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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)-,
basic fibroblast growth factor (bFGF), insulin-like growth factor I
(IGF-I), two trefoil peptides, and interleukin (IL)-1
further
enhanced migration. The specific EGF receptor (EGFR) monoclonal antibody, MAb-528, inhibited both basal and TGF-
- or
IL-1
-stimulated migration, but not the response to trefoil peptide,
bFGF, or IGF-I. Exogenous TGF-
inhibited cell proliferation but did
not alter migration. Immunoneutralization with anti-TGF-
blocked the
response to exogenous TGF-
and produced a small enhancement of basal
thymidine incorporation but did not attenuate basal or
TGF-
-stimulated migration. In conclusion, endogenous EGFR ligands
regulate proliferation and migration. TGF-
inhibits mitogenesis; it
did not upregulate migration in these cultures. Although bFGF, IGF-I,
and IL-1
enhance gastric epithelial migration, only IL-1
acted in
a TGF-
-dependent fashion.
gastric mucosa; thymidine incorporation; epidermal growth factor receptors; epidermal growth factor receptor antibody; peptic ulcer; cytokines
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)- as a
critical modulator of migration. For example, expression of TGF-
was
induced by TGF-
, and the action of TGF-
was blocked by
immunoabsorption with TGF-
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-
potently inhibited thymidine incorporation, it did not enhance
migration. Furthermore, immunoneutralization with antibody to TGF-
did not impair migration in untreated cells or reduce the response to
TGF-
. 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. Materials for cell
preparation and culture were obtained from sources outlined previously
(7). Human recombinant EGF and TGF- were purchased, respectively,
from Amgen (Thousand Oaks, CA) and Bachem (Torrance, CA); porcine
TGF-
1 was purchased from R&D Systems (Minneapolis, MN); turkey
anti-TGF-
1 was from Collaborative Research (Bedford, MA); sheep
anti-TGF-
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-
(IFN-
) and
tumor necrosis factor-
(TNF-
) were from Biosource (Camarillo, CA); human recombinant interleukin (IL)-1
, IL-1
, 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-1, IL-1
, IL-6, IFN-
, and TNF-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EGF/TGF- 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.
|
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- 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).
|
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).
|
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- (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).
|
|
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- (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.
|
We also utilized a sheep anti-TGF- antiserum from East Acres to
assess regulation of migration. TGF-
antiserum (1:125 dilution) attenuated migration in response to exogenous TGF-
in a fashion surmountable by higher doses of TGF-
(n = 4, P < 0.05, not illustrated). In
contrast, EGF effects on migration were not inhibited by this TGF-
antiserum (n = 4, P > 0.2, data not illustrated),
consistent with the specificity of this antiserum against TGF-
and
not EGF (6). However, in contrast to MAb-528 (Fig.
4B), TGF-
antiserum did not
reduce basal migration (n = 4, P > 0.2, data not illustrated). The
failure of TGF-
antiserum to reduce basal migration may indicate that TGF-
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-
antisera
enhances migration, masking effects of immunoneutralizing TGF-
.
Serum is likely to contain sufficient quantities of components, such as
IGF-I, to obviate interpretation. Also in contrast to MAb-528, this
TGF-
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-
. Clarifying the role of TGF-
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- (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-
(1 nM)-stimulated
thymidine uptake (Fig. 4A).
TGF- regulates proliferation but also inhibits
migration at high doses. TGF-
1 effectively inhibited
thymidine incorporation by nonconfluent cultures (Fig.
5A). The
effects of exogenous TGF-
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-
1 did not stimulate migration. To the contrary, TGF-
1 (10-100 pM)
significantly inhibited cell migration by ~20-40% (Fig.
5B).
|
To test possible effects of endogenous TGF-1 present in these
cultures, we utilized an immunoneutralizing monoclonal antibody against
TGF-
1 that blocks action of endogenous TGF-
in IEC-6 cells (10,
12). As a positive control, we confirmed that this monoclonal antibody
attenuated inhibition of proliferation by exogenous TGF-
1 (Fig.
6A).
TGF-
1 immunoneutralization caused a small but statistically
significant enhancement of basal thymidine uptake (Fig.
