Biliary atresia is associated with CD4+ Th1 cell-mediated portal tract inflammation

Abstract

A proposed mechanism in the pathogenesis of biliary atresia involves an initial virus-induced, progressive T cell-mediated inflammatory obliteration of bile ducts. The aim of this study was to characterize the inflammatory environment present within the liver of infants with biliary atresia to gain insight into the role of a primary immune-mediated process versus a nonspecific secondary response to biliary obstruction. Frozen liver tissue obtained from patients with biliary atresia, neonatal giant cell hepatitis, total parenteral nutrition (TPN)-related cholestasis, choledochal cysts, and normal control subjects was used for fluorescent immunohistochemistry studies of cellular infiltrates, cytokine mRNA expression, and in situ hybridization for localization of cytokine-producing cells. Immunohistochemistry revealed increases in CD8(+) and CD4(+) T cells and Kupffer cells (CD68(+)) in the portal tracts of biliary atresia. Reverse transcription-PCR analysis of biliary atresia tissue showed a Th1-type cytokine profile with expression of IL-2, interferon-gamma, tumor necrosis factor-alpha, and IL-12. This profile was not seen in normal, neonatal hepatitis or choledochal cyst livers but was present in TPN-related cholestasis. In situ hybridization revealed that the Th1 cytokine-producing cells were located in the portal tracts in biliary atresia and in the parenchyma of TPN-related cholestasis. A distinctive portal tract inflammatory environment is present in biliary atresia, involving CD4(+) Th1 cell-mediated immunity. The absence of similar inflammation in other pediatric cholestatic conditions suggests that the portal tract inflammation in biliary atresia is not a secondary response to cholestasis but rather indicates a specific immune response involved in the pathogenesis of biliary atresia.

Figure 1

Fluorescent immunohistochemistry reveals an increase in CD8⁺ T cells, CD3⁺/CD4⁺ T cells, and Kupffer cells in the portal tracts in biliary atresia. Frozen sections from normal liver (n = 4), other neonatal cholestatic liver diseases (TPN, n = 4; CDC, n = 1; NH, n = 3), and biliary atresia (n = 8) were incubated with MAb, amplified with fluorescein-labeled substrate (green), and counterstained with nuclear stain DAPI (blue; ×200 magnification). Portal tracts were identified by characteristic vessel formation and bile duct epithelial nuclear staining and verified by serial sections with cytokeratin 7 staining, which is specific for bile duct epithelium. In normal livers, the portal tracts are seen in the center of each photograph. In other cholestatic diseases and biliary atresia, the portal tracts are expanded and encompass the entire field at ×200 magnification. (A) Portal tract regions were verified by bile duct epithelium surface expression of cytokeratin 7. (B) CD8⁺ T cells are more abundant in biliary atresia portal tracts. (C) CD4⁺ cell surface expression is found not only on T cells but also on sinusoidal cells and Kupffer cells. For delineating the CD4⁺ T cells, the tissue was double stained with anti-CD3 ⁺ antibody (T cell–specific marker; red) and CD4⁺ antibody (green). Double-positive cells (CD3⁺CD4⁺) are yellow. There is an increase in the number of CD3⁺CD4⁺ cells in biliary atresia portal tracts. The single-stained red cells (CD3⁺CD4⁻) in biliary atresia reflect the CD8⁺ T cells. Also note the single staining CD4⁺ sinusoidal cells in the parenchyma surrounding the portal tract in normal liver controls. (D) CD68⁺ cells (macrophage/Kupffer cells) are increased in the portal tracts of biliary atresia. Note the CD68⁺ staining of cells in the parenchyma of controls.

Figure 2

Quantification of cell number in portal tracts reveals significant increases in CD8⁺, CD3⁺CD4⁺, and CD68⁺ cells in biliary atresia. Frozen sections from normal liver (n = 4), other cholestatic liver diseases (n = 8), and biliary atresia (n = 8) were analyzed for total number of cells per portal tract staining positive for CD8⁺ (A), CD3⁺CD4⁺ (B), CD68⁺ (C), CD20⁺ (B cell; D), and NK1 (E). Every portal tract from each frozen section was analyzed. The number of portal tracts per section ranged from two to seven. Mean cell counts per portal tract from each patient group are shown with SDs. Statistically significant (p < 0.01) increases in CD8⁺, CD3⁺CD4⁺, and CD68⁺ cells were found in the portal tracts of biliary atresia patients compared with both normal liver controls and other neonatal cholestatic diseases.

Figure 3

Biliary atresia is associated with a Th1 inflammatory cytokine profile. RNA from liver of biliary atresia patients (BA, n = 10), normal controls (N; n = 4), CDC patients (n = 4), NH (n = 3), and TPN-related cholestasis patients (n = 4) were analyzed for the presence of cytokine-specific mRNA transcripts by RT-PCR. Th1 cytokine (IL-2, IFN-γ, TNF-α, and IL-12) and Th2 cytokine (IL-4 and IL-5) PCR products are shown for each patient. Biliary atresia and TPN-related cholestasis were associated with a Th1 inflammatory cytokine profile that was not present in normal, NH, or CDC livers.

Figure 4

Fluorescent immunohistochemistry reveals an increase in CD8⁺ and CD68⁺ cells in the parenchyma of TPN-related cholestasis. Frozen sections from normal livers, TPN-related cholestasis, and biliary atresia were analyzed for cell surface expression of CD4, CD8, CD20, CD68, and NK1 within the parenchyma of the liver. Increased expression of CD8⁺ and CD68⁺ cells was observed in the parenchyma (hepatic lobule) of patients with TPN-related cholestasis compared with both normal and biliary atresia livers (×100 magnification). There was no observable difference among the groups in the expression of CD4⁺, CD20⁺, or NK1 cells in the parenchyma (data not shown).

Figure 5

In situ hybridization reveals that cells in the portal tracts of biliary atresia produce Th1 cytokines. Frozen sections of biliary atresia and TPN-related cholestatic liver tissue were hybridized with cytokine-specific, digoxin-labeled antisense probes followed by anti-digoxin/HRP conjugation and cyanine 3 (red) labeled substrate. Sections were counterstained with the nuclear stain DAPI (blue) and viewed by fluorescent microscopy (×200 magnification). Expression for IL-2, IFN-γ, and TNF-α mRNA was present in the portal tracts of biliary atresia liver tissue but was absent in the portal tracts of TPN-related cholestatic liver tissue.