Functional Modulation of Crohn’s Disease Myofibroblasts by Anti-Tumor Necrosis Factor Antibodies
2007; Elsevier BV; Volume: 133; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.04.069
ISSN1528-0012
AutoresAntonio Di Sabatino, Sylvia L. F. Pender, Claire L. Jackson, Joanna D. Prothero, J Gordon, Lucia Picariello, L. Rovedatti, Guillermo Docena, Giovanni Monteleone, David S. Rampton, Francesco Tonelli, Gino Roberto Corazza, Thomas T. MacDonald,
Tópico(s)Autoimmune and Inflammatory Disorders
ResumoBackground & Aims: Infliximab induces immune cell apoptosis by outside-to-inside signaling through transmembrane tumor necrosis factor-α (mTNF). However, in inflamed gut, myofibroblasts also produce TNF-α, and the affects of anti-TNF antibodies on these structural cells are unknown. We investigated the action of infliximab on apoptosis, the production of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP)-1, and migration of Crohn’s disease (CD) myofibroblasts. Methods: Colonic myofibroblasts were isolated from patients with active CD and controls. mTNF was evaluated by Western blotting and flow cytometry. Infliximab-treated myofibroblasts were analyzed for apoptosis by Annexin V staining and caspase-3. TIMP-1 and MMPs were measured by Western blotting, and fibroblast migration was assessed by using an in vitro wound-healing scratch assay. Results: CD myofibroblasts showed higher mTNF expression than control myofibroblasts. Infliximab had no effect on CD myofibroblast apoptosis, caspase-3 activation, and production of MMP-3 and MMP-12. However, infliximab induced a significant dose-dependent increase in TIMP-1 production, which was inhibited by the p38 mitogen-activated protein kinase inhibitor SB 203580. The anti-TNF agents adalimumab, etanercept, and p55 TNF-receptor–human IgG fusion protein also increased TIMP-1 production. The migration of CD myofibroblasts was enhanced significantly by infliximab and recombinant human TIMP-1, and infliximab-induced migration was inhibited by anti–TIMP-1 neutralizing antibody. Infliximab also decreased CD myofibroblast collagen production. Conclusions: Our findings show a novel therapeutic pathway for anti-TNF therapies in enhancing TIMP-1 production and myofibroblast migration, which may reduce MMP activity and facilitate the wound healing. Background & Aims: Infliximab induces immune cell apoptosis by outside-to-inside signaling through transmembrane tumor necrosis factor-α (mTNF). However, in inflamed gut, myofibroblasts also produce TNF-α, and the affects of anti-TNF antibodies on these structural cells are unknown. We investigated the action of infliximab on apoptosis, the production of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP)-1, and migration of Crohn’s disease (CD) myofibroblasts. Methods: Colonic myofibroblasts were isolated from patients with active CD and controls. mTNF was evaluated by Western blotting and flow cytometry. Infliximab-treated myofibroblasts were analyzed for apoptosis by Annexin V staining and caspase-3. TIMP-1 and MMPs were measured by Western blotting, and fibroblast migration was assessed by using an in vitro wound-healing scratch assay. Results: CD myofibroblasts showed higher mTNF expression than control myofibroblasts. Infliximab had no effect on CD myofibroblast apoptosis, caspase-3 activation, and production of MMP-3 and MMP-12. However, infliximab induced a significant dose-dependent increase in TIMP-1 production, which was inhibited by the p38 mitogen-activated protein kinase inhibitor SB 203580. The anti-TNF agents adalimumab, etanercept, and p55 TNF-receptor–human IgG fusion protein also increased TIMP-1 production. The migration of CD myofibroblasts was enhanced significantly by infliximab and recombinant human TIMP-1, and infliximab-induced migration was inhibited by anti–TIMP-1 neutralizing antibody. Infliximab also decreased CD myofibroblast collagen production. Conclusions: Our findings show a novel therapeutic pathway for anti-TNF therapies in enhancing TIMP-1 production and myofibroblast migration, which may reduce MMP activity and facilitate the wound healing. Infliximab promotes rapid closure of fistulas and sustained mucosal healing in active