6A). Although this antibody blocked
action of TGF-
1 regulating proliferation, it did not reduce
migration either in the basal state or in response to TGF-
treatment
(Fig. 6B). The control antibody
(CURE S6) had no effect in these studies
(n = 4, data not shown).
|
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.
|
|
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).
|
Variable effects on migration and proliferation by
cytokines. Effects on migration and proliferation of
IL-1, IL-1
, TNF-
, IFN-
, and IL-6 were tested over dose
ranges from 5 to 100 U/ml. Only IL-1
, at concentrations from 1 to 50 U/ml, significantly enhanced migration (Fig.
10B).
TNF-
(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.
|
|
MAb-528 EGFR antibody (20 nM) attenuated stimulation of thymidine
incorporation by cytokines IL-1, IL-1
, TNF-
, and IFN-
, each
studied at 5 U/ml (data not shown). Furthermore, IL-1
stimulation of
migration was completely blocked by MAb-528 (Fig.
10C). In contrast, the control
antibody (CURE S6) did not block IL-1
-enhanced migration (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- 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-
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-
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-. 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-. TGF-
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-
antiserum
that we used probably has actions independent of TGF-
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- potently and effectively inhibited cell proliferation in our
system, confirming well-recognized actions in other epithelial cells
(3, 33). Although exogenous TGF-
was clearly active in these
preparations downregulating proliferation, it inhibited cell migration
only at high concentrations. Additionally, immunoneutralization with
TGF-
antibody blocked the response to exogenous TGF-
but did not
alter the high rate of migration in control cells or attenuate the
response to TGF-
. These findings are in direct contrast to studies
with IEC-6 cells in which TGF-
stimulated migration and TGF-
immunoneutralization reduced the response to exogenous TGF-
(12).
Several factors might explain the failure to detect TGF--positive
actions on migration in our canine gastric cultures. The antibody we
utilized, generated against human TGF-
1, may not fully cross-react
with canine peptide. However, this antibody does cross-react with
porcine TGF-
and did produce a trend suggesting enhanced basal
thymidine incorporation, suggesting effective immunoneutralization of
canine peptide. This latter finding also indicates that TGF-
is
probably activated under these conditions. We conclude that TGF-
receptors present on canine gastric cells downregulate thymidine incorporation, and we speculate that endogenous TGF-
exerts negative control on cell replication. However, we do not know whether the contrast to TGF-
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-. 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--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-1 is a proinflammatory cytokine
produced by monocytes, macrophages, platelets, fibroblasts, and
endothelial cells. IL-1
suppresses gastric acid secretion and
reduces experimental gastric injury when administered peripherally or
in the brain (39, 49). Previous studies have demonstrated that IL-1
acted as an autocrine growth stimulator on thyroid carcinoma cells and
normal fibroblasts and gastric carcinoma cells (23). On the other hand,
IL-1
inhibits the growth of the breast carcinoma cell line MCF-7,
and IL-4 inhibits growth of gastric carcinoma cells (32). IL-1
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- may be involved in actions of
IL-1
. We found that IL-1
enhanced migration, whereas IL-1
, IL-6, and IFN-
were inactive. Conversely, TNF-
modestly inhibited migration. In contrast to these variable effects on migration, each of
these cytokines (IL-1
, IL-1
, IL-6, TNF-
, and IFN-
) 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-1
-enhanced migration. IL-1
appears to exert its migratory
effects in a fashion mediated by or dependent on the activity of
endogenous ligands for EGFR, presumably TGF-
.
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-, 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-
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- 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-, 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-1
, TNF-
, IFN-
, and IL-6)
may stimulate growth, whereas others (i.e., IL-1
) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, A.,
G. Flemstrom,
A. Garner,
and
E. Kivilaakso.
Gastroduodenal mucosal protection.
Physiol. Rev.
73:
823-857,
1993
2.
Ando, Y.,
and
P. J. Jensen.
Epidermal growth factor and insulin-like growth factor I enhance keratinocyte migration.
J. Invest. Dermatol.
100:
633-639,
1993[Abstract].
3.
Attisano, L.,
J. L. Wrana,
F. Lopez-Casillas,
and
J. Massague.
TGF-beta receptors and actions.
Biochim. Biophys. Acta
1222:
71-80,
1994[Medline].
4.
Barnard, J. A.,
R. D. Beauchamp,
W. E. Russell,
R. N. Dubois,
and
R. J. Coffey.
Epidermal growth factor-related peptides and their relevance to gastrointestinal pathophysiology.
Gastroenterology
108:
564-580,
1995[Medline].
5.
Chen, M. C.,
A. Chang,
T. Buhl,
M. Tanner,
and
A. H. Soll.
Apical acidification induces paracellular injury in canine gastric mucosal monolayers.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G1012-G1020,
1994
6.
