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Targeted Deletion of Metalloproteinase 9 Attenuates Experimental Colitis in Mice: Central Role of Epithelial-Derived MMP

2005; Elsevier BV; Volume: 129; Issue: 6 Linguagem: Inglês

10.1053/j.gastro.2005.09.017

1528-0012

Florencia E. Castaneda, Baljit Walia, Matam Vijay–Kumar, Neal Patel, Susanne Roser, Vasantha L. Kolachala, Mauricio Rojas, Lixin Wang, Gabriela Oprea, Pallavi Garg, Andrew T. Gewirtz, Jesse Roman, Didier Merlin, Shanthi V. Sitaraman,

Protease and Inhibitor Mechanisms

Background & Aims: There is mounting evidence that matrix metalloproteinases are the predominant proteinases expressed in the gut mucosa during active inflammatory bowel disease. We investigated the role of metalloproteinase 9 (MMP-9), a secreted gelatinase that is consistently up-regulated in both animal models and human inflammatory bowel disease and is associated with disease severity, in the pathogenesis of colitis by using mice containing a targeted deletion of the MMP-9 gene. Methods: Dextran sodium sulfate–induced colitis and Salmonella typhimurium–induced enterocolitis were used as animal models to study colitis. Results: MMP-9 activity and protein expression were absent from normal colonic mucosa but were up-regulated during experimental colitis. MMP-9−/− mice exposed to dextran sodium sulfate or salmonella had a significantly reduced extent and severity of colitis. Immunohistochemical studies showed that MMP-9 was localized to epithelial cells and granulocytes during active colitis. The immune response to systemic administration of Salmonella typhimurium was not affected in MMP-9−/− mice. Neutrophil transmigration studies and bone marrow chimeras showed that neutrophil MMP-9 is neither required for its migration nor sufficient to induce tissue damage during colitis and that epithelial MMP-9 is important for tissue damage. MMP-9 inhibited cell attachment and wound healing in the model intestinal epithelial cell line, Caco2-BBE. Conclusions: Taken together, our data suggest that MMP-9 expressed by epithelial cells may play an important role in the development of colitis by modulating cell–matrix interaction and wound healing. Thus, strategies to inhibit MMP-9 may be of potential therapeutic benefit. Background & Aims: There is mounting evidence that matrix metalloproteinases are the predominant proteinases expressed in the gut mucosa during active inflammatory bowel disease. We investigated the role of metalloproteinase 9 (MMP-9), a secreted gelatinase that is consistently up-regulated in both animal models and human inflammatory bowel disease and is associated with disease severity, in the pathogenesis of colitis by using mice containing a targeted deletion of the MMP-9 gene. Methods: Dextran sodium sulfate–induced colitis and Salmonella typhimurium–induced enterocolitis were used as animal models to study colitis. Results: MMP-9 activity and protein expression were absent from normal colonic mucosa but were up-regulated during experimental colitis. MMP-9−/− mice exposed to dextran sodium sulfate or salmonella had a significantly reduced extent and severity of colitis. Immunohistochemical studies showed that MMP-9 was localized to epithelial cells and granulocytes during active colitis. The immune response to systemic administration of Salmonella typhimurium was not affected in MMP-9−/− mice. Neutrophil transmigration studies and bone marrow chimeras showed that neutrophil MMP-9 is neither required for its migration nor sufficient to induce tissue damage during colitis and that epithelial MMP-9 is important for tissue damage. MMP-9 inhibited cell attachment and wound healing in the model intestinal epithelial cell line, Caco2-BBE. Conclusions: Taken together, our data suggest that MMP-9 expressed by epithelial cells may play an important role in the development of colitis by modulating cell–matrix interaction and wound healing. Thus, strategies to inhibit MMP-9 may be of potential therapeutic benefit. Chronic inflammatory bowel diseases (IBD) of the intestine, including Crohn’s disease and ulcerative colitis, result in considerable morbidity and are associated with an increased risk of cancer of the colon and rectum.1Loftus Jr, E.V. Clinical epidemiology of inflammatory bowel disease incidence, prevalence, and environmental influences.Gastroenterology. 