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From the Department of Gastroenterology*
and
Institute of Pathology,
University Hospital
Benjamin Franklin, Free University; Berlin; the Department of
Gastroenterology,
University of Erlangen,
Erlangen; the Department of Internal Medicine
II,§
University Hospital, Homburg/Saar; the
Institute of Pathology,¶
University of Freiburg
im Breisgau; and the Institute of Pathology,||
University Hospital Eppendorf, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1(I) and
1(IV), matrix-metalloproteinase (MMP)-1 and -13, and
tissue inhibitor of metalloproteinase (TIMP)-1 genes was
semiquantitated by in situ hybridization on serial biopsies
of 12 patients with collagenous colitis and compared to controls.
Collagen types I, III, IV, and VI,
tenascin, undulin/collagen XIV, and -actin were
localized by immunohistology. The superficial collagen layer stained
strongly for collagen types I, III, and VI, and
particularly for tenascin, but not for undulin. Elevated
procollagen 1(I), procollagen 1(IV), and TIMP-1
transcript levels were found in -actin-positive cells with linear
distribution underneath the superficial collagenous layer,
whereas MMP-1 RNA expression was variable and restricted to cell
clusters. MMP-13 expression was undetectable. The patterns of
procollagen 1(I)- and 1(IV)-specific labeling, combined
with an intense tenascin- but absent undulin-specific staining,
indicate deposition of an immature interstitial matrix that may be
susceptible to degradation. The restricted MMP-1 RNA
expression, counteracted by increased TIMP-1
expression, suggests locally impaired fibrolysis as a relevant
factor in the pathogenesis of collagenous colitis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies on patients subjected to surgical fecal stream diversion12 as well as anecdotal evidence13 point to a potentially rapid reversibility of the excess collagenous deposits. Thus, these deposits seem to reflect a local disturbance of ECM turnover resulting in the formation of a provisional ECM. Moreover, this suggests that not only the formation, but also the degradation of excess ECM may be relevant in the pathogenesis of CC.
Among ECM degrading enzymes, matrix-matalloproteinases (MMPs) have a
central role. MMPs are a family of zinc-dependent neutral proteinases
with overlapping but distinct substrate spectra. Four subfamilies of
MMPs are known: collagenases, gelatinases, stromelysins, and
membrane-MMPs.14-16
Together with MMP-8 (collagenase-2,
neutrophil collagenase), MMP-1 (collagenase-1, interstitial
collagenase) and MMP-13 (collagenase-3) form the MMP-subfamily of
collagenases, which are instrumental for the degradation of native
interstitial collagens, in particular collagen types I, II, and
III.14-16
Because of their destructive potential, MMPs are
regulated at three levels, namely transcription, zymogen activation,
and inhibition of the active enzyme. In vitro, stromelysin-1
(MMP-3) may enhance the activity of MMP-1 by a factor of 5 to 12 by
additional N-terminal proteolysis, whereas MMP-13 may be activated by
gelatinase A (MMP-2) and the membrane type MT1-MMP
(MMP-14).16-18
The details of MMP-1 and -13 activation
in vivo, however, are largely unknown. Inhibition of active
MMP-1 is a major function of tissue inhibitor of metalloproteinases
(TIMP)-1.14-16
In body fluids, MMP-1 may also be inhibited
by 2-macroglobulin.19
CC is diagnosed by histology and, unlike ulcerative colitis, does not require continuous histological re-evaluation. Thus, in a common clinical setting, CC biopsies are usually available only as single time-point formalin-fixed specimens. Because of their size, extractive procedures cannot be performed, making analysis of enzyme activities impossible. The study of ECM and MMPs in CC biopsies depends, therefore, on localizing a limited number of relevant gene products.
