ARTICLE |
Correspondence to: Christine V. Whiting, Div. of Molecular & Cellular Biology, Dept. of Clinical Veterinary Science, University of Bristol, Langford, Bristol, UK BS40 5DU. E-mail: c.v.whiting@bris.ac.uk
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Summary |
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Using a CD4+ T-cell-transplanted SCID mouse model of colitis, we have analyzed TGF-ß transcription and translation in advanced disease. By in situ hybridization, the epithelium of both control and inflamed tissues transcribed TGF-ß1 and TGF-ß3 mRNAs, but both were expressed significantly farther along the crypt axis in disease. Control lamina propria cells transcribed little TGF-ß1 or TGF-ß3 mRNA, but in inflamed tissues many cells expressed mRNA for both isoforms. No TGF-ß2 message was detected in either control or inflamed tissues. Immunohistochemistry for latent and active TGF-ß1 showed that all cells produced perinuclear latent TGF-ß1. The epithelial cell basal latent protein resulted in only low levels of subepithelial active protein, which co-localized with collagen IV and laminin in diseased and control tissue. Infiltrating cells expressed very low levels of active TGF-ß. By ELISA, very low levels (069 pg/mg) of soluble total or active TGF-ß were detected in hypotonic tissue lysates. TGF-ß1 and TGF-ß3 are produced by SCID mouse colon and transcription is increased in the colitis caused by transplantation of CD4+ T-cells, but this does not result in high levels of soluble active protein. Low levels of active TGF-ß may be a factor contributing to unresolved inflammation. (J Histochem Cytochem 49:727738, 2001)
Key Words: TGF-ß, inflammatory bowel disease, in situ hybridization, basal lamina
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Introduction |
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Transforming growth factor-b (TGF-b) is a multifunctional growth factor that influences growth and differentiation in many cell types. There are three isoforms in mammals (reviewed in
Although the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, are of unknown etiology, an inappropriate and uncontrolled immune response involving infiltrating T-cells and macrophages, probably responding to intestinal luminal antigens, is believed to be involved. A feature of chronic disease is increased matrix deposition, probably modulated via myofibroblast TGF-ß ( and modulation by TGF-ß. On the other hand, levels of TGF-ß message and protein are increased in active human IBD (
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Materials and Methods |
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Animals and Development of Disease
C.B-17+/+ mice and congenic C.B-17scid/scid (SCID) mice were bred and housed under specific pathogen-free conditions in the animal colonies of the University of Ulm, Germany and the Panum Institute, University of Copenhagen, Denmark. Experiments conformed to local and national guidelines. Transplanted and non-transplanted mice were monitored under identical conditions. Mice were injected IP with 5 x 105 non-fractionated CD4+ splenic T-cells and monitored for signs of the disease as previously described (
Immunohistochemistry, Antibodies, and Reagents
Colon tissue samples were taken from 11 CD4+ T-cell-transplanted, diseased SCID mice and from five non-transplanted age- and sex-matched control SCID mice. Samples were placed on labeled cork discs (RA Lamb; London, UK), covered with OCT (RA Lamb), and snap-frozen in isopentane cooled over liquid nitrogen. Samples were stored at -70C. Sections for immunohistochemistry (5 µm) and in situ hybridization (10 µm) were cut at -20C and air-dried. Spleen tissue from transplanted mice was used as a positive control for TGF-ß staining.
Sections for ß1-LAP immunohistology were fixed in 100% methanol at -20C for 10 min. Sections to be stained with other antibodies were fixed in acetone at 4C for 10 min and then rehydrated in PBS for 10 min. Nonspecific staining was blocked with 50% (v/v) methanol in PBS plus 0.6% (v/v) hydrogen peroxide, followed by 10% (v/v) normal goat serum plus 5% (w/v) non-fat milk powder (for TGF-ß) or 10% normal horse serum (for ß1-LAP, collagen, and laminin) in PBS for 1 hr at 20C, followed by an avidinbiotin block (Vector Laboratories; Peterborough, UK). Chicken anti-TGF-ß1 (10 µg/ml) (R&D Systems Europe; Abingdon, UK), goat anti-ß1-LAP (10 µg/ml) (R&D), or rabbit anti-mouse collagen IV, or rabbit anti-mouse laminin (both 1 µg/ml) (TCS Biologicals; Buckingham, UK) were applied in PBS. The anti-ß1-LAP antibody was centrifuged at 10,000 x g for 5 min to remove debris, possibly caused by interaction between the antibody and latent TGF-ß bound to immunoglobulin. Biotinylated secondary antibodies were goat anti-chicken 1:500 (Vector), donkey anti-rabbit (Jackson ImmunoResearch Laboratories; West Grove, PA) 1:1000 or donkey anti-goat (Jackson) 1:1000. After washing in PBS, StreptABComplex/HRP peroxidase complexes (DAKO; Glostrup, Denmark) were added to sections following the manufacturer's instructions. To amplify the TGF-ß stain, biotin-conjugated anti-streptavidin (Vector) 1:100 was applied for 1 hr, followed by a second round of ABC complexes. After further washing, staining was visualized with 1.67 mM diaminobenzidene-4HCl in 0.05 M Tris-HCl, pH 7.5, plus 0.06% (v/v) H2O2. The slides were then either counterstained with Mayer's hematoxylin (Merck; Poole, UK) and mounted in DPX or mounted without counterstain which tended to mask the delicate TGF-ß stain. For double immunofluorescence staining, the above secondaries were used sequentially with an avidinbiotin blocking step. TGF-ß was detected with streptavidinFITC (Southern Biotechnology; Birmingham, AL) and collagen IV or laminin with streptavidinTexas Red (Vector). Chicken IgY, goat IgG, or rabbit IgG isotype controls were applied to sections at the same concentration as primary antibodies. All reagents were from Sigma, (Poole, UK) except where indicated.