Crohn’s disease (CD).1Targan S.R. Hanauer S.B. van Deventer S.J. Mayer L. Present D.H. Braakman T. DeWoody K.L. Schaible T.F. Rutgeerts P.J. Crohn’s Disease cA2 Study GroupA short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s disease.N Engl J Med. 1997; 337: 1029-1035Google Scholar, 2Present D.H. Rutgeerts P. Targan S. Hanauer S.B. Mayer L. van Hogezand R.A. Podolsky D.K. Sands B.E. Braakman T. DeWoody K.L. Schaible T.F. van Deventer S.J. Infliximab for the treatment of fistulas in patients with Crohn’s disease.N Engl J Med. 1999; 340: 1398-1405Google Scholar, 3Geboes K. Rutgeerts P. Opdenakker G. Olson A. Patel K. Wagner C.L. Marano C.W. Endoscopic and histologic evidence of persistent mucosal healing and correlation with clinical improvement following sustained infliximab treatment for Crohn’s disease.Curr Med Res Opin. 2005; 21: 1741-1754Google Scholar The effectiveness of infliximab is linked not only to the neutralization of soluble tumor necrosis factor (TNF)-α and transmembrane TNF-α (mTNF), but to the induction of apoptosis by reverse signaling through mTNF.4van Deventer S.J.H. Transmembrane TNF-α, induction of apoptosis, and the efficacy of TNF-targeting therapies in Crohn’s disease.Gastroenterology. 2001; 121: 1242-1246Google Scholar, 5Mitoma H. Horiuchi T. Hatta N. Tsukamoto H. Harashima S. Kikuchi Y. Otsuka J. Okamura S. Fujita S. Harada M. Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha.Gastroenterology. 2005; 128: 376-392Abstract Full Text Full Text PDF Scopus (247) Google Scholar Monocytes and T lymphocytes, which express high amounts of mTNF, are particularly susceptible to infliximab-induced caspase-dependent apoptosis.6Lugering A. Schmidt M. Lugering N. Pauels H.G. Domschke W. Kucharzik T. Infliximab induces apoptosis in monocytes from patients with chronic active Crohn’s disease by using a caspase-dependent pathway.Gastroenterology. 2001; 121: 1145-1157Google Scholar, 7Van den Brande J.M. Braat H. van den Brink G.R. Versteeg H.H. Bauer C.A. Hoedemaeker I. van Montfrans C. Hommes D.W. Peppelenbosch M.P. van Deventer S.J. Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease.Gastroenterology. 2003; 124: 1774-1785Google Scholar, 8Di Sabatino A. Ciccocioppo R. Cinque B. Millimaggi D. Morera R. Ricevuti L. Cifone M.G. Corazza G.R. Defective mucosal T cell death is sustainably reverted by infliximab in a caspase-dependent pathway in Crohn’s disease.Gut. 2004; 53: 70-77Google Scholar Myofibroblasts are key cells in the process of tissue injury and wound healing in the gut.9Powell D.W. Mifflin R.C. Valentich J.D. Crowe S.E. Saada J.I. West A.B. Myofibroblasts II. Intestinal subepithelial myofibroblasts.Am J Physiol. 1999; 277: C183-C201Google Scholar They cause gut damage by secreting matrix metalloproteinases (MMPs),10Baugh 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: 814-822Google Scholar which are calcium ion–dependent and zinc ion–containing neutral endopeptidases involved in extracellular matrix (ECM) degradation.11Pender S.L. MacDonald T.T. Matrix metalloproteinases and the gut—new roles for old enzymes.Curr Opin Pharmacol. 2004; 4: 546-550Google Scholar MMP activity is under tight physiologic control by tissue inhibitors of metalloproteinases (TIMPs).12Nagase H. Woessner J.F. Matrix metalloproteinases.J Biol Chem. 1999; 274: 21491-21494Google Scholar Tissue-degrading MMPs act as end-stage effectors of several disorders in which there is an excess of TNF-α,11Pender S.L. MacDonald T.T. Matrix metalloproteinases and the gut—new roles for old enzymes.Curr Opin Pharmacol. 2004; 4: 546-550Google Scholar, 13Davies D.E. Wicks J. Powell R.M. Puddicombe S.M. Holgate S.T. Airway remodeling in asthma: new insights.Allergy Clin Immunol. 2003; 111: 215-225Google Scholar, 14Vandooren B. Kruithof E. Yu D.T. Rihl M. Gu J. De Rycke L. Van Den Bosch F. Veys E.M. De Keyser F. Baeten D. Involvement of matrix metalloproteinases and their inhibitors in peripheral synovitis and down-regulation by tumor necrosis factor alpha blockade in spondylarthropathy.Arthritis Rheum. 2004; 50: 2942-2953Google Scholar and their increase in the inflamed gut has been associated with mucosal degradation, ulcerations, and fistulas.15von Lampe B. Barthel B. Coupland S.E. 