ORIGINAL ARTICLE: GASTROENTEROLOGY
MMPs-2 and -14 Are Elevated in Eosinophilic Esophagitis and Reduced Following Topical Corticosteroid Therapy
Lisa Beppu, Tom Yang, Michelle Luk, yRobert O. Newbury, Jacob Palmquist, y Ranjan Dohil, zRichard C. Kurten, David H. Broide, and Seema S. Aceves
See ‘‘Eosinophilic Esophagitis: Current Concepts’’ by Putnam on page 156.
ABSTRACT Objectives: Eosinophilic esophagitis (EoE) is a chronic, antigen-mediated disease in children and adults associated with substantial esophageal remodeling and fibrosis. The expression of the remodeling-associated matrix metalloproteinases (MMPs) has not been previously detailed in EoE. Methods: MMP-2 and -14 expression and cellular localization were assessed using real-time quantitative polymerase chain reaction and immunohistochemistry/immunofluorescence in EoE fibroblasts, active and inactive pediatric EoE biopsies, and nondiseased control biopsies. The effect of transforming growth factor (TGF)-b1 treatment on MMP-2 expression in cultured esophageal epithelial (HET1A) cells was analyzed. Results: MMP-2 and -14 mRNA were expressed in EoE fibroblasts and biopsies. Proliferating epithelial cells produced MMP-14 more abundantly in EoE than in controls (P < 0.001) and the degree of epithelial MMP-14 expression correlated positively with basal zone hyperplasia (r ¼ 0.65, P ¼ 0.002). EoE lamina propria had higher numbers of MMP-2- and -14positive cells (906 167 and 701 93 cells/mm2) as compared with controls (258 93 cells/mm2, P < 0.01 and 232 54 cells/mm2, P < 0.01), and MMP-14 expression correlated with the severity of fibrosis. Following therapy with topical corticosteroids, MMP-14 and -2 were significantly
Received July 6, 2014; accepted October 4, 2014. From the Division of Allergy, Immunology, Center for Immunity, Infection, and Inflammation, the yDepartment of Pediatrics, University of California, and the zDepartment of Physiology and Biophysics, University of Arkansas Medical Center, Little Rock. Address correspondence and reprint requests to Seema S. Aceves, MD, PhD, Division of Allergy, Immunology, Center for Immunity, Infection, and Inflammation, University of California, 9500 Gilman Drive, MC0760, La Jolla, CA 92093-0760 (e-mail: [email protected]). Supplemental digital content is available for the present article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of the present article on the journal’s Web site (www.jpgn.org). The study was funded by NIH/NIAID AI 092135 (S.S.A.), APFED/AAAAI HOPE/ARTrust Pilot Grant (S.S.A.), DOD FA100044 (D.H.B., S.S.A.), NIH/NIAID AI 107779 (D.H.B.), AI 70535 (D.H.B.), AI 72115 (D.H.B.), NIH/NCRR/NCATS UL1TR000039 (R.C.K.), and Hearst Foundation (R.D.). The present project was also partially supported by the NIH grant ULRR031980 for years 1 and 2 or CTSA funding and/ or ULT1TR0001000 during year 3 and beyond of CTSA funding (support of the EoE database). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000668
diminished (P < 0.01). TGF-b1 increased the expression and secretion of MMP-2 from esophageal epithelial HET1A cells. Conclusions: MMP-2 and -14 are elevated in pediatric patients with EoE and significantly decrease following topical corticosteroid therapy. TGF-b1 increases MMP-2 in esophageal epithelial cells. This alludes to previously unappreciated role for MMPs in EoE-associated esophageal remodeling and a potential positive feedback loop via TGF-b1. Key Words: eosinophilic esophagitis, fibrosis, matrix metalloproteinase, remodeling, transforming growth factor-b1
(JPGN 2015;61: 194–199)
E