In Situ Hybridization
Plasmids containing cDNA for TGF-ß1, -ß2, and -ß3 (
In situ hybridization and detection of hybrids were performed essentially as previously published (
Sections were desalted before the stringent wash in 0.1 x SSC at 65C for 60 min in a Hybaid Omnislide wash module. Slides were then cooled and stabilized in 2 x SSC for 5 min before treatment with RNase A (Boehringer-Mannheim) (1 µg/ml) at 37C for 30 min followed by extensive washing. DIG-labeled transcripts were then detected as previously (
Microscopy and Image Analysis
Images for analysis were viewed through an F15 Panasonic CCD video camera, grabbed with Neotech software, and analyzed using GaugeGem software (both from ME Electronics; Reading, UK) or captured using a Colour CoolView camera (Photonic Science; Robertsbridge, E. Sussex, UK) and Image Pro Plus software (Media Cybernetics; Baltimore, MD). Crypt lengths were measured using the x10 objective. Images were calibrated using a graticule.
ELISA
Colons were taken from different groups of mice from those examined histologically, but transplanted mice showed similar levels of disease. Colons were frozen in liquid nitrogen after removal of fecal contents by extrusion under gentle pressure. Tissues were weighed while frozen, pulverized in a freezer mill, and then subjected to hypotonic lysis at a ratio of 100 mg tissue:500 µl lysis buffer (20 mM Tris buffer, pH 7.5) on ice, in the presence of protease inhibitors (2 mM PMSF, 1.66 µM aprotonin, 0.5 µM soybean trypsin inhibitor, 20 µM leupeptin, 10 mM EDTA). Samples were centrifuged at 20,000 x g for 20 min at 4C and lysates were used immediately. Protein concentration of the supernatant was determined by the bicinchoninic acid (BCA) method (Pierce; Rockford, IL) using BSA for standard curve preparation. Lysates were first diluted to an equal protein concentration of 6.8 mg/ml and then to 1.7 mg/ml in 0.2 x PBS, acidified to pH 2 with 0.5 M HCl, and neutralized with 0.5 M NaOH to pH 7.5 (pH was confirmed using pH-fix 0-14 dipsticks; Fisher, Loughborough, UK). PBSTween (0.05% v/v) (PBT) was then added to take samples to a dilution of 1.36 mg/ml and globulin-free BSA (Sigma) was added to a final concentration of 0.05% (w/v). The rabbit and chicken anti-TGF-ß antibodies used in the ELISA had previously been shown to detect mouse splenic TGF-ß by Western blotting (data not shown). Wells were coated with catching antibody (5 µg/ml rabbit anti-TGF-ß; R&D) in carbonate buffer (pH 9.5) overnight at 4C. Plates were washed and then blocked with 0.1% BSA in PBT. After one wash, triplicate samples and TGF-ß standards (R&D) were added to wells and incubated overnight at 4C. Plates were washed and then chicken anti-TGF-ß (0.1 µg/ml; R&D) was added in 0.05% globulin-free BSA in PBT. Biotinylated goat anti-chicken (1:1000) was added after washes, then the complexes (1:50), followed by TMB substrate. Plates were read at 650 nm and concentrations of TGF-ß calculated against a standard curve using Labsystems Genesis software (Life Sciences International). Positive controls included spleen lysates and mouse plasma (with and without acid treatment). Negative controls included omission of sample and replacement of chicken detection antibody with chicken IgY. The sensitivity of the assay was 10 pg/mg soluble protein. In some experiments, lysates or BSA (1.36 mg/ml) in the same buffer as added to wells were spiked with 100 pg TGF-ß. Spiked samples were added to wells after 15-min incubation on ice. Results are expressed as expected TGF-ß (100 pg spike plus any TGF-ß present in unspiked sample) minus actual sample reading calculated from the standard curve.
