ARTICLE |
Correspondence to: Christine V. Whiting, Div. of Veterinary Pathology, Infection and Immunity, Dept. of Clinical Veterinary Science, University of Bristol, Bristol BS40 5DU, UK. E-mail: c.v.whiting@bris.ac.uk
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Summary |
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Transforming growth factor-ß (TGF-ß) depresses mucosal inflammation and upregulates extracellular matrix (ECM) deposition. We analyzed TGF-ß receptors RI and RII as well as ECM components using the CD4+ T-cell-transplanted SCID mouse model of colitis. The principal change in colitis was an increased proportion of TGF-ß RII+ mucosal mesenchymal cells, predominantly -smooth muscle actin (SMA)+ myofibroblasts, co-expressing vimentin and basement membrane proteins, but not type I collagen. TGF-ß RII+ SMA- fibroblasts producing type I collagen were also increased, particularly in areas of infiltration and in ulcers. Type IV collagen and laminin were distributed throughout the gut lamina propria in disease but were restricted to the basement membrane in controls. In areas of severe epithelial damage, type IV collagen was lost and increased type I collagen was observed. To examine ECM production by these cells, mucosal mesenchymal cells were isolated. Cultured cells exhibited a similar phenotype and matrix profile to those of in vivo cells. The data suggested that there were at least two populations of mesenchymal cells responsible for ECM synthesis in the mucosa and that ligation of TGF-ß receptors on these cells resulted in the disordered and increased ECM production observed in colitic mucosa.
(J Histochem Cytochem 51:11771189, 2003)
Key Words: extracellular matrix, TGF-ß colitis, mouse model, TGF-ß receptors, intestine, wound healing
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Introduction |
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TRANSFORMING GROWTH FACTOR-ß (TGF-ß) is a ubiquitous cytokine that controls cell growth, differentiation, and death. It regulates proteolytic enzyme and extracellular matrix (ECM) production, neutrophil chemotaxis, and macrophage phagocytosis. It is considered to be important in the initial recruitment phase of an immune response but then to be one of the factors responsible for downregulating activated immune cells and promoting tissue repair (
TGF-ß activity is tightly regulated both during proteolytic generation of the biologically active cytokine (reviewed
There are contradictory reports on intestinal TGF-ß receptor expression. In human gut, RI and RII have been reported to be expressed by colon surface epithelial cells (
The human inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, are chronic diseases of unknown etiology but are probably caused by a loss of immunological tolerance, mediated by T-cells, to the normal indigenous flora (
The transfer of CD4+ T-cells into severe combined immunodeficient (SCID) mice results in a Th1-mediated colitis with similarities to Crohn's disease (transmural involvement, granuloma formation, mucosal hyperplasia, and T-cell accumulation in the mucosa) (
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Materials and Methods |
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Animals and Colitis Model
C.B-17+/+ mice (wild-type) and congenic C.B-17scid/scid mice (control SCID) were bred and housed under specific pathogen-free conditions. Experiments conformed to local and national guidelines. Control and transplanted SCID (colitic SCID) mice were housed under identical conditions. Colitic SCID mice were monitored daily for signs of disease and weights were recorded every week. C.B-17 mice are congenic with Balb/c mice. The CD4+ T cell-transplanted C.B-17 SCID model of colitis was first described by
Human Samples
Samples from four control human colons taken at tumor resection and three Crohn's disease and two ulcerative colitis resection specimens were collected from Southmead Hospital, (Bristol UK) and Southampton General Hospital (Southhampton, UK). Tissues were collected with approval of local ethical committees.