osinophilic esophagitis (EoE) is a chronic, antigen-driven allergic disease that is associated with substantial tissue remodeling (1–5). Esophageal remodeling is the mechanism for EoE complications including fibrosis and smooth muscle dysfunction, which ultimately cause esophageal rigidity, strictures, and dysmotility (1,3,6–10). Tissue remodeling involves collagen accumulation and leads to the entrapment of cells and growth factors in a fibrotic milieu. Angiogenesis and vascular activation provide increased numbers of blood vessels for inflammatory cell trafficking into the esophagus (3,11). Matrix metalloproteinases (MMPs) are a family of proteins that are integral to collagen breakdown, angiogenesis, and tissue remodeling. They, however, have not been previously studied systematically in EoE. MMPs function as gelatinases and collagenases that remodel extracellular matrix and activate other MMPs and growth factors such as transforming growth factor (TGF)-b1 (12,13). TGF-b1 is overexpressed in EoE and has direct effects on esophageal fibroblasts, smooth muscle cells, and epithelium, causing increased fibrotic gene expression, smooth muscle cell contraction, and epithelial mesenchymal transformation (3,6,7,9,14). In particular, MMP-14 and -2 can act in sequence to activate one another and cause subsequent release of TGF-b1 from its latency complex (12). At present, a single preliminary report describes MMP-14 mRNA in EoE (15). In asthma, some of TGF-b1’s profibrotic effects can be mediated through MMP-2 (16). In the present study, we aimed to delineate the expression pattern and potential roles of MMP-2 and -14 in pediatric EoE. We report increased esophageal levels of MMP-2 and -14 in pediatric EoE and demonstrate that proliferating epithelial cells and EoE fibroblasts are cellular sources of both MMP-2 and -14. Cultured esophageal epithelial cells treated with TGF-b1 increased the production and release of MMP-2. As such, MMPs appear to play a previously unreported role in EoE.
METHODS Patients With EoE/Biopsies The University of California, San Diego (UCSD)/Rady Children’s Hospital, San Diego (RCHSD) EoE database was
194 JPGN Volume 61, Number 2, August 2015 Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 61, Number 2, August 2015
screened to randomly select a cohort of patients with EoE who met the following criteria: EoE defined as histologic criteria (15 eosinophils per high-power field [hpf] on hematoxylin and eosin stain at 400 magnification on light microscopy); the presence of typical symptom and endoscopic features; successful treatment (defined as
MMPs-2 and -14 Are Elevated in Eosinophilic Esophagitis and Reduced Following Topical Corticosteroid Therapy
Lisa Beppu, Tom Yang, Michelle Luk, yRobert O. Newbury, Jacob Palmquist, y Ranjan Dohil, zRichard C. Kurten, David H. Broide, and Seema S. Aceves
See ‘‘Eosinophilic Esophagitis: Current Concepts’’ by Putnam on page 156.
ABSTRACT Objectives: Eosinophilic esophagitis (EoE) is a chronic, antigen-mediated disease in children and adults associated with substantial esophageal remodeling and fibrosis. The expression of the remodeling-associated matrix metalloproteinases (MMPs) has not been previously detailed in EoE. Methods: MMP-2 and -14 expression and cellular localization were assessed using real-time quantitative polymerase chain reaction and immunohistochemistry/immunofluorescence in EoE fibroblasts, active and inactive pediatric EoE biopsies, and nondiseased control biopsies. The effect of transforming growth factor (TGF)-b1 treatment on MMP-2 expression in cultured esophageal epithelial (HET1A) cells was analyzed. Results: MMP-2 and -14 mRNA were expressed in EoE fibroblasts and biopsies. Proliferating epithelial cells produced MMP-14 more abundantly in EoE than in controls (P < 0.001) and the degree of epithelial MMP-14 expression correlated positively with basal zone hyperplasia (r ¼ 0.65, P ¼ 0.002). EoE lamina propria had higher numbers of MMP-2- and -14positive cells (906 167 and 701 93 cells/mm2) as compared with controls (258 93 cells/mm2, P < 0.01 and 232 54 cells/mm2, P < 0.01), and MMP-14 expression correlated with the severity of fibrosis. Following therapy with topical corticosteroids, MMP-14 and -2 were significantly