These considerations and restrictions led us to use immunohistology and semiquantitative in situ hybridization to establish the expression patterns of procollagens type I and IV as representative molecules for interstitial and basement membrane ECM, respectively. Furthermore, immunostaining for tenascin and undulin/collagen XIV was performed to assess the maturity of the matrix deposits. Expression of MMP-1, MMP-13, and TIMP-1 as pivotal elements in collagen type I degradation was assessed by in situ hybridization to obtain indicators for the local fibrolytic potential.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Paraffin blocks of mucosal biopsies from 12 patients with a
clinical history of chronic watery diarrhea were drawn from the files
of the University Institutes of Pathology in Berlin, Hamburg, and
Freiburg. Biopsies had been immersed in formalin immediately after
removal and submitted for histopathological diagnosis. Criteria for the
diagnosis of CC were as recently described.7
The thickness
of the superficial collagen layer was measured with an ocular
micrometer on well-oriented tissue sections. For each biopsy the
minimal, maximal, and mean thickness of the superficial ECM layer were
recorded along a range of 5 to 10 consecutive intercryptal spaces. The
patient data, the biopsy sites, and the histopathological
characteristics are listed in Table 1
. As
controls, we used colonic tissues with regular histology (at a distance
of >10 cm from the focal lesions, n = 7), with
ulcerative colitis (n = 4), and with Crohn's
disease (n = 3), and squamous cell carcinomas of
the head and neck region (n = 9).
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Sections (5 µm) were collected onto 3-aminopropyl triethoxysilane-coated slides, dried for several hours at 37°C, dewaxed in xylene, rehydrated in graded alcohols, and immediately processed.
Immunohistology
Collagens type I, III, and VI were detected using
affinity-purified rabbit antibodies specific for monkey collagen type I
and monkey procollagen type III aminoterminal propeptide20
as well as a rabbit antibody against human collagen type
VI.21
Tenascin was localized with a monoclonal antibody
specific for human tenascin (MAB1927, Chemikon, Temecula, CA).
Immunohistology for undulin was performed with a monoclonal antibody
against human undulin (clone 15/III/D6).22
Collagen type IV
and smooth-muscle -actin were detected with the antibodies CIV22 and
1A4, respectively (Dako, Glostrup, Denmark). The monoclonal antibodies
required pretreatment of paraffin sections with pronase (Boehringer
Mannheim, Mannheim, Germany) at a concentration of 0.5 mg/ml in
phosphate buffered saline, pH 7.2, for 10 minutes at 37°C. The
immunohistochemical detection of MMP-1 was performed with
affinity-purified polyclonal rabbit antibodies specific for human MMP-1
(Quartett, Berlin, Germany). The alkaline phosphatase anti-alkaline
phosphatase (APAAP) method was used for the detection of immobilized
antibodies, using affinity-purified mouse anti-rabbit immunoglobulin
serum (Dianova, Hamburg, Germany), affinity-purified rabbit anti-mouse
immunoglobulin serum (Dako), and APAAP complex (1:20 dilution, Dako).
Alkaline phosphatase was developed with new fuchsin.
Preparation and Labeling of Probes
pGEM1 (Promega, Madison, WI)-based run-off transcription
expression vectors were constructed by subcloning of the 1300-bp Pst
fragment of p1R1 corresponding to the triple helical region of rat
1(I) procollagen,23
the 500-bp BamHI fragment of pHT21
corresponding to the triple helical region of human 1(IV)
procollagen,24
as well as the 735 bp
SstI-EcoRI and the 730 bp
EcoRI-SstI fragments of pCllaseI (#57685,
American Type Culture Collection, Manassas, VA) harboring a human MMP-1
cDNA.25
The TIMP-1 probe was a reverse
transcription-polymerase chain reaction-derived cDNA
fragment.26
All of these probes had been extensively
characterized in previous studies.26-29
The MMP-13 probe
was generated by reverse transcription and subsequent amplification of
positions 24 to 495 in the reported sequence30
using
placental RNA as well as the primers 5'-CTG CCT TCC TCT TCT TGA GCT GG
and 5'-ATG ATG TCA GCA ATG CCA TCG. All constructs were subjected to
nucleic acid sequence analysis and conformed to the published
sequences. After linearization of the plasmids with either
HindIII or EcoRI restriction endonuclease, T7 or
SP6 RNA-polymerase (Gibco-BRL, Eggenstein, Germany), respectively, were
employed to obtain run-off transcripts of either the anti-sense
(complementary to mRNA), or sense (anti-complementary, negative
control) strands. Transcription and labeling of RNA probes were
performed as described previously using
[35S]-uridine-5'-(-thio)-triphosphate (1250 Ci/mmol,
New England Nuclear, Dreieich, Germany).26,27
The specific
activity routinely obtained was 1.2–1.4 x 109
cpm/µg.
