Growth Hormone Reduces the Severity of Fibrosis Associated With Chronic Intestinal Inflammation
2005; Elsevier BV; Volume: 129; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2005.05.019
ISSN1528-0012
AutoresArianne L. Theiss, C. Randall Fuller, James G. Simmons, Bo Liu, R. Balfour Sartor, P. Kay Lund,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoBackground & Aims: Growth hormone (GH) is used to treat growth delay in children with Crohn's disease and in patients with short-bowel syndrome. GH can increase collagen accumulation in intestinal mesenchymal cells, raising concern that GH therapy could exacerbate fibrosis in patients with Crohn's disease. We tested if GH treatment altered inflammation or fibrosis during chronic, experimental granulomatous enterocolitis. Methods: Ileum and cecum of Lewis rats were subserosally injected with peptidoglycan-polysaccharide (PG-APS) or control human serum albumin. At the onset of chronic PG-APS-induced inflammation, rats were administered recombinant human GH or vehicle for 14 days. Fibrosis and inflammation were quantified by gross gut disease scoring, histologic scoring, type I collagen, and cytokine expression in cecum. Abundance and localization of suppressor of cytokine signaling-3 (SOCS-3) messenger RNA and/or protein were determined in cecum. Effect of GH, cytokines, or PG-APS on SOCS-3 synthesis was measured in intestinal myofibroblasts. Myofibroblasts overexpressing SOCS-3 were used to test whether SOCS-3 inhibits collagen accumulation. Results: In PG-APS-injected rats, GH modestly reduced gross adhesions and mesenteric contractions, cecal fibrosis score, and collagen expression, but had no effect on intestinal inflammation. GH increased SOCS-3 messenger RNA and protein abundance in PG-APS rats and SOCS-3 messenger RNA was localized to the periphery of granulomas. GH in combination with cytokines or PG-APS, but not alone, induced SOCS-3 synthesis in intestinal myofibroblasts. Myofibroblasts overexpressing SOCS-3 showed reduced cytokine-induced collagen accumulation. Conclusions: GH modestly reduces intestinal fibrosis associated with chronic experimental enterocolitis and stimulates expression of antifibrogenic SOCS-3, suggesting that GH therapy in inflammatory bowel disease should not exacerbate fibrosis. Background & Aims: Growth hormone (GH) is used to treat growth delay in children with Crohn's disease and in patients with short-bowel syndrome. GH can increase collagen accumulation in intestinal mesenchymal cells, raising concern that GH therapy could exacerbate fibrosis in patients with Crohn's disease. We tested if GH treatment altered inflammation or fibrosis during chronic, experimental granulomatous enterocolitis. Methods: Ileum and cecum of Lewis rats were subserosally injected with peptidoglycan-polysaccharide (PG-APS) or control human serum albumin. At the onset of chronic PG-APS-induced inflammation, rats were administered recombinant human GH or vehicle for 14 days. Fibrosis and inflammation were quantified by gross gut disease scoring, histologic scoring, type I collagen, and cytokine expression in cecum. Abundance and localization of suppressor of cytokine signaling-3 (SOCS-3) messenger RNA and/or protein were determined in cecum. Effect of GH, cytokines, or PG-APS on SOCS-3 synthesis was measured in intestinal myofibroblasts. Myofibroblasts overexpressing SOCS-3 were used to test whether SOCS-3 inhibits collagen accumulation. Results: In PG-APS-injected rats, GH modestly reduced gross adhesions and mesenteric contractions, cecal fibrosis score, and collagen expression, but had no effect on intestinal inflammation. GH increased SOCS-3 messenger RNA and protein abundance in PG-APS rats and SOCS-3 messenger RNA was localized to the periphery of granulomas. GH in combination with cytokines or PG-APS, but not alone, induced SOCS-3 synthesis in intestinal myofibroblasts. Myofibroblasts overexpressing