2004; 126: 1504-1517Abstract Full Text Full Text PDF PubMed Scopus (2384) Google Scholar IBD is defined as chronic, relapsing inflammation of the intestinal tract of unknown origin characterized by inflammatory cell infiltration, epithelial cell destruction, connective tissue defects, and ulceration of the lining of the colon and intestine resulting in a marked disruption of the mucosal architecture.1Loftus Jr, E.V. 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Riecken E.O. Rosewicz S. Differential expression of matrix metalloproteinases and their tissue inhibitors in colon mucosa of patients with inflammatory bowel disease.Gut. 2000; 47: 63-73Crossref PubMed Scopus (303) Google Scholar, 6Pender S.L. Tickle S.P. Docherty A.J. Howie D. Wathen N.C. MacDonald T.T. A major role for matrix metalloproteinases in T cell injury in the gut.J Immunol. 1997; 158: 1582-1590PubMed Google Scholar, 19Baugh M.D. Evans G.S. Hollander A.P. Davies D.R. Perry M.J. Lobo A.J. Taylor C.J. Expression of matrix metalloproteases in inflammatory bowel disease.Ann N Y Acad Sci. 1998; 859: 249-253Crossref PubMed Scopus (28) Google Scholar, 20Baugh M.D. Perry M.J. Hollander A.P. Davies D.R. Cross S.S. Lobo A.J. Taylor C.J. Evans G.S. Matrix metalloproteinase levels are elevated in inflammatory bowel disease.Gastroenterology. 1999; 117: 814-822Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 21Kirkegaard T. Pedersen G. Saermark T. Brynskov J. 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Increased activity and expression of matrix metalloproteinase-9 in a rat model of distal colitis.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G116-G122PubMed Google Scholar, 25McKaig B.C. McWilliams D. Watson S.A. Mahida Y.R. Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases by intestinal myofibroblasts in inflammatory bowel disease.Am J Pathol. 2003; 162: 1355-1360Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 26Gao Q. Meijer M.J. Kubben F.J. Sier C.F. Kruidenier L. van Duijn W. van den Berg M. van Hogezand R.A. Lamers C.B. Verspaget H.W. Expression of matrix metalloproteinases-2 and -9 in intestinal tissue of patients with inflammatory bowel diseases.Dig Liver Dis. 2005; 37: 584-592Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar MMP-3 and MMP-9 expression correlate with fistulas in IBD, and MMP-9 is associated with active inflammation associated with fistulas.27Kirkegaard T. Hansen A. Bruun E. Brynskov J. Expression and localisation of matrix metalloproteinases and their natural inhibitors in fistulae of patients with Crohn’s disease.Gut. 2004; 53: 701-709Crossref PubMed Scopus (161) Google Scholar It is interesting to note that MMP-2 and MMP-9 activity have been detected in lower crypt epithelial cells surrounding degraded matrix and in underlying ulcerations.8Seifert W.F. Wobbes T. Hendriks T. Divergent patterns of matrix metalloproteinase activity during wound healing in ileum and colon of rats.Gut. 1996; 39: 114-119Crossref PubMed Scopus (45) Google Scholar, 10Schuppan D. Hahn E.G. MMPs in the gut inflammation hits the matrix.Gut. 2000; 47: 12-14Crossref PubMed Scopus (50) Google Scholar, 23Tarlton J.F. Whiting C.V. Tunmore D. Bregenholt S. Reimann J. Claesson M.H. Bland P.W. The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis.Am J Pathol. 2000; 157: 1927-1935Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Furthermore, serum and tissue MMP-9 levels, but not MMP-2 levels, correlate with disease activity in ulcerative colitis.20Baugh M.D. Perry M.J. Hollander A.P. Davies D.R. Cross S.S. Lobo A.J. Taylor C.J. Evans G.S. Matrix metalloproteinase levels are elevated in inflammatory bowel disease.Gastroenterology. 1999; 117: 814-822Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 28Kossakowska A.E. Medlicott S.A. Edwards D.R. Guyn L. Stabbler A.L. Sutherland L.R. Urbanski S.J. Elevated plasma gelatinase A (MMP-2) activity is associated with quiescent Crohn’s disease.Ann N Y Acad Sci. 1999; 878: 578-580Crossref PubMed Scopus (12) Google Scholar MMP inhibitors ameliorate trinitrobenzene sulfonic acid and DSS colitis.29Di Sebastiano P. di Mola F.F. Artese L. Rossi C. Mascetta G. Pernthaler H. Innocenti P. Beneficial effects of Batimastat (BB-94), a matrix metalloproteinase inhibitor, in rat experimental colitis.Digestion. 