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: 63-73Google Scholar, 16Louis E. Ribbens C. Godon A. Franchimont D. De Groote D. Hardy N. Boniver J. Belaiche J. Malaise M. Increased production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by inflamed mucosa in inflammatory bowel disease.Clin Exp Immunol. 2000; 120: 241-246Google Scholar, 17Saarialho-Kere U.K. Vaalamo M. Puolakkainen P. Airola K. Parks W.C. Karjalainen-Lindsberg M.L. Enhanced expression of matrilysin, collagenase, and stromelysin-1 in gastrointestinal ulcers.Am J Pathol. 1996; 148: 519-526Google Scholar, 18Vaalamo M. Karjalainen-Lindsberg M.L. Puolakkainen P. Kere J. Saarialho-Kere U. Distinct expression profiles of stromelysin-2 (MMP-10), collagenase-3 (MMP-13), macrophage metalloelastase (MMP-12), and tissue inhibitor of metalloproteinases-3 (TIMP-3) in intestinal ulcerations.Am J Pathol. 1998; 152: 1005-1014Google Scholar, 19Kirkegaard 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: 701-709Google Scholar, 20Heuschkel R.B. MacDonald T.T. Monteleone G. Bajaj-Elliott M. Smith J.A. Pender S.L. Imbalance of stromelysin-1 and TIMP-1 in the mucosal lesions of children with inflammatory bowel disease.Gut. 2000; 47: 57-62Google Scholar TNF-α blockade prevents ECM degradation concomitant with inhibition of MMP production.21Pender S.L. Fell J.M. Chamow S.M. Ashkenazi A. MacDonald T.T. A p55 TNF receptor immunoadhesin prevents T cell-mediated intestinal injury by inhibiting matrix metalloproteinase production.J Immunol. 1998; 160: 4098-4103Google Scholar Myofibroblast migration is an important component of intestinal wound healing.22Leeb S.N. Vogl D. Falk W. Scholmerich J. Rogler G. Gelbmann C.M. Regulation of migration of human colonic myofibroblasts.Growth Factors. 2002; 20: 81-91Google Scholar Myofibroblasts become activated and proliferate in the early stage of wounding. They respond to proinflammatory cytokines with elaboration of ECM proteins and additional growth factors.9Powell D.W. Mifflin R.C. Valentich J.D. Crowe S.E. Saada J.I. West A.B. Myofibroblasts II. Intestinal subepithelial myofibroblasts.Am J Physiol. 1999; 277: C183-C201Google Scholar, 11Pender S.L. MacDonald T.T. Matrix metalloproteinases and the gut—new roles for old enzymes.Curr Opin Pharmacol. 2004; 4: 546-550Google Scholar, 22Leeb S.N. Vogl D. Falk W. Scholmerich J. Rogler G. Gelbmann C.M. Regulation of migration of human colonic myofibroblasts.Growth Factors. 2002; 20: 81-91Google Scholar Recently, persistent mucosal wounding and ulcerations have been associated with a reduced migratory potential of intestinal myofibroblasts in CD, and TNF-α appears to have a role in inhibiting this migration.23Leeb S.N. Vogl D. Gunckel M. Kiessling S. Falk W. Goke M. Scholmerich J. Gelbmann C.M. Rogler G. Reduced migration of fibroblasts in inflammatory bowel disease: role of inflammatory mediators and focal adhesion kinase.Gastroenterology. 2003; 125: 1341-1354Google Scholar Although most attention in inflammatory bowel disease (IBD) has focused on TNF production by T cells and macrophages, TNF also is made by other cell types, including myofibroblasts.24Beil W.J. Weller P.F. Peppercorn M.A. Galli S.J. Dvorak A.M. Ultrastructural immunogold localization of subcellular sites of TNF-alpha in colonic Crohn’s disease.J Leukoc Biol. 1995; 58: 284-298Google Scholar However, there is no information on the effect of infliximab on myofibroblasts. Thus, in this study we have determined whether CD myofibroblasts express mTNF, and whether infliximab and other anti-TNF reagents alter myofibroblast function. Endoscopic biopsies or surgical specimens were taken from macroscopically and microscopically inflamed and unaffected colonic mucosa of 15 patients affected by active CD (mean age, 35.6 y; range, 20–59 y). The diagnosis of CD was ascertained according to the usual clinical criteria,25Best W.R. Becktel J.M. Singleton J.W. Kern Jr, F. Development of a Crohn’s disease activity index: National Cooperative Crohn’s Disease Study.Gastroenterology. 