Immunohistochemistry
Samples were placed on cork discs (RA Lamb; London, UK), covered with OCT (RA Lamb), snap-frozen in isopentane cooled over liquid nitrogen, and stored at -70C. Five-µm sections for immunohistochemistry (IHC) from all groups of mice were cut at -20C on to the same slide and air-dried. Sections were fixed in acetone at 4C for 10 min and then rehydrated in PBS for 10 min. Staining for RII, -smooth muscle actin (SMA), plasminogen, or vimentin was enhanced by a 10-sec pretreatment in 50% (v/v) methanol/PBS. Nonspecific binding was blocked with 10% (v/v) normal goat serum (rat monoclonals) or 10% normal donkey serum (goat or rabbit polyclonal antibodies) in PBS for 1 hr at 20C, followed by an avidin/biotin block (Vector Laboratories; Peterborough, UK). To block endogenous mouse immunoglobulins when mouse primary antibodies were used, the MOM blocking kit (Vector) was used according to the manufacturer's instructions, with either the supplied biotinylated secondary or with isotype-specific fluorochrome-conjugated goat secondary antibodies (Southern Biotechnology; Birmingham, AL). Primary rat and mouse monoclonal or rabbit and goat polyclonal antibodies used are given in Table 1. Primary antibodies or isotype-matched control immunoglob- ulins were diluted in PBS and usually applied at 4C overnight. As a further negative control for RI and RII, primary antibody was incubated with a fivefold excess of immunizing peptide for 3 hr at 20C before application to the sections. Secondary antibodies were biotinylated goat anti-rat (HarlanSeraLab; Crawley Down, UK) 1:200, donkey anti-rabbit (Stratech; West Grove, PA) 1:500, and donkey anti-goat (Stratech) 1:250. Where possible, multiple primary or secondary antibodies for dual or triple immunoflorescence were added together. However, when two biotinylated secondaries were used, one was applied first followed by streptavidinFITC (Southern Biotechnology) (1:300), then a second avidin/biotin block, and then the second biotinylated secondary was added, followed by streptavidinTexas red (TXRD) (Vector) (1:100), or avidin AMCA (Vector) (1:100). When two rabbit primary antibodies were used, the antigen giving the weakest signal under optimized conditions was incubated first with the relevant primary overnight and then developed with goat anti-rabbit (Fab fragment) conjugated with FITC (Stratech) (1:200). The second rabbit primary was then added to the sections and incubated at 20C for 1 hr and then incubated with biotinylated donkey anti-rabbit IgG (1:1000) for 1 hr followed by streptavidinTexas red. Some slides were developed using ABComplexes (DAKO; Glostrup, Denmark) and peroxidase with DAB as substrate, as previously described (
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Image Capture and Analysis
Images were viewed using a Leica DMRB microscope (Leica UK; Milton Keynes, UK) and grabbed using a Colour Coolview CCD camera (Photonic Sciences; Robertsbridge, E. Sussex, UK) and Image-Pro Plus software (Media Cybernetics; Baltimore, MD). Multiple fluorescent images were viewed using a triple-pass filter. Sensitivity within the red, green, and blue channels and other camera settings was set at the start of each of the six analyses of images and kept constant for all images captured. Six to eight stored images of non-ulcerated colon, from each colon region for each group, were then analyzed automatically using Image Pro-Plus software (
Measurements were made of the distance between SMA+ cells and the basement membrane in non-ulcerated colon. In six experiments, tissue sections from all groups of mice and colon regions were double stained for SMA and type IV collagen. Two mice from each group were randomly selected and then one image from each region was randomly selected for analysis, with a total of six images analyzed for each group. The image was magnified by two levels and a minimum of 10 cell:BM distances were determined by drawing lines along the proximal (to the nearest BM) cell surface and then along the epithelial surface of the BM opposite the same cell, for the length of that cell. Image analysis was then used to determine the mean distance between the lines and data for the images from each group were pooled.
