Bacterial cell wall polymers promote intestinal fibrosis by
direct stimulation of myofibroblasts
Eric A. F.
van Tol1,
Lisa
Holt1,
Feng Ling
Li1,
Feng-Ming
Kong2,
Richard
Rippe1,
Mitsuo
Yamauchi3,
Jolanta
Pucilowska1,
P. Kay
Lund1, and
R. Balfour
Sartor1
1 Center for Gastrointestinal
Biology and Disease and 3 Dental
Research Center, University of North Carolina, Chapel Hill
27599-7080; and 2 Department of
Radiation Oncology, Duke University Medical Center, Durham, North
Carolina 27710
 |
ABSTRACT |
Normal luminal bacteria and bacterial cell wall
polymers are implicated in the pathogenesis of chronic intestinal
inflammation. To determine the direct involvement of
bacteria and their products on intestinal fibrogenesis, the effects of
purified bacterial cell wall polymers on collagen and cytokine
synthesis were evaluated in intestinal myofibroblast cultures
established from normal fetal and chronically inflamed cecal tissues.
In this study, the intestines of Lewis rats were intramurally injected
with peptidoglycan-polysaccharide polymers. Collagen and transforming
growth factor (TGF)-
1 mRNA levels were measured and correlated with
mesenchymal cell accumulation by immunohistochemistry. The direct
effects of cell wall polymers on fibrogenic cytokine and collagen
1
(type I) expression were evaluated in intestinal myofibroblast
cultures. We found that intramural injections of bacterial cell wall
polymers induced chronic granulomatous enterocolitis with markedly
increased collagen synthesis and concomitant increased TGF-
1 and
interleukin (IL)-6 expression. Intestinal myofibroblast cultures were
established, which both phenotypically and functionally resemble the
mesenchymal cells that are involved in fibrosis in vivo. Bacterial cell
wall polymers directly stimulated collagen
1 (I), TGF-
1, IL-1
,
and IL-6 mRNA expression in the intestinal myofibroblasts derived from
both normal and inflamed cecum. Neutralization of endogenous TGF-
1
inhibited in vitro collagen gene expression. From our results, we
conclude that increased exposure to luminal bacterial products can
directly activate intestinal mesenchymal cells, which accumulate in
areas of chronic intestinal inflammation, thus stimulating intestinal
fibrosis in genetically susceptible hosts.
intestinal myofibroblast; Lewis rats; experimental colitis
 |
INTRODUCTION |
THE CHRONIC INFLAMMATORY bowel diseases, ulcerative
colitis and Crohn's disease, are multifactorial processes
characterized by abnormally aggressive immune responses in genetically
susceptible hosts (39). Recent studies implicate normal intestinal
bacteria in the development of chronic enterocolitis (35, 40), possibly through a mechanism involving the breakdown of peripheral tolerance to
autologous intestinal flora (9). Intestinal bacteria and their products
induce inflammation in different models of experimental enterocolitis,
and increased immune reactivity against resident luminal bacteria has
been shown in patients with inflammatory bowel disease (3, 9, 19, 23,
25). In the present study, we investigated the role of the bacterial
cell wall polymers peptidoglycan-polysaccharide (PG-PS) in
immune-mediated intestinal fibrosis and activation of intestinal
myofibroblasts. PG-PS are structural components of almost all bacteria
and share many immunologic properties with lipopolysaccharides (LPS)
(43). Intramural injection of PG-PS from group A streptococci (PG-APS)
causes a biphasic, spontaneous relapsing granulomatous enterocolitis
and fibrosis in genetically susceptible Lewis rats with
immunopathological features resembling Crohn's disease (41, 60). This
development of chronic granulomatous enterocolitis and associated
fibrosis depends on the long-term presence of these poorly degradable
cell wall polymers (42).
Stricture formation with subsequent obstruction is one of the most
common complications of Crohn's disease. In these patients, abundant
collagen deposition is found in stenotic segments (14), accompanied by
increased transcription and submucosal concentrations of various types
of collagen, including collagen type I (14, 27). Mesenchymal cells
isolated from the strictured intestinal segments of Crohn's disease
patients have increased spontaneous and transforming growth factor
(TGF)-
1-stimulated collagen synthesis (45). TGF-
1 is one of the
most potent fibrogenic cytokines implicated in arthritis, hepatic
cirrhosis, glomerulonephritis, pulmonary fibrosis, and pancreatitis (5,
26, 29, 31, 38, 44, 53, 59). TGF-
1 stimulates extracellular matrix formation, is a potent chemoattractant for monocytes and fibroblasts, and stimulates interleukin (IL)-1
mRNA expression (16, 29, 31, 34,
56, 57). Local administration of TGF-
1 stimulates fibrosis
associated with chronic inflammation in various organs (7, 38, 44, 53),
whereas neutralization of TGF-
1 reduces inflammation and matrix
deposition (4, 6, 55). Hence, despite its beneficial role in tissue
regeneration and wound healing, chronic TGF-
1 production in
inflammatory foci may cause pathogenic fibrosis.