1976; 70: 439-444Abstract Full Text PDF Scopus (3019) Google Scholar and the site and extent of the disease were confirmed by endoscopy, histology, and enteroclysis in all patients. Disease activity was assessed by the Crohn’s Disease Activity Index. Patients with scores of less than 150 were classified as being in remission, whereas patients with scores higher than 450 had severe disease.25Best W.R. Becktel J.M. Singleton J.W. Kern Jr, F. Development of a Crohn’s disease activity index: National Cooperative Crohn’s Disease Study.Gastroenterology. 1976; 70: 439-444Abstract Full Text PDF Scopus (3019) Google Scholar In 9 patients the primary site of involvement was ileocolonic, and colonic in the remaining 6 patients. Four were untreated at the time of biopsy, being at the first disease presentation; 5 were treated with mesalazine, steroids, or antibiotics, and 6 were treated with only mesalazine at the time of biopsy and had suspended the steroid treatment at least 3 months earlier. None of them had ever been treated with cyclosporine, methotrexate, or infliximab. Mucosal samples also were collected from the colon of 7 subjects who turned out to have functional diarrhea at the end of their diagnostic work-up, from macroscopically and microscopically unaffected colonic areas of 7 patients undergoing colectomy for colon cancer (mean age, 37.8 y; range, 22–65 y), and from macroscopically and microscopically inflamed and unaffected colonic areas of 7 patients affected by active ulcerative colitis (UC) (mean age, 31.4 y; range, 19–53 y), used as disease control group. Two UC patients had pancolitis, the remaining 5 had left-sided colitis. Three of them were untreated at the time of biopsy, being at the first disease presentation; 2 were treated with mesalazine and topical steroids; and 2 were treated with only mesalazine at the time of biopsy, and had suspended the steroid treatment at least 3 months earlier. Some of the mucosal samples were used to isolate myofibroblasts, some others for organ culture experiments. Each patient who took part in the study was recruited after appropriate local ethics committee approval (both in London and Southampton) and informed consent was obtained in all cases. Mucosal myofibroblasts were isolated as previously described.26Mahida Y.R. Galvin A.M. Gray T. Makh S. McAlindon M.E. Sewell H.F. Podolsky D.K. Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells.Clin Exp Immunol. 1997; 109: 377-386Google Scholar Briefly, the epithelial layer was removed by 1 mmol/L ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, Poole, UK) for two 30-minute periods at 37°C. After EDTA treatment, mucosal samples were denuded of epithelial cells, and subsequently were cultured at 37°C in a humidified CO2 incubator in Dulbecco’s modified Eagle medium (Sigma-Aldrich) supplemented with 20% fetal calf serum, 1% nonessential amino acids (Invitrogen, Paisley, UK), 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamycin, and 1 μg/mL amphotericin (Sigma-Aldrich). During culture, numerous cells appeared both in suspension and adherent to the culture dish. The cells in suspension were removed after every 24- to 72-hour culture period, and the denuded mucosal tissue was maintained in culture for up to 6 weeks. Established colonies of myofibroblasts were seeded into 25-cm2 culture flasks and cultured in Dulbecco’s modified Eagle medium supplemented with 20% fetal calf serum and antibiotics. At confluence, the cells were passaged using trypsin-EDTA in a 1:2 to 1:3 split ratio. Cells were grown to at least passage 4 before they were used in stimulation experiments, and were characterized by immunocytochemical staining as previously described.27Pender 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: 1582-1590Google Scholar The following antibodies were used for the myofibroblast characterization: anti–α-smooth muscle cell actin (clone 1A4; DAKO, High Wycombe, UK), antivimentin (clone V9; Santa Cruz Biotechnology, Wiltshire, UK), anti-PR2D3 (a kind gift from Dr P. Richman, Imperial Cancer Research Fund, London, UK), antidesmin (clone D33; DAKO), anti–cytokeratin-18 (clone CY-90; AbCam, Cambridge, UK), anti-CD3 (clone UCHT1; DAKO), anti-CD68 (clone PG-M1; DAKO), and appropriate isotype-matched controls (Sigma-Aldrich). After 24-hour culture in serum-free