Cell Culture
Colons were taken from three 6-week-old Balb/c mice, cut open, and vigorously washed in cold PBS. The epithelium and mucus were scraped off with a scalpel blade and discarded. The remaining tissue was minced with two scalpel blades in serum-free RPMI culture medium containing antibiotics (penicillin 200 U/ml, streptomycin 200 µg/ml, gentamycin 100 µg/ml, and amphotericin B 0.25 µg/ml) and HEPES (20 mM). The pieces were washed with two changes of medium. Six-well plates were seeded with one sixth of a colon per well in 1 ml -MEM containing antibiotics, ribonucleosides, and 10% FCS and cultured in a humidified CO2 incubator. Half the medium was replaced after 4 days, and by day 6 outgrowing fibroblasts were observed. Cells were allowed to grow in six-well plates with weekly medium changes until about day 21, when cells were removed with trypsinEDTA and placed in 25-cm2 flasks. Thereafter, cells were trypsinized when confluent and flasks reseeded at 100 cells/mm2 either in tissue culture flasks or chamber slides (Falcon). Each trypsinization was considered as an increase in passage number. All culture media and supplements were from Invitrogen (Glasgow, UK).
Statistics
For analysis of image data from tissue sections, differences in untransformed cell size or area measurements were determined by analysis of variance using mouse group, colon region, and mucosal site as factors. For the analysis of distance of cells from the basement membrane, analysis of variance was carried out on log-transformed measurement data followed by a post hoc analysis to determine the level of significance between groups. Results were considered significant at p0.05.
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Results |
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Disease Scores
Tissues from all regions of inflamed SCID mouse colons were moderately diseased, with colon region scores ranging from 4 to 13 (average = 8.6/18 maximum), similar to those previously reported by our laboratory (
TGF-ß Receptor I and II Distribution in Wild-type, Control SCID, and Colitis SCID Mouse Colon
The distribution of RI was similar in wild-type (Fig 1A), control SCID (Fig 1B), and colitic SCID (Fig 1C) tissue. Subjectively, RI was expressed at high intensity by epithelial cells (Fig 1A1C) and all muscle cells, including smooth muscle of the outer muscle layers (Fig 1A1C), the muscularis mucosa (Fig 1B and Fig 1C) and surrounding vessels (Fig 1B). Cells in the LP showed lower intensity expression. Epithelial RI was expressed to the same intensity along the length of the crypt (Fig 1A1C). RII was also expressed at high intensity by all smooth muscle cells, including smooth muscle of the outer muscle layers and muscularis mucosa (Fig 1E and Fig 1G) and surrounding vessels (Fig 1E), in all groups of mice. Epithelial cells along the length of the crypt expressed RII and, subjectively, the intensity of epithelial expression was lower than that observed on muscle cells. Many LP cells expressed low levels of RII, but scattered spindle-shaped cells expressed RII at a particularly high intensity in controls (Fig 1E and Fig 1F) and colitic SCID (Fig 1G). The enlarged and elongated polymorphic cells were particularly prominent in colitis (Fig 1G and Fig 1I). With high magnification, both RI and RII were observed throughout the cytoplasm of expressing cells but not in the nucleus (data not shown). The antibodies were raised against conserved intracellular epitopes, so the intracellular expression probably represents a combination of newly synthesized, endocytosed, and inner membrane proteins. The specificity of these antibodies, which has been reported previously (
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In summary, there was no change in RI expression with disease, but the large LP spindle-shaped cells seen in disease showed very high-intensity expression of RII. Our next series of experiments sought to phenotype this polymorphic TGF-ß RII+ cell.