2001; 63: 234-239Crossref PubMed Scopus (64) Google Scholar, 30Naito Y. Takagi T. Kuroda M. Katada K. Ichikawa H. Kokura S. Yoshida N. Okanoue T. Yoshikawa T. An orally active matrix metalloproteinase inhibitor, ONO-4817, reduces dextran sulfate sodium-induced colitis in mice.Inflamm Res. 2004; 53: 462-468Crossref PubMed Scopus (62) Google Scholar Despite extensive characterization of the MMP profile in IBD, little is known about the role of MMP in the pathogenesis of intestinal inflammation. To directly examine the role of MMP-9 in the pathogenesis of colitis, we compared mice genetically deficient in MMP-9 with wild-type (WT) littermates for the development of colitis in response to oral administration of DSS or Salmonella typhimurium. Furthermore, we addressed the potential mechanism by which MMP-9 modulates intestinal inflammation. The Animal Care Committee of the Emory University, Atlanta, Georgia, approved all procedures performed on animals. In addition, all experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US Public Health Service. MMP-9−/− mice and age- and sex-matched WT littermates were purchased from Jackson Laboratories (Bar Harbor, ME) and bred at our facility. Mice lacking MMP-9 show a normal phenotype except for developmental defects in long bone formation caused by a delay in vascularization of the growth plate, which is compensated for by the time of birth resulting in normal bone.31Vu T.H. Shipley J.M. Bergers G. Berger J.E. Helms J.A. Hanahan D. Shapiro S.D. Senior R.M. Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1497) Google Scholar The homozygous MMP-9–deficient mice used were progeny of heterozygous breeding pairs of FVB background with disruption of the MMP-9 gene that were backcrossed for more than 6 generations. To confirm the absence of MMP-9 gene expression, genomic DNA was isolated, and the disruption of the MMP-9 gene was confirmed via polymerase chain reaction by using primers designed to specifically detect heterozygote and homozygote mice.25McKaig B.C. McWilliams D. Watson S.A. Mahida Y.R. Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases by intestinal myofibroblasts in inflammatory bowel disease.Am J Pathol. 2003; 162: 1355-1360Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar For MMP-9, we used a sense oligonucleotide primer (5′-GCA TAC TTG TAC CGC TAT GG-3′) and an antisense primer (5′-TAA CCG GAG GTC CAA ACT GG-3′). For the neomycin cassette, we also used a sense oligonucleotide primer (5′-GAA GGG ACT GGC TGC TAT TG-3′) and an antisense primer (5′-AAT ATC ACG GGT AGC CAA CG-3′). The lack of MMP-9 protein and activity was confirmed by Western blot and gelatin zymography, respectively. WT and MMP-9−/− littermates used in the study were between 6 and 8 weeks old at the beginning of the experimental protocol, were maintained on a 12-hour dark/light cycle, and were allowed free access to pelleted nonpurified diet and tap water under conditions of controlled temperatures (25°C ± 2°C). Colitis was induced in 2 groups of age- and sex-matched male and female WT and MMP-9−/− littermates by oral administration of DSS (molecular weight, 50000; ICN Biomedicals, Aurora, OH) at 3% (wt/vol) in tap water ad libitum for 6 days. Age-matched male and female WT and MMP-9−/− littermates receiving tap water served as controls. Mice were observed daily and evaluated for changes in body weight and development of clinical symptoms. Gut-restricted S typhimurium infection was induced as described previously.32Barthel M. Hapfelmeier S. Quintanilla-Martinez L. Kremer M. Rohde M. Hogardt M. Pfeffer K. Russmann H. Hardt W.D. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.Infect Immun. 