Despite strong indications for a pathogenic role of normal intestinal
bacteria in clinical and experimental intestinal inflammation, little
is known about the role of bacteria and their products in the
development of fibrosis associated with chronic enterocolitis. We
therefore investigated whether bacterial cell wall polymers used to
induce chronic granulomatous enterocolitis directly affect the
synthesis of collagen
1 (type I) in cultured intestinal
myofibroblasts and the in vivo and in vitro expression of cytokines
involved in fibrosis such as TGF-
1, IL-1
, and IL-6. We present
evidence for the direct action of normal luminal bacterial products in the activation of intestinal myofibroblasts, which could contribute to
the development of fibrosis during chronic intestinal inflammation.
 |
MATERIALS AND METHODS |
Induction of enterocolitis.
Sterile PG-APS polymers were prepared from group A, type 3 strain D58
Streptococcus pyogenes (46). The
preparation was sonicated immediately before use, and the concentration
was calculated based on rhamnose content (8). Experiments were
performed with female, inbred, specific pathogen-free Lewis rats
(145-160 g, Charles River Laboratories, Raleigh, NC) in compliance
with regulations from our Institutional Animal Care and Use Committee.
Animals were anesthetized with 1.3 ml/kg body wt innovar (Pitman-Moore, Washington Crossing, NJ). Intestines were exposed by aseptic laparotomy and subserosally injected with PG-APS
(n = 6, 12.5 µg rhamnose/g body wt)
or human serum albumin (HSA; n = 5, 37.5 µg/g body wt, Baxter Health Care) into seven sites within the
distal ileum and cecum, including the lymphoid aggregate at the cecal
tip, the midcecum, the junction of the mesentery and distal ileum, and two distal ileal Peyer's patches, causing chronic granulomatous enterocolitis as described previously (28, 41). The animals were killed
during the chronic phase of inflammation (17 and 26 days after PG-APS
injection), and both macroscopic and histological inflammation were evaluated.
Tissue collection and processing.
Cecal tissue samples were snap frozen and stored at
80°C for
isolation of RNA and protein. Formalin-fixed, paraffin-embedded sections from the cecal tip were used to study collagen deposition using Masson's trichrome staining and for immunocytochemical
localization of TGF-
1,
-smooth muscle actin (
-SMA), and
vimentin (as detailed in
Immunohistochemistry). Total RNA was isolated
from cecal tissue by the guanidine thiocyanate method (28).
Intestinal (myo)fibroblast cultures and Rat-1 cell line.
Normal intestinal tissue from neonatal Lewis rats was dissected and
washed in DMEM-F12 with 2 mM
L-glutamine and 10 mM HEPES with
10 times the usual concentration of antibiotics [penicillin (1,000 U/ml), streptomycin (1 mg/ml), and amphotericin B (2.5 µg/ml)]. The tissue was cut into small pieces, washed, and
centrifuged in a tabletop centrifuge at 500 rpm for 1 min to select for
tissue fragments, and the supernatant was then decanted. This procedure was repeated four times to minimize bacterial contamination, and the
remaining small explants were seeded into
75-cm2 tissue culture flasks in
RPMI 1640 medium supplemented with antibiotics (5×) and 20% FCS
in 5% CO2 at 37°C. After 24 h, the tissue fragments were collected, transferred to a new flask, and
cultured for two to three more days, after which adherent and remaining
floating tissue fragments were rinsed off and discarded. The outgrowing cells were cultured in DMEM-F12 growth medium with
L-glutamine, HEPES, 10% FCS,
and antibiotics (1×). After at least four passages, clusters of
cells were selected based on a more discoid than spindle-shaped morphology and grown on culture chamber slides (Lab-Tek, Nunc, Naperville, IL) to permit phenotypic characterization. Cells were characterized by immunostaining with specific antibodies to
-SMA, vimentin, and TGF-
1.
Myofibroblasts from chronically inflamed tissues were isolated from
cecal granulomas of Lewis rats 24 days after PG-APS injection. Granulomas were carefully dissected from tissues and washed in medium
as described above. Explants were seeded on collagen IV-coated filters
in hormonally defined medium (36) supplemented with 5% FCS and
antibiotics (1×). Outgrowing cells were cultured in DMEM-F12 with
L-glutamine, HEPES, 2% FCS, and
antibiotics (1×) as described above. A control Rat-1 embryonic
fibroblast cell line was maintained in DMEM-F12 culture medium with 5%
FCS, L-glutamine, HEPES, and antibiotics.
Immunohistochemistry.