Statistical Analysis
The MannWhitney U-test was used to determine the significance of differences between groups of data. An initial multiple regression analysis was done using a general linear model to determine if any disease score indicators correlated with TGF-ß transcription. Final analysis was carried out using Pearson correlation.
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Results |
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Disease Status
All T-cell-transplanted mice showed symptoms of advanced disease. All diseased colons were macroscopically thickened at autopsy. Histologically, the colon tissues exhibited various degrees of crypt hyperplasia and leukocyte infiltration. Infiltrating cells were principally CD3+ T-cells, macrophages, and polymorphonuclear cells (data not shown).
In Situ Hybridization
Distribution of TGF-ß mRNA.
The hybridization conditions used were sufficiently stringent to differentiate the three isoforms. All three TGF-ß isoform probes gave strong hybridization signals using splenic megakaryocytes as a positive control (Fig 1K, Fig 1L, and Fig 1M).
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Epithelium. TGF-ß1 and TGF-ß3 mRNA showed similar distribution in the epithelium. Transcription by crypt epithelial cells was observed in tissues from both control (Fig 1B and Fig 1H), diseased (Fig 1A and Fig 1G), and C.B-17+/+ (data not shown) animals. Expression in epithelial cells was clearly cytoplasmic (Fig 1J). No TGF-ß2 mRNA was detected in either control or inflamed SCID mouse colon (Fig 1D and Fig 1E) or C.B-17+/+ colon (data not shown).
In inflamed tissues, transcription of TGF-ß1 and TGF-ß3 by crypt epithelium showed an extended distribution along the crypt axis (compare Fig 1A and Fig 1G to Fig 1B and Fig 1H). In some areas, message was observed in all crypt epithelial cells, including surface epithelium. By image analysis, the average length of crypts in control mice was 199 µm (range 180214 µm) compared to 470 µm (range 356633 µm) in diseased animals. In control mice, crypt epithelium expressed TGF-ß1 mRNA over an average of 63% of crypt length before the signal became undetectable, whereas in tissues from inflamed mice transcription of TGF-ß1 extended further along the crypt axis (82%) (Fig 2). Similarly, extension of TGF-ß3 transcription along the crypt axis was also seen (control 53%, inflamed 73%) (Fig 2). The difference between control and inflamed tissue was significant for both TGF-ß1 and TGF-ß3 (p<0.01). However, there was no correlation between degree of crypt hyperplasia (crypt length) and the distance along the crypt of epithelial cells expressing TGF-ß1 or TGF-ß3 mRNA. TGF-ß1 transcription was significantly correlated with the sum of the scores for crypt branching and mononuclear cell infiltration (p<0.02) (Table 1).
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Lamina Propria. Colon lamina propria of control SCID mice showed low-level infiltration by mononuclear cells which were almost all F4/80+ (data not shown), with occasional clusters of presumed pre-B-cells in immature follicles. There were very few TGF-ß mRNA-positive cells. However, inflamed colon was infiltrated at variable levels throughout mucosa and serosa by cells expressing both TGF-ß1 and TGF-ß3 mRNA (Fig 1A, Fig 1G, and Fig 1J).
Determination of TGF-ß Protein
Immunohistochemistry.
TGF-ß is produced as a latent precursor protein, with the active molecule maintained in the latent state by electrostatic interaction with its latency-associated peptide (LAP). This study used goat anti-ß1-LAP to detect latent protein and chicken anti-TGF-ß antibody, which has been reported to detect only active TGF-ß (
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Immunohistology of colon tissues showed weak but distinct expression of active TGF-ß1 at the junction between the epithelium and lamina propria in both non-transplanted (Fig 4A and Fig 4C) and diseased mice (Fig 4B and Fig 4D). This basal protein co-localized with both collagen IV (Fig 5A and Fig 5D) and laminin (Fig 5B) in the basal lamina in both controls and colitis and was strongest on the epithelial side of the basement membrane. Although this basal lamina TGF-ß was observed along the entire length of the crypt, expression was strongest beneath luminal epithelium. In controls, laminin (Fig 5B) and collagen IV (Fig 5D) showed a similar distribution and were restricted to basement membranes bordering crypts or lamina propria vessels. As can be seen in Fig 5A, there was disordered collagen IV deposition within the expanded lamina propria of diseased mice, and both matrix components were more widely distributed throughout the tissue. Within epithelial cells, TGF-ß expression was generally weak, confined to the apical cytoplasm, and was expressed by cells throughout the length of the crypt. Only rarely were clusters of strongly stained epithelial cells seen in diseased mice in the deep crypts or near the lumen (Fig 4G). These clusters were always surrounded by infiltrating cells, but no conclusions could be drawn because of their infrequent occurrence. In the lamina propria, mononuclear cells were only weakly positive for active TGF-ß, whereas endothelium on some large and small vascular structures was positive in both controls (Fig 4A and Fig 4C) and colitis (data not shown).
