IGF-I and procollagen
1(I) are
coexpressed in a subset of mesenchymal cells in active Crohn's
disease
Jolanta B.
Pucilowska1,2,
Kirk K.
McNaughton1,
Nirupama K.
Mohapatra1,2,
Eileen C.
Hoyt1,2,
Ellen M.
Zimmermann3,
R. Balfour
Sartor2,4, and
P. Kay
Lund1,2
1 Department of Cell and Molecular Physiology,
2 Center for Gastrointestinal Biology and Disease, and
4 Department of Medicine, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599-7545; and
3 Department of Medicine, University of Michigan, Ann Arbor,
Michigan 48109-0586
 |
ABSTRACT |
This study tested
the hypothesis that insulin-like growth factor I (IGF-I) expression is
increased at sites of fibrosis in diseased intestine of patients with
Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection
assay in uninvolved and involved intestine of 13 CD patients (10 ileum,
3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ
hybridization histochemistry compared the localization of IGF-I and
procollagen
1(I) mRNAs. Masson's trichrome staining and
immunohistochemistry for IGF-I precursor,
-smooth muscle actin (A),
vimentin (V), desmin (D), and c-kit were used to examine the
mesenchymal cell subtypes that express IGF-I and collagen in uninvolved
and involved ileum and colon of CD patients and "normal" ileum and
colon from noninflammatory controls. IGF-I mRNA was elevated in
involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen
1(I) mRNA showed
overlapping distribution within fibrotic submucosa and muscularis
propria of involved CD ileum and colon. In involved CD intestine,
increased IGF-I precursor expression localized to mesenchymal cells in
regions of tissue disorganization and fibrosis in muscularis mucosa,
submucosa, and muscularis propria. In these regions, there were
increased numbers of V+ cells relative to normal or
uninvolved intestine. Increased IGF-I expression was localized to cells
with a phenotype typical of fibroblasts
(V+/A
/D
), myofibroblasts
(V+/A+/D+), and, to a lesser
extent, cells with normal enteric smooth muscle phenotype
(V
/A+/D+). We conclude that
increased IGF-I expression in multiple mesenchymal cell subtypes and
increased numbers of cells with fibroblast/myofibroblast phenotype are
involved in fibrosis associated with CD.
intestinal fibrosis; mesenchymal cells
 |
INTRODUCTION |
CROHN'S DISEASE
(CD) and ulcerative colitis (UC) are immunologically mediated
inflammatory diseases of the gastrointestinal tract that are
characterized by chronic inflammation, mucosal damage, and epithelial
cell destruction (26, 31). CD differs from UC, in that it
may involve other regions of the gastrointestinal tract in addition to
the colon and is characterized by transmural, granulomatous
inflammation and fibrosis (7, 26, 31). Fibrosis frequently
leads to stricture and bowel obstruction, and these are major causes of
surgery and bowel resection in CD (7). Fibrosis in this
disorder can be variable in presentation but is typically associated
with mesenchymal cell hyperplasia, tissue disorganization, and
fibrillar collagen deposition that can occur in lamina propria,
muscularis mucosa, submucosa, and muscularis propria (7, 26,
31). Fibrosis in CD is thought to develop as a result of
aberrant tissue repair processes, yet the cellular and molecular
mechanisms that underlie fibrosis are not well defined.
A number of lines of evidence implicate insulin-like growth factor I
(IGF-I) in the fibrogenic process in CD. IGF-I expression is increased
in areas of fibrosis in a number of tissues and disease states,
including bleomycin-induced pulmonary fibrosis (14), nephrosclerosis (12), and hypertrophic scarring of the
skin (5). IGF-I stimulates proliferation of fibroblasts
(19, 29), myofibroblasts (24, 29), and smooth
muscle cells (13) that each are implicated as cellular
mediators of fibrosis in CD (21, 22). IGF-I also
stimulates collagen synthesis in intestinal fibroblasts and
myofibroblasts (24) and intestinal smooth muscle cells
(37). In animal models, local IGF-I expression is
increased in the colon during experimental enterocolitis induced by
sodium dextran sulfate (28), ethanol trinitrobenzene
sulfonic acid (TNBS) (36), or
peptidoglycan-polysaccharides (PG-PS) (38, 39). In the
PG-PS and TNBS models, the elevated IGF-I expression occurs at sites of
increased collagen deposition and fibrosis (36-38).
Recent findings suggest that these observations in animal models are
relevant to CD. IGF-I immunoreactivity is elevated in lavage fluid
obtained from patients with CD but not UC (6). We reported
preliminary data that IGF-I mRNA is upregulated in regions of active
disease in the intestine of patients with CD (3, 15, 23).
One aim of the present study was to compare IGF-I mRNA abundance in
involved and uninvolved ileum and colon from patients with CD and
involved and uninvolved colon of patients with UC to establish
definitively whether local IGF-I overexpression is a particular
characteristic of active CD. In addition, we sought to establish
whether the cellular sites of IGF-I overexpression colocalize with
sites of increased collagen gene expression, collagen deposition, and
fibrosis. We therefore examined surgical specimens of ileum or colon
obtained from patients with CD or UC for IGF-I and collagen mRNA
localization using in situ hybridization histochemistry and
histological stains for collagen.
The primary sites of IGF-I expression in the normal or diseased
intestine are mesenchymal cells (17-19, 36-38),
but the precise mesenchymal cell subtype is not well defined.
Mesenchymal cell subtypes can be broadly classified into fibroblasts,
smooth muscle cells, or myofibroblasts on the basis of immunostaining
properties with antibodies to vimentin (V) and
-smooth muscle
(
SM) actin (A) (21, 22, 27). Typically, fibroblasts are
V+/A
, smooth muscle cells are
V
/A+, and myofibroblasts are
V+/A+ (21, 22, 27). Although
SM-actin and vimentin are useful as phenotypic markers, it is
increasingly evident that mesenchymal cells in the intestine and other
systems may represent a more heterogeneous population than previously
suspected (21, 22). Not all myofibroblasts stain
positively for
SM-actin (21, 22). Desmin, an
intermediate-filament protein, is typically found in phenotypically
normal smooth muscle but may represent a marker of myofibroblasts in
some tissues or disease states (21, 22). Interstitial
cells of Cajal (ICC) represent a myofibroblast-related mesenchymal
subtype specific to the intestine. ICC are typically located between
enteric smooth muscle layers and regulate motility (21,
22). The c-kit receptor, which binds the
protooncogene stem cell, or steel factor, is a phenotypic
marker of ICC, but immunostaining characteristics with other
mesenchymal cell antigens are not well defined, nor is the role of ICC
in fibrosis associated with CD (21, 22). In the present
study we compared immunostaining patterns for IGF-I precursor,
SM-actin, vimentin, desmin, and c-kit in uninvolved and
involved ileum and colon of patients with CD and in ileum and colon of
patients with no history of inflammatory bowel disease (IBD). Our
primary aim was to identify the mesenchymal cell subtypes that show
increased IGF-I or collagen expression in involved ileum or colon of
patients with CD. We aimed also to assess whether there were
qualitative or obvious quantitative differences in mesenchymal cell
subtypes in diseased/fibrotic intestine of patients with CD relative to
uninvolved or normal intestine. The results demonstrate that expression
of IGF-I mRNA and encoded precursor is increased at sites of fibrosis
in active CD. Furthermore, the sites of increased IGF-I precursor
expression and collagen deposition in ileum and colon are populated by
cells that have fibroblast or myofibroblast phenotype and, to a lesser extent, by cells with the phenotype of normal enteric smooth muscle. Compared with normal or uninvolved bowel, active CD and fibrosis are
associated with increases in the number of V+ cells in
muscularis mucosa, submucosa, and muscularis propria, indicating an
involvement of fibroblasts and myofibroblasts in the pathophysiology of fibrosis.
 |
MATERIALS AND METHODS |
Tissue collection.