SOCS-3 showed reduced cytokine-induced collagen accumulation. Conclusions: GH modestly reduces intestinal fibrosis associated with chronic experimental enterocolitis and stimulates expression of antifibrogenic SOCS-3, suggesting that GH therapy in inflammatory bowel disease should not exacerbate fibrosis. Intestinal fibrosis is a common complication of Crohn's disease (CD) and generally is considered an excessive, irreversible, wound-healing response to chronic transmural inflammation.1Zimmermann E. Lund P.K. Fibrogenesis.in: Sartor R.B. Sandborn W.J. Kirsner's inflammatory bowel diseases. 6th ed. Elsevier, Philadelphia2004: 219-229Google Scholar Fibrosis in this disorder involves overgrowth of the muscularis mucosa and muscularis propria,2Graham M.F. Diegelmann R.F. Elson C.O. Lindblad W.J. Gotschalk N. Gay S. Gay R. Collagen content and types in the intestinal strictures of Crohn's disease.Gastroenterology. 1988; 94: 257-265Abstract PubMed Google Scholar excessive collagen deposition,2Graham M.F. Diegelmann R.F. Elson C.O. Lindblad W.J. Gotschalk N. Gay S. Gay R. Collagen content and types in the intestinal strictures of Crohn's disease.Gastroenterology. 1988; 94: 257-265Abstract PubMed Google Scholar, 3Matthes H. Herbst H. Schuppan D. Stallmach A. Milani S. Stein H. Riecken E.O. 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Surgical therapy for ulcerative colitis and Crohn's disease.Gastroenterol Clin North Am. 1999; 28 (viii-ix): 371-390Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar Recombinant human growth hormone (rhGH), alone or in combination with glutamine and modified diet, has been tested as therapy in SBS patients in a number of clinical trials and was reported to improve weight gain and lean body mass and reduce the need for parenteral nutrition.9Scolapio J.S. Camilleri M. Fleming C.R. Oenning L.V. Burton D.D. Sebo T.J. Batts K.P. Kelly D.G. Effect of growth hormone, glutamine, and diet on adaptation in short-bowel syndrome a randomized, controlled study.Gastroenterology. 1997; 113: 1074-1081Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 10Ellegard L. Bosaeus I. Nordgren S. 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Tales from the crypt.Gastroenterology. 2003; 124: 561-564Abstract Full Text PDF PubMed Scopus (13) Google Scholar Some SBS patients treated with rhGH had CD, but no information was reported regarding rhGH effects on inflammation or fibrosis.9Scolapio J.S. Camilleri M. Fleming C.R. Oenning L.V. Burton D.D. Sebo T.J. Batts K.P. Kelly D.G. Effect of growth hormone, glutamine, and diet on adaptation in short-bowel syndrome a randomized, controlled study.Gastroenterology. 1997; 113: 1074-1081Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 10Ellegard L. Bosaeus I. Nordgren S. Bengtsson B.A. Low-dose recombinant human growth hormone increases body weight and lean body mass in patients with short bowel syndrome.Ann Surg. 1997; 225: 88-96Crossref PubMed Scopus (124) Google Scholar rhGH therapy improved linear growth in pediatric CD patients with growth delay.18Mauras N. George D. Evans J. Milov D. Abrams S. Rini A. Welch S. Haymond M.W. 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However, potential effects of GH on fibrosis, a serious complication of CD, have not been analyzed in the clinical studies or in animal models of inflammatory bowel disease (IBD). Beneficial effects of GH on intestinal inflammation have been documented in animal models of colitis. Administration of rhGH reduced inflammation in the trinitrobenzene sulfonic acid rat model of IBD.21Christensen H. Flyvbjerg A. Orskov H. Laurberg S. Effect of growth hormone on the inflammatory activity of experimental colitis in rats.Scand J Gastroenterol. 