Chen, M. C.,
A. T. Lee,
W. E. Karnes,
D. Avedian,
M. Martin,
J. M. Sorvillo,
and
A. H. Soll.
Paracrine control of gastric epithelial cell growth in primary culture by transforming growth factor-.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G390-G396,
1993
7.
Chen, M. C.,
A. T. Lee,
and
A. H. Soll.
Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor and insulin-like growth factor 1.
J. Clin. Invest.
87:
1716-1723,
1991[Medline].
8.
Chen, M. C.,
M. J. Sanders,
D. A. Amirian,
L. P. Thomas,
G. Kauffman,
and
A. H. Soll.
Prostaglandin E2 production by dispersed canine fundic mucosal cells: contribution of macrophages and endothelial cells as major sources.
J. Clin. Invest.
84:
1536-1549,
1989[Medline].
9.
Chuang, C. N.,
M. Tanner,
K. K. C. Lloyd,
H. Wong,
and
A. Soll.
Inhibition by endogenous somatostatin of histamine release from canine oxyntic mucosal cells in primary culture.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G521-G525,
1993
10.
Ciacci, C.,
S. E. Lind,
and
D. K. Podolsky.
Transforming growth factor beta regulation of migration in wounded rat intestinal epithelial monolayers.
Gastroenterology
105:
93-101,
1993[Medline].
11.
Corden-Carlo, C.,
I. Vlodavsky,
and
A. Haimovitz-Friedman.
Expression of basic fibroblast growth factor in normal human tissues.
Lab. Invest.
63:
832-840,
1990[Medline].
12.
Dignass, A. U.,
and
D. K. Podolsky.
Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor .
Gastroenterology
105:
1323-1332,
1993[Medline].
13.
Dignass, A. U.,
S. Tsunekawa,
and
D. K. Podolsky.
Fibroblast growth factors modulate intestinal epithelial cell growth and migration.
Gastroenterology
106:
1254-1262,
1994[Medline].
14.
Dvorak, B.,
A. L. Stephana,
H. Holubec,
C. S. Williams,
A. F. Philipps,
and
O. Koldovskoy.
Insulin-like growth factor-I (IGF-I) mRNA in the small intestine of suckling and adult rats.
FEBS Lett.
388:
155-160,
1996[Medline].
15.
Ennis, B. W.,
E. M. Valverius,
S. E. Bates,
M. E. Lippman,
F. Bellot,
R. Kris,
J. Schlessinger,
H. Masui,
A. Goldenberg,
and
J. Mendelsohn.
Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.
Mol. Endocrinol.
3:
1830-1838,
1989[Abstract].
16.
Folkman, J.,
S. Szabo,
M. Stovroff,
P. McNeil,
W. Li,
and
Y. Shing.
Duodenal ulcer: discovery of a new mechanism and development of angiogenic therapy which accelerates healing.
Ann. Surg.
214:
414-427,
1991[Medline].
17.
Gartner, M. H.,
J. D. Benson,
and
M. D. Caldwell.
Insulin-like growth factors I and II expression in the healing wound.
J. Surg. Res.
52:
389-394,
1992[Medline].
18.
Gill, G. N.,
T. Kawamoto,
C. Cochet,
A. Le,
J. D. Sato,
H. Masui,
C. McLeod,
and
J. Mendelsohn.
Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity.
J. Biol. Chem.
259:
7755-7760,
1984
19.
Hanby, A. M.,
R. Poulsom,
S. Singh,
G. Elia,
R. E. Jeffery,
and
N. A. Wright.
Spasmolytic polypeptide is a major antral peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach.
Gastroenterology
105:
1110-1116,
1993[Medline].
20.
Ho, S. B.,
A. M. Roberton,
L. L. Shekels,
C. T. Lyftogt,
G. A. Niehans,
and
N. W. Toribara.
Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression.
Gastroenterology
109:
735-747,
1995[Medline].
21.
Ho, S. B.,
L. L. Shekels,
N. W. Toribara,
Y. S. Kim,
C. Lyftogt,
D. L. Cherwitz,
and
G. A. Niehans.
Mucin gene expression in normal, preneoplastic, and neoplastic human gastric epithelium.
Cancer Res.
55:
2681-2690,
1995[Abstract].
22.
Hsu, S.-M.,
L. Raine,
and
H. Fanger.
Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures.
J. Histochem. Cytochem.
29:
577-580,
1981[Abstract].