Excess surgical tissue from resected ileum or colon obtained from
patients undergoing medically indicated surgery for complications of CD
and UC was used in all analyses. This tissue was not required for
pathology and would otherwise have been discarded. Use of this tissue
is exempt from required approval by the Institutional Review Board for
studies on human subjects, but the Institutional Review Board was
informed of use of the tissue for these studies. Diagnoses of CD and UC
were based on clinical, radiological, endoscopic, and histological
criteria (26, 31). Samples from each CD or UC patient were
separated into grossly involved, actively diseased tissue and grossly
uninvolved tissue at the margins of diseased intestine. Small samples
of uninvolved and involved tissue were fixed in 4% paraformaldehyde to
allow histological evaluation of disease activity and
immunohistochemistry. Additional, adjacent samples of each region were
embedded in capsules of optimum cutting temperature compound (Miles,
Elkhard, IN) and stored at
80°C for in situ hybridization
histochemistry. The remainder of each sample was snap frozen in liquid
nitrogen for RNA extraction. Samples were processed further for in situ
hybridization histochemistry, immunohistochemistry, or RNA extraction
only if histology confirmed the initial categorization as grossly
involved or uninvolved. To verify appropriate sample categorization,
coded sections of uninvolved and involved ileum and colon, stained with
hematoxylin and eosin and Masson's trichrome, were scored for
histological abnormalities. Surface epithelial damage, lamina propria
inflammation, thickness of muscularis propria, and fibrosis were
assigned a score of 0-2, where 0 represents normal, 1 represents
mild abnormality, and 2 represents severe abnormality. Total histology
score was the sum of these values for each bowel layer. Thickness of
muscularis propria was also measured in well-oriented sections as an
additional measure of disease. By comparing uninvolved and involved
samples from the same patient, we aimed to minimize the impact of
interpatient variability on our measured parameters.
The characteristics of the CD and UC patients from whom tissues were
subjected to detailed analyses are shown in Table
1. Of the 13 CD patients studied, ileum
was obtained from 10 patients and colon from 3 patients. All samples
obtained from UC patients were colon. The colon samples from CD
patients provided an important subset of samples to permit direct
comparisons of the same bowel segment in patients with CD and UC. Table
1 also lists the characteristics of four patients with no history of
IBD (noninflammatory controls). Fixed samples of ileum or colon from
these noninflammatory controls were used only in immunohistochemical
analyses to assess the phenotypic characteristics of mesenchymal cells
in normal human intestine.
RNA extraction and analyses.
Total RNA was extracted from snap-frozen samples by homogenization in
guanidine isothiocyanate and centrifugation over 5.7 M cesium chloride,
as previously described (37). The abundance of IGF-I mRNA
was measured by RNase protection assay (RPA; RPA II kit, Ambion,
Austin, TX) as specified by the manufacturer's protocol. Briefly, 40 µg of total RNA were hybridized with 32P-labeled
antisense RNA probes complementary to human IGF-I mRNA (11,
15) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
mRNA (Ambion). After hybridization and RNase treatment, reactions were
denatured and then electrophoresed on 5% polyacrylamide-urea gels.
Gels were dried, and the intensity of protected bands was visualized
and quantified by phosphoimager analysis (Image Quant, Molecular
Dynamics, Sunnyvale, CA). IGF-I mRNA abundance in each sample was
normalized to the abundance of constitutively expressed GAPDH mRNA to
control for RNA loading. The normalized values for IGF-I mRNA abundance
were used for comparisons and statistical analyses.
mRNA localization.
For in situ hybridization, tissue fragments were cryosectioned at 10 µm and processed as previously described (39). Briefly, sections were fixed in 4% paraformaldehyde, treated with proteinase K
(0.5 µg/ml) for 10 min, and then acetylated with triethanolamine (0.1 M) and 0.25% (vol/vol) acetic anhydride. Dehydrated and air-dried slides were incubated with 50 µl of hybridization buffer containing 75% formamide and 1 × 106 counts/min of
35S-labeled antisense or sense RNA probes derived from
human IGF-I (9, 11) or procollagen
1(I) (20,
30) cDNAs. Sections were hybridized for 18 h at 55°C,
treated with RNase, and extensively washed in low-salt buffers
(39). Slides were exposed to Ilford K.5F radiographic
emulsion (Polysciences, Warrington, PA) at 4°C for 3-14 days.
Developed slides were counterstained with hematoxylin and photographed
under dark- and bright-field optics. Positive hybridization was defined
as clusters of silver grains observed over cells at higher densities
than in sections hybridized with sense probes. Adjacent sections were
counterstained with Sirius red (1) to localize collagen
and with hematoxylin and eosin for histology.
Immunohistochemistry.
Immunohistochemistry was performed on fixed, paraffin-embedded samples
sectioned at 6 µm. Serial sections were incubated with antibodies
specific for pro-IGF-I,
SM-actin, desmin, vimentin, or
c-kit. A rabbit polyclonal antiserum specific for a
carboxy-terminal precursor peptide, or E-domain of pro-IGF-I
(anti-human IGF-I Ea), was used at a dilution of 1:500. The pro-IGF-I
antibody localizes an intermediate in IGF-I biosynthesis, providing a
useful tool to localize sites of increased IGF-I expression (18,
19, 39). Available data indicate that at least a portion of
newly synthesized IGF-I is secreted as an E-domain extended form, and
so IGF-I precursor may be intracellular or secreted (18, 19,
39). Prior studies have established that the pro-IGF-I antibody
yields immunostaining superior to that obtained with antibodies to
mature IGF-I, probably because IGF-I is rapidly secreted or associates
with tissue IGF binding proteins (IGFBPs) that mask the epitopes
recognized by available IGF-I antibodies (18, 19, 39).
Mouse monoclonal antibodies to human
SM-actin (clone A4), human
vimentin (clone V9), and human desmin (clone D33) were purchased from
DAKO (Carpinteria, CA) and used at a dilution of 1:200. A rabbit
polyclonal antibody to c-kit (SC 168) was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA) and used at a dilution
of 1:200. Binding of the primary antibodies was detected by the
avidin-biotinylated peroxidase method (VectaStain kit, Vector
Laboratories, Burlingame, CA). Negative controls, which consisted of
omission of primary antiserum, were uniformly negative. Sections
adjacent to those used for immunohistochemistry were counterstained
with Masson's trichrome to reveal tissue histology and to localize collagen.
Statistical analyses.
Mean thickness of muscularis propria was compared in involved and
uninvolved intestine of patients with CD and UC using Student's t-test. IGF-I mRNA abundance in each sample of involved
intestine was expressed as a ratio of the abundance in uninvolved
tissue from the same patient. These ratios were compared for a
significant difference from 1 using the Mann-Whitney U test.
To directly compare IGF-I mRNA abundance in colon of patients with CD
and UC, values in uninvolved or involved colon of UC patients were
expressed as a ratio of the mean value for uninvolved colon from CD
patients assayed on the same gels and compared for a difference from 1 using the mean Mann-Whitney U test. P < 0.05 was considered statistically significant.
 |
RESULTS |
Histological verification of tissue categorization.
Histological scoring verified that samples of CD and UC intestine that
were categorized as involved showed significantly greater evidence of
disease than those categorized as uninvolved. In ileum and colon from
CD patients, histological scores for the uninvolved samples were
0-2, and scores >0 primarily reflected inflammation within the
lamina propria. In involved ileum and colon samples from CD patients,
scores were 7-8, indicating significant transmural disease. In
addition, the muscularis propria was significantly thicker
(P < 0.0001) in involved ileum and colon from patients with CD [4.8 ± 0.39 (SE) mm, range 3-8 mm] than in
uninvolved samples (2.4 ± 0.15 mm, range 2-3 mm) from the
same patients. For UC patients, histological scores for uninvolved
colon were 0-2, and for involved colon the scores were 3-4.
In UC samples, mean thickness of muscularis propria did not differ in
uninvolved and involved colon (2.8 ± 0.22 and 2.7 ± 0.18 mm, respectively).
IGF-I mRNA expression is upregulated
in active CD but not in active UC.
RPA detected IGF-I mRNA in all specimens of ileum or colon from CD
patients and all colon samples from UC patients. Representative autoradiograms are shown in Fig.
1A. For each patient, IGF-I
mRNA abundance in involved intestine was expressed as a ratio of the abundance in adjacent, uninvolved samples of the same region (i.e., ileum or colon). The mean ratio of IGF-I mRNA abundance in involved to
that in uninvolved samples from patients with CD was 2.73 ± 0.7 and was significantly (P < 0.05) greater than 1.0. In
10 of 13 CD patients studied, IGF-I mRNA abundance was higher in
involved segments of the ileum or colon than in the uninvolved sample
from the same patient (Fig. 1B). The 10 involved samples
with elevated IGF-I mRNA included 7 of the ileum samples and all 3 of
the colon samples. Analyses of CD ileum and colon on the same gels
revealed no major differences in IGF-I mRNA abundance in the two
segments (data not shown). The mean ratio of IGF-I mRNA abundance in
involved to that in uninvolved colon of patients with UC was 1.13 ± 0.11 and did not differ significantly (P = 0.43)
from 1. IGF-I mRNA abundance in samples from individual patients with
UC is shown in Fig. 1B. Relative to IGF-I mRNA abundance in
uninvolved colon from CD patients assayed on the same gels, IGF-I mRNA
abundance was 1.05 ± 0.29 for uninvolved UC colon and 1.13 ± 0.35 for involved UC colon. IGF-I mRNA abundance did not differ
significantly in involved (P = 0.56) or uninvolved
(P = 1.0) colon of UC patients compared with uninvolved
colon of patients with CD.

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Fig. 1.
Insulin-like growth factor I (IGF-I) mRNA abundance in
uninvolved and involved intestine of patients with Crohn's disease
(CD) and ulcerative colitis (UC). IGF-I and control
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were quantified
by RNase protection assay on 40 µg of total RNA extracted from
uninvolved and involved samples of intestine from patients with UC and
CD. A: autoradiogram of a representative RNase protection
assay. Probes, undigested probes; probes + RNase, probes alone
incubated with RNase to show complete digestion; other lanes show
representative samples of RNA from uninvolved (U) and involved (I)
colon from patients with UC or CD. Arrows at right indicate
protected fragments corresponding to IGF-I (578 nt) and GAPDH (295 nt)
mRNAs. These were evident in each sample of RNA from UC or CD colon
shown and all other samples tested. B: IGF-I mRNA abundance
in individual uninvolved or involved samples from 7 patients with UC
and 13 patients with CD. Values are densitometric units for IGF-I mRNA,
normalized to values for the GAPDH mRNA internal control.
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Colocalization of IGF-I and procollagen
1(I) mRNA
expression at sites of fibrosis in active CD.
In situ hybridization histochemistry was used to assess the cellular
sites of IGF-I mRNA expression in sections of involved and uninvolved
ileum of nine patients with CD and uninvolved and involved colon of
three patients with CD. Ileum samples from the 13th CD patient were not
analyzed because of poor morphology. In situ hybridization was also
performed on samples of uninvolved and involved colon from four
patients with UC. Data from the UC samples are not presented, inasmuch
as hybridization signals for IGF-I mRNA were low and not convincingly
above background in most samples. When detected in UC samples, IGF-I
mRNA was observed in scattered cells within the lamina propria (data
not shown). In the samples from the 12 CD patients studied,
hybridization signals for IGF-I were barely above background in
uninvolved ileum or colon. When detected in uninvolved bowel, IGF-I
mRNA was localized to submucosa, muscularis propria, or serosa (Table
2). Consistent with RPA data, strong
hybridization signals for IGF-I mRNA were observed in sections of
involved ileum and colon of the majority of patients with CD. The sites
of IGF-I mRNA expression did not differ in involved ileum and colon.
Weak but detectable hybridization signals were observed in the lamina
propria and muscularis mucosa of the majority of patients (Table 2).
The strongest hybridization signals were observed in the thickened,
disorganized muscularis propria and submucosa of all patients studied
(Table 2). Photomicrographs of regions of uninvolved and involved
submucosa (Fig. 2) and muscularis propria
(Figs. 3 and
4) of selected CD patients
illustrate typical hybridization signals for IGF-I. In uninvolved
intestine, hybridization signals for IGF-I mRNA were barely above
background levels. In involved submucosa, hybridization signals for
IGF-I mRNA were typically detected in mesenchymal cells surrounding
aggregates of lymphoid cells or granulomas (Fig. 2). In involved
muscularis propria, IGF-I mRNA was localized to areas of tissue
disorganization, particularly to cells within or surrounding
hypertrophied/hyperplastic smooth muscle cell bundles, rather than to
cells with typical smooth muscle morphology (Figs. 3 and 4).
Photomicrographs of adjacent sections stained with Sirius red or
hybridized with the procollagen
1(I) probe are shown in Figs.
2-4 to permit comparisons between sites of IGF-I mRNA and
procollagen
1(I) mRNA expression and collagen deposition.
Photomicrographs of procollagen
1(I) mRNA shown in Figs. 2-4
represent short exposure times that reveal only sites of high-level
expression to illustrate overlap with sites of IGF-I mRNA expression in
involved intestine. In submucosa and muscularis propria of involved CD
ileum or colon, sites of procollagen
1(I) mRNA upregulation
overlapped with sites of collagen deposition visualized by Sirius red
staining and sites of IGF-I expression (Figs. 2-4 ). In submucosal
granulomas found in involved ileum of patients with CD, high-level
procollagen
1(I) mRNA expression was observed in connective tissue
surrounding the granulomas (Fig. 2). In the involved muscularis
propria, high-level procollagen
1(I) mRNA was observed in regions of
fibrosis (Figs. 3 and 4), and this pattern was typical of all samples
of involved CD ileum or colon. In uninvolved CD ileum or colon,
procollagen
1(I) mRNA expression was not evident at the short
exposure times illustrated in Figs. 2-4, but longer exposure
revealed low-level procollagen
1(I) mRNA expression in submucosa and
serosa, as would be expected for these connective tissue layers (data
not shown). Specificity of hybridization signals detected with
antisense IGF-I and procollagen
1(I) probes was verified by the
absence of hybridization signal when the same sections were hybridized
with sense probes (Fig. 4).