1993; 28: 503-511Crossref PubMed Scopus (18) Google Scholar, 22Kara E. Sungurtekin H. Sungurtekin U. Alkanat M. Ilkgul O. The effect of recombinant human growth hormone (rhGH) on trinitrobenzene sulfonic acid-induced colitis in rats an experimental study.Inflamm Bowel Dis. 2004; 10: 112-115Crossref PubMed Scopus (21) Google Scholar Transgenic mice overexpressing GH exhibited improved mucosal repair after acute colitis induced by dextran sodium sulfate compared with wild-type mice.23Williams K.L. Fuller C.R. Dieleman L.A. DaCosta C.M. Haldeman K.M. Sartor R.B. Lund P.K. Enhanced survival and mucosal repair after dextran sodium sulfate-induced colitis in transgenic mice that overexpress growth hormone.Gastroenterology. 2001; 120: 925-937Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Improved repair in transgenic mice overexpressing GH was associated with more rapid but transient increases in crypt cell proliferation. However, GH action on intestinal fibrosis was not analyzed in any of these studies. Overall, fibrosis during chronic inflammation has been less well studied than inflammation. This reflects the fact that many animal models of IBD do not exhibit fibrosis and analyses in clinical trials generally are limited to the mucosa and not the submucosal layers, which are predominant sites of fibrosis. Concerns that GH treatment could exacerbate fibrosis stem from preliminary observations that GH stimulates collagen accumulation in cultured intestinal myofibroblasts,24Fruchtman S. Simmons J.G. Fuller C.R. Greenhalgh C.J. Lund P.K. Suppressors of cytokine signaling (SOCS) limit fibrogenic actions of growth hormone and insulin-like growth factor-I in intestinal myofibroblasts.Gastroenterology. 2004; 126: A-138Google Scholar the primary mesenchymal cell type thought to mediate intestinal fibrosis.6Pucilowska J.B. McNaughton K.K. Mohapatra N.K. Hoyt E.C. Zimmermann E.M. Sartor R.B. Lund P.K. IGF-I and procollagen alpha1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease.Am J Physiol. 2000; 279: G1307-G1322PubMed Google Scholar GH also induces insulin-like growth factor-I (IGF-I) expression in some tissues and considerable evidence suggests that IGF-I may play a role in mediating fibrosis in CD.1Zimmermann E. Lund P.K. Fibrogenesis.in: Sartor R.B. Sandborn W.J. Kirsner's inflammatory bowel diseases. 6th ed. Elsevier, Philadelphia2004: 219-229Google Scholar This raises the possibility that GH therapy in IBD patients may increase directly or indirectly intestinal collagen deposition or fibrosis. This study tested the hypothesis that therapeutically administered GH exacerbates fibrosis in a rat model of chronic intestinal inflammation induced by peptidoglycan-polysaccharide polymers derived from group A streptococci (PG-APS), one of the few animal models characterized by fibrosis associated with chronic intestinal inflammation.25van Tol E.A. Holt L. Li F.L. Kong F.M. Rippe R. Yamauchi M. Pucilowska J. Lund P.K. Sartor R.B. Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts.Am J Physiol. 1999; 277: G245-G255PubMed Google Scholar When injected subserosally into ileum and cecum of susceptible rat strains, PG-APS polymers induce acute inflammation for 24–48 hours followed by remission of inflammation and then spontaneous reactivation between 10–20 days after injection with progression to chronic granulomatous inflammation.26Sartor R.B. Herfarth H. Van Tol E.A.F. Bacterial cell wall polymer-induced granulomatous inflammation.Methods. 1996; 9: 233-247Crossref PubMed Scopus (16) Google Scholar, 27Sartor R.B. Cromartie W.J. Powell D.W. Schwab J.H. Granulomatous enterocolitis induced in rats by purified bacterial cell wall fragments.Gastroenterology. 1985; 89: 587-595PubMed Google Scholar This model particularly is relevant to CD for a number of reasons. Reactivation and chronic inflammation is T-cell mediated and accompanied by systemic responses including arthritis, which is also a complication in a significant number of CD patients.28Greig E.R. Rampton D.S. et al.Management of Crohn's Disease. Martin Dunitz, New York2003Google Scholar Importantly, chronic inflammation induced by PG-APS is known to involve severe transmural fibrosis and smooth muscle hyperplasia,25van Tol E.A. Holt L. Li F.L. Kong F.M. Rippe R. Yamauchi M. Pucilowska J. Lund P.K. Sartor R.B. Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts.Am J Physiol. 1999; 277: G245-G255PubMed Google Scholar making this model particularly useful for studying the possible effects of therapeutic interventions on fibrosis. In the present study, we tested whether GH given therapeutically after reactivation of chronic inflammation altered the severity of fibrosis associated with chronic PG-APS-induced enterocolitis. As well as testing the effects of GH on fibrosis, we examined whether GH altered the severity of intestinal inflammation, key cytokine mediators of inflammation, or local expression of IGF-I, which is up-regulated at sites of fibrosis in the PG-APS model29Zimmermann E.M. Sartor R.B. McCall R.D. Pardo M. Bender D. Lund P.K. Insulinlike growth factor I and interleukin 1 beta messenger RNA in a rat model of granulomatous enterocolitis and hepatitis.Gastroenterology. 1993; 105: 399-409Abstract PubMed Google Scholar and in involved intestine of patients with CD.6Pucilowska J.B. McNaughton K.K. Mohapatra N.K. Hoyt E.C. Zimmermann E.M. Sartor R.B. Lund P.K. IGF-I and procollagen alpha1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease.Am J Physiol. 2000; 279: G1307-G1322PubMed Google Scholar, 30Lawrance I.C. Fiocchi C. Chakravarti S. 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Hilton D.J. Roberts A.W. Alexander W.S. SOCS3 negatively regulates IL-6 signaling in vivo.Nat Immunol. 2003; 4: 540-545Crossref PubMed Scopus (679) Google Scholar, 33Lovato P. Brender C. Agnholt J. Kelsen J. Kaltoft K. Svejgaard A. Eriksen K.W. Woetmann A. Odum N. Constitutive STAT3 activation in intestinal T cells from patients with Crohn's disease.J Biol Chem. 2003; 278: 16777-16781Crossref PubMed Scopus (166) Google Scholar Given the unexpected antifibrogenic actions of GH and its induction of SOCS-3, follow-up studies were performed in intestinal myofibroblasts to examine directly whether GH induces SOCS-3 in intestinal mesenchymal cells and to test whether SOCS-3 negatively modulates the effects of GH or IGF-I on collagen accumulation. Increasing evidence suggests a role for tumor necrosis factor α (TNFα) in inflammation-induced fibrosis in other organs including lung and kidney34Ortiz L.A. Lasky J. Gozal E. Ruiz V. Lungarella G. Cavarra E. Brody A.R. Friedman M. Pardo A. Selman M. Tumor necrosis factor receptor deficiency alters matrix metalloproteinase 13/tissue inhibitor of metalloproteinase 1 expression in murine silicosis.Am J Respir Crit Care Med. 2001; 163: 244-252Crossref PubMed Scopus (48) Google Scholar, 35Guo G. Morrissey J. McCracken R. Tolley T. Liapis H. Klahr S. Contributions of angiotensin II and tumor necrosis factor-alpha to the development of renal fibrosis.Am J Physiol. 2001; 280: F777-F785Google Scholar and that TNFα is a key proinflammatory mediator in CD.28Greig E.R. Rampton D.S. et al.Management of Crohn's Disease. Martin Dunitz, New York2003Google Scholar Therefore, we tested whether SOCS-3 influenced TNFα action on collagen accumulation in intestinal myofibroblasts. PG-APS fragments from cell walls of group A, type 3, strain D58 streptococci (Streptococcus pyogenes) were isolated and prepared as previously described.36Fox A. Brown R.R. Anderle S.K. Chetty C. Cromartie W.J. Gooder H. Schwab J.H. Arthropathic properties related to the molecular weight of peptidoglycan-polysaccharide polymers of streptococcal cell walls.Infect Immun. 