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ELISA
Increased TGF-ß transcription in disease did not lead to obvious differences in the levels of TGF-ß protein by immunohistology. Therefore, to determine changes in protein levels, tissue lysates were first examined by Western blotting, but no protein was detected in colon tissue, whereas it was readily detected in spleen (data not shown). Therefore, an ELISA was developed that detected mouse TGF-ß at a sensitivity of 10 pg/mg (700-fold more sensitive than Western blotting). Acid-activated spleen lysates and mouse plasma contained 830 pg/mg and 5220 pg/ml, respectively, with much lower levels (44 pg/mg and 180 pg/ml) in non-activated samples (plasma data not shown). In lysates of control and diseased colon (Fig 6), levels of TGF-ß were near the limits of detection of the assay and many samples were negative. There was no difference in either acid-activated or non-activated samples between controls and diseased mice. To investigate possible sequestration of TGF-ß by soluble matrix components, colon lysates were spiked with 100 pg TGF-ß. Detection of the TGF-ß spike in lysis buffer containing BSA as a protein source at an equivalent protein concentration to cell extracts gave 102.4 ± 4.6 pg recovered (mean ± SD of three determinations). In contrast, only 30% of the TGF-ß spike was detectable when spiked colon lysates were used in the ELISA (mean 28.7 ± 6.8 pg recovered).
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Discussion |
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This study has shown that, in severe colon inflammation, TGF-ß1 and TGF-ß3 transcription by cells in all layers of the intestine is increased, but particularly in the epithelium. Although mRNA was translated to ß1-LAP, this did not result in a concomitant elevation of active TGF-ß protein. To our knowledge, there have been no other studies in the rodent large intestine of TGF-ß transcription detected by in situ hybridization in combination with the determination of latent and active protein by immunohistology. First, with regard to the epithelium, we have shown that, in control SCID mice (and in C.B-17+/+ mice), TGF-ß1 and TGF-ß3 mRNA were readily detected in colon lower crypt epithelial cells. This agrees with the majority of reports on rodent small intestine (
In summary, we suggest that, in the normal adult rodent, concordant transcription of TGF-ß1 and TGF-ß3 takes place in colon immature crypt epithelial cells and that, as differentiation takes place, transcription is suppressed. This is consistent with the known role of TGF-ß in enterocyte differentiation and with TGF-ß2 being essential for epithelialmesenchymal interactions during embryogenesis, but not for epithelial differentiation in the adult. In inflammation-induced crypt hyperplasia, as the proliferative zone extends and the proportion of immature enterocytes in the crypt increases, so the transcription of TGF-ß1 and TGF-ß3 is extended in distribution, with no effect on TGF-ß2 transcription. Indeed, no change was found in epithelial TGF-ß2 transcription in radiation-induced enteropathy (
Second, with regard to the inflammatory infiltrate, many cells were observed that transcribed both TGF-ß1 and TGF-ß3. They were seen in all layers of the intestine, including the muscle wall. Similarly, in human IBD (
In contrast to transcription, little evidence for elevated levels of latent or active TGF-ß protein was found in this study by immunohistology, Western blotting, or ELISA. All cells produced perinuclear ß1-LAP, with strongest epithelial expression in deep crypt epithelium. However, there was no obvious increase in translation of LAP in colitis, even though transcription of TGF-ß1 was greatly increased, most notably in the epithelium. There may be post-transcriptional controls that prevent increased translation to LAP.
Active TGF-ß1 protein was clearly demonstrated at the interface between the epithelium and the lamina propria. In both controls and inflamed tissue, it co-localized with basement membrane collagen IV and laminin. Collagen IV has been shown to bind TGF-ß (
Only weak expression of TGF-ß by infiltrating mononuclear cells was seen. These findings of low levels of active TGF-ß expression in both the epithelium and the inflammatory infiltrate suggest that levels are tightly controlled in the gut. The absorption of active TGF-ß by colon lysates suggests the presence of an inactivating protein such as decorin, which has been shown to tightly bind TGF-ß and block its biological activity (
Other studies of the distribution of TGF-ß protein (
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Footnotes |
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1 Current address: Novo Nordisk A/S Novo Alle DK-2880, Bagsværd, Denmark.
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Acknowledgments |
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Supported by grants from the European Union no. BMH4-96-0612 and QLG1-CT-1999-00050.
Received for publication July 19, 2000; accepted January 19, 2001.
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