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Fig. 2.
Localization of IGF-I and procollagen 1(I) mRNAs in
submucosa of uninvolved and involved ileum from a patient with CD.
Bright-field (BF and SR) and dark-field (IGF-I and Col I)
photomicrographs illustrate in situ hybridization data. IGF-I and Col
I, dark-field image of sections of uninvolved or involved ileal
submucosa from a CD patient hydbridized with IGF-I or procollagen
1(I) antisense RNA probes, respectively. Exposure times were 14 days
for sections hybridized with the IGF-I probe and 3 days for sections
hybridized with the procollagen 1(I) probe. BF, hematoxylin-stained
section after hybridization with the IGF-I antisense probe to show
histology and submucosal immune cell aggregates in involved ileum; SR,
Sirius red-stained section adjacent to that hybridized with procollagen
1(I) probe to indicate localization of collagen protein (appears
darker) surrounding the submucosal lymphoid aggregates in involved
submucosa. Note higher-level expression of IGF-I mRNA in the involved
ileum adjacent to the lymphoid aggregate and high-level expression of
procollagen 1(I) mRNA and collagen deposition in the same region.
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Fig. 3.
Localization of IGF-I and procollagen 1(I) mRNAs in
muscularis propria of uninvolved and involved ileum of a patient with
CD. Bright-field (BF and SR) and dark-field (IGF-I and Col I)
photomicrographs illustrate in situ hybridization data for uninvolved
and involved ileal muscularis propria from a CD patient. IGF-I and Col
I, dark-field images of sections hybridized with IGF-I or procollagen
1(I) antisense RNA probes, respectively. Exposure times were 14 days
for sections hybridized with the IGF-I probe and 3 days for sections
hybridized with the procollagen 1(I) probe. BF, hematoxylin-stained
section after hybridization with the IGF-I antisense probe to show
histology; SR, Sirius red-stained section adjacent to that hybridized
with the procollagen (I) probe to indicate localization of collagen
protein (appears darker). Note increased IGF-I and procollagen 1(I)
mRNA expression in the intermuscular connective tissue of involved
muscularis propria, where increased collagen deposition is also
evident.
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Fig. 4.
Localization of IGF-I and procollagen 1(I) mRNAs
in muscularis propria of uninvolved and involved colon of a patient
with CD and sense controls. Bright-field (BF and SR) and dark-field
(IGF-I and Col I) photomicrographs illustrate in situ hybridization
data. IGF-I and Col I at left and middle,
dark-field images of sections of muscularis propria of uninvolved and
involved colonic muscularis propria from a CD patient hybridized with
IGF-I or procollagen 1(I) antisense RNA probes, respectively; IGF-I
and Col I at right (Sense), dark-field images of sections of
involved colonic muscularis propria from the same CD patient hybridized
with IGF-I or procollagen 1(I) sense RNA probes, respectively.
Exposure times were 14 days for sections hybridized with the IGF-I and
3 days for sections hybridized with the procollagen 1(I) antisense
or sense probes. BF, hematoxylin-stained section after hybridization
with the IGF-I probe to show histology; SR, Sirius red-stained section
adjacent to that hybridized with the procollagen 1(I) probe to
indicate localization of collagen protein (appears darker). Note
increased IGF-I and procollagen 1(I) mRNA expression in the regions
between 2 muscle layers and throughout 1 muscle layer in involved
muscularis propria. SR shows increased collagen deposition in these
same regions. Lack of detectable hybridization signal with sense
control IGF-I and procollagen 1(I) probes indicates specificity of
signals obtained with the antisense probes.
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Colocalization of IGF-I precursor immunoreactivity
and collagen in intestine of patients with CD.
Immunohistochemistry was used to establish whether pro-IGF-I, like the
mRNA encoding the IGF-I precursor, was colocalized to areas of fibrosis
in involved intestine from patients with CD. It was not possible to
directly compare localization of IGF-I mRNA and encoded protein on the
same tissue sections. This is because IGF-I mRNA localization has
proven successful only on frozen, postfixed sections using isotopically
labeled probes, whereas immunohistochemistry to localize IGF-I
precursor has proven successful only on fixed, paraffin-embedded
tissue. As illustrated in Fig. 5,
pro-IGF-I was detected within regions of collagen deposition in the
submucosa and muscularis propria of involved ileum or colon of patients
with CD. Within the submucosa, IGF-I precursor immunoreactivity was
observed in mesenchyme-like cells surrounding lymphoid aggregates similar to the distribution of IGF-I mRNA (cf. Figs. 2 and 5). Within
the muscularis propria, distribution of pro-IGF-I was particularly striking. As shown in examples of involved ileum or colon in Fig. 5,
strong immunostaining for IGF-I precursor was observed in regions of
disorganized/fibrotic muscularis propria visualized by Masson's trichrome stain on adjacent sections. Immunostaining for IGF-I precursor was less extensive or intense in regions of muscularis propria that appeared relatively normal.

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Fig. 5.
Localization of IGF-I precursor immunoreactivity and collagen
within involved submucosa and muscularis propria of patients with CD.
Bright-field photomicrographs of involved ileal submucosa
(left), involved ileal muscularis propria
(middle), and involved colonic muscularis propria
(right) of patients with CD: H + E,
hematoxylin-and-eosin-stained sections to show histology; MT, Masson's
trichrome-stained adjacent sections to show histology and collagen as
blue stain; IGF-I, adjacent section incubated with an antibody to IGF-I
precursor followed by localization of sites of antibody binding (brown
stain) using the avidin-biotin-peroxidase method. Note that IGF-I
precursor localizes predominantly to the same sites as collagen.
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Distribution of phenotypic markers of mesenchymal cell subtypes and
IGF-I precursor in normal intestine and uninvolved and
involved intestine of patients with CD.
We wished to identify the mesenchymal cells associated with increased
IGF-I expression and fibrosis in involved intestine of patients with
CD. To achieve this we performed immunohistochemistry on involved and
uninvolved CD ileum and colon using antibodies to pro-IGF-I in
conjunction with a panel of antibodies to antigens associated with
different mesenchymal cell subtypes. Inasmuch as information was
limited about the localization of these different mesenchymal cell
antigens in normal compared with CD intestine, we included in the
immunohistochemical analyses samples of ileum and colon from patients
with no history of IBD as a "normal" comparison group.
Representative data in Figs.