In Situ Hybridization
Pre-hybridization, hybridization, washing procedures including removal of non-specifically bound probe by ribonuclease A digestion, and autoradiography of slides, as well as the procedure for combined immunohistology and radioactive in situ hybridization, were as described in detail.26,27
Statistical Evaluation
Autoradiographic signals were evaluated by counting silver grains attributable to individual cells. The background labeling was determined on adjacent serial sections subjected to sense control hybridization. In each tissue section the number of positively labeled cells and the relative transcript levels, ie, grains per cell, were determined in 10 microscopic fields at a magnification of 1:400. For this purpose the lamina propria was divided into three compartments: the superficial subepithelial myofibroblast (sSEMF) sheet, the pericryptal subepithelial myofibroblast (pcSEMF) sheet, and the remainder of the lamina propria (LP). The sSEMF sheet was defined as the double cell layer immediately under the superficial collagenous band extending to the crypt opening. The pcSEMF sheet consisted of the double cell layer immediately adjacent to the necks and bases of crypts. The standard error of the mean (SE) was calculated and statistical evaluation was performed using the Mann-Whitney U-test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Colon biopsies diagnosed as CC displayed a characteristic sharply
delineated, band-like linear deposition of ECM immediately underneath
the surface epithelium without extension along the crypts. The
subepithelial layer showed an average thickness between 11.1 and 44.2
µm. In many cases, the surface epithelium appeared flattened. The
number of intraepithelial lymphocytes was small. These findings were
accompanied by a mild, uncharacteristic inflammatory infiltrate of the
lamina propria, composed mainly of small lymphocytes, plasma cells, and
few eosinophils (Table 1)
.
Immunohistology
In all cases immunostaining for procollagen type III (Figure 1, A and B)
, and collagens type I and VI was
found in the extracellular space of the lamina propria, around blood
vessels, and under the surface and crypt epithelium. In CC, staining
with these antibodies was most intense in the superficial collagenous
layer. In ulcerative colitis and Crohn's disease, a moderate and
partially intense staining for procollagen type III and collagens type
I and VI was seen in the submucosal stroma. Collagen type IV was
localized in basement membranes of epithelium and blood vessels but did
not accumulate in the superficial collagenous layer. Tenascin-specific
staining was largely restricted to a thin layer underneath the surface
epithelium in normal mucosa (Figure 1C)
. In CC biopsies,
tenascin-specific staining was most prominent within the subepithelial
layer in its entire thickness (Figure 1D)
. In ulcerative colitis,
staining with this antibody was seen in areas with a dense inflammatory
infiltrate of the lamina propria, particularly around blood vessels.
The undulin-specific antibody displayed a weak staining in the lamina
propria, decorating densely packed fiber bundles in the submucosal
stroma (Figure 1E)
. The subepithelial ECM in normal controls and,
remarkably, in CC specimens did not display undulin-specific staining
(Figure 1F)
. In contrast, areas of transmural fibrosis in Crohn's
disease showed intense staining for undulin but not for tenascin. Some
of the stromal cells beneath the superficial collagenous layer stained
for smooth-muscle -actin, indicating a myofibroblastic phenotype of
these cells. Immunostaining for MMP-1 displayed a weak diffuse staining
within the lamina propria with accentuation around scattered
mononuclear cells.
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Procollagen 1(I) transcripts were detectable within the lamina
propria of all specimens. In normal colonic tissue, the signal
intensity, ie, the number of procollagen 1(I)-specific silver grains
attributable to individual cells, did not differ significantly between
sSEMF, pcSEMF, and other lamina propria cells (Figures 2A and 3)
. In
contrast, CC specimens displayed a significantly enhanced signal
intensity (P < 0.001) in sSEMF cells in an
almost linear, uninterrupted distribution (Figures 2B, 2C, and 3)
. The
relative number of cells expressing procollagen 1(I), however, was
similar in normal mucosa and CC specimens. Pericryptal subepithelial
cells and some of the superficial subepithelial cells stained for
smooth-muscle -actin, identifying these cells as myofibroblasts. By
combining immunohistology and in situ hybridization, we
found that at least a proportion of approximately 30% of the
procollagen 1(I)-expressing cells of the sSEMF sheet were
-actin-positive myofibroblastic cells (Figure 1, G and H)
.