Immunostaining was performed on paraformaldehyde-fixed,
paraffin-embedded sections of the cecal tip from PG-PS- or HSA-injected Lewis rats or on intestinal myofibroblasts that were cultured in
chamber slides and postfixed in 4% paraformaldehyde. Primary antibodies used were as follows: anti-TGF-
1 rabbit polyclonal antibody for immunohistochemistry (21) and immunofluorescence using
confocal imaging (SC146; Santa Cruz Biotechnology, Santa Cruz, CA) at a
dilution of 1:50-1:100; anti-
-SMA and anti-vimentin mouse
monoclonal antibodies (Dako, Carpenteria, CA) at dilutions of
1:25-1:100. Normal rabbit serum and mouse IgG were used as negative controls for antibody specificity. In addition, detailed characterization of the specificity of the TGF-
antibody has been
reported previously, including selective blockade of immunostaining with TGF-
1 and not other TGF-
family members as well as specific recognition of TGF-
1 on Western blots (20). Indirect immunostaining was performed using an avidin-biotin peroxidase complex (ABC) kit
(Vecstatin, Vector Lab, Burlingame, CA) or by indirect
immunofluorescence and confocal microscopy. In each case, sections were
preincubated with 2% blocking serum (normal goat serum) before
incubation with primary antibodies overnight at 4°C. Sections were
then washed in PBS. For ABC immunostaining, sections were incubated
with biotinylated goat anti-rabbit IgG or biotinylated rat adsorbed
horse anti-mouse IgG (1:400 dilution; Vector Lab) for 45 min, washed in
PBS, and then incubated with the avidin and biotin
components of the Vectastain kit for 45 min. Red
precipitation product was then developed using an
aminoethylcarbazole substrate kit (Zymed Labs, San Francisco, CA).
Sections were counterstained with Gill's hematoxylin (Fisher Scientific) and coverslipped with Crystal/Mount (Biomeda, Foster City,
CA). For indirect immunofluorescence and colocalization of TGF-
and
-SMA, sections were incubated simultaneously with both primary
antibodies and then primary antibodies were localized using
Cy2-conjugated AffiniPure goat anti-rabbit IgG and Cy5-conjugated AffiniPure goat anti-mouse IgG, both from Jackson Immunoresearch (West
Grove, PA). Digital images of immunofluorescent staining were obtained
via a Leica TCS 4D confocal microscope.
In vitro stimulation of myofibroblasts with PG-APS and
TGF-
1.
Cultures of subconfluent intestinal myofibroblasts were switched from
growth medium to DMEM-F12 medium supplemented with 0.5% serum,
L-glutamine, HEPES, and
antibiotics for 24 h. The cultures were incubated in the same medium
supplemented with TGF-
1 (2 ng/ml; R&D Systems, Minneapolis, MN) or
with different concentrations (5, 20, 50 µg/ml) of the sterile PG-APS
used to induce chronic granulomatous enterocolitis in vivo. Cells were
harvested to evaluate collagen and cytokine gene expression, and total
RNA was isolated using the TRIzol method (GIBCO).
In vitro blockade of endogenous TGF-
1 in
PG-APS-stimulated myofibroblasts.
Subconfluent intestinal myofibroblasts from neonatal Lewis rats were
cultured in DMEM-F12 medium supplemented with 10% serum, antibiotics,
L-glutamine, and HEPES. Cells
were exposed to rabbit anti-TGF-
1 polyclonal antibody (0-100
ng/ml, R&D Systems) for 1 h and then stimulated with or without
PG-APS (40 µg/ml) for 8 h. Total RNA was isolated from the cells
by the TRIzol method and used to evaluate collagen gene expression.
Northern blotting.
Samples of 2.5-10 µg of total RNA extracted from cecal tissues
and myofibroblast and Rat-1 cultures were electrophoresed in a 1.4%
agarose-formaldehyde gel. The size-fractioned RNA was transferred to a
nylon membrane (Hybond-N, Amersham Life Sciences) and hybridized with a
[32P]dCTP-labeled
(3,000 Ci/mmol; ICN, Costa Mesa, CA) 1300-bp cDNA probe encoding rat
collagen
1 (I). Northern hybridization of rat mRNA reveals two
different molecular weight transcripts of rat
1 (I) collagen of 4.7 and 5.7 kb, which is characteristic for rat collagen
1 (I) as
described by Genovese et al. (12). Hybridizations were performed in
Rapid-Hyb buffer (Amersham) for 2-3 h at 65°C followed by
washing under high-stringency conditions to reduce background using
variable (2-0.1×) concentrations of sodium chloride-sodium
phosphate-EDTA buffer with 0.1% SDS. The membranes were exposed to
Kodak X-OMAT film (Kodak, Rochester, NY) at
80°C using an
intensifier screen. Blots were reprobed with a
32P-labeled commercially available
rat pTri-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe
(Ambion, Austin, TX).
RT-PCR.