6-8
reflect findings in samples of normal ileum from two
patients, normal colon from two patients, uninvolved and involved ileum
from three CD patients, and uninvolved and involved colon from two CD
patients. Figures 6 and 7 show data obtained in normal, uninvolved and
involved mucosa/submucosa of the ileum (Fig. 6) or colon (Fig. 7) using
all antibodies except c-kit, which did not immunostain cells
in the mucosa/submucosa. Muscularis propria of uninvolved ileum and
colon is also represented in Figs. 6 and 7. Figure 8 compares
immunostaining of muscularis propria in ileum from a noninflammatory
control and involved ileum from a CD patient. Immunostaining patterns
in normal and involved colonic muscularis propria were essentially the
same as those observed in ileum, and so data are shown only for ileum.
Figure 9 shows
high-power views of selected regions of involved ileum from different
CD patients to illustrate colocalization of particular antigens and
IGF-I immunostaining in samples with particularly severe fibrosis of
the muscularis propria.

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Fig. 6.
Mesenchymal cell subtypes and IGF-I precursor localization in
ileum. Bright-field photomicrographs show sections of ileum of a
patient with no history of imflammatory bowel disease and sections of
uninvolved and involved ileum from a patient with CD. Serial sections
were treated as follows: MT, Masson's trichrome-stained sections to
show histology and collagen as blue stain; other sections show
immunostaining with antibodies to -smooth muscle ( SM) actin,
desmin (des), vimentin (vim), and IGF-I precursor (IGF-I). mm,
Muscularis mucosa; sm, submucosa; mp, muscularis propria (represented
only in uninvolved ileum). White arrows in normal and uninvolved ileum
point to the typical location of subepithelial myofibroblasts, where
positive immunostaining for SM-actin (A), vimentin (V), and IGF-I
precursor is evident, but only a few cells are desmin (D) positive.
Muscularis mucosa of normal and uninvolved ileum and muscularis propria
of uninvolved ileum are A+ and D+ but
V . Note increased numbers of V+ cells in
muscularis mucosa and submucosa of involved ileum. In involved ileum,
black arrows indicate a region populated by
V+/A cells (see Fig. 9A), and
white arrows point to regions with V+/A+ cells.
Note increase in IGF-I precursor immunostaining in muscularis mucosa
and submucosa of involved ileum.
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Fig. 7.
Mesenchymal cell subtypes and IGF-I precursor localization in
colon. Bright-field photomicrographs show sections of colon from a
patient with no history of inflammatory bowel disease (normal) and
sections of uninvolved and involved colon from a patient with CD. Some
disease activity is evident in uninvolved colon. Serial sections were
treated as follows: Masson's trichrome-stained sections show histology
and collagen as blue stain; other sections show immunostaining with
antibodies to SM-actin, desmin, vimentin, and IGF-I precursor. mm,
Muscularis mucosa; sm, submucosa; mp, muscularis propria (represented
only in uninvolved and involved colon). In normal and uninvolved colon,
white arrows point to the typical location of subepithelial
myofibroblasts that are A+ and V+ but
D . Normal muscularis mucosa is A+ but
D and V ; uninvolved muscularis mucosa is
A+ and contains some D+ and V+
cells. In involved colon the mucosa is largely destroyed, and it is not
possible to clearly delineate the epithelial layer, muscularis mucosa,
or submucosa. Note the greater number of V+ cells in
involved mucosa/submucosa than in normal tissue. In the involved
sample, black arrows indicate an area populated largely by
V+/A cells that is strongly positive for IGF
precursor, and white arrows point to regions at the surface of the
mucosa where SM-actin and vimentin immunostaining overlap.
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Fig. 8.
Mesenchymal cell phenotypes and IGF-I localization in
muscularis propria of normal and involved CD ileum. Bright-field
photomicrographs show sections of muscularis propria from a sample of
normal ileum and from involved ileum from a patient with CD. Serial
sections were treated as follows: Masson's trichrome-stained sections
show histology and collagen as blue stain; other sections show
immunostaining with antibodies to SM-actin, desmin,
c-kit, vimentin, and IGF-I precursor. Normal muscularis
propria is A+ and D+ but largely
V . Note c-kit-positive/V+ cells at
the junction between circular and longitudinal muscle layer in normal
muscularis propria, typical of the location of interstitial cells of
Cajal. IGF-I precursor may weakly immunostain these
c-kit-positive cells, but this was not definitive in any
sample of normal ileum or colon studied. In involved muscularis
propria, note the larger number of V+ cells and no
c-kit-positive cells. IGF-I precursor immunostaining is
clearly higher in involved muscularis propria than in normal muscularis
propria, especially in the more disorganized/fibrotic layer, throughout
the V+/A /D region between
circular and longitudinal layers and in collagenous septa between
muscle bundles.
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Fig. 9.
Colocalization of IGF-I precursor and antigens for
particular mesenchymal cell subtypes in involved ileum of patients with
CD. Bright-field photomicrographs of sections of muscularis mucosa
(A) and muscularis propria (B-D) of involved
CD ileum are shown. Masson's trichrome-stained sections show histology
and collagen as blue stain. Other sections show immunostaining with
antibodies to SM-actin, vimentin, and IGF-I precursor. Black arrows
indicate regions that are V+/A ; white arrows
indicate regions that are V+/A+. A:
comparison of SM-actin and vimentin immunostaining in involved
muscularis mucosa indicates a majority of V+/A+
cells as well as some V+/A cells. Note strong
IGF-I precursor immunoreactivity in the region of
V+/A cells but also IGF-I precursor located
in regions containing V+/A+ cells.
B: involved muscularis propria sample showing intense IGF-I
immunostaining in a highly fibrotic region containing more
V+ than A+ cells and in
V+/A cells located between muscle layers.
C and D: low- and higher-power views,
respectively, of a highly fibrotic involved muscularis propria. Note
intense IGF-I immunostaining in smooth muscle-like bundles populated by
V+/A+ cells (1 indicated by white arrow) as
well as the surrounding fibrotic area that contains
V+/A cells. Desmin immunostaining of these
sections revealed patterns identical to SM-actin immunostaining
(data not shown).
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Immunostaining patterns of mesenchymal cell subtypes in normal and
uninvolved ileum.
In normal and uninvolved ileum (Figs. 6 and 8), the patterns of
immunostaining indicate that vimentin and
SM-actin antibodies can
distinguish subepithelial myofibroblasts (SEMF), which are V+/A+, enteric smooth muscle cells in
muscularis mucosa and muscularis propria, which are
V
/A+, and scattered fibroblasts in the
submucosa or lying between smooth muscle bundles in muscularis propria,
which are V+/A
.
SM-actin immunostaining
was sometimes not observed or was not very strong in the circular or
the longitudinal layer of the muscularis propria, as shown for the
longitudinal muscle layer of the normal ileum in Fig. 8. Across
different samples analyzed, the relative intensity of
SM-actin
immunostaining of the two layers of muscularis propria varied depending
on the orientation of smooth muscle fibers in the section, and this
seemed to reflect the orientation/accessibility of antigen in the plane
of the section rather than a true difference in relative expression of
SM-actin in circular and longitudinal muscle layers. Desmin
antibodies immunostained cells in the lamina propria within villi and
in pericryptal regions of normal and uninvolved ileum, but these were
fewer in number than observed with
SM-actin or vimentin antibodies
(Fig. 6). This indicates that either a subset of ileal SEMF are
V+/A+/D+ or desmin antibodies
immunostain only smooth muscle cells in villus lamina propria or
pericryptal regions. Desmin antibodies immunostained the muscularis
mucosa and muscularis propria in normal and uninvolved ileum (Figs. 6
and 8), consistent with a consensus in the literature that normal
enteric smooth muscle has V
/A+/D+
phenotype (21, 22). Cells positive for c-kit
were detected between the muscle layers of normal ileal muscularis
propria, typical of the location of ICC, and these were also
V+/A
/D
(Fig. 8).