Dulbecco’s modified Eagle medium, subconfluent monolayers of myofibroblasts seeded in 12-well plates at 3 × 105 cells per well were incubated for 24 hours with infliximab (Remicade; Schering-Plough, Milan, Italy) added to the culture medium at different concentrations (10 and 100 μg/mL) or its isotype-matched control (human IgG1, Sigma-Aldrich). In parallel experiments, cells treated with infliximab or IgG1 were incubated with 10 μmol/L mitogen-activated protein kinase (MAPK) p38 inhibitor SB 203580 (SB 203580 hydrochloride; Calbiochem, La Jolla, CA), or 1 ng/mL recombinant human interleukin-1β (R&D Systems, Abingdon, UK). Additional experiments were performed by incubating cells for 24 hours with recombinant human transforming growth factor (TGF)-β1 (10 ng/mL; R&D Systems), etanercept (10 μg/mL; Enbrel; Wyeth Europa, Maidenhead, UK), p55 TNF-receptor–human IgG fusion protein (10 μg/mL, p55-TNFR-IgG; Genentech, San Francisco, CA), and adalimumab (10 μg/mL, Humira; Abbott Laboratories, Chicago, IL). The human Jurkat T-cell line was stimulated in anti-CD3–coated 96-well plates (BD Biosciences, Oxford, UK) with anti-CD28 antibody (0.5 μg/mL; eBioscience, San Diego, CA), and then incubated for 24 hours with 10 μg/mL infliximab or human IgG1. Cell lysates were used in Western blotting as control for caspase-3 detection. Myofibroblast migration was assessed according to the method of Rodriguez et al28Rodriguez L.G. Wu X. Guan J.L. Wound-healing assay.Methods Mol Biol. 2005; 294: 23-29Google Scholar and modified by us. Briefly, cells (2 × 105) were seeded into Nunc cell culture dishes (Nalge Nunc International, Rochester, NY) with 2-mm grids, size 35 × 10 mm, in 2 mL of Dulbecco’s modified Eagle medium supplemented with 20% fetal calf serum and antibiotics. The cells were maintained at 37°C and 5% CO2 until confluent. Once confluent, each dish of monolayer cells was given a mechanical wound by scoring with a 200-μL pipette tip, parallel to the grid bars along the central grid line. This permits easy viewing of the cells growing back together, and ensures that the 2-mm grid may be used as a reference so that the wound areas can be measured and compared. Wound placement was checked with an Olympus inverted CK2 microscope (Olympus UK Ltd., London, UK). The medium then was removed, and the cells were washed 5 times with HL-1 serum-free medium (Cambrex Bio Science, Nottingham, UK) supplemented with antibiotics, and then replaced with 1.5 mL HL-1 medium with the following treatments: human IgG1 (100 μg/mL), infliximab (10 and 100 μg/mL), adalimumab (100 μg/mL), etanercept (100 μg/mL), p55-TNFR-IgG (100 μg/mL), neutralizing anti–TIMP-1 antibody (5 μg/mL, Calbiochem, Nottingham, UK), control mouse IgG (100 μg/mL, Sigma-Aldrich), and recombinant human TIMP-1 (10-8 mol/L; a kind gift from Dr A. Docherty, Celltech Pharmaceuticals, Slough, UK). Some of the wells were pretreated with TNF-α (5 ng/mL, National Institute for Biological Standards and Controls, Hertfordshire, UK) for 24 hours, then washed 5 times with HL-1 serum-free medium to remove TNF-α. Cells then were treated with infliximab (100 μg/mL) or human IgG1 (100 μg/mL). Photographs of the cells in each grid along the induced wound were taken at 0, 2, 4, 8, 16, and 24 hours using a digital camera (Olympus Camedia 34-40 zoom, 20× magnification) attached to a light microscope. The computer program Image J (National Institutes of Health, Bethesda, MD) was used to measure the area of initial damage (images taken at time 0) and of the remaining damage at subsequent time points. Each grid image was observed separately; 2 points per grid at the same position at every time point were measured using imaging software at the same magnification. The percentage of wound repair then was calculated. Myofibroblasts were washed twice in phosphate-buffered saline containing 2% fetal calf serum and incubated at 4°C for 30 minutes with infliximab at a concentration of 1 μg/mL. A fluorescein isothiocyanate (FITC)-labeled anti-human IgG1 antibody (DAKO) was used as secondary antibody. Appropriate isotype-matched control antibodies (BD Biosciences) were included in all experiments. After washing twice with 250 μL