Matrix metalloproteinases in the restorative proctocolectomy pouch of pediatric ulcerative colitis

Abstract

Aim: To investigate matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in pouch mucosa of pediatric onset ulcerative colitis (UC).

Methods: In this cross-sectional study, 28 patients with pediatric onset UC underwent ileal pouch biopsy 13 years (median) after proctocolectomy. Expression of MMPs-3, -7, -8, -9, -12 and -26 and TIMPs-1, -2 and -3 in samples was examined using immunohistochemichal methods, and another biopsy was used to evaluate the grade of histological inflammation. Two investigators independently graded the immunohistochemical specimens in a semiquantitative fashion, using a scale marking staining intensity as follows: 0 = less than 20 positive cells; 1 = 20-50 positive cells; 2 = 50-200 positive cells; 3 = over 20 positive cells. Fecal calprotectin and blood inflammatory markers [serum C-reactive protein (CRP) and erythrocyte sedimentation rate] were determined during a follow-up visit to examine correlations between these markers and the expression of MMPs and TIMPs.

Results: Of the 28 patients with pediatric onset UC, nine had not experienced pouchitis, whereas thirteen reported a single episode, and six had recurrent pouchitis (≥ 4 episodes). At the time of the study, six patients required metronidazole. In all of the others, the most recent episode of pouchitis had occurred over one month earlier, and none were on antibiotics. Only four samples depicted no sign of inflammation, and these were all from patients who had not had pouchitis. Two samples were too small to determine the grade of inflammation, but both had suffered pouchitis, the other recurrent. No sample depicted signs of colonic metaplasia. Most pouch samples showed expression of epithelial (e) and stromal (s) MMP-3 (e, n = 22; s, n = 20), MMP-7 (e, n = 28; s, n = 27), MMP-12 (e, n = 20; s, n =24), TIMP-2 (e, n = 23; s, n = 23) and MMP-3 (e, n = 23; s, n = 28) but MMP-8 (e, n = 0; s, n = 1), MMP-9 (e, n = 0; s, n = 9) and MMP-26 (e, n = 0; s, n = 3) and TIMP-1 (n = 0, both) were lacking. In samples with low grade of inflammatory activity, the epithelial MMP-3 and MMP-7 expression was increased (r = -0.614 and r = -0.472, respectively, P < 0.05 in both). MMPs and TIMPs did not correlate with the markers of inflammation, fecal calprotectin, erythrocyte sedimentation rate, or CRP, with the exception of patients with low fecal calprotectin (< 100 μg/g) in whom a higher expression of epithelial MMP-7 was found no differences in MMP- or TIMP-profiles were seen in patients with a history of pouchitis compared to ones with no such episodes. Anastomosis with either straight ileoanal anastomosis or ileoanal anastomosis with J-pouch did depict differences in MMP- or TIMP-expression.

Conclusion: The expression of MMPs pediatric UC pouch in the long-term shares characteristics with inflammatory bowel disease, but inflammation cannot be classified as a reactivation of the disease.

Keywords: Children; Matrix metalloproteinase 3; Matrix metalloproteinase 7; Pouchitis; Tissue inhibitor of matrix metalloproteinase 3; Ulcerative colitis.

Figure 1

Matrix metalloproteinases-3, matrix metalloproteinases-7 and tissue inhibitor of matrix metalloproteinase-2 in lower (A, C, E1, respectively) and higher grade of inflammation and calprotectin levels (B1, D1, F, respectively), tissue inhibitor of matrix metalloproteinase-3 in pouch (G1). Black solid arrows plasma cells, black dotted arrows macrophages, red solid arrows eosinophils and red dotted arrows endothelium. Scale bars: 15 μm (A, B1, C, D1, E1, F, G1); 7.5 μm (B2-B4, D2, D3, E2, G2, G3). Stainings were performed using diaminobenzidine or NovaRED as chromogenic substrates and Mayer’s hematoxylin as counterstain. Images were obtained using a light-field microscope, and edited using Adobe Photoshop 7.0 (Adobe Systems Incorporated).

Figure 2

matrix metalloproteinases-8 (A1), matrix metalloproteinases-9 (D1, E1), matrix metalloproteinases-12 (F1, G), matrix metalloproteinases-26 (B1) and tissue inhibitor of matrix metalloproteinase-1 (C) in pouch. Inset g’ added to figure from another sample, not shown in here in lesser magnification. Arrowheads depict neutrophils, black solid arrows plasma cells, black dotted arrows macrophages, red solid arrows eosinophils. Scale bars: 15 μm (A1, B1, C, D1, E1, F1,G); 7.5 μm (A2, B2, B3, D2, D3, E2, F2, F3). Stainings were performed using diaminobenzidine or NovaRED as chromogenic substrates and Mayer’s hematoxylin as counterstain. Images were obtained using a light-field microscope, and edited using Adobe Photoshop 7.0 (Adobe Systems Incorporated).