Immunostaining patterns of mesenchymal cell subtypes in normal and
uninvolved colon.
In normal colonic mucosa, patterns of immunostaining with vimentin and
SM-actin were similar to those observed in normal and uninvolved
ileal mucosa, in that SEMF were V+/A+ and the
muscularis mucosa was V
/A+ (Fig. 7). Normal
colonic mucosa did, however, differ from normal ileal mucosa in
patterns of desmin immunostaining. In normal colon, the mucosa was
essentially D
, indicating that colonic SEMF and
muscularis mucosa do not express desmin (Fig. 7). The uninvolved CD
colon samples had more inflammatory cells in lamina propria and
somewhat thickened muscularis mucosa relative to normal colon,
indicating some mild disease activity (Fig. 7). In contrast to normal
colon, the samples of uninvolved colon showed D+ cells in
the muscularis mucosa (Fig. 7). As well as desmin staining, the
thickened muscularis mucosa in uninvolved colon showed increased numbers of V+ cells relative to normal muscularis mucosa.
These findings in uninvolved colonic mucosa, albeit on a small sample
number, support a possibility that in CD a subset of smooth muscle
cells of the colonic muscularis mucosa may change phenotype to express
desmin or transform toward a
V+/A+/D+ myofibroblast phenotype.
Vimentin,
SM-actin, desmin, and c-kit immunostaining
patterns in normal and uninvolved colonic muscularis propria were
indistinguishable from those in ileal muscularis propria (data not shown).
Immunostaining patterns of mesenchymal cell subtypes in involved
CD ileum and colon.
Masson's trichrome staining of involved CD ileal mucosa revealed major
disease activity (Figs. 6 and 9A). The muscularis mucosa was
thickened and disorganized, with islands of what appeared to be smooth
muscle bundles surrounded by collagen. The most striking feature was
the large number of V+ cells in the subepithelial region of
ileal mucosa and the disorganized muscularis mucosa. V+
cells were evident even in A+/D+ smooth muscle
bundles in the muscularis mucosa of involved CD ileum (Figs. 6 and Fig.
9A). Figure 9A clearly reveals coexpression of
SM-actin and vimentin in the bundles of smooth muscle-like cells
evident in sections stained with Masson's trichrome. In addition,
there were clearly regions of fibrotic ileal muscularis mucosa
surrounding the smooth muscle-like cells that were V+ but
A
and D
(Figs. 6 and 9A). The
patterns shown for involved ileal mucosa in Figs. 6 and 9A
were similar to those in ileum of all other CD patients analyzed.
Although they are not strictly quantitative, these data provide
qualitative/semiquantitative data to indicate an increase in relative
numbers of V+/A+/D+ myofibroblasts
and V+/A
/D
fibroblasts in
diseased ileal muscularis mucosa.
As illustrated in Fig. 7, the involved CD colon samples examined in the
present study showed more severe mucosal damage than involved ileal
mucosa. The colonic mucosa was so damaged that it was not possible to
clearly delineate mucosal epithelium, muscularis mucosa, and submucosa
(Fig. 7). Most striking, however, was the large number of
V+ cells throughout the colonic mucosa/submucosa, with
bundles of strongly V+ but A
/D
cells being readily apparent (Fig. 7). Involved colonic mucosa contained only a few A+ cells and no detectable
D+ cells. Some A+ cells were located at the
mucosal surface, and some of these regions stained positively with
vimentin antibodies (Fig. 7). Thus the involved colonic mucosa samples
analyzed showed a higher prevalence of fibroblasts and fewer cells with
obvious myofibroblast phenotype or enteric smooth muscle phenotype than
involved ileal mucosa. This could reflect a difference in mesenchymal
cell phenotypes between colon and ileum during active CD but could also
reflect the fact that fibroblasts predominate in the mucosa of samples with more severe mucosal destruction/fibrosis. We favor this latter possibility, inasmuch as fibroblasts were also more prevalent in
severely fibrotic muscularis propria, as described below.
Immunostaining of the muscularis propria of involved ileum from CD
patients, similar to involved mucosa, revealed a larger number of
V+ cells than in normal or uninvolved muscularis propria
(Figs. 8 and 9). This was true for colonic muscularis propria as well (data not shown). In regions of moderately fibrotic muscularis propria
that contained thickened but relatively normal-appearing A+/D+ smooth muscle layers, V+
cells were most apparent at the boundary between smooth muscle bundles
and collagenous septa coursing through the muscle (Fig. 8). It was not
possible using adjacent sections to definitively establish whether
these V+ cells coexpressed actin or desmin, and so they may
have fibroblast or myofibroblast phenotype. Attempts to colocalize
vimentin and
SM-actin or vimentin and desmin on the same sections
using different chromogens or confocal microscopy have proved
difficult, because available antibodies that yield positive
immunostaining are each mouse monoclonal antibodies. There were,
however, V+/A
/D
cells in
involved CD muscularis propria that lay between circular and
longitudinal smooth muscle layers (Figs. 8 and 9B),
indicating fibroblast phenotype. These
V+/A
/D
cells occupied a region
where c-kit/vimentin-positive ICC cells were evident in
normal muscularis propria, yet none of the samples of involved ileal or
colonic muscularis propria examined contained c-kit-positive
cells at the boundaries of circular and longitudinal muscle layers
(Fig. 8). A trivial explanation for this finding is that the increased
mass and disorganization of enteric smooth muscle during active CD
leads to reduced representation of ICC in tissue sections. More
interesting possibilities are that active CD involves loss or damage of
ICC or induces a phenotypic change in ICC, so that they no longer
express c-kit but assume a more fibroblast-like phenotype.
These findings of a possible change in phenotype of ICC during active
CD should be considered preliminary, inasmuch as they represent data
from a relatively small number of patients. The findings do suggest,
however, that further analyses of the phenotype and function of ICC
during intestinal inflammation may prove interesting. Figure 9,
B-D, illustrates some samples of severely fibrotic
ileal muscularis propria from patients with CD that contain large
numbers of V+ cells (Fig. 9, B and
C). In Masson's trichrome-stained sections of one severely
fibrotic layer of involved muscularis propria (Fig. 9, C and
D), there were bundles that appeared to be islands of smooth
muscle cells embedded in collagen. Immunostaining revealed that these
islands of smooth muscle-like cells contained cells that coexpressed
vimentin and
SM-actin (Fig. 9, C and D).
Together, the immunostaining data provide qualitative/semiquantitative
evidence that fibrosis of muscularis propria in active CD is associated with increases in the relative numbers of cells with fibroblast or
myofibroblast phenotype and that the more severe the fibrosis, the more
prevalent are fibroblasts.
IGF-I precursor immunoreactivity in normal,
uninvolved and involved ileum and colon.