fluorescence-activated cell sorter buffer (phosphate-buffered saline containing 1 mmol/L EDTA and 0.02% sodium azide), cells were fixed with 2% paraformaldehyde, and analyzed by flow cytometry using a FACSCalibur Flow Cytometer (BD Biosciences). The level of mTNF on cell populations was determined by geometric mean fluorescence intensity with subtraction of values for isotype-matched controls. Apoptosis was quantified using FITC–Annexin V (Zymed Laboratories, San Francisco, CA). Cells were stained with 5 μL of FITC–Annexin V diluted 1:10 in buffer. After incubation for 15 minutes, the cells were analyzed by flow cytometry using a FACSCalibur Flow Cytometer (BD Biosciences). Colonic biopsy specimens were placed on iron grids in the central well of an organ culture dish and the dishes were placed in a tight chamber with 95% O2/5% CO2 at 37°C, at 1 bar. Infliximab (10 and 100 μg/mL) or human IgG1 were added and, after 24 hours, proteins were extracted from the tissue and determined by Western blotting. Western blotting was performed according to a modified method described previously.27Pender 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: 1582-1590Google Scholar In brief, cells or tissue samples were lysed in ice-cold lysate buffer (10 mmol/L EDTA, 50 mmol/L pH 7.4 Tris-HCl, 150 mmol/L sodium chloride, 1% Triton X-100, 2 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L sodium orthovanadate, 10 mg/mL leupeptin, and 2 mg/mL aprotinin) and the amount of protein was determined by the Bio-Rad Protein assay (Bio-Rad Laboratories, Hemel Hempstead, UK). A total of 10 μg of protein or 15 μL of cell culture supernatants were loaded in each lane and were run on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions. After electrophoresis, protein was transferred to nitrocellulose (Bio-Rad); a sheep anti-human MMP-3 polyclonal antibody (1:500 dilution; The Binding Site, Birmingham, UK), a mouse anti-human MMP-12 monoclonal hemopexin-like domain antibody (1:200 dilution; R&D Systems), a mouse anti-human TIMP-1 monoclonal antibody (1 μg/mL; Calbiochem), a rabbit anti-human caspase-3 monoclonal antibody (1:200 dilution; Upstate, Lake Placid, NY), and infliximab (1 μg/mL) were used as primary antibodies. Rabbit anti-sheep, rabbit anti-mouse, goat anti-rabbit, or rabbit anti-human antibodies conjugated to horseradish peroxidase (DAKO) were used as secondary antibodies, and the reaction was developed with the ECL plus kit (Amersham Biosciences, Little Chalfont, UK). When required, blots were stripped and analyzed for β-actin, as an internal loading control, using a polyclonal rabbit anti-human β-actin (1:5000 dilution, AbCam). Bands were quantified by scanning densitometry using an LKB Ultrascan XL Laser Densitometer (Kodak Ltd., Hemel Hempstead, UK). Reverse gelatin zymography was performed to detect TIMPs in the myofibroblast culture supernatants. Twenty-five microliters of each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 12% gels containing 1 mg/mL gelatin and 0.2 μg/mL recombinant human Pro-MMP-2 (R&D Systems). Samples were electrophoresed at 120 V for 70 minutes. The gels were washed in a washing buffer (50 mmol/L Tris, 0.2 mol/L NaCl, and 2.5% Triton X-100) for 15 minutes 2 times, and then incubated in a digestion buffer (50 mmol/L Tris, 0.2 mol/L NaCl, 10 mmol/L CaCl2, 0.02% Brij-35) at 37°C for 20 hours. The gels were stained with 0.5% Coomassie blue R-250 in 50% methanol and 10% acetic acid solution for 10 minutes, and destained in 30% methanol and 10% acetic acid solution until the background of the gel became clear. Recombinant human TIMP-1 (Celltech Pharmaceuticals) was used as control protein for reverse zymography at 3 different concentrations (0.025 mol/L, 0.05 mol/L, and 0.01 mol/L). Total soluble forms of collagen were measured in myofibroblast supernatants using the Sircol Collagen Assay kit (Biocolor Ltd, Belfast, UK) according to the manufacturer’s instructions. The Sircol dye reagent has been formulated to bind specifically to the [Gly-X-Y]n helical structure found in collagen types I–XIV. The cell supernatant (200 μL) was mixed with 1 mL of Sircol dye reagent, and the tubes were shaken for 30 minutes at room temperature to