For semiquantitative analysis of TGF-
1, IL-1
, IL-6, and actin
mRNAs, 1 µg of total RNA from cecal tissues and cell cultures was
reverse transcribed in a volume of 50 µl containing 200 units of
Moloney murine leukemia virus RT and transcription buffer (GIBCO BRL,
Gaithersburg, MD), 10 mM dithiothreitol (GIBCO), 1 mM dNTP (Perkin-Elmer/Applied Biosystems, Foster City, CA), 1 mM random hexamers (Perkin-Elmer), and 0.5 U/µl RNase inhibitor (Perkin-Elmer). PCR amplifications were performed using a GeneAmp PCR system 9600 (Perkin-Elmer) in a 50-µl reaction volume containing 1 µl of
first-strand cDNA, 0.1-0.25 µM of each antisense and sense
primer, 0.1-0.25 mM dNTP (Perkin-Elmer), 1.5 units of
Taq polymerase (Perkin-Elmer), and
buffer (Perkin-Elmer). For each PCR reaction, the optimal amplification
conditions for primer and dNTP concentrations were worked out within
the indicated ranges. Primers for TGF-
1 (product size 405 bp) were
sense 5'-CGTCGAGGTGACCTGGGC and antisense
5'-CTCCACCTTGGGCTTGCG, for IL-1
(size 543 bp) sense
5'-GCTACCTATGTCTTGCCCGT and antisense 5'-GACCATTGCTGTTTCCTAGG, for IL-6 (size 504 bp) sense
5'-CTTCCAGCCAGTTGCCTTCT and antisense
5'-GAGAGCATTGGAAGTTGGGG, and for
-actin (size 281 bp) sense
5'-ACCACAGCTGAGAGGGAAATCG and antisense
5'-AGAGGTCTTTACGGATGTCAACG. Conditions for amplification were
94°C for 45 s and 58°C for 1 min, followed by 72°C for 1.5 min, and samples were evaluated and compared in the linear range of
amplification. Optimal cycle number for each cytokine is listed in
Figs. 6 and 7. PCR products were analyzed in agarose gels.
-Actin
mRNA served as a control to ensure that any observed changes in
abundance of TGF-
1, IL-1
, or IL-6 mRNAs were
specific. Specificity was confirmed by size and restriction enzyme
digests and by including a water control instead of the first-strand cDNA.
Statistics.
Data are presented as means ± SE, and differences between the
groups were analyzed using the two-tailed Student's
t-test.
 |
RESULTS |
Collagen
1 (I) expression during chronic
granulomatous enterocolitis.
Collagen
1 (I) mRNA expression was examined in cecal tissues by
Northern blot hybridization (Fig. 1).
Marked increases of tissue collagen
1 (I) synthesis occurred in all
animals with PG-APS (n = 6)-induced
chronic intestinal inflammation compared with low constitutive
expression levels in the cecum of noninflamed HSA
(n = 4)-injected control animals
(Fig. 1). Quantitation of the collagen
1 (I) mRNA
expression in the Northern blot experiments revealed a sixfold increase
(3.28 ± 0.52 inflamed vs. 0.53 ± 0.1 control,
P = 0.0026) in chronically
inflamed cecal tissues when normalized for GAPDH expression. This
increase in active collagen synthesis during PG-APS-induced chronic
granulomatous enterocolitis in Lewis rats is in agreement with our
previous findings of significantly increased fibrillar collagen in
chronically inflamed cecal tissue homogenates (54).

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Fig. 1.
Bacterial cell wall injection induces in vivo fibrosis associated with
granulomatous enterocolitis in genetically susceptible rats.
Autoradiogram of a Northern blot hybridization experiment showing
markedly increased collagen 1 (type I) mRNA in inflamed cecal
tissues of Lewis rats 26 days after subserosal injection of
peptidoglycan-polysaccharide polymers obtained from group A
streptococci (PG-APS) compared with the noninflamed human serum albumin
(HSA)-injected control animals. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as a control.
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Localization of collagen, TGF-
1, and mesenchymal
cells in chronically inflamed cecal tissues of PG-APS-treated rats.
Masson's trichrome stain showed collagen deposition to be abundant in
the submucosal layer of chronically inflamed cecum with distinct
accumulation of collagen in the mesenchymal rim of granulomas (Fig.
2A).
Immunohistochemistry showed TGF-
1 staining to be localized adjacent
to areas of collagen deposition surrounding the granulomas in areas of
mesenchymal cell accumulation (Fig.
2B). In addition, aggregates of
intense TGF-
1-positive mononuclear cells with a macrophage-like
morphology were found scattered throughout the mucosa and submucosa
(Fig. 2C) as well as in areas of
subserosal granulomatous inflammation (data not shown). Control
incubations using normal rabbit serum or rabbit IgG were uniformly
negative.

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Fig. 2.
Histochemical and immunohistochemical staining of chronic granulomatous
inflammation in the cecum of a Lewis rat 26 days after PG-APS
injection demonstrating increased collagen deposition, transforming
growth factor- (TGF)- production, and myofibroblast infiltration.
A: abundant collagen deposition (blue
staining) is visible in areas of mesenchymal cell accumulation
surrounding a granuloma (Masson's trichrome staining).
B: TGF- 1 immunoreactive cells
(brown staining) are prominent within the granuloma and are localized
adjacent to or coincident with a layer of mesenchymal cells (arrow),
which also stain positive for -smooth muscle actin ( -SMA) (see
D).
C: TGF- 1 staining was also
prominent in mononuclear phagocyte-like cells in the submucosa.