23.
Ito, R.,
Y. Kitadai,
E. Kyo,
H. Yokozaki,
W. Yasui,
U. Yamashita,
H. Nikai,
and
E. Tahara.
Interleukin 1 alpha acts as an autocrine growth stimulator for human gastric carcinoma cells.
Cancer Res.
53:
4102-4106,
1993[Abstract].
24.
Jones, J. I.,
A. Gockerman,
W. H. Busby, Jr.,
G. Wright,
and
D. R. Clemmons.
Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence.
Proc. Natl. Acad. Sci. USA
90:
10553-10557,
1993[Abstract].
25.
Jones, J. I.,
T. Prevette,
A. Gockerman,
and
D. R. Clemmons.
Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.
Proc. Natl. Acad. Sci. USA
93:
2482-2487,
1996
26.
Kato, K.,
M. C. Chen,
F. S. Lehmann,
M. Nguyen,
and
A. H. Soll.
Restitution in primary canine gastric epithelial cells reflects endogenous TGF-alpha rather than TGF-beta (Abstract).
Gastroenterology
108:
A129,
1995.
27.
Konturek, S. J.,
T. Brzozowski,
I. Majka,
W. Pawlik,
and
J. Stachura.
Omentum and basic fibroblast growth factor in healing of chronic gastric ulcerations in rats.
Dig. Dis. Sci.
39:
1064-1071,
1994[Medline].
28.
Lacy, E. R.,
and
S. Ito.
Rapid epithelial restitution of the rat gastric mucosa after ethanol injury.
Lab. Invest.
51:
573-583,
1984[Medline].
29.
Madara, J. L.,
and
J. Stafford.
Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers.
J. Clin. Invest.
83:
724-727,
1989[Medline].
30.
McCormack, S. A.,
M. J. Viar,
and
L. R. Johnson.
Migration of IEC-6 cells: a model for mucosal healing.
Am. J. Physiol.
263 (Gastrointest. Liver Physiol. 26):
G426-G435,
1992
31.
McGee, D. W.,
K. W. Beagley,
W. K. Aicher,
and
J. R. McGhee.
Transforming growth factor-beta and IL-1 beta act in synergy to enhance IL-6 secretion by the intestinal epithelial cell line IEC-6.
J. Immunol.
151:
970-978,
1993
32.
Morisaki, T.,
D. H. Yuzuki,
R. T. Lin,
L. J. Foshag,
D. L. Morton,
and
D. S. Hoon.
Interleukin 4 receptor expression and growth inhibition of gastric carcinoma cells by interleukin 4.
Cancer Res.
52:
6059-6065,
1992[Abstract].
33.
Moses, H. L.
TGF-beta regulation of epithelial cell proliferation.
Mol. Reprod. Dev.
32:
179-184,
1992[Medline].
34.
Murayama, Y.,
J. Miyagawa,
S. Higashiyama,
S. Kondo,
M. Yabu,
K. Isozaki,
Y. Kayanoki,
S. Kanayama,
Y. Shinomura,
and
N. Taniguchi.
Localization of heparin-binding epidermal growth factor-like growth factor in human gastric mucosa.
Gastroenterology
109:
1051-1059,
1995[Medline].
35.
Murugesan, G.,
G. Sa,
and
P. L. Fox.
High-density lipoprotein stimulated endothelial cell movement by a mechanism distinct from basic fibroblast growth factor.
Circ. Res.
74:
1149-1156,
1996[Abstract].
36.
Nakao-Hayashi, J.,
H. Ito,
T. Kanayasu,
I. Morita,
and
S. Murota.
Stimulatory effects of insulin and insulin-like growth factor I on migration and tube formation by vascular endothelial cells.
Atherosclerosis
92:
141-149,
1992[Medline].
37.
Nusrat, A.,
C. Delp,
and
J. L. Madara.
Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells.
J. Clin. Invest.
89:
1501-1511,
1992[Medline].
38.
Paimela, H.,
P. J. Goddard,
K. Carter,
R. Khakee,
P. L. McNeil,
S. Ito,
and
W. Silen.
Restitution of frog gastric mucosa in vitro: effect of basic fibroblast growth factor.
Gastroenterology
104:
1337-1345,
1993[Medline].
39.
Perretti, M.,
K. G. Mugridge,
J. L. Wallace,
and
L. Parente.
Reduction of aspirin-induced gastric damage in rats by interleukin-1 beta: possible involvement of endogenous corticosteroids.