In normal and uninvolved ileum and colon, IGF-I precursor
immunostaining was generally weak but was present in SEMF (Figs. 6 and
7). Little, if any, specific immunoreactivity was observed in mucosal
epithelial cells, muscularis mucosa, or muscularis propria (Figs.
6-8). Samples of uninvolved colon that had mild disease activity
showed stronger immunostaining for IGF-I precursor in submucosa than in
normal colon or normal and uninvolved ileum (Figs. 6 and 7).
In involved ileal mucosa from patients with CD, increased
immunostaining for IGF-I precursor was evident in the disorganized fibrotic muscularis mucosa/submucosa. Strong IGF-I precursor
immunostaining was observed in
V+/A
/D
cells adjacent to
A+/D+ smooth muscle bundles but also was
present in V+/A+/D+ regions of
muscularis mucosa (Figs. 6; see also high-power view in Fig.
9A). Foci of V+/A
/D
cells that were strongly positive for IGF-I precursor immunoreactivity were particularly evident in the samples of severely diseased, involved
colonic mucosa (Fig. 7), and similar regions were observed in some
severely diseased samples of ileal mucosa (data not shown). It thus
appears that cells with fibroblast or myofibroblast phenotype are the
primary cell types expressing IGF-I in involved CD mucosa.
Regions of strongest IGF-I immunostaining were always observed in
disorganized, fibrotic muscularis propria. As shown in Fig. 8, IGF-I
precursor was sometimes localized throughout one layer of
thickened/moderately fibrotic smooth muscle and was expressed in
A+/D+ muscle bundles as well as in the
collagenous septa running through or lying between muscle layers that
contained V+ cells. Figure 9, B-D, shows
severely fibrotic muscularis propria in samples of ileum from two
patients with CD, where strong IGF-I precursor immunostaining is
evident in the outer longitudinal layer and most cells in the layer are
V+/A
/D
(Fig. 9, B
and C). In one of these samples, pro-IGF-I was localized strongly to collagen-embedded bundles of smooth muscle-like cells with
V+/A+ myofibroblast phenotype as well as to
cells surrounding these bundles. We conclude that, during active CD
associated with fibrosis of the muscularis propria, pro-IGF-I is
expressed in V+/A+/D+
myofibroblasts, V+/A
/D
fibroblasts, and V
/A+/D+ smooth
muscle cells. Although it was not possible to strictly quantify the
relative numbers of these different mesenchymal cell types that express
IGF-I precursor, overall it appeared that samples with the most severe
disorganization and fibrosis of muscularis propria showed localization
of IGF-I precursor to more cells with fibroblast
(V+/A
/D
) or myofibroblast
(V+/A+/D+) phenotype than to cells
with normal enteric smooth muscle
(V
/A+/D+) phenotype.
 |
DISCUSSION |
Accumulating evidence from animal models (19, 28, 36,
39) indicates that upregulation of IGF-I mRNA occurs locally within the intestine during inflammation, particularly inflammation associated with fibrosis. The present findings extend on preliminary data (3, 15, 23) that elevated IGF-I mRNA expression
occurs in involved intestine of patients with active CD. Our study, in an independent patient population, provides definitive evidence that
the majority of patients with CD leading to resection exhibit elevated
IGF-I mRNA expression in diseased intestinal segments and that this
characteristic is not shared by the other major IBD, UC.
Proinflammatory cytokines may induce IGF-I mRNA in involved intestine
of patients with CD. Interleukin-1
and tumor necrosis factor-
have, for example, been shown to increase IGF-I expression in some cell
types (19, 25). However, increased expression of these
proinflammatory cytokines would be expected in CD and UC and cannot
readily account for the specific increase in IGF-I expression in CD.
IGF-I expression may increase in CD, because transmural inflammation
exposes mesenchymal cells in submucosa or muscularis propria to
proinflammatory cytokines. Support for this possibility stems from our
observations that submucosa and muscularis propria are the sites of
strongest and most frequently observed IGF-I mRNA upregulation (Table
2).
Our data demonstrate that the increase in IGF-I mRNA in active CD is
accompanied by increased expression of IGF-I precursor. This occurs in
mesenchymal cells in regions of fibrosis within the muscularis mucosa,
submucosa, and muscularis propria and overlaps with sites of increased
procollagen
1(I) mRNA and collagen deposition. Increased IGF-I
synthesis may therefore contribute to the development of fibrosis or
represent an epiphenomenon of fibrosis. Support for the former
possibility stems from in vitro studies in which IGF-I was shown to
induce collagen expression in intestinal myofibroblasts (24) and intestinal smooth muscle cells (37),
and IGF-I was shown to be a potent mitogen for these cells (13,
29). In CD, locally expressed IGF-I could contribute to fibrosis
by expanding a population of phenotypically modified,
collagen-producing intestinal mesenchymal cells and/or by directly
stimulating collagen synthesis. Our data suggest that it will be of
interest to develop strategies to experimentally modulate local IGF-I
expression or action in intestinal mesenchymal cells in vivo to
determine the functional relevance of local upregulation of IGF-I
during intestinal inflammation. Transgenic mice have been developed
recently in which the
SM-actin promoter was used to target
overexpression of IGF-I to enteric smooth muscle and myofibroblasts in
vivo (17, 33). Such models could prove useful to better
define whether IGF-I overexpression in these two mesenchymal cell
subtypes alters mesenchymal cell hyperplasia and hypertrophy or
collagen synthesis and fibrosis in response to experimental
enterocolitis. If so, this would point to avenues for future
therapeutic intervention in CD. A number of high-affinity IGFBPs can
inhibit IGF-I action (18, 19) and could possibly be useful
as experimental or therapeutic agents to inhibit IGF-I action during
inflammation-induced fibrosis in the intestine. Defining the potential
of IGFBPs as therapeutic agents requires a better understanding of the
complex interplay between IGF-I and endogenous IGFBPs during intestinal
inflammation and fibrosis. Most information about intestinal IGFBPs has
been obtained in animal models. IGFBP3, IGFBP4, and IGFBP5 are the primary IGFBPs expressed in rodent intestine postnatally (18, 19). Expression of IGFBPs is altered in some situations of
intestinal inflammation. The mRNA encoding IGFBP4, an IGFBP that
inhibits IGF-I action in most systems tested (18, 19), is
elevated in colon during TNBS-induced enterocolitis (36).
Expression of colonic IGFBP5 mRNA also is increased at similar sites as
IGF-I in TNBS and PG-PS models of entercolitis and in active CD
(15, 36, 37). IGF-I is known to induce IGFBP5 in colonic
smooth muscle cells in culture (37). IGFBP5 generally
potentiates IGF-I action in mesenchymal cells (2, 18, 19,
37) and so could amplify any effects of IGF-I during
inflammation-induced fibrosis. More information about the in vivo
actions and interactions of IGF-I and IGFBPs in intestinal mesenchymal
cells is clearly required before their role in inflammation-induced
fibrosis can be defined. A transgenic mouse line with
SM-actin
promoter-mediated overexpression of IGFBP4 in intestinal myofibroblasts
and enteric smooth muscle represents a promising model to address the
in vivo consequences of IGFBP4 upregulation in experimental
entercolitis (32). Indeed, in light of our findings about
the mesenchymal cell subtypes associated with fibrosis and IGF-I
overexpression in active CD, use of promoters to target overexpression
of particular peptides to specific mesenchymal cell subtypes in
transgenic models represents a generally attractive strategy to analyze
the molecular mediators of inflammation-induced fibrosis in the
intestine (17).