D-F:
in adjacent sections, myofibroblasts accumulating around the granuloma
are positive for -SMA (D) (arrows
show myofibroblasts and arrowheads show vascular smooth muscle) and
vimentin (E), respectively, whereas
isotype control staining (F) was
uniformly negative. G-I: confocal
images of immunofluorescent staining for TGF- 1 (G) and
-SMA (H). Comparison of the images and overlay
(I) indicate coexpression of -SMA and TGF- 1 in a
subpopulation of mesenchymal cells.
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Antibodies against cytoskeletal filaments were used on adjacent
sections of cecum from PG-APS-injected rats to determine the phenotype
of the mesenchymal cells in the capsule surrounding the mature
granulomas in areas of dense collagen deposition. These mesenchymal
cells stained positive for
-SMA (Fig.
2D) and vimentin (Fig.
2E), which is typical of
myofibroblasts, whereas the control staining with mouse IgG isotype was
negative (Fig. 2F). Vascular smooth
muscle cells (arrowheads in Fig. 2D)
stain only for
-SMA.
Because initial immunohistochemistry indicated that sites of TGF-
1
localization corresponded to areas of mesenchymal cell accumulation and
overlapped with sites of
-SMA staining, confocal microscopy was
performed to localize these antigens on the same section incubated
simultaneously with
-SMA and TGF-
1. As shown in Fig. 2,
G-I,
this staining revealed colocalization of TGF-
and
-SMA in
mesenchymal cells at the periphery of granulomas.
RT-PCR analysis of TGF-
1 and IL-6 mRNA expression.
RT-PCR analysis of TGF-
1 and IL-6 mRNA expression in the cecum of
HSA- or PG-APS injected animals is shown in Fig.
3. Cecal tissue samples taken from animals
with PG-APS-induced chronic granulomatous enterocolitis had 2.5-fold
higher TGF-
1 mRNA levels when normalized for actin mRNA compared
with HSA-injected control animals [(in densitometry units) 23 ± 4 (n = 6) vs. 9 ± 2 (n = 4);
P = 0.02]. The increased
TGF-
1 mRNA expression corresponded with the increased abundance of
TGF-
1-positive cells in the chronically inflamed cecum as determined
by immunohistochemical staining. The PG-APS-induced chronic
granulomatous inflammation was also characterized by dramatic induction
or upregulation of IL-6 mRNA compared with the low or undetectable IL-6
mRNA levels in HSA-injected animals that had no macroscopic or
histological signs of intestinal inflammation. Interestingly,
relatively low levels of IL-6 mRNA in two of the PG-APS-injected rats
(Fig. 3, lanes 5 and
6) corresponded with low cecal
granuloma scores.

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Fig. 3.
Semiquantitative RT-PCR analysis reveals increased TGF- 1 and
interleukin (IL)-6 mRNA expression in the cecal tissues of Lewis rats
with PG-APS-induced chronic granulomatous enterocolitis
(lanes 1-6) compared with the
noninflamed (HSA-injected) control group (lanes
7-10). Numbers of PCR cycles used are listed in
parentheses (right).
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Primary intestinal myofibroblast cultures from neonatal Lewis rats.
To address the direct effects of the bacterial cell wall polymers on
the function of intestinal mesenchymal cells, we established intestinal
myofibroblast culture systems from both normal and chronically inflamed
cecum from Lewis rats. Immunohistochemical studies with these
mesenchymal cells were done after at least four passages in culture to
ensure uniformity of the phenotype of the cells tested. The functional
experiments were all done between passages
6 and 12 and only
after phenotyping the cultures for cytoskeletal filament expression.
The cells were found to have a typical stellate to discoid appearance
when cultured at low density and showed a stratified growth pattern on
reaching confluency. There were vast bundles of cytoplasmic
microfilaments and abundant expression of
-SMA (Fig.
4A) and
vimentin (Fig. 4B) as shown by
immunohistochemical staining. This confirms a myofibroblast phenotype
similar to that described by Valentich et al. (51) for differentiated
human intestinal myofibroblasts. There was no staining when sections
were incubated with control mouse IgG (Fig.
4C) or rabbit IgG (not shown).

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Fig. 4.
Phenotypic characterization of cultured intestinal mesenchymal cells
isolated from the cecum of a neonatal Lewis rat after 4 passages shows
the abundant expression of both -SMA-positive
(A, ×200) and vimentin-positive
(B, ×200) filaments. All cells
were found to express these markers, although intensity of the staining
varied between cells as shown in this representative picture. Isotype
control antibody staining (C,
×100) was uniformly negative.
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In vitro effects of PG-APS and TGF-
1 on collagen and
fibrogenic cytokine synthesis.
Functional studies tested the ability of PG-APS and TGF-
1 to
activate intestinal myofibroblasts. Rat-1 fibroblasts were first used
as positive controls to confirm the capacity of TGF-
1 to stimulate
collagen gene expression (Fig.
5A).