TGF-ß RII Was Strongly Expressed Predominantly on Myofibroblasts and Fibroblasts
Double immunofluorescence was carried out to identify the spindle-shaped cells that expressed high levels of RII. Expression of RII on these cells was observed to mainly co-localize with SMA (Fig 2A) and plasminogen (Fig 2B). Lamina propria SMA also co-localized with vimentin (Fig 2D), an intermediate filament found in mesenchymal cells, indicating that most of these cells were myofibroblasts (MFB). As expected, vimentin was also expressed on all other LP cells, but epithelial and muscle cells were vimentin-negative. There were cells, or parts of cells, showing SMA expression alone, but because vimentin tended to be perinuclear both in vivo and in vitro (Fig 6F) this probably represented SMA staining of peripheral parts of MFB. However, the presence of SMCs in the mucosa in colitis cannot be discounted. A small population of RII+ cells not expressing SMA was observed in the submucosa and LP of all groups of mice, in colon mucosal lymphoid aggregates of wild-type mice, and in heavily infiltrated areas of diseased colon mucosa (Fig 2E). Using serial sections and double staining with combinations of antibodies against RII, SMA, type I collagen (CI) (Fig 2E2G), and vimentin (data not shown), these cells were defined as RII+ CI+vimentin+ SMA- fibroblasts expressing membrane RII and pericellular type I collagen. ECM proteins are rapidly secreted and deposited as insoluble fibers, so although cell-associated type I collagen was observed in tissue sections (Fig 5F) and in cultured cells (Fig 6D), some of the type I collagen staining represented matrix fibers. Because there is no marker for mouse fibroblasts, this population was further characterized to eliminate other cell types that may have co-localized with the spindle-shaped type I collagen deposition, but no co-localization was observed with immune cells, endothelium, or SMC (data not shown). To a lesser extent, RII also co-localized with endothelium (Fig 2H), MadCAM-1+ HEV (Fig 2I), F4/80+ macrophages (Fig 2K), and MHC class II+ (Fig 2L) cells. Although Fig 2I demonstrates quite high levels of RII expression by MadCAM-1+ HEV, such extensive co-localization was a relatively uncommon observation. The number of MadCAM-1+ vessels did increase in SCID mouse colitis (data not shown), but not to a level that could account for the number of RII+ spindle-shaped cells. The vessels were also morphologically quite distinct from MFB.
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In summary, the major change in TGF-ß receptor expression observed in colitis was the emergence of two populations of fibroblasts: (a) widespread, large LP myofibroblasts with an RII+ SMA+ vimentin+ plasminogen+ phenotype, and (b) fibroblasts with a RII+ SMA- vimentin+ type I collagen+ phenotype associated with mucosa, submucosa, and leukocyte aggregates in controls, and spreading to areas of intense inflammatory leukocyte infiltration in colitis.
TGF-ß RII+ Myofibroblasts Were Increased in Size and Occupied an Increased Mucosal LP in Colitis
It was not possible to count individual MFB, so short axis diameter of RII+ SMA+ cells was used as a measure of size, and area of mucosa occupied was used as a measure of the relative proportion of these cells in the tissue. Image analysis showed that, in inflamed tissue, RII+ SMA+ MFB were larger than in either of the control groups (Fig 3A), as determined by short axis maximal diameter. Mean cell size was greater in colitis in all regions of the colon, with 14/15 sections in the superficial mucosa and 10/15 sections in the deep mucosa having a mean short axis diameter larger than SCID control means (p<0.0001, p<0.02, and p=0.001 for proximal, mid- and distal regions, respectively). Mean cell size was also greater in colitis than in wild-type controls in the mid- and distal regions (p<0.02 and p<0.01 respectively). Random orientation of these cells in the tissue precluded measurement of the long axis. In colitis, RII+ SMA+ cells were found throughout the mucosa in contrast to controls, in which they were mostly concentrated in the deep mucosa. There was a significantly greater area of superficial mucosa occupied by RII+ SMA+ cells in colitis compared to either of the control groups (p<0.0001 for both comparisons) (Fig 3B). In the superficial mucosa, an increased area of double-stained cells was observed in all regions of the colon, and 13/15 sections from colitic tissue had mean values greater than controls. The increased size and volume occupied by RII+ cells is particularly obvious in Fig 1G, and Fig 1I vs 1E and 1F (note the scale difference).
In summary, the SMA+ RII+ LP MFB are enlarged and occupy an increased proportion of the mucosa in the inflamed colon.
SMA+ MFB Dissociate from the Basement Membrane in Colitis
Using image analysis on a random sample of colitis and control tissues, MFB were shown to spatially separate from the basement membrane (Fig 4). In control tissues, the median distance from the proximal edge of MFB, defined by SMA expression, to the epithelial surface of the BM was 1.85 µm (control SCID range 0.66.6 µm) and 2.3 µm (wild-type range 1.16.6 µm). The median distance observed in colitic SCID colon tissues (median 4.41) was significantly greater than both wild-type and SCID control tissue (p<0.0005 for both comparisons). There were many cells in colitic SCID colon that were close to the BM, but the range was greatly increased (117 µm). Wild-type control MFB were at a significantly greater distance from the BM than SCID control MFB (p=0.033).