1982; 35: 1003-1010Crossref PubMed Google Scholar The preparation was sonicated immediately before use to disperse aggregates. The final PG-APS concentration based on rhamnose measurements was 12 mg/mL. Female inbred specific pathogen-free Lewis rats (140–170 g) were obtained from Charles River Laboratories (Raleigh, NC). All animals were housed in standard cages with 6 animals per cage and were allowed food and water ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill and conformed to National Institutes of Health guidelines. Animals were anesthetized with 1.5 mL/kg body weight Innovar (Pitman-Moore, Washington Crossing, NJ) and 80 mg/kg body weight ketamine hydrochloride, and their intestines were exposed by laparotomy using aseptic technique. A total of 24 rats were injected subserosally with PG-APS (12.5 μg rhamnose/g body weight) into 7 sites of the distal ileum and cecum. A control group of rats (n = 16) were injected with human serum albumin (HSA; 37.5 μg/g body weight, Baxter Healthcare Corporation, Glendale, CA). The rats were monitored for arthritis and were weighed daily. Of the 24 rats injected with PG-APS, 16 rats showed spontaneous reactivation of a chronic inflammatory response to PG-APS indicated by an increase in joint diameter of .6 mm from the previous measurement accompanied by edema and redness in 1 or both hind ankle joints. On the day of reactivation, rats were implanted with osmotic minipumps (Durect Corporation, Cupertino, CA) to administer rhGH (500 μg/kg body weight/day, Genentech Inc., San Francisco, CA) or vehicle (saline) for 14 days. The dose of GH was chosen to match the low doses reported in the literature for clinical trials in humans.17Scolapio J.S. Tales from the crypt.Gastroenterology. 2003; 124: 561-564Abstract Full Text PDF PubMed Scopus (13) Google Scholar This dose is at the low end of a range of doses of GH tested for effects on the intestine in rodent models.21Christensen H. Flyvbjerg A. Orskov H. Laurberg S. Effect of growth hormone on the inflammatory activity of experimental colitis in rats.Scand J Gastroenterol. 1993; 28: 503-511Crossref PubMed Scopus (18) Google Scholar, 22Kara E. Sungurtekin H. Sungurtekin U. Alkanat M. Ilkgul O. The effect of recombinant human growth hormone (rhGH) on trinitrobenzene sulfonic acid-induced colitis in rats an experimental study.Inflamm Bowel Dis. 2004; 10: 112-115Crossref PubMed Scopus (21) Google Scholar, 37Dahly E.M. Miller M.E. Lund P.K. Ney D.M. Postreceptor resistance to exogenous growth hormone exists in the jejunal mucosa of parenterally fed rats.J Nutr. 2004; 134: 530-537PubMed Google Scholar, 38Park J.H. Vanderhoof J.A. Growth hormone did not enhance mucosal hyperplasia after small-bowel resection.Scand J Gastroenterol. 1996; 31: 349-354Crossref PubMed Scopus (35) Google Scholar PG-APS-injected rats showing reactivation were paired based on comparable joint diameter at onset of reactivation and one of each pair was administered GH and the other was administered vehicle. The aim in pairing animals for comparable joint diameter was to minimize any potential effects of differences in disease severity at the onset of treatment. An experienced laboratory research analyst assigned the paired animals to vehicle or GH treatment groups and assigned a code number to each animal such that experimenters performing all subsequent analyses and evaluation or scoring of disease severity were blinded to the treatment groups. A total of 8 pairs of GH-treated and vehicle-treated PG-APS-injected rats were studied. HSA control rats were administered GH (n = 8) or vehicle (n = 8) in parallel with PG-APS-treated groups. Joint diameter and body weight were monitored daily throughout the experiment. After the 14-day treatment period, rats were killed by an intramuscular injection of sodium pentobarbital (100 μg/g body weight, Abbot Laboratories, Chicago, IL). The abdomen was opened by a midline incision and a gross gut disease score was derived as one measure of disease severity. Well-established criteria for gross gut disease score have been detailed in previous reports using the PG-APS model.39McCall R.D. Haskill S. Zimmermann E.M. Lund P.K. Thompson R.C. Sartor R.B. Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats.Gastroenterology. 