inducing TGF-alpha . Because IGF-I is present in serum, enterocytes (14), mesenchymal, and polymorphonuclear leukocytes (17), it is a good candidate as an endogenous regulator of growth and migration in gastric epithelial cells.

bFGF also enhanced migration and replication of these primary canine cultures. bFGF was originally recognized as a growth factor expressed in endothelial cells, fibroblasts, and macrophages and acting on a variety of cell types, including fibroblasts and smooth muscle cells. FGF is bound to the extracellular matrix in basement membranes and released in an active form to stimulate tissue repair and healing. Accumulating evidence implicates FGF peptides in repair of mucosal injury and ulcer healing (27, 43, 48). bFGF is present in amphibian gastric epithelial cells; immunoneutralization indicated that bFGF played a role in rapid epithelial repair after surface injury (38). Biologically active bFGF is present in human and rat gastric and duodenal mucosa (11, 16). bFGF has also been identified in the bed of acetic acid-induced gastric ulcers (42) and cysteamine-induced duodenal ulcers (16), suggesting that bFGF is released from the cytosol of leaky injured cells. Healing of acetic acid-induced gastric ulcers in rat is retarded by intravenous injection of monoclonal antibody for bFGF and is accelerated by treatment with exogenous bFGF (42). These data do not establish whether bFGF acts directly on epithelial migration, replication, and/or other wound healing mechanisms, such as angiogenesis. Our data indicate that bFGF exerts regulatory effects both on replication and migration of normal gastric epithelial cells and acts independently of the EGF-related peptides, since EGFR blockade did not attenuate these actions.

Trefoil peptides, a family of epithelial mucin-associated molecules, are abundantly expressed in the gastrointestinal epithelium. pS2, which was first identified and purified from a human breast cancer cell line, shares homology with pancreatic spasmolytic polypeptide and its human counterpart hSP. pS2 is expressed in proximal stomach and hSP in distal stomach (19, 40). Recently, another trefoil peptide, ITF, has been identified in small intestine (40). Trefoil peptides are found in abundance in the "ulcer-associated cell lineage," indicating that regenerative epithelium expresses these peptides. One pattern has emerged consistently: these peptides are present in mucin-secreting cells. Although trefoil peptides have been hypothesized to be growth factors, prominent proliferative actions have not been established. Trefoil peptides have been reported to induce migration through a TGF-beta -independent pathway in IEC-6 and HT-29 cells (13); however, direct effects of these trefoil peptides on proliferation and migration in primary cells, particularly from gastric mucosa, remain to be established.

We found that hSP and rITF stimulated cell migration over the concentration range from 0.1 to 1.0 mg/ml. Similar concentrations were also required for enhanced migration in IEC-6 cells (13). This may be the appropriate physiological range because these peptides are present at very high concentrations in gastric juice and in the mucus layer of the antrum, reflecting their high content in the apical portion of epithelial cells. hSP also produced an increase in [3H]thymidine incorporation in our cells; however, the response was small and biphasic. Thus, consistent with findings with IEC-6 and HT-29 cells, our data indicate that trefoil peptides induce migration in primary gastric cells. Our data also suggest that trefoil peptides can exert mitogenic effects, although additional studies are required to establish the dose range and magnitude of response. These findings, taken together with the observations that hSP and pS2 are expressed in the gastric mucosa and increased in expression in injured mucosa, implicate a role of trefoil peptides in mucosal repair and healing.

The physiological role of cytokines in normal gastric mucosa and in response to injury and wound healing remains to be defined. Various cytokines are found in normal intestinal mucosa and appear to serve as paracrine and/or autocrine modulators of cell function (29, 31). Various cytokines have also been identified in Helicobacter pylori-infected tissue, raising a possible role as mediator of injury and/or repair. For example, IL-1beta is a proinflammatory cytokine produced by monocytes, macrophages, platelets, fibroblasts, and endothelial cells. IL-1beta suppresses gastric acid secretion and reduces experimental gastric injury when administered peripherally or in the brain (39, 49). Previous studies have demonstrated that IL-1alpha acted as an autocrine growth stimulator on thyroid carcinoma cells and normal fibroblasts and gastric carcinoma cells (23). On the other hand, IL-1alpha inhibits the growth of the breast carcinoma cell line MCF-7, and IL-4 inhibits growth of gastric carcinoma cells (32). IL-1beta increased proliferation of IEC-18 and Caco-2 cells (47, 50); it also stimulated gastric epithelial cell proliferation through stimulating hepatocyte growth factor release (54).

