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. 1999 Jan;44(1):17–25. doi: 10.1136/gut.44.1.17

Increased expression of mRNA for matrix metalloproteinases-1 and -3 and tissue inhibitor of metalloproteinases-1 in intestinal biopsy specimens from patients with coeliac disease

S Daum 1, U Bauer 1, H Foss 1, D Schuppan 1, H Stein 1, E Riecken 1, R Ullrich 1
PMCID: PMC1760063  PMID: 9862821

Abstract

Background—Extracellular matrix (ECM) degradation may play a role in villus atrophy in coeliac disease (CD). 
Aims—To compare the cellular expression of mRNA transcripts for the two major matrix degrading proteases, matrix metalloproteinase (MMP)-1 and MMP-3, their inhibitor, tissue inhibitor of metalloproteinases (TIMP)-1, and procollagen I in the intestinal mucosa of patients with untreated and treated CD and normal controls. 
Patients/Methods—Duodenal biopsy specimens from ten untreated CD patients, from six of these after a gluten free diet, and from ten control patients were hybridised with 35S-labelled RNA probes. The number of positive cells in the subepithelial region and lamina propria were counted microscopically. 
Results—The numbers of cells positive for MMP-1 (p<0.005), MMP-3 (p<0.01), and TIMP-1 (p<0.05) mRNA were higher in the subepithelial region of CD mucosa than in that from controls. In the lamina propria, only cells positive for MMP-1 mRNA were increased in CD patients compared with controls (p<0.01). MMP-1 and MMP-3 mRNA expression returned to normal in CD patients after treatment with a gluten free diet (p<0.05), while TIMP-1 mRNA expression remained elevated. The number of procollagen I mRNA expressing cells did not change. Expression of MMP-1 and MMP-3 mRNA was mainly localised to subepithelial fibroblasts and macrophages. 
Conclusions—The decreased ratio of collagen I and TIMP-1 mRNA expressing cells to MMP-1 and MMP-3 mRNA expressing cells in untreated CD suggests a shift towards ECM degradation. ECM degradation by activated subepithelial fibroblasts and macrophages may be an important mechanism driving mucosal transformation in CD. 



Keywords: coeliac disease; extracellular matrix; villus atrophy; matrix metalloproteinases; TIMP-1; collagen I

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Figure 1 .

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The number (A) and proportion (B) of cells expressing matrix metalloproteinase-1 (MMP-1) mRNA is significantly increased in patients with coeliac disease (CD) compared with the controls, in both the subepithelial layer (SEL) and the lamina propria (LP). A significant reduction in MMP-1 transcripts in the subepithelial cell layer was found in biopsy specimens taken from CD patients on a gluten free diet (GFD) in comparison with untreated patients. The number of positive cells in the subepithelial region and the lamina propria were counted per high power field at a 400-fold magnification. (C) MMP-1 mRNA expression per positive cell is also increased in untreated CD compared with controls and compared with after treatment with a gluten free diet. Results are expressed as number of positive cells per high power field (HPF) (A), percentage of positive cells per total number of cells (B), and number of grains per positive cell (C). Results are from 10 patients with CD, 10 controls, and six of the CD patients after treatment with a gluten free diet. 


Figure 2 .

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(A) and (B) Expression of matrix metalloproteinase-3 (MMP-3) mRNA is significantly increased in the subepithelial layer (number of positive cells per high power field (HPF) and percentage of total number of cells that were positive) and in the lamina propria in patients with coeliac disease (CD) compared with controls (positive cells per high power field). The reduction in MMP-3 transcripts in CD patients after a gluten free diet (GFD) is only significant in the lamina propria (B). (C) In biopsy specimens from untreated CD patients compared with controls and after treatment with a gluten free diet, the MMP-3 mRNA expression per positive cell is increased in the subepithelial layer, but not in the lamina propria. For further explanations, see fig 1. 


Figure 3 .

Figure 3

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Localisation of matrix metalloproteinase (MMP)-1 (A), MMP-3 (B), tissue inhibitor of metalloproteinases (TIMP)-1 (C), and collagen I (D) mRNA in the duodenal mucosa in coeliac disease (CD) (left) and uninflamed control tissue (right) by in situ hybridisation. Black (white in dark field photographs in CD) grains indicate hybridisation of RNA antisense probes to mRNA in the tissue. In CD, expression of MMP-1 and MMP-3 mRNA was found almost exclusively in the subepithelial cell layer and the adjacent region of the lamina propria. MMP-1 and MMP-3 mRNA positive cells were morphologically assessed as macrophages or fibroblasts and myofibroblasts. In addition, expression of MMP-1 mRNA was detected in epithelial cells (E). TIMP-1 mRNA localised to the same regions as MMP-1 and MMP-3 mRNA but without epithelial staining (C). Positive staining for collagen I mRNA can be seen over subepithelial myofibroblasts and fibroblasts (D). (F) Sense hybridisation for procollagen I. Original magnification × 100 (dark field photographs and E) and × 200. 


Figure 4 .

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(A, B) The expression of matrix metalloproteinase (MMP)-1 mRNA in untreated coeliac disease (CD) epithelial cells is significantly increased per high power field and per total cells compared with controls and treated CD. (C) MMP-1 mRNA expression per positive cells is also increased in untreated CD compared with controls and treated CD. For further explanations, see fig 1. 


Figure 5 .

Figure 5

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Expression of tissue inhibitor of metalloproteinases (TIMP)-1 mRNA is significantly increased in the subepithelial layer of biopsy specimens from patients with coeliac disease (CD) (as percentage of positive cells per total number of cells (B) as well as positive cells per high power field (HPF) (A)) and in the lamina propria (A) compared with controls. (C) TIMP-1 mRNA expression is not altered by the gluten free diet in CD biopsy specimens, as TIMP-1 mRNA expression per positive cell is also not increased in untreated CD compared with controls and treated CD. For further explanations, see fig 1. 


Figure 6 .

Figure 6

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Expression of procollagen α1(I) mRNA in the duodenal mucosa of patients with active and treated CD and in uninflamed control tissue. Only the percentages of procollagen α1(I) mRNA positive cells per total cells are significantly reduced in CD biopsy specimens compared with controls. After a gluten free diet, procollagen α1(I) mRNA expression per positive cell in the subepithelial layer is increased compared with before the gluten free diet. For further explanations, see fig 1. 


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