PG-APS stimulated collagen
1 (I) mRNA levels in the Rat-1 embryonic
fibroblasts, whereas IL-1
and corticotropin-releasing hormone,
representing a cytokine and neuropeptide that are upregulated in the
inflamed cecal tissue (28, 54), had no effect (Fig.
5A). The abilities of PG-APS and
TGF-
1 to simulate collagen and cytokine mRNA expression in
intestinal myofibroblast cultures were then evaluated. Myofibroblasts from a normal cecum showed high constitutive levels of collagen
1
(I) mRNA expression, but PG-APS and TGF-
1 both stimulated an
increase in collagen
1 (I) expression in a time-dependent fashion
(Fig. 5, B and
C). Myofibroblasts cultured from
PG-APS-induced cecal granulomas were also examined for effects of
PG-APS and TGF-
1 on expression of collagen mRNA. PG-APS and TGF-
1
stimulated collagen
1 (I) mRNA expression in these myofibroblasts,
showing similar kinetics of upregulation as the fetal cecal
myofibroblasts (Fig. 5D). Peak
collagen stimulation at 8 h was approximately twofold the unstimulated
values for PG-APS and threefold for TGF-
.


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Fig. 5.
Upregulation of collagen gene expression in cultured intestinal
myofibroblasts from neonatal and inflamed cecal tissues by bacterial
cell wall polymers and TGF- 1. Autoradiogram of Northern blots
demonstrating collagen 1 (I) mRNAs in control Rat-1 cells
(A) and intestinal myofibroblasts
after 6-12 passages
(B-D).
Collagen mRNA expression is increased in control Rat-1 cells
(A) stimulated for 12 h with PG-APS
(20 µg/ml). Intestinal myofibroblasts derived from normal neonatal
colon (B and
C) show a time-dependent increase of
collagen 1 (I) mRNA after TGF- 1 (2 ng/ml) or PG-APS stimulation.
A similar pattern of collagen expression was seen following PG-APS or
TGF- stimulation of myofibroblasts derived from inflamed cecal
tissues (D). Densitometry values are
normalized for -actin. Blots are representative of at least 3 different experiments.
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The effects of PG-APS on the expression of fibrogenic cytokines by
cultured myofibroblasts were further investigated by RT-PCR. The
stimulatory effect of TGF-
1 was also addressed, since this cytokine
was found to be abundantly present in areas of myofibroblast accumulation during chronic granulomatous inflammation. PG-PS increased
the expression of TGF-
1, IL-1
, and IL-6 mRNAs in intestinal myofibroblast cultures from fetal tissues, and TGF-
1 induced expression of TGF-
1 and IL-1
mRNA (Fig.
6). The kinetics and degree of cytokine
responses were similar in the fetal and inflamed myofibroblast cells in
that PG-APS also rapidly induced expression of IL-1
, TGF-
1, and
IL-6 mRNA in myofibroblasts isolated from granulomas (Fig.
7). TGF-
upregulated IL-1
and IL-6
mRNA in these cells (Fig. 7) as observed in myofibroblast
cultures from normal cecum (Fig. 6). Peak induction at 8 h after
TGF-
stimulation was 14.8 ± 6.7-fold (IL-1
) and 13.6 ± 6.6-fold (IL-6) higher than unstimulated values in three separate
experiments in myofibroblasts isolated from granulomas. TGF-
autoinduction of TGF-
mRNA in these cells was, however, modest (2.7 ± 1.0-fold increased relative to unstimulated).

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Fig. 6.
Stimulation of cytokine and growth factor expression in cultured
intestinal myofibroblasts by bacterial cell wall polymers or TGF- 1.
RT-PCR analysis of cytokine mRNA expression in intestinal
myofibroblasts (passages 6-12)
from normal cecum was performed. TGF- 1 and IL-1 mRNA are
upregulated by direct stimulation of the cells with TGF- 1 (2 ng/ml)
or PG-APS (20 µg/ml) (A). IL-6
mRNA expression is upregulated by PG-APS
(B). Results are representative of 3 different experiments.
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Fig. 7.
RT-PCR analysis of cytokine expression in myofibroblasts from inflamed
cecum reveals upregulation of IL-1 , IL-6, and TGF- mRNA by PG-APS
(20 µg/ml) and TGF- (2 ng/ml). Results are representative of 3 different experiments.
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Because PG-APS upregulated both TGF-
1 and collagen mRNA expression
and TGF-
1 independently stimulated collagen expression in intestinal
myofibroblasts, we next determined whether induction of TGF-
1 was
required for PG-APS-stimulated collagen gene expression in vitro.
Neutralization of endogenous TGF-
1 inhibited both constitutive and
PG-APS-stimulated collagen
1 (I) mRNA expression in a
dose-responsive fashion (Fig. 8).
PG-APS-stimulated collagen expression was inhibited to a greater extent
than constitutive collagen synthesis. Blockade of endogenous TGF-
1
inhibited PG-APS-stimulated collagen synthesis by 70% to below
constitutive levels.

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Fig. 8.