2003; 71: 2839-2858Crossref PubMed Scopus (731) Google Scholar To prepare S typhimurium inocula, bacteria (SL3201) were grown overnight at 37°C in 10 mL of LB broth in a 20-mL container with shaking (150 rpm) and were then used to inoculate fresh medium (1:100) and were grown under the same conditions for 2 to 3 hours until an optical density at 550 nm of 0.35 to 0.6 was reached. Bacterial cultures were then diluted in normal saline, and the colony-forming units were enumerated by plating a dilution series of the inoculum. Water and food were withdrawn 4 hours before oral treatment with 7.5 mg of streptomycin (75 μL of sterile water containing streptomycin or 75 μL of sterile water by gavage). Afterward, animals were supplied with food and water ad libitum. At 20 hours after streptomycin treatment, food and water were withdrawn again for 4 hours before mice were infected with 108 colony-forming units of S typhimurium (50 μL of suspension in phosphate-buffered saline [PBS], orally) or treated with vehicle (control). Thereafter, food and water were offered immediately. Mice were killed by carbon dioxide inhalation, and tissue samples were processed as described for the DSS colitis model. Systemic salmonellosis was induced by the administration of S typhimurium SL3201 (104 colony-forming units/mouse) intraperitoneally. Mice were weighed daily and followed up for clinical signs of sepsis, as well as mortality. Assessment of body weights, stool consistency, and the presence of occult/gross blood by a guaiac test (Hemoccult Sensa; Beckman Coulter, Fullerton, CA) were determined daily for each mouse. Colitis was quantified with a clinical score, as described by Cooper et al,33Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar by using the parameters of weight loss, stool consistency, and fecal blood. Briefly, no weight loss was considered as 0 points, weight loss of 1% to 5% was scored as 1 point, loss of 5% to 10% was scored as 2 points, loss of 10% to 20% was scored as 3 points, and a loss of more than 20% was scored as 4. The stool was characterized as normal (score 0), soft with well-formed pellets (score 1), soft without pellets (score 2), or diarrhea (score 4). For occult blood, no blood was scored 0, positive hemoccult was scored as 2 points, and gross bleeding was scored as 12. These scores were added to get a clinical activity score ranging from 0 to 4. Six days after the induction of colitis, mice were killed by carbon dioxide/hypothermia. The abdominal cavity was exposed by a midline laparotomy, and the entire colon was removed from the cecum to the anus. The colon was flushed with PBS and opened longitudinally for morphological studies. The length and weight of the colon were measured, and tissue obtained from each colon was processed for further assays. Colonic specimens obtained as described previously were fixed in formalin and coded for blind microscopic assessment of mucosal lesions (descending colon for DSS colitis and cecum for S typhimurium colitis). Sections were stained with H&E. Microscopic sections were analyzed by a pathologist (G.O.), and histological scoring was performed as described by Cooper et al33Cooper H.S. Murthy S.N. Shah R.S. Sedergran D.J. Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar on the basis of 3 variables, according to the severity of the induced damage. Briefly, for inflammation, rare inflammatory cells in the lamina propria were counted as 0, increased numbers of granulocytes in the lamina propria as 1, confluence of inflammatory cells extending into the submucosa were counted as 2, and a score of 3 was given for transmural extension of the infiltrate. For crypt damage, an intact crypt was scored as 0, loss of the basal one third counted as 1, loss of the basal two thirds was counted as 2, entire crypt loss was scored as 3, a change of epithelial surface with erosion was scored as 4, and a score of 5 was given for confluent erosion. For evaluation of ulcers, an absence of ulcer was scored 0, 1 or 2 foci of ulcerations were scored as 1, 3 or 4 foci of ulcerations were scored as 2, and confluent or extensive ulceration was scored as 3. These values were added to give a total histological score of 11. Neutrophil infiltration into the colon was quantified by measuring myeloperoxidase (MPO) activity as described previously.34Gewirtz A.T. Collier-Hyams L.S. Young A.N. Kucharzik T. Guilford W.J. Parkinson J.F. Williams I.R. Neish A.S. Madara J.L. Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis.J Immunol. 