RESUMO
Elevated cytokines, especially TNF-alpha, have been implicated in the pathogenesis of necrotising enterocolitis (NEC). We have previously shown that TNF-alpha drives the production of matrix degrading enzymes, the matrix metalloproteinases (MMPs), in the gut wall. In this study we have therefore investigated the role of MMPs in the pathogenesis of NEC in neonates. Nine newborn infant nonnecrotic resected bowels with confirmed NEC were studied and 8 newborn infants with neonatal bowel obstructions were used as controls. Immunostaining was used to identify the numbers of monocytes, macrophages, neutrophils, and T cells in the tissue. We used quantitative, competitive RT-PCR to analyze the number of TNF-alpha, IFN-gamma, MMP, and TIMP mRNA transcripts and western blotting to analyze MMP and TIMP protein production. Double labeling (immunostaining and in situ hybridization) was used to identify the phenotype of MMP mRNA expressing cells. We found increased numbers of monocytes, macrophages, and neutrophils in NEC tissue compared with controls. The number of T cells was unexpectedly low in NEC as was the number of IFN-gamma transcripts in comparison with the control samples. Increased numbers of transcripts for TNF-alpha were detected in NEC tissue, as was mRNA expression and protein production for stromelysin-1 and TIMP-1 but not collagenase, gelatinases, or TIMP-2. The cellular source of stromelysin-1 in NEC was alpha-smooth muscle actin positive cells. These results suggest that stromelysin-1, which has the ability to degrade the mucosal extra-cellular matrix, may be responsible for the extensive tissue injury in infants with NEC.
Assuntos
Enterocolite Necrosante/genética , Mucosa Intestinal/enzimologia , Metaloproteinase 3 da Matriz/genética , Enterocolite Necrosante/imunologia , Enterocolite Necrosante/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Recém-Nascido , Interferon gama/genética , Mucosa Intestinal/imunologia , Metaloproteinase 2 da Matriz/genética , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/imunologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Pancreatic stellate cells mediate fibrosis in chronic pancreatitis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 are crucial modulators of fibrosis. Transforming growth factor-beta (TGF-beta) is a key regulator of extracellular matrix production and myofibroblast proliferation. We have examined MMP and TIMP synthesis by transformed cultured pancreatic stellate cells and their regulation by TGF-beta 1. By Northern analysis they expressed mRNAs for procollagen 1, TIMP-1, TIMP-2, and MMP-2. Expression of membrane type-1 MMP was confirmed by Western blotting. By immunohistochemistry these enzymes localized to fibrotic areas in human chronic pancreatitis. Active TGF-beta 1 constitutes 2 to 5% of total TGF-beta 1 secreted by pancreatic stellate cells; they express TGF-beta receptors I and II. Exogenous TGF-beta 1 (10 ng/ml) significantly increased procollagen-1 mRNA by 69% and collagen protein synthesis by 34%. Similarly TGF-beta 1 at 0.1, 1, and 10 ng/ml significantly reduced cellular proliferation rate by 37%, 44%, and 44%, respectively, whereas pan-TGF-beta-neutralizing antibody increased proliferation by 40%. TGF-beta1 (10 ng/ml) down-regulated MMP-9 by 54% and MMP-3 by 34% whereas TGF-beta 1-neutralizing antibody increased MMP-9 expression by 39%. Pancreatic stellate cells express both mediators of matrix remodeling and the regulatory cytokine TGF-beta 1 that, by autocrine inhibition of MMP-3 and MMP-9, may enhance fibrogenesis by reducing collagen degradation.