Our studies indicate that TGF-alpha may be involved in actions of IL-1beta . We found that IL-1beta enhanced migration, whereas IL-1alpha , IL-6, and IFN-gamma were inactive. Conversely, TNF-alpha modestly inhibited migration. In contrast to these variable effects on migration, each of these cytokines (IL-1alpha , IL-1beta , IL-6, TNF-alpha , and IFN-gamma ) produced modest, but significant, increases in thymidine incorporation. These mitogenic effects required a prolonged incubation period (72 h) rather than the 24- to 36-h periods studied for other factors. In contrast to the effects on IGF-I and bFGF, antibody to EGFR markedly attenuated IL-1beta -enhanced migration. IL-1beta appears to exert its migratory effects in a fashion mediated by or dependent on the activity of endogenous ligands for EGFR, presumably TGF-alpha .

In this model, migration occurs at a high rate in untreated cells. Our data indicate that the ligand(s) for the EGFR, presumably primarily TGF-alpha , is one set of important endogenous factors maintaining gastric mucosal integrity via regulating migration and cell proliferation. However, even in the setting of receptor blockade by a concentration of anti-EGFR antibody that blocks >95% of 125I-labeled EGF binding to these cell populations (Chen and Soll, unpublished observation), considerable migration and DNA synthesis still occurred in MAb-528-blocked cultures. We do not know whether this residual activity represents endogenous ligands acting at a small proportion of unblocked EGFR, other factors acting by EGFR-independent mechanisms, or regulation-independent, "spontaneous" migration and proliferation. Our data support the view that TGF-beta downregulates replication but does not alter migration in gastric cells. Because migration and replication are likely to be under redundant control by multiple mediators, we speculate that other endogenous regulators are expressed in our system that exert control over these vital processes.

Prior studies have demonstrated that migration and cell replication are distinct processes (10, 12, 37, 51). Several lines of evidence from our system also served to delineate the independence of migration and cell proliferation. Hydroxyurea inhibited EGF-stimulated cell proliferation but not migration. Cells that had undergone DNA synthesis (BrdU positive) were underrepresented in the migrating population, suggesting that dividing and immature cells may be less inclined to migrate. TGF-beta markedly inhibited proliferation but only minimally reduced migration.

The characterization of the cells in our cultures is important to interpreting results. All of the cells in these cultures are epithelial cells, positive for epithelial cell cytokeratin (7). In addition, these cultures form tight monolayers that resist apical acidification to apical pH values below 2.0 (5), confirming their identity as functional gastric epithelial cells. We have not detected any small endocrine, vascular endothelial, and surface epithelial cells in the monolayers. The major cell types present in these monolayers at the time they just become confluent are parietal cells (25 ± 6.5%), pepsinogen-containing cells (55 ± 9.2%), which will include chief and some mucous neck cells, and some replicating cells that did not stain with H+-K+-ATPase and pepsinogen I. Immunohistochemical studies with antimucin antibodies (MUC5 and MUC6) showed findings similar to human studies (20, 21), which show that surface mucous cells of canine fundus expressed MUC5 peptide and mucous neck cells expressed MUC6 peptide. We have found that 5 ± 1.6% of cells were positive for MUC6, and we found no detectable MUC5-positive cells at the time of initial confluence. The number of MUC6-positive cells decreased to 2.8 ± 0.7% (n = 3 preparations) 48 h after initial confluence.

We found that the proportion of parietal and pepsinogen-positive cells was comparable in the migrating and nonmigrating populations both basally and in response to EGF, therefore providing indirect evidence that each of these cell types participates in the migratory response to EGF. Further studies with specific antibodies are needed to establish the role of specific cell types in the migratory response to the various factors.

As with all other important physiological processes, migration and replication are likely to be regulated by multiple endogenous factors delivered by distinct pathways. Our studies provide support for the concept that migration and growth in normal gastric epithelium are regulated by TGF-alpha , presumably delivered via a paracrine route from parietal cells and by bFGF delivered by diffusion primarily from stromal cells in the lamina propria. IGF-I may be an important serum and possibly mucosal factor mediating both migration and replication. The trefoil peptides are likely to act topically to enhance migration; effects on thymidine incorporation are much less certain. Our data also indicate that certain cytokines (IL-1alpha , TNF-alpha , IFN-gamma , and IL-6) may stimulate growth, whereas others (i.e., IL-1beta ) enhance growth and migration. This redundancy reflects a familiar theme in regulation of gastric mucosal secretion and defense.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-19984, DK-30444, and DK-41557 and by the Medical and Research Services of Veterans Affairs.


    FOOTNOTES

Address for correspondence and reprint requests: A. H. Soll, CURE: VA/UCLA Digestive Diseases Research Center, Bldg. 115, Rm. 215 (W151H), West Los Angeles VA Medical Center, 13100 Wilshire Blvd., Los Angeles, CA 90073.

Received 1 October 1996; accepted in final form 27 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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