Neutralization of endogenous TGF- 1 blocks PG-APS-stimulated collagen
expression in cultured intestinal myofibroblasts. Preincubation of
myofibroblasts derived from neonatal rat cecal tissues with varying
concentrations of anti-TGF- 1 antibody (0-100 ng/ml) for 1 h
blocked collagen gene expression induced by 8-h exposure to PG-APS (40 µg/ml), as measured by Northern blot. Results are normalized for
actin expression and are representative of 4 separate experiments.
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DISCUSSION |
The present observations in PG-APS-induced chronic granulomatous
enterocolitis in Lewis rats and in cultured intestinal myofibroblasts prompt us to propose a role for bacterial cell wall products in inflammation-induced intestinal fibrosis. We suggest that activated cells with a myofibroblast phenotype typified by expression of
-SMA
and vimentin are directly responsive to bacterial stimulation and are
important mediators of increased collagen synthesis and deposition in
the chronically inflamed rat intestine. In vitro cultures were set up
with myofibroblasts isolated from both normal fetal cecum and
PG-APS-induced cecal granulomas. These intestinal myofibroblasts can be
stimulated to synthesize collagen
1 (I), TGF-
1, IL-1
, and IL-6
by direct actions of bacterial products such as PG-APS as well as in
response to TGF-
. Stimulation of collagen expression by PG-APS in
these myofibroblasts was dependent on autocrine production of TGF-
1.
Our results are consistent with other studies that demonstrate that
LPS, IL-1
, or tumor necrosis factor-
can induce mesenchymal cells
to secrete proinflammatory cytokines. However, these previous studies
did not relate to intestinal fibrogenesis. For example, LPS stimulates
granulocyte monocyte-colony stimulating factor, IL-1
, IL-1
, IL-6,
IL-8, and intercellular adhesion molecule 1 expression in human
duodenal fibroblasts (32) and stimulates IL-6 and platelet-activating
factor secretion in hepatic myofibroblasts (33, 49). Similarly, LPS
from Bacteroides stimulates IL-1
and IL-6 secretion by human gingival fibroblasts (48). Mourelle et al.
(30) found that colonic anaerobic bacteria, including Bacteroides species, injected into the
bowel wall, stimulate TGF-
1 secretion and collagen deposition. Our
studies demonstrate that bacterial products directly stimulate
expression of TGF-
and collagen in intestinal mesenchymal cells in
vitro. Although group A streptococci are not found in the intestine,
PG-APS is used as a prototype of poorly degradable PG-PS polymers.
PG-PS from group D streptococci
(Enterococci) and
Eubacterial species found in the
intestine are also capable of inducing chronic granulomatous inflammation (43, 46).
The effects of TGF-
1 stimulation on collagen mRNA expression in the
intestinal myofibroblasts are consistent with other cell culture
systems in which TGF-
1 upregulates collagen synthesis (2, 13, 38).
Moreover, we found that TGF-
1 increases its own expression in
intestinal myofibroblasts, probably through the previously documented
autoregulatory stimulation of distinct TGF-
1 promotor sites (18).
Constitutive expression of IL-1
mRNA in the cultured intestinal
myofibroblasts is low; however, the kinetics of IL-1
mRNA upregulation by PG-APS and TGF-
1 resemble IL-1
expression by these stimuli in monocytes and macrophages (28, 56). In addition, there
was a marked stimulation of IL-6 mRNA by PG-APS in cultured myofibroblasts from normal or chronically inflamed cecal tissues. These
observations suggest that activated myofibroblasts in areas of
intestinal inflammation may attract other mesenchymal cells due to
increased secretion of chemotactic IL-6 on bacterial stimulation. Further studies will be of interest to assess whether TGF-
1 or IL-1
plays an intermediate role in PG-APS stimulation
of IL-6 mRNA expression. Increased IL-6 synthesis may not only
aggravate inflammation but may also enhance fibrosis through its
inhibitory effect on collagen-degrading enzymes (50).
The PG-APS-induced stimulation of TGF-
1 mRNA in cultured
myofibroblasts and protein synthesis in monocytes (26), together with
the in vivo production of TGF-
1 by mononuclear cells and myofibroblasts in areas of collagen deposition, point to a regulatory role of TGF-
1 in intestinal fibrosis. Indeed, our data show that the
increased collagen
1 (I) gene expression during PG-APS-induced chronic enterocolitis was accompanied by increased cecal TGF-
1 and
IL-6 mRNA expression. Moreover, abundant TGF-
1 immunoreactivity was
detected in clusters of macrophage-like cells and in inflammatory cells
associated with the granulomas, which is consistent with studies in
hepatic granulomas by Manthey et al. (26). Locally increased TGF-
1
expression has also been described in other models of
inflammation-induced fibrosis, with mesenchymal cells and
monocytes/macrophages being the most abundant sources of endogenous
TGF-
1 (1, 17, 26, 53, 59).