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1(IV) procollagen transcript levels
were near the threshold of detection and required extended
autoradiographic exposure of up to 36 days to be clearly labeled. These
cells were found underneath the surface and crypt epithelium and along
small blood vessels (Figure 2E)
. In CC, procollagen 1(IV) RNA
expression showed a significantly elevated number of labeled cells in
all compartments of the LP and an increased signal intensity in the
sSEMF (P < 0.01) (Figures 2F and 4)
. The procollagen 1(IV)-expressing
cells displayed a procollagen 1(I)-like linear distribution pattern
with increased transcript levels in sSEMF cells.
|
. sSEMF
cell clusters with elevated MMP-1 transcript levels alternated with
extended regions devoid of any specific signal. The peculiarity of this
labeling pattern was evident when compared to procollagen
1(I)-specific hybridizations on neighboring serial sections (Figure 5D)
.
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|
. In the sSEMF sheet of
CC biopsies, TIMP-1 expression was increased as well
(P < 0.025), both with regard to the number of
cells and the signal intensity per cell (Figures 5F and 7)
.
TIMP-1-positive cells showed an almost linear distribution in the sSEMF
sheet including clusters of strongly labeled sSEMF cells. In serial
sections, these clusters of strongly TIMP-1 RNA-expressing cells
colocalized with clusters of cells displaying excess MMP-1 transcripts.
Thus, the cellular distribution of TIMP-1 RNA
appeared as a superimposition of both the procollagen 1(I) and the
MMP-1 characteristic patterns.
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MMP-13 RNA transcripts were not detectable in CC specimens and in three
cases of ulcerative colitis. The MMP-13 probe produced, however, a
clear signal in few submucosal mesenchymal cells of 1 of 3 cases
of Crohn's disease (Figure 6A)
, and a very intense autoradiographic
signal in peritumoral mesenchymal cells in 4 of 9 squamous cell
carcinomas of the head and neck region (Figure 6B)
. Hybridizations with
sense (control) probes revealed a weak, nonspecific background labeling
(Figure 2D)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The enhanced immunostaining for tenascin in the absence of any undulin/collagen XIV-specific labeling within the ECM layer may provide an explanation for these findings. Tenascin is considered a marker of mesenchymal cell proliferation and migration, and undulin is usually found in dense and differentiated collagen matrices. The tenascin/undulin dichotomy has previously been described in the context of matrix deposits that are subject to rapid remodeling, such as the ECM of tumors or in hepatic fibrosis.22,32 Thus, an ECM that is subject to rapid turnover, regardless of the collagen composition, is made up of loosely connected interstitial collagen fibers with a high tenascin content. In contrast, matrices of long-standing scar tissue such as those present in Crohn's disease or cirrhotic liver display a high undulin but a low tenascin content.22,33-36 Apparently, undulin condenses interstitial ECM by binding and interconnecting collagen type I, III, V, and VI fibers and decorin.37,38 These staining patterns can explain some aspects of the above-mentioned clinical observations.
In situ hybridization demonstrated synthesis of procollagen
1(I) and 1(IV) at significantly increased transcript levels by
resident sSEMF in all of our CC specimens. In case of procollagen
1(I), an almost linear distribution of sSEMF cells with high
transcript levels became evident. For procollagen 1(IV), an
essentially identical distribution of labeled cells was found, albeit
with lower autoradiographic signal intensity requiring extended
autoradiographic exposure. These observations indicate a continuously
up-regulated synthesis of both prototypic ECM molecules during the
course of CC. Our findings are well in keeping with the recently
published immunostaining patterns for collagens type I, III, IV, and
VI.39
The nonradioactive in situ hybridization
patterns for the corresponding procollagens reported in the latter
study are largely congruent with the distribution of (myo-)
fibroblasts displaying >30 silver grains in our hybridizations.