Changes in the ECM in Colitis
Myofibroblasts and fibroblasts contribute to ECM protein production and are responsible for all of the stromal type I collagen deposition in the mucosa. By double immunofluorescence, SMA+ MFB co-localized with both type IV collagen (Fig 5A5C) and laminin (Fig 5D and Fig 5E), two major BM proteins. Endothelium also co-localized with these BM proteins (Fig 5D and Fig 5E), but there was no co-localization of laminin and type IV collagen with epithelial cytoplasm. Type I collagen also formed part of the BM, but SMA did not co-localize with either large spindle-shaped type I collagen or the dense bands of type I collagen observed in ulcers. This can be most clearly seen in Fig 2F, where type I collagen (labeled with FITC) is just visible surrounding each crypt but there was no co-localization of SMA (TXRD) with the type I collagen cluster. The anti-type I collagen antibody used did not crossreact with type IV collagen or laminin, as determined by ELISA (data not shown). Spindle-shaped cells expressing both type I and type IV collagens were observed in ulcer beds (Fig 5G, arrow). In wild-type and control SCID mouse tissue, the BM was thin and continuous, underlying the epithelium (Fig 5A, Fig 5B, and Fig 5D). However, in disease both type IV collagen (Fig 5C) and laminin (Fig 5E) were distributed throughout the LP and the BM was either more diffuse and occasionally thickened (Fig 5C and Fig 5E) or was absent bordering crypt abscesses (Fig 5C). Occasionally, at the luminal surface (Fig 5F) and in ulcers (Fig 5G), normal BM, as defined by type IV collagen deposition, was absent or incomplete and was replaced by a dense band of type I collagen (insets in Fig 5F and Fig 5G). In Fig 5F, cells expressing nascent, intracellular, or pericellular type I collagen (arrows, inset) can be seen just below the dense band of type I collagen that has been deposited in an area where the BM and epithelium appears to be dissociating from the underlying LP. The data suggested that there were at least two populations of mesenchymal cells synthesizing matrix within the mucosa. Although these results suggested that RII+ mucosal mesenchymal cells contribute to the de novo synthesis of laminin, type IV collagen, and type I collagen, it is also possible that co-localization simply indicates deposited ECM proteins surrounding or adhering to these cells. To investigate ECM synthesis by mucosal mesenchymal cells and to determine the relationship between SMA+ cells and ECM synthesis, we designed experiments using isolated mucosal cells.
Ex Vivo Cultured MFB Have a Similar Phenotype to In Vivo Cells
Cultures of colon mesenchymal cells were established and analyzed for the expression of the same proteins as analyzed in frozen sections. Cells from passage 1 to 3 expressed vimentin, RI, RII, type IV collagen, and laminin (Fig 6B, Fig 6C, and Fig 6F6H) but were a mixture of -SMA positive and negative phenotype (Fig 6A) and exhibited variable type I collagen expression (Fig 6D). The majority of cells synthesized type I collagen, whereas only 30% of cells synthesized SMA at passage 1 (4 weeks of culture). Fig 6I and Fig 6J show cells that expressed (a) both SMA stress fibers and type I collagen at passage 1 (arrows), (b) neither protein (open arrowheads), or (c) only type I collagen (closed arrowheads). Cells varied in their morphology, from fine spindles, which were usually SMA-, to large polymorphic cells that usually expressed SMA. Cultures did not contain endothelial cells or immune cells because there was no expression of either the pan-endothelial antigen or CD45. Further evidence that myofibroblasts and fibroblasts expressed functional TGF-ß receptors was obtained from experiments in which the addition of TGF-ß to cultured cells resulted in altered ECM synthesis (unpublished observations).