Received July 6, 2014; accepted October 4, 2014. From the Division of Allergy, Immunology, Center for Immunity, Infection, and Inflammation, the yDepartment of Pediatrics, University of California, and the zDepartment of Physiology and Biophysics, University of Arkansas Medical Center, Little Rock. Address correspondence and reprint requests to Seema S. Aceves, MD, PhD, Division of Allergy, Immunology, Center for Immunity, Infection, and Inflammation, University of California, 9500 Gilman Drive, MC0760, La Jolla, CA 92093-0760 (e-mail: [email protected]). Supplemental digital content is available for the present article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of the present article on the journal’s Web site (www.jpgn.org). The study was funded by NIH/NIAID AI 092135 (S.S.A.), APFED/AAAAI HOPE/ARTrust Pilot Grant (S.S.A.), DOD FA100044 (D.H.B., S.S.A.), NIH/NIAID AI 107779 (D.H.B.), AI 70535 (D.H.B.), AI 72115 (D.H.B.), NIH/NCRR/NCATS UL1TR000039 (R.C.K.), and Hearst Foundation (R.D.). The present project was also partially supported by the NIH grant ULRR031980 for years 1 and 2 or CTSA funding and/ or ULT1TR0001000 during year 3 and beyond of CTSA funding (support of the EoE database). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000668
diminished (P < 0.01). TGF-b1 increased the expression and secretion of MMP-2 from esophageal epithelial HET1A cells. Conclusions: MMP-2 and -14 are elevated in pediatric patients with EoE and significantly decrease following topical corticosteroid therapy. TGF-b1 increases MMP-2 in esophageal epithelial cells. This alludes to previously unappreciated role for MMPs in EoE-associated esophageal remodeling and a potential positive feedback loop via TGF-b1. Key Words: eosinophilic esophagitis, fibrosis, matrix metalloproteinase, remodeling, transforming growth factor-b1
(JPGN 2015;61: 194–199)
E
osinophilic esophagitis (EoE) is a chronic, antigen-driven allergic disease that is associated with substantial tissue remodeling (1–5). Esophageal remodeling is the mechanism for EoE complications including fibrosis and smooth muscle dysfunction, which ultimately cause esophageal rigidity, strictures, and dysmotility (1,3,6–10). Tissue remodeling involves collagen accumulation and leads to the entrapment of cells and growth factors in a fibrotic milieu. Angiogenesis and vascular activation provide increased numbers of blood vessels for inflammatory cell trafficking into the esophagus (3,11). Matrix metalloproteinases (MMPs) are a family of proteins that are integral to collagen breakdown, angiogenesis, and tissue remodeling. They, however, have not been previously studied systematically in EoE. MMPs function as gelatinases and collagenases that remodel extracellular matrix and activate other MMPs and growth factors such as transforming growth factor (TGF)-b1 (12,13). TGF-b1 is overexpressed in EoE and has direct effects on esophageal fibroblasts, smooth muscle cells, and epithelium, causing increased fibrotic gene expression, smooth muscle cell contraction, and epithelial mesenchymal transformation (3,6,7,9,14). In particular, MMP-14 and -2 can act in sequence to activate one another and cause subsequent release of TGF-b1 from its latency complex (12). At present, a single preliminary report describes MMP-14 mRNA in EoE (15). In asthma, some of TGF-b1’s profibrotic effects can be mediated through MMP-2 (16). In the present study, we aimed to delineate the expression pattern and potential roles of MMP-2 and -14 in pediatric EoE. We report increased esophageal levels of MMP-2 and -14 in pediatric EoE and demonstrate that proliferating epithelial cells and EoE fibroblasts are cellular sources of both MMP-2 and -14. Cultured esophageal epithelial cells treated with TGF-b1 increased the production and release of MMP-2. As such, MMPs appear to play a previously unreported role in EoE.
METHODS Patients With EoE/Biopsies The University of California, San Diego (UCSD)/Rady Children’s Hospital, San Diego (RCHSD) EoE database was
194 JPGN Volume 61, Number 2, August 2015 Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 61, Number 2, August 2015
screened to randomly select a cohort of patients with EoE who met the following criteria: EoE defined as histologic criteria (15 eosinophils per high-power field [hpf] on hematoxylin and eosin stain at 400 magnification on light microscopy); the presence of typical symptom and endoscopic features; successful treatment (defined as