In CC, up-regulated MMP-1 expression was restricted to few sSEMF cells in clustered, patchy distribution, contrasting with ECM molecules, whereas its physiological inhibitor, TIMP-1, displayed a partially superimposable expression pattern. Degradation of the various ECM components is a function of an array of enzymes with diverse, partially overlapping substrate specificities.14-16 The observation of MMP-1 RNA suggests local enzyme synthesis, but does not prove the presence of active enzyme. Similarly, the immunostaining pattern for MMP-1 was inconclusive as it does not distinguish among zymogen, active, and inhibited forms of the enzyme. TIMP-1 expression seems to occur independently of MMP-1, as demonstrated by the weak, linear distribution of TIMP-1 RNA along the subepithelial collagenous layer. In areas with expression of MMP-1, MMP-1 protein, if activated, may be inhibited by TIMP-1 and other inhibitors unless, at balance, MMP-1 molecules prevail. Local and temporary changes of this balance in favor of active MMP-1 may then result in a rapid increase of the fibrolytic potential.
The expression of MMP-13 has previously been documented in ulcerative colitis and Crohn's disease at the base of ulcerations and proved to be absent within intact mucosa.40 MMP-13 was also found in squamous cell carcinomas of the head and neck region.41 Our in situ hybridization results confirm these patterns. In our CC biopsies, however, MMP-13 transcript levels were below the threshold of detection, suggesting that this collagenase may not play a major role in nonulcerating intestinal lesions such as CC. This finding and the observation of only few and scattered granulocytes as potential sources of MMP-8 (neutrophil collagenase) in the LP underline the suggested central role of MMP-1 expression for interstitial collagen type I degradation in CC. A future approach to the study of collagen degradation in CC should analyze the MMP-1 activation cascade including expression patterns of plasmin-generating molecules and stromelysins (MMP-3 and -10), which serve as co-activators of MMP-1.15,16
MMP expression in inflammatory bowel diseases has recently been studied by several groups reporting enhanced expression of MMP-1, -3, -7, -9, -10, -12, and -13 in ulcerative lesions of Crohn's disease, ulcerative and ischaemic colitis, and in wound healing.40,42-44 However, analysis of such lesions does not warrant conclusions about CC, because CC differs from the former lesions and entities by its peculiar clinical presentation and histology as a non-ulcerating, potentially reversible intestinal disorder.
In summary, the patterns of procollagen 1(I)-specific labeling in
CC, which is accompanied by an enhanced deposition of tenascin in the
absence of undulin, indicate synthesis of an immature, loosely
connected interstitial collagenous matrix that may be rapidly degraded.
The restricted occurrence of MMP-1 transcripts in the presence of
increased TIMP-1 and interstitial procollagen expression, however,
suggests a local imbalance of fibrogenesis and fibrolysis in CC,
resulting in the characteristic, linear ECM deposits. Thus, future
therapeutic strategies may be directed at influencing the levels of
TIMP-1 produced by sSEMF cells.
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Acknowledgements |
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Footnotes |
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Supported by the Deutsche Forschungsgemeinschaft (grants He 1330/2-1 and SFB366 C5) and the Werner-Otto-Stiftung. D. S. was a recipient of a Hermann-und-Lilly-Schilling professorship.
Accepted for publication April 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 1984, 23:6210-6216[Medline]
1(IV) chain of human type IV procollagen reveal an unusual homology of amino acid sequences in two halves of the carboxyl-terminal domain. J Biol Chem 1985, 260:7681-7687This article has been cited by other articles:
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  C. Medina and M. W. Radomski Role of Matrix Metalloproteinases in Intestinal Inflammation J. Pharmacol. Exp. Ther., September 1, 2006; 318(3): 933 - 938. [Abstract] [Full Text] [PDF]
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 C. V. Whiting, J. F. Tarlton, M. Bailey, C. L. Morgan, and P. W. Bland Abnormal Mucosal Extracellular Matrix Deposition Is Associated with Increased TGF-{beta} Receptor-expressing Mesenchymal Cells in a Mouse Model of Colitis J. Histochem. Cytochem., September 1, 2003; 51(9): 1177 - 1189. [Abstract] [Full Text] [PDF]
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 A Stallmach, C C Chan, K-W Ecker, G Feifel, H Herbst, D Schuppan, and M Zeitz Comparable expression of matrix metalloproteinases 1 and 2 in pouchitis and ulcerative colitis Gut, September 1, 2000; 47(3): 415 - 422. [Abstract] [Full Text] [PDF]
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D SCHUPPAN and E G HAHN MMPs in the gut: inflammation hits the matrix Gut, July 1, 2000; 47(1): 12 - 14. [Full Text] [PDF]
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