1994; 106: 960-972Abstract PubMed Scopus (113) Google Scholar Briefly, 4 independent parameters consisting of extent of cecal wall thickening, severity of adhesions, severity of mesenteric contractions, and number of cecal nodules were each given a score ranging from 0 to 4, where 4 indicates the most severe disease. The scores for each parameter were totaled to derive an overall gross gut disease score, with a maximum possible score of 16.39McCall R.D. Haskill S. Zimmermann E.M. Lund P.K. Thompson R.C. Sartor R.B. Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats.Gastroenterology. 1994; 106: 960-972Abstract PubMed Scopus (113) Google Scholar Blood was collected by cardiac puncture for hematologic assays (performed by the Animal Clinical Core Facility at the University of North Carolina). Plasma was separated by centrifugation and plasma IGF-I concentrations were measured by enzyme-linked immunosorbent assay (Diagnostic Systems Laboratories Inc., Webster, TX). Samples were pretreated as described in the enzyme-linked immunosorbent assay protocol to remove IGF-binding proteins complexed to IGF-I. Briefly, enzyme-linked immunosorbent assay samples were incubated with biotin-labeled rat IGF-I and goat anti-rat IGF-I antiserum in wells coated with rabbit anti-goat γ globulin. Unlabeled IGF-I in plasma and biotin-labeled IGF-I compete for limited anti-rat IGF-I binding sites. Streptavidin-horseradish peroxidase binds the antibody-free biotinylated rat IGF-I, whose concentration in standards and each sample is estimated based on enzymatic turnover of the substrate tetramethylbenzidine and absorbance at 450 and 620 nm. The entire cecum was dissected and the contents were flushed with ice-cold .9% saline. The cecal tip was embedded in frozen tissue-embedding medium (Fisher Scientific, Fair Lawn, NJ) for in situ hybridization histochemistry. Adjacent samples were formalin-fixed and paraffin-embedded; sections were used for histologic evaluation of inflammation and fibrosis. The remaining cecum was cut longitudinally into 2 portions and homogenized immediately in 4 mol/L guanidine thiocyanate for RNA extraction or in protein extraction buffer (50 mmol/L Hepes, 150 mmol/L NaCl, 20 mmol/L Na pyrophosphate, 100 mmol/L NaF, 1.5% Triton X-100, and 100 mmol/L ethylenediaminetetraacetic acid containing protease inhibitors: 1 μg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, and 2 mmol/L vanadate; pH 7.4). Coded paraffin-embedded, formalin-fixed sections of cecum were stained with H&E, Masson's trichrome, or Sirius red. H&E-stained sections were used to obtain a histologic score for inflammation using criteria previously described.27Sartor R.B. Cromartie W.J. Powell D.W. Schwab J.H. Granulomatous enterocolitis induced in rats by purified bacterial cell wall fragments.Gastroenterology. 1985; 89: 587-595PubMed Google Scholar, 39McCall R.D. Haskill S. Zimmermann E.M. Lund P.K. Thompson R.C. Sartor R.B. Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats.Gastroenterology. 1994; 106: 960-972Abstract PubMed Scopus (113) Google Scholar A score ranging from 0 to 4 (4 being the most severe) was assigned for both acute and chronic inflammation of each layer of the cecal wall. The acute and chronic scores were summed to derive an overall inflammatory score for each rat, and the maximum possible score was 32. The acute inflammatory score was based on hemorrhage, edema, polymorphonuclear leukocytic infiltration, and necrosis. The chronic inflammatory score was based on the number of mononuclear cells present. Masson's trichrome- and Sirius red-stained sections of cecum were used to obtain a histologic score for fibrosis. Masson's trichrome stains collagen, whereas Sirius red stains only fibrillar collagen. Experimental sections were compared with sections from normal untreated control rats to derive a score r
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