Contribution of hepatic parenchymal and nonparenchymal cells to hepatic fibrogenesis in biliary atresia

Abstract

Extrahepatic biliary atresia is a severe neonatal liver disease resulting from a sclerosing cholangiopathy of unknown etiology. Although biliary obstruction may be surgically corrected by a "Kasai" hepatoportoenterostomy, most patients still develop progressive hepatic fibrosis, although the source of increased collagen deposition is unclear. This study examined the role of hepatic stellate cells (HSCs) and assessed the source of transforming growth factor-beta (TGF-beta) production in hepatic fibrogenesis in patients with biliary atresia. Liver biopsies from 18 biliary atresia patients (including 5 pre- and post-Kasai) were subjected to immunohistochemistry for alpha-smooth muscle actin and in situ hybridization for either procollagen alpha1 (I) mRNA or TGF-beta1 mRNA. Sections were also subjected to immunohistochemistry for active TGF-beta1 protein. The role of Kupffer cells in TGF-beta1 production was assessed by immunohistochemistry for CD68. Procollagen alpha1 (I) mRNA was colocalized to alpha-smooth muscle actin-positive HSCs within the region of increased collagen protein deposition in fibrotic septa and surrounding hyperplastic bile ducts. The number of activated HSCs was decreased in only one post-Kasai biopsy. TGF-beta1 mRNA expression was demonstrated in bile duct epithelial cells and activated HSCs and in hepatocytes in close proximity to fibrotic septa. Active TGF-beta1 protein was demonstrated in bile duct epithelial cells and activated HSCs. This study provides evidence that activated HSCs are responsible for increased collagen production in patients with biliary atresia and therefore play a definitive role in the fibrogenic process. We have also shown that bile duct epithelial cells, HSCs, and hepatocytes are all involved in the production of the profibrogenic cytokine, TGF-beta1.

Figure 1.

Identification of activated HSCs as cellular source of increased collagen production in biliary atresia. A: Colocalization of SMA (brown) and procollagen α₁ (I) mRNA (blue) within stellate-shaped activated HSCs in a liver biopsy from an infant with biliary atresia, using immunohistochemistry and in situ hybridization, respectively. Original magnification, ×400. B: Bile duct hyperplasia within fibrotic bands in a liver biopsy from an infant with biliary atresia. Intense staining for SMA (brown) and procollagen α₁ (I) mRNA (blue), colocalized in activated HSCs. Original magnification, ×400. C: Immunohistochemistry for SMA (brown) within activated HSCs surrounding hyperplastic bile ducts in a pre-Kasai HPE. D: A post-Kasai HPE liver biopsy from the same infant shown in C with biliary atresia. Original magnification, ×100.

Figure 2.

Colocalization of activated HSCs to collagen protein deposition in biliary atresia liver. A: Collagen protein deposition surrounding two bile ducts in liver biopsy from an infant with biliary atresia (pink). B: Activated HSCs surrounding bile ducts, showing colocalization of SMA (brown) and procollagen α₁ (I) mRNA (blue). Original magnification, ×1000. C and D: Fibrotic region in liver biopsy from an infant with biliary atresia showing deposition of collagen protein fibrils (C, pink) and activated HSCs (D), as evidenced by colocalization of SMA (brown) and procollagen α₁ (I) mRNA (blue). Original magnification, ×200.

Figure 3.

Identification of cellular source of TGF-β. A: Immunohistochemistry for TGF-β protein (brown) predominantly expressed within bile duct epithelial cells and also in activated HSCs in fibrotic extracellular matrix (arrows) and, to a lesser extent, in hepatocytes at the interface of fibrotic scar tissue (B, arrowheads). Original magnification, ×400. C: In situ hybridization for TGF-β₁ mRNA (blue) expressed in bile duct epithelial cells and colocalized to SMA (brown) within activated HSCs (arrows). D: TGF-β₁ mRNA (blue) was also demonstrated in hepatocytes at the interface of the regenerative nodule and fibrous scar tissue. Original magnification, ×1000. E: In situ hybridization for TGF-β₁ mRNA (blue) expressed in bile duct epithelial cells and HSCs (arrows) within scar tissue. Immunohistochemistry for CD68, used as a marker of Kupffer cells (brown), demonstrated that Kupffer cells were present in the sinusoidal and perisinusoidal regions of the regenerative hepatocyte nodules, but they did not express TGF-β₁ mRNA. In addition, CD68-positive macrophages within scar tissue did not contribute to TGF-β₁ mRNA expression. Original magnification, ×400. F: TGF-β₁ mRNA signal specificity for the antisense probe in E was demonstrated by the absence of signal over bile duct epithelial cells and HSCs using the sense probe. This serial section of E was also subjected to immunohistochemistry for CD68.