The results of this study demonstrate that the portal tract cellular infiltrate present in biliary atresia at the time of diagnosis represents a CD4+ Th1 cell–mediated inflammatory process. Within biliary atresia livers, the portal tracts are infiltrated with a characteristic inflammatory cell population, consisting of Kupffer cells, CD4+ T cells, and CD8+ T cells with local production of IL-2, IFN-γ, and TNF-α. This process is distinctive to biliary atresia and was not found in liver from normal control subjects or CDC patients (representing another cause of extrahepatic bile duct obstruction) or from infants with NH or TPN-related cholestasis (representing similar portal tract cholestatic histology in infancy). The absence of this inflammatory process in other neonatal cholestatic conditions suggests that the portal tract inflammation in biliary atresia is not simply an immune response to the presence of cholestasis but rather is an important pathophysiologic event involved in the pathogenesis of biliary atresia.
Our hypothesis that Th1 cell–mediated inflammation is central to the pathogenesis of biliary atresia is supported by recent studies. Urushihara et al.
) observed increased numbers and size of Kupffer cells in the liver associated with elevated serum IL-18 at the time of diagnosis of biliary atresia. IL-18 (IFN-γ
–inducing factor) is a macrophage-derived cytokine that works in concert with IL-12 to promote Th1 cell differentiation in the inflammatory setting. Bezerra et al.
) used microarray analysis to show that liver of infants with biliary atresia expressed several genes involved in lymphocyte differentiation, with an overexpression of osteopontin, another cytokine in the Th1 pathway. They further determined by RT-PCR that 65% of biliary atresia livers expressed IFN-γ
compared with none of controls with idiopathic neonatal intrahepatic cholestasis. However, tissue localization of cells expressing IFN-γ
was not possible with the techniques used, no other cytokines were investigated, and the only controls were infants with idiopathic neonatal hepatitis, unlike the CDC control subjects in the present study.
The predominance of CD8+
T cells in the portal tracts of biliary atresia livers supports a role for cytotoxic CD8+
T cells in mediating the bile duct epithelial damage. In this regard, Ohi et al.
) examined CD8+
T cells in biliary atresia livers and found that CD8+
T cells infiltrated the proliferating portal tract bile ducts in 95% of the biliary atresia cases. However, they concluded that the CD8+
T cells were not actively cytotoxic because immunohistochemistry staining for granzyme, perforin, and FasL were negative. However, CD8+
cytotoxic T cells also produce cytokines and chemokines with effector functions that do not directly involve target cell death. For example, CD8+
T cells produce IFN-γ
and the chemokines MIP-1α
, and RANTES. Taken together, these molecules mediate the promotion of inflammatory responses, activation of macrophages, and direct antiviral effects to infected cells (27
). The role of the CD8+
T cells in the ongoing destruction of bile duct epithelium in biliary atresia warrants further investigation.
Our findings support the role of immune-mediated destruction of bile ducts within the liver of patients with the perinatal/acquired form of biliary atresia. This study adds to the observations of Ohya et al.
), who described an association in biliary atresia between degeneration of intrahepatic bile ducts and the lymphocytic infiltration of biliary epithelial cells. In that study, 31 patients with biliary atresia had these findings compared with only 2 of 9 patients with CDC and none of 5 patients with idiopathic neonatal hepatitis. From our study, it can be inferred further that this lymphocytic infiltrate is primarily composed of CD4+
T cells. Further investigation will be needed to determine whether the lymphocytes are clonal and reactive to a specific antigen (e.g.
viral or self-bile duct epithelial antigen) and the nature of the antigenic epitopes.
The findings in this study may shed light on the potential therapeutic use of immunosuppressive therapy in the treatment of biliary atresia. Recent noncontrolled studies using corticosteroid therapy after the Kasai procedure suggested an improved overall survival rate compared with historical controls (29
). First, corticosteroids regulate the expression of many genes with a net anti-inflammatory effect, including a reduction in inflammatory mediator secretion, such as TNF-α
and nitric oxide. Second, corticosteroids inhibit inflammatory cell migration to sites of inflammation by inhibiting the expression of adhesion molecules (32
). The ultimate result of these anti-inflammatory effects would be to inhibit further bile duct injury and to slow the development of biliary cirrhosis. Carefully conducted controlled studies are needed to determine the effects of corticosteroid therapy on immune injury and outcome in biliary atresia.
In conclusion, this study offers strong evidence for a Th1-mediated immune response focused within the portal tracts in biliary atresia that is not present in other, similar neonatal cholestatic disorders. This unique immune pattern suggests that the observed immune repertoire is not merely a stereotypic response to the accumulation of biliary constituents in the neonatal cholestatic liver but rather an indicator of specific immune processes involved in the pathogenesis of biliary atresia. Further elucidation of the nature and the source of antigenic stimulation of the Th1 response in the biliary tree of these patients will be essential for advancing our understanding of this disorder and developing new strategies toward effective treatment.