Fibrosis in CD is associated with hyperplasia and disorganization of
enteric smooth muscle layers and excessive collagen deposition around
and within the smooth muscle. The present study suggests that in active
CD the muscularis mucosa and muscularis propria show an increase in the
relative numbers of cells with fibroblast (V+/A
/D
) or myofibroblast
(V+/A+/D+) phenotype. Although our
findings indicate that V+/A
/D
and/or V+/A+/D+ cells are
associated with regions of fibrosis in diseased intestine of patients
with CD, they do not define the lineage that gives rise to this
fibrogenic cell population. In the mucosa, expansion of the SEMF and/or
transformation of smooth muscle cells within the muscularis mucosa
toward collagen-expressing fibroblasts/myofibroblasts may contribute to
fibrosis. In muscularis propria, our findings support a concept that
excessive collagen deposition in CD may result from a phenotypic switch
of resident enteric smooth muscle cells toward fibroblast/myofibroblast
phenotype or infiltration/proliferation of collagen-producing
fibroblasts/myofibroblasts within enteric smooth muscle layers. Our
inability to detect normal c-kit/vimentin-positive ICC cells in involved muscularis propria is intriguing. This finding is
consistent with a study in isolated canine circular smooth muscle, in
which damage and structural alterations in ICC were reported in
response to inflammatory stimuli (16). Our detection of
V+ cells in regions of collagen deposition between circular
and longitudinal smooth muscle layers raises the possibility that transformation of ICC toward fibroblast phenotype may accompany fibrosis of muscularis propria in CD. Further analyses of phenotypic or
functional changes in ICC during IBD and experimental enterocolitis are
warranted, inasmuch as this could contribute to motility disorders in
IBD as well as fibrosis (16, 21, 22).
Even the most comprehensive immunohistochemical analyses of resected
bowel from patients with CD can provide only a snapshot at one
particular point in time and cannot define the phenotypic or functional
changes in mesenchymal cells during initiation or progression of
fibrosis. To gain a better understanding of the cellular basis of
inflammation-induced fibrosis of the intestine, it will be necessary to
first study animal models in which disease is more homogeneous and
phenotype of mesenchymal cells may be better correlated with the onset
and progression of fibrosis. In this regard, it is noteworthy that
mesenchymal cell phenotype has not been examined in detail in any
animal model of experimental enterocolitis and fibrosis, such as the
rat PG-PS and TNBS models. Furthermore, it is clearly desirable to
develop mouse models of inflammation-induced intestinal fibrosis so
that the power of mouse genetics may be used to define mechanisms of
fibrosis. There is no mouse model of intestinal inflammation in which
fibrosis has been well documented. Our present findings in clinical
samples provide important information that there are indeed changes in the phenotype of intestinal mesenchymal cells during active CD and
suggest that more detailed studies of the cellular basis of fibrosis
are warranted in appropriate animal models.
Mature 70-residue IGF-I is the predominant form of IGF-I in the
circulation (18, 19), probably derived primarily from liver (35). However, available evidence indicates that a
higher-molecular-weight form of IGF-I extended at the carboxy terminus
by an E-domain peptide present in pro-IGF-I is the predominant form of
IGF-I secreted from nonhepatic cells (10, 18, 19),
including mesenchymal cells in the intestine (38, 39).
Most reports documenting the sites of IGF-I precursor expression using
immunohistochemistry have localized IGF-I precursor at sites of injury
or inflammation in a number of nonhepatic tissues (18, 19,
39). Little or no IGF-I precursor is detected in the
circulation. Neither IGF-I nor IGF-I precursor is readily detected or
detected at high levels by immunohistochemistry in normal tissues, even
though the IGF-I mRNA is expressed (18, 19, 39). We have
speculated that the IGF-I precursor may accumulate at sites of tissue
damage due to association with extracellular matrix (ECM) as well as
increased synthesis by mesenchymal cells (18, 19, 39).
Indirect support for this possibility stems from the present findings
of high levels of IGF-I precursor localized within regions of increased
ECM deposition in involved intestine of patients with CD. It also is
known that some IGFBPs associate with ECM (2, 18, 19).
Upregulation of IGFBPs during enterocolitis could serve to sequester
IGF-I precursor onto ECM and/or modulate IGF-I action on mesenchymal cells. Ultrastructural or in vitro studies will be required to establish whether IGF-I precursor associates with ECM. In vitro studies
could establish whether this is direct or mediated by IGFBPs and its
biological relevance. Our present data demonstrate increased IGF-I
precursor expression or accumulation at regions of fibrosis that were
populated by cells with fibroblast and myofibroblast phenotype and, to
a lesser extent, regions containing phenotypically normal enteric
smooth muscle cells. High levels of IGF-I precursor could also be
expressed in modified ICC in involved CD intestine, inasmuch as
particularly high levels of IGF-I precursor were observed in
V+ cells between circular and longitudinal layers of
fibrotic muscularis propria. These findings suggest that future
analyses aimed at defining the role of IGF-I precursor or IGF-I in
conversion of mesenchymal cells to fibrogenic phenotype or in
regulating proliferation of different intestinal mesenchymal cell
subtypes are warranted. In this regard, it is of interest that
preliminary studies indicate that IGF-I does stimulate proliferation of
intestinal fibroblasts that were previously converted to myofibroblast
phenotype by pretreatment with transforming growth factor-
(24). Additional in vitro studies and analyses of
mesenchymal cell responses to inflammation in the intestine of
transgenic mice with targeted overexpression of IGF-I precursor in
enteric smooth muscle and myofibroblasts (17, 33) should
provide further insights into the functional significance of IGF-I
precursor overexpression in intestinal mesenchymal cells during active CD.
In conclusion, increased IGF-I expression in mesenchymal cells at sites
of fibrosis is a feature of active CD. In CD, severe fibrosis is
associated with increased numbers of cells that exhibit fibroblast or
myofibroblast phenotype in regions of intestine usually populated by
smooth muscle cells. IGF-I precursor is localized primarily to regions
of diseased and fibrotic bowel populated by fibroblasts and
myofibroblasts and, to a lesser extent, phenotypically normal smooth
muscle. IGF-I may regulate fibrosis in CD by actions on mesenchymal
cell phenotype, proliferation, or collagen expression.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Louis E. Underwood for providing the IGF-I precursor
antibody, Drs. E. O. Riecken and D. Schuppan for the collagen probes, and Deborah Carver for secretarial work.
 |
FOOTNOTES |
This work was facilitated by the Molecular Biology and Histopathology
Core Facilities of the Center for Gastrointestinal Biology and Disease
(National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-34987) and supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants DK-02402 (to J. B. Pucilowska),
DK-40249 (to R. B. Sartor), and DK-47769 (to P. K. Lund).
Address for reprint requests and other correspondence: J. B. Pucilowska, Dept. of Cell and Molecular Physiology, University of
North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: jola{at}med.unc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 December 1999; accepted in final form 14 July 2000.
 |
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