Volume 117, Issue 4, Pages 814-822 (October 1999) Matrix metalloproteinase levels are elevated in inflammatory bowel disease  Mark D. Baugh, Michael J. Perry, Anthony P. Hollander, D.Rhodri Davies, Simon S. Cross, Alan J. Lobo, Christopher J. Taylor, Gareth S. Evans  Gastroenterology  Volume 117, Issue 4, Pages 814-822 (October 1999) DOI: 10.1016/S0016-5085(99)70339-2 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Total proteolytic activity measured in zymographs of ulcerative colitis and Crohn's disease vs. control biopsy specimens. Supernatants of biopsy homogenates were subjected to SDS-PAGE zymography against various substrates on 10% polyacrylamide gels. Each sample lane was loaded as 5 μg of protein. After separation, gels were stained with Coomassie blue and enzyme activity was identified by cleared areas of lysis. Gels were analyzed using a Kodak DC-40 camera and Kodak 1D image analysis software (each value is the sum of the net band intensities for each lane of sample). (A) Total gelatinolytic activity and (B) total serine protease activity for the inflamed and noninvolved biopsy specimen pairs of ulcerative colitis patients, each control biopsy specimen, and the ulcerated and nonulcerated biopsy specimen pairs of the Crohn's disease patients. I, biopsy from inflamed site; NI, biopsy from noninvolved area. Significant differences between inflamed and noninvolved ulcerative colitis biopsy specimens were observed (P = 0.0051), as well as between inflamed vs. control (P = 0.0002) and noninvolved vs. control (P = 0.0061). No significant differences were found between the 2 Crohn's disease sites, but there were significant differences between the ulcerated vs. control (P < 0.01) and nonulcerated vs. control (P < 0.03). Gastroenterology 1999 117, 814-822DOI: (10.1016/S0016-5085(99)70339-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Zymographic activity of ulcerative colitis and Crohn's disease biopsy specimen pairs. Each individual biopsy specimen was loaded at a concentration of 5 μg protein/lane and subjected to zymography on 10% polyacrylamide gels containing (A) 0.1% gelatin or (B) 0.1% casein. After separation, gels were incubated overnight at 37°C in buffer selective for metalloproteases or serine protease-like enzymes. A-D, Separate pairs of biopsy specimens from ulcerative colitis patients; E, a biopsy pair from a patient with Crohn's disease; I, biopsy specimens from the inflamed region; N, biopsy specimens from the noninvolved regions. Cleared bands indicate areas of enzymatic activity. The enzyme activity on gelatin-containing zymographs is shown in A, and the serine protease on the casein substrate in B. Right margin indicates the relative molecular weight in kilodaltons. Gastroenterology 1999 117, 814-822DOI: (10.1016/S0016-5085(99)70339-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of enzyme inhibitors on the gelatinolytic activity of 2 inflamed biopsy specimens. Samples were loaded at 5 μg protein/lane on gelatin-containing gels and incubated overnight in developing buffer with or without inhibitors. Lane I, activity without inhibitors; lane II, activity with developing buffer containing 2 μg/mL aprotinin (serine protease inhibitor); lane III, activity with developing buffer containing 10 mmol/L EDTA (metalloprotease inhibitor). (A) Sample A: activity of a biopsy specimen with elevated serine protease levels; (B) Sample B: a biopsy specimen with elevated MMP levels. Right margin indicates relative molecular weight in kilodaltons. Gastroenterology 1999 117, 814-822DOI: (10.1016/S0016-5085(99)70339-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Western immunoblotting for detection of MMPs in biopsy specimen pairs. Each sample was loaded as 10 μg/lane in reducing and denaturing conditions onto 10% polyacrylamide gels. Each biopsy specimen pair was probed with antibodies to MMP-1, -2, -3, and -9 using chemiluminescent detection. Lanes I and N, inflamed and noninvolved biopsy specimens, respectively; lane Std, purified MMP standards: 12.5 ng/lane MMP-1, -2, and -3, and 25 ng/lane MMP-9. Right and left margins indicate relative molecular weight in kilodaltons. Gastroenterology 1999 117, 814-822DOI: (10.1016/S0016-5085(99)70339-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Comparison of gelatinolytic activity of an inflamed ulcerative colitis biopsy specimen (lane I) with conditioned media from neutrophils treated with 10−8 mol/L phorbol myristate acetate (lane II), which produce MMP-9 (245, 145, and 89 kilodaltons). Right margin indicates relative molecular weight in kilodaltons. Gastroenterology 1999 117, 814-822DOI: (10.1016/S0016-5085(99)70339-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Frozen section of descending colon in a woman (age, 49 years) with active ulcerative colitis showing the presence of MMP-9 (intense yellow/green fluorescence) in the lamina propria around the crypts. (B) Control section of descending colon from the same patient as in A: an adjacent area but without the primary antibody. (C) Localization of MMP-9 in frozen sections of noninvolved transverse colon of a boy (age, 14 years) with mild ulcerative colitis and little staining for MMP-9. (D) Control section of transverse colon from the same patient as in C but without the primary antibody; some green autofluorescent mast cells are present. All sections were fixed in acetone/methanol and stained with sheep anti–MMP-9 and donkey anti-sheep immunoglobulin G FITC; the nuclei were counterstained with propidium iodide. Original magnification: A and B, 550×; C and D, 1100×. Gastroenterology 1999 117, 814-822DOI: (10.1016/S0016-5085(99)70339-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions