Does a betaretrovirus infection trigger primary biliary cirrhosis?

Abstract

Patients with primary biliary cirrhosis develop progressive ductopenia associated with the production of antimitochondrial antibodies that react with a protein aberrantly expressed on biliary epithelial cells and peri-hepatic lymph nodes. Although no specific microbe has been identified, it is thought that an infectious agent triggers this autoimmune liver disease in genetically predisposed individuals. Previous serologic studies have provided evidence to suggest a viral association with primary biliary cirrhosis. Here we describe the identification of viral particles in biliary epithelium by electron microscopy and the cloning of exogenous retroviral nucleotide sequences from patients with primary biliary cirrhosis. The putative agent is referred to as the human betaretrovirus because it shares close homology with the murine mammary tumor virus and a human retrovirus cloned from breast cancer tissue. In vivo, we have found that the majority of patients with primary biliary cirrhosis have both RT-PCR and immunohistochemistry evidence of human betaretrovirus infection in lymph nodes. Moreover, the viral proteins colocalize to cells demonstrating aberrant autoantigen expression. In vitro, we have found that lymph node homogenates from patients with primary biliary cirrhosis can induce autoantigen expression in normal biliary epithelial cells in coculture. Normal biliary epithelial cells also develop the phenotypic manifestation of primary biliary cirrhosis when cocultivated in serial passage with supernatants containing the human betaretrovirus or the murine mammary tumor virus, providing a model to test Koch's postulates in vitro.

Fig. 1.

Electron microscopy studies reveal virus-like particles in vivo and in vitro in samples from patients with PBC. (a and b) Freshly isolated biliary epithelial cells from a PBC patient showing virus-like particles in the extracellular space with a distinct envelope and electrondense core (Inset shows particle at ×5 magnification; white bar represents 100 nm). (c and d) Similar particles ranging in size from 110 to 120 nm and showing features consistent with B-type particles (25) were observed in negatively stained pellets from PBC-conditioned media in BEC supernatants. The negative stain has penetrated the structure in d to reveal the nucleoprotein core.

Fig. 2.

Immunohistochemistry studies show viral capsid protein in PBC patient's peri-portal lymph nodes in the same distribution as aberrant autoantigen expression. (a) A PBC lymph node displays a peri-sinusoidal distribution of anti-p27^CA reactivity where macrophage/monocyte cells are located. (b) Anti-p27^CA reactivity is absent from a control lymph node from a patient with primary sclerosing cholangitis (PSC). (c and d) Aberrant expression of PDC-E2 in the PBC lymph node but not in the PSC lymph node control. Plasma membrane localization of mitochondrial antigen can be seen by fast red stain of AMA in c, but only mitochondrial staining can be seen in d. Cellular colocalization of FITC-stained (green) anti-p27^CA in the cytoplasm is observed with aberrant AMA staining in c. (Original magnification: ×100 for a and b and ×400 for c and d.)

Fig. 3.

Normal BEC develop the phenotypic manifestation of PBC when incubated with PBC patient's lymph node homogenates. (a–c) Before coculture, studies in normal BEC show no AMA staining by immunofluorescence (a), no cell surface expression of PDC-E2 by immunoelectron microscopy (b), and no evidence of viral proteins by immunofluorescence (c). (d–g) After coculture with PBC patient's lymph node homogenates, the BEC develop aberrant PDC-E2 expression after 7 days in culture (d), with cell surface AMA reactivity on BEC (e) that is similar to that seen in PBC BEC (g), and evidence of cytoplasmic localization of betaretrovirus capsid protein (f). (h) Normal BEC incubated with supernatant from MMTV-producing MM5MT cells also show a similar punctate, cytoplasmic signal from the anti-p27^CA immunofluorescence.