2002; 168: 5260-5267Crossref PubMed Scopus (239) Google Scholar Five animals were studied in each group. Briefly, a portion of colon was homogenized in 1:20 (wt/vol) 50 mmol/L phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide on ice by using a Polytron homogenizer. The homogenate was sonicated for 10 seconds, freeze-thawed 3 times, and centrifuged at 14,000 rpm for 15 minutes. Supernatant (14 μL) was added to 1 mg/mL of o-dianisidine hydrochloride and 0.0005% hydrogen peroxide, and the change in absorbance at 460 nm was measured. One unit of MPO activity was defined as the amount that degraded 1 μmol of peroxidase per minute at 25°C. The results were expressed as absorbance per gram of tissue. Polymorphonuclear neutrophils (PMNs) were isolated from the bone marrow.35Cowland J.B. Borregaard N. Isolation of neutrophil precursors from bone marrow for biochemical and transcriptional analysis.J Immunol Methods. 1999; 232: 191-200Crossref PubMed Scopus (67) Google Scholar Briefly, the femur and tibia were removed and stripped of all muscle and sinew, and the bones were placed in Hank’s balanced salt solution (HBSS) without Ca2+ or Mg2+ (HBSS−) on ice. The bone marrow was flushed from each bone with HBSS−. Cells were pelleted, and mature erythrocytes were removed by hypotonic lysis. PMNs were confirmed by Giemsa staining and fluorescence-activated cell-sorting analysis by using anti-CD11b. Isolated PMNs were resuspended in HBSS− and used within 4 hours of isolation. Neutrophil transmigration experiments across filters or the T84 monolayer were performed as previously described.36Si-Tahar M. Sitaraman S. Shibahara T. Madara J.L. Negative regulation of epithelium-neutrophil interactions via activation of CD44.Am J Physiol Cell Physiol. 2001; 280: C423-C432PubMed Google Scholar Transmigration was initiated by adding 1–10 μmol/L n-formyl methionine leucine phenylalanine (fMLP) in HBSS into the lower chamber of 5-μm-pore transwell filters (Corning Costar, Cambridge, MA) coated with collagen followed by incubation at 37°C for 2 hours. PMN migration across filters or epithelial monolayers into the fMLP-containing lower chambers was quantified by MPO assay as described previously.36Si-Tahar M. Sitaraman S. Shibahara T. Madara J.L. Negative regulation of epithelium-neutrophil interactions via activation of CD44.Am J Physiol Cell Physiol. 2001; 280: C423-C432PubMed Google Scholar A total of 1 × 106 neutrophils per well were used for transmigration studies, and typically 1%–5% of neutrophils migrated in response to fMLP 10 μmol/L.36Si-Tahar M. Sitaraman S. Shibahara T. Madara J.L. Negative regulation of epithelium-neutrophil interactions via activation of CD44.Am J Physiol Cell Physiol. 2001; 280: C423-C432PubMed Google Scholar The capacity of PMNs to migrate through Matrigel (Becton-Dickinson Labware, Bedford, MA), a prototype basement membrane secreted by the Engelbreth–Holm–Swarm tumor, was assessed in a modified Boyden chamber assay.37Betsuyaku T. Shipley J.M. Liu Z. Senior R.M. Neutrophil emigration in the lungs, peritoneum, and skin does not require gelatinase B.Am J Respir Cell Mol Biol. 1999; 20: 1303-1309Crossref PubMed Scopus (127) Google Scholar PMNs were isolated from bone marrow and suspended at 1.5 × 106/mL in HBSS with Ca2+ and Mg2+ containing 0.1% bovine serum albumin and placed in the upper compartment. The lower compartment contained fMLP 10 μmol/L in HBSS. The 2 compartments were separated by an 8-μm-pore membrane coated with Matrigel. After a 3-hour incubation at 37°C in humidified 5% carbon dioxide/air, the filter was stained with LeukoStat (Fisher Scientific, Fairlawn, NJ) and mounted on a glass slide. The number of cells that migrated to the underside of the filter in 5 random high-power fields (400×) was determined for each of the filters in experimental condition. Bone marrow cells were harvested from both femurs and tibias by flushing the bone cavity with basal bone marrow medium: Iscove’s medium (Cambrex, Walkersville, MD) supplemented with 30% fetal bovine serum (Stem Cell Technologies, Vancouver, BC, Canada), 1% bovine serum albumin (Sigma, St Louis, MO), 100 mmol/L 2-mercaptoethanol (Sigma), 2 mmol/L l-glutamine, 1% penicillin/streptomycin, and 1% fungisone (Irvine Scientific, Santa Ana, CA).38Rojas M. Xu J. Woods C.R. Mora A.L. Spears W. Roman J. Brigham K.