J. Pharmacol. Exp. Ther.
261:
1238-1243,
1992[Abstract].
40.
Podolsky, D. K.,
K. Lynch-Devaney,
J. L. Stow,
P. Oates,
B. Murgue,
M. De-Beaumont,
B. E. Sands,
and
Y. R. Mahida.
Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion.
J. Biol. Chem.
268:
12230,
1993
41.
Rutten, M. J.,
P. J. Dempsey,
T. E. Solomon,
and
R. J. Coffey, Jr.
TGF-alpha is a potent mitogen for primary cultures of guinea pig gastric mucous epithelial cells.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G361-G369,
1993
42.
Satoh, H.,
A. Shino,
F. Sato,
S. Asano,
I. Murakami,
N. Inatomi,
H. Nagaya,
K. Kato,
S. Szabo,
and
J. Folkman.
Role of endogenous basic fibroblast growth factor in the healing of gastric ulcers in rats.
Jpn. J. Pharmacol.
73:
59-71,
1997[Medline].
43.
Schmassmann, A.,
A. Tarnawski,
B. M. Peskar,
L. Varga,
B. Flogerzi,
and
F. Halter.
Influence of acid and angiogenesis on kinetics of gastric ulcer healing in rats: interaction with indomethacin.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G276-G285,
1995
44.
Sgagias, M. K.,
A. Kasid,
and
D. N. J. Danforth.
Interleukin-1 alpha and tumor necrosis factor-alpha (TNF alpha) inhibit growth and induce TNF messenger RNA in MCF-7 human breast cancer cells.
Mol. Endocrinol.
5:
1740-1747,
1991[Abstract]. [Corrigenda. Mol. Endocrinol. 6: December 1992, p. 620.]
45.
Silen, W.
Gastric mucosal defense and repair.
In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1987, p. 1055-1069.
46.
Silen, W.,
and
S. Ito.
Mechanisms for rapid re-ephithelialization of the gastric mucosal surface.
Annu. Rev. Physiol.
47:
217-229,
1985[Medline].
47.
Sutherland, D. B.,
G. W. Varilek,
and
G. A. Neil.
Identification and characterization of the rat intestinal epithelial cell (IEC-18) interleukin-1 receptor.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1198-C1203,
1994
48.
Szabo, S.,
J. Folkman,
P. Vattay,
R. E. Morales,
G. S. Pinkus,
and
K. Kato.
Accelerated healing of duodenal ulcers by oral administration of a mutein of basic fibroblast growth factor in rats.
Gastroenterology
106:
1106-1111,
1994[Medline].
49.
Uehara, A.,
T. Okumura,
S. Kitamori,
Y. Takasugi,
and
M. Namiki.
Interleukin-1: a cytokine that has potent antisecretory and anti-ulcer actions via the central nervous system.
Biochem. Biophys. Res. Commun.
173:
585-590,
1990[Medline].
50.
Varilek, G. W.,
G. A. Neil,
and
W. P. Bishop.
Caco-2 cells express type I interleukin-1 receptors: ligand binding enhances proliferation.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G1101-G1107,
1994
51.
Watanabe, S.,
M. Hirose,
X. E. Wang,
K. Maehiro,
T. Murai,
O. N. A. Kobayashi,
and
N. Sato.
Hepatocyte growth factor accelerates the wound repair of cultured gastric mucosal cells.
Biochem. Biophys. Res. Commun.
199:
1453-1460,
1994[Medline].
52.
Wong, H. C.,
J. H. Walsh,
H. Yang,
Y. Tache,
and
A. M. Buchan.
A monoclonal antibody to somatostatin with potent in vivo immunoneutralizing activity.
Peptides
11:
707-712,
1990[Medline].
53.
Wright, N. A.,
C. M. Pike,
and
G. Elia.
Ulceration induces a novel epidermal growth factor-secreting cell lineage in human gastrointestinal mucosa.
Digestion
46, Suppl. 2:
125-133,
1990[Medline].
54.
Yasunaga, Y.,
Y. Shinomura,
S. Kanayama,
Y. Higashimoto,
M. Yabu,
Y. Miyazaki,
S. Kondo,
Y. Murayama,
H. Nishibayashi,
S. Kitamura,
and
Y. Matsuzawa.
Increased production of interleukin 1 beta and hepatocyte growth factor may contribute to foveolar hyperplasia in enlarged fold gastritis.
Gut
39:
787-794,
1996[Abstract].