allow collagen-dye binding to complete. After centrifugation, the supernatant of unbound dye was removed, and the collagen-bound dye pellet was dissolved in the alkali reagent. The recovered dye concentration was measured by a spectrometer with the absorbance of 540 nm. The calibration curve for the spectrometer had been drawn previously using the supplied collagen standard. The collagen content in each sample of cell supernatant was obtained as an average of 3 readings. Data were analyzed in the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA) using the paired t test and the Mann–Whitney U test. A P value of less than .05 was considered statistically significant. First, phenotypic characterization of CD and control myofibroblasts was performed by immunocytochemistry (Table 1).Table 1Immunocytochemical Characterization of Colonic Myofibroblasts Isolated From 8 CD Patients and 8 Control SubjectsPercentage positive cells (mean ± SD)Anti-α-SMAVimentinPR2D3DesminCytokeratinCD3CD68CD86.0 ± 3.294.9 ± 1.224.2 ± 8.434.6 ± 10.77.0 ± 2.200Controls85.7 ± 2.295.5 ± 1.828.8 ± 9.938.7 ± 6.16.1 ± 2.300Anti–α-SMA, α-smooth muscle actin marker; antivimentin, cytoplasmic intermediate filament marker; anti-PR2D3, pericryptal mesenchymal cell marker; antidesmin, smooth muscle cell marker; anti-cytokeratin, cytokeratin-18 marker; anti-CD3, T-cell marker; anti-CD68, macrophage marker. Open table in a new tab Anti–α-SMA, α-smooth muscle actin marker; antivimentin, cytoplasmic intermediate filament marker; anti-PR2D3, pericryptal mesenchymal cell marker; antidesmin, smooth muscle cell marker; anti-cytokeratin, cytokeratin-18 marker; anti-CD3, T-cell marker; anti-CD68, macrophage marker. Second, to determine if intestinal myofibroblasts might be a target of infliximab, mTNF expression was analyzed by Western blotting and flow cytometry using myofibroblasts isolated from both inflamed and uninvolved areas of CD and UC patients, and from control subjects. mTNF expression detected by Western blotting on myofibroblasts was increased significantly (P < .0001) in both CD and UC inflamed areas in comparison with uninvolved areas and controls. No significant difference was found between myofibroblasts from IBD uninvolved areas and normal myofibroblasts (Figure 1A). These results were confirmed by flow cytometric analysis showing a significantly (P < .0001) higher mean fluorescence intensity of mTNF expression in myofibroblasts from both CD and UC lesions in comparison with myofibroblasts from uninvolved areas or control myofibroblasts. Moreover, a significantly (P < .001) higher number of mTNF-positive myofibroblasts was found in CD lesions (mean, 24.4% ± 4.8%) and UC lesions (mean, 20.1% ± 4.3%) in comparison with both uninvolved CD areas (mean, 5.9% ± 1.8%) and UC areas (mean, 4.2% ± 1.4%), and controls (mean, 2.8% ± 0.4%) (Figure 1B). No significant difference was observed in the expression of mTNF between UC and CD myofibroblasts, both in involved and uninvolved sites. To investigate the influence of infliximab on apoptosis, myofibroblasts were cultured with increasing concentrations of infliximab (10 and 100 μg/mL) or IgG1, and apoptosis was analyzed by flow cytometry with FITC–Annexin V staining. Representative results of CD patients show no effect of both concentrations of infliximab on myofibroblast apoptosis (Figure 2A). No significant difference was found in CD in the percentage of apoptotic myofibroblasts when cultured with IgG1 (mean, 5.2% ± 1.2%) or with infliximab at 10 μg/mL (mean, 4.7% ± 1.7%) or 100 μg/mL (mean, 5.5% ± 1.5%). The same trend was observed in control myofibroblasts (mean, 5.4% ± 1.6%, 5.9% ± 1.9%, and 4.8% ± 1.4%, respectively). Western blotting showed that infliximab did not activate caspase-3 in CD myofibroblasts, as indicated by the absence of the active forms of caspase-3, the p12 and p17 subunits that instead were evident in anti–CD3/CD28-stimulated Jurkat T cells treated with infliximab (Figure 2B). As expected, interleukin-1β increased MMP-3 production by CD myofibroblasts. Infliximab did not change MMP-3 levels in the supernatants of both unstimulated and interleukin-1β–s
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