RII-expressing MFB in Human Control and Colitis Tissue
In a limited study of five inflamed and four control human colons, SMA co-localized with RII expression on LP spindle-shaped cells in both controls (Fig 7A) and in colitis (Fig 7B). Fig 7 is representative of findings for all control and inflamed tissue studied. As observed in mouse colitis, RII+ SMA+ cells were greatly enlarged in colitis.
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Discussion |
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Transforming growth factor-ß has crucial roles in regulating both the immune response and ECM deposition in the intestine, and its activity is, therefore, tightly regulated. A major facet of this regulation is control of expression of the two main signaling receptors, RI and RII. This study shows that, in the colon, expression of these receptors in the normal state is concentrated on smooth muscle elements and the epithelium, where they presumably function in matrix homeostasis (
It is perhaps surprising that, given the role of TGF-ß in regulating colitis in this model (
Our observation that colon mucosal MFB also co-localized with plasminogen gives a clear indication that one of the most important mechanisms for the local activation of TGF-ß is associated with these cells. Plasminogen, when cleaved by plasminogen activator to plasmin, is known to activate latent TGF-ß, and its co-localization with RII makes it likely that these cells are able to generate and respond to active TGF-ß. In further support of this,
Myofibroblasts were much enlarged in colitis, in all regions of the colon. It is likely that increased size of MFB equates with increased activation status. LPS-activated gut MFB exhibit increased proliferation and ECM synthesis and produce elevated levels of inflammatory mediators (-stimulated MFB produced increased levels of activated MMP (
in areas of crypt destruction and tissue infiltration (
Myofibroblasts are important in the intestine to help maintain barrier function and are also involved in the immune response. Our studies have shown that in vivo, and in isolated cells, MFB co-localize with the BM proteins type IV collagen and laminin. BM proteins have previously been detected in cultured human MFB (
The deposition of type I collagen in areas in which laminin and type IV collagen have been lost probably represents attempted wound healing. In both healthy and inflamed intestine, type I collagen was deposited at a low level in the epithelial BM, similar to findings in the human colon (
To characterize the mesenchymal cell population for future analysis of the effects of TGF-ß in vitro and to determine the relationship between SMA and type I collagen, we carried out an analysis of cell phenotype and matrix synthesis by ex vivo cultured mucosal MFB. Although the elevated deposition of BM proteins throughout the lamina propria could have been due to endothelium, only fibroblasts and myofibroblasts expressed high levels of TGF-ßRII. The isolated cells represented a mixed population with a similar phenotype to the mesenchymal cells in the mucosa in tissue sections, i.e., all early-passage cells expressed vimentin, type IV collagen and laminin, in agreement with data for adult human MFB (
TGF-ß-mediated pathology in the colon will depend on the level of biologically active protein and the level of intracellular signaling after ligation of target cell receptors. MFB and fibroblasts certainly produce type I collagen and BM proteins, and their expression of high levels of TGF-ßRII indicates that they are capable of responding to TGF-ß in vivo, probably by upregulating matrix synthesis. It is probable that, in normal uninflamed intestine, MFB are in a quiescent state and BM protein synthesis is controlled by crosstalk with the epithelium, including control by TGF-ß. MFB response to TGF-ß could be altered on activation during inflammation, potentially losing the control of directed synthesis provided by the epithelium. Alternatively, due to tissue architecture disruption, MFB spatially separate from the high level of BM TGF-ß and produce BM matrix in a non-directed fashion throughout the mucosa, resulting in decreased deposition at the BM. On the basis of the data, one of the consequences to disease pathogenesis of changed activation status and ECM production of the mesenchymal cell population is likely to be compromised epithelial barrier function.
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Acknowledgments |
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Supported by European Union grant QLGI-CT-1999-00050.
We would like to thank Dr Jorg Reimann (Ulm University, Germany) for providing mouse tissue, and Prof Thomas T. MacDonald (Southampton University, UK) for human IBD tissue.
Received for publication September 3, 2002; accepted March 26, 2003.
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