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. 2000 Dec;157(6):1927-35.
doi: 10.1016/S0002-9440(10)64831-6.

The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis

J F Tarlton  1 , C V Whiting, D Tunmore, S Bregenholt, J Reimann, M H Claesson, P W Bland
Affiliations

The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis

J F Tarlton et al. Am J Pathol. 2000 Dec.

Abstract

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

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Figures

Figure 1.
Figure 1.
Up-regulation of tissue serine proteinases in SCID mice with colitis. Zymography was performed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing casein as substrate and incubated overnight at 37oC in the presence of EDTA. A: Colonic extracts from eight SCID mice transplanted with CD4+ T cells. B: Colonic extracts from four C.B-17 mice 9–12 and two nontransplanted SCID mice. 13,14 Arrowheads depict rat neutrophil elastase marker. Three samples per mouse: distal colon, left; mid-colon, middle; proximal colon, right. Relative mobility of the major protease species is given. C: Comparison of proteases in an extract from a colitic colon from a transplanted mouse (C) with those in a mouse pancreatic extract (P), with trypsin (T) and chymotrypsin (Ch) markers. D: Relative activity of total serine proteases and of the three major protease species (23 kd, 30 kd, and 45 kd) in the three groups of mice, mean densitometry values of all samples in each group + SE. E: Relative activity of total and individual serine proteases in distal, mid, and proximal regions of the colon in CD4+ T-cell-transplanted SCID mice only, mean of densitometry values from all eight mice + SE.
Figure 2.
Figure 2.
Up-regulation of matrix MMPs in SCID mice with colitis. Zymography was performed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing gelatin as substrate and incubated overnight at 37oC in the presence of PMSF, leupeptin, and soybean trypsin inhibitor. A: Colonic extracts from eight SCID mice transplanted with CD4+ T cells. B: Colonic extracts from four C.B-17 mice 9–12 and two nontransplanted SCID mice. 13,14 Arrowheads depict MMP-2 standard. Three samples per mouse: distal colon, left; mid colon, middle; proximal colon, right. Relative mobility of the major protease species is given. C: Relative activity of total MMPs and of pro- and active forms of MMP-2 and MMP-9 in the three groups of mice, mean densitometry values of all samples in each group + SE. D: Proportion of MMP-2 in its activated form in colon tissues from mice transplanted with CD4+ T cells (n = 8), nontransplanted SCID mice (n = 2) and C.B-17 mice (n = 4), + SE, as determined by densitometry from gelatin sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. E: Relative activity of total MMPs and of pro- and active forms of MMP-2 and MMP-9 in distal, mid, and proximal regions of the colon in CD4+ T-cell-transplanted SCID mice only, mean of densitometry values from all eight mice + SE.
Figure 3.
Figure 3.
Incubation of proMMP-2 and proMMP-9, derived from acute wound fluids, with fecal extracts and defined serine proteinases. A: Activated MMP forms were not generated by the action of fecal proteases during extraction conditions (4°C), but activation did occur under near-physiological conditions (37°C), with particular formation of a 75-kd-activated form of MMP-9 (arrow). B: Pancreatic trypsin was able to mediate processing of proMMP-2 and proMMP-9, even at 4°C, with the formation of a characteristic 75-kd active form of MMP-9. C: Pancreatic chymotrypsin was also able to process MMP-2 and MMP-9, with the formation of a characteristic 80-kd active form of MMP-9. D: Wound fluids incubated alone (UT) and MMP-2 standard (arrowhead).
Figure 4.
Figure 4.
In situ zymography on casein substrate showing location of protease activities in colonic tissues. A: Nontransplanted SCID mouse colon showing minimal substrate degradation associated with luminal and upper crypt epithelium (arrows), but none with basal crypt epithelium (arrowhead). B: C.B-17 mouse colon, mucosal lymphoid aggregate, showing substrate degradation under epithelium and within the lymphoid tissue. C: Nontransplanted SCID mouse colon, lymphoid aggregate, showing no substrate degradation. D: CD4+ T-cell-transplanted SCID mouse colon, showing extensive areas of substrate degradation under inflammatory infiltrate. E: Nontransplanted SCID mouse colon showing relative lack of substrate degradation, compared with CD4+ T-cell-transplanted SCID mouse colon (F–H). F: No inhibitors, showing substrate degradation particularly under epithelium and under pericryptal lamina propria. G: With soybean trypsin inhibitor to block serine proteases, showing maintenance of substrate degradation. H: With peptidyl hydroxamate to block MMPs, showing reduced proteolysis. Original magnification, ×128 (A–C) and (E–H); ×64 (D). Yellow is autofluorescence.
Figure 5.
Figure 5.
Epithelial and stromal proteolytic activity associated with infiltrating cells. Serial sections of a region of inflammatory infiltrate from the colon of a CD4+ T-cell-transplanted SCID mouse (proximal segment, mouse 4). A and B: In situ zymograms, casein substrate, of area of infiltrate shown boxed in C. A: No inhibitors, showing substrate degradation associated with the epithelium (arrow) and the infiltrate (arrowheads). B: With cocktail of inhibitors blocking both serine proteases and MMPs, relative lack of substrate degradation. C–F: Indirect immunohistology showing distribution of CD3+ T cells (C), CD11b myeloid cells (D), F4/80+ myeloid cells (E), activated macrophages, mAb 158.2 (F). Original magnifications, ×128 (A and B); ×64 (C–F).
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