We found that areas of collagen deposition within PG-APS-induced
granulomas colocalize with mesenchymal cells expressing
-SMA and
vimentin, suggesting that intestinal myofibroblasts mediate in vivo
collagen deposition and fibrosis. In situ, these mesenchymal cells have
the same myofibroblast characteristics, i.e., expression of
-SMA and
vimentin, because the cells were isolated and characterized from normal
and inflamed colon. Myofibroblasts are implicated as mediators of
fibrosis in various models of inflammation in other organs (7, 21, 44,
59). Consistent with the concept that TGF-
1 causes differentiation
of fibroblasts into myofibroblasts (7, 11, 58), we found abundant
TGF-
1 immunoreactivity in areas adjacent to or coincident with
-SMA and vimentin staining mesenchymal cells in the capsule of
intestinal granulomas. Confocal microscopic analysis colocalized
TGF-
1 and
-SMA in a subpopulation of myofibroblasts in the
fibrotic rim surrounding granulomas, indicating that myofibroblasts
themselves are a source of TGF-
1. This observation supports the
concept of autocrine regulatory effects of this cytokine in situ, which
is further supported by the inhibition of PG-APS-induced collagen gene
expression in vitro by neutralization of endogenous TGF-
1. Thus an
intestinal milieu of excessive local TGF-
1 production as we
documented may promote the differentiation of mesenchymal cells into a
myofibroblast phenotype that then becomes committed to collagen deposition.
The cellular mediators of intestinal fibrosis in vivo are not yet
precisely defined. It has been reported that IL-1
stimulates the
proliferation of cultured intestinal smooth muscle cells (15, 47),
whereas this cytokine inhibits collagen synthesis (15). Similar to our
findings, Graham et al. (13) demonstrated TGF-
1 stimulated collagen
deposition in human intestinal smooth muscle cells. Fritsch et al. (11)
have shown that TGF-
1 causes differentiation of rat intestinal
fibroblasts into myofibroblasts and that this growth factor may inhibit
proliferation, depending on the cell line used. Furthermore, Stallmach
and colleagues (45) found that fibroblasts isolated from strictured,
inflamed intestines from patients with Crohn's disease synthesize more
collagen and show increased responsiveness to TGF-
1 stimulation
compared with fibroblasts from control intestines. However,
myofibroblasts isolated from normal fetal or PG-APS-induced
granulomatous cecal tissues displayed similar responses to fibrogenic
stimuli. These similar responses probably reflect their identical
genetic background (Lewis rat) and the lack of exposure to fibrogenic
stimuli such as TGF-
1 during multiple in vitro passages, causing
them to revert to their basal unstimulated state. The lack of complete
blockade of PG-APS-induced collagen expression in cultured
myofibroblasts by neutralization of endogenous TGF-
1 suggests that
other growth factors, including other TGF-
isoforms and insulin-like
growth factor (60), contribute to fibrogenesis.
In conclusion, we present evidence that myofibroblasts accumulate in
areas of fibrosis associated with PG-APS-induced chronic enterocolitis.
In vitro studies demonstrated that these myofibroblasts are directly
stimulated by poorly degradable PG-APS and soluble products of
activated macrophages, thereby increasing the synthesis of collagen and
fibrogenic cytokines. PG-APS-stimulated collagen synthesis, and to a
lesser extent constitutive collagen gene expression, is dependent on
autocrine production of TGF-
1 by intestinal myofibroblasts. Inflammation and fibrogenesis may further be perpetuated by sustained activation of macrophages and mesenchymal cells by TGF-
1, IL-1
, and PG-APS in an autocrine and paracrine fashion and through continued exposure to luminal bacterial products due to enhanced mucosal translocation (23, 40). Indeed, the strategic localization of
subepithelial myofibroblasts in the normal human colon, as described by
Valentich and Powell (52), may subject these cells to bacterial
stimulation during episodes of inflammation. Finally, this persistent
activation of myofibroblasts by bacterial products and fibrogenic
cytokines during chronic enterocolitis may cause excessive collagen
deposition leading to pathogenic fibrosis.
 |
ACKNOWLEDGEMENTS |
We appreciate the support of Kirk McNaughton and Curtis Connor for
immunohistology, the expert computer graphics by Eileen Hoyt, and the
essential advice of Dr. Lola Reid, Director of the Advanced Cell
Technologies and Tissue Engineering (ACT) Core of the Center for
Gastrointestinal Biology and Disease, University of North Carolina,
Chapel Hill who identified the ex vivo expansion conditions for the
intestinal-derived stromal cells using hormonally defined medium and
extracellular matrix substrata, which were subsequently made by the
staff of the ACT core.
 |
FOOTNOTES |
This work was supported by grants from the Crohn's and Colitis
Foundation of America and National Institutes of Health Grants DK-40249, DK-34987, DK-47769, K08-DK-2402, and DE-10489.
Address for reprint requests and other correspondence: R. B. Sartor,
Division of Digestive Diseases & Nutrition, UNC School of Medicine, Rm.
030 Glaxo Bldg., CB# 7080, Chapel Hill, NC 27599-7080 (E-mail:
rbs{at}med.unc.edu).
Received 30 September 1997; accepted in final form 23 April 1999.
 |
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0002-9513/99 $5.00
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