Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis

Abstract

Aim: To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis (PBC).

Methods: Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B (HBV) virus, 3 patients with hepatitis C virus (HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis (AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targeted by autoantibodies present in the patients sera was performed by indirect immunofluorescence (IIF) analysis using paraffin-embedded liver sections as a substrate. Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy.

Results: In total, 18/21 (85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21 (47.6%) in lymphocytes, 8/21 (38%) in cholangiocytes and 7/21 (33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G (IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining (20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody, positive staining was observed in 75% of lymphocytes, 62.5% of cholangiocytes, 37.5% of hepatocytes and 50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections, weak positive staining of cholangiocytes was observed in 3/21 (14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients.

Conclusion: In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

Keywords: Autoantibodies; Cholangiocytes; Liver tissue; Primary biliary cirrhosis; Sinusoidal cells.

Figure 1

Immunofluorescence of primary biliary cirrhosis sections incubated with primary biliary cirrhosis serum and fluorescein isothiocyanate -conjugated IgG or IgM secondary antibody. A-C: Negative control incubated with IgG secondary antibody; D-F: Primary biliary cirrhosis (PBC) section incubated with homologous PBC serum and IgG secondary antibody, showing staining of cholangiocytes, sinusoidal cells (weak staining) and mononuclear cells, probably lymphocytes; G-I: PBC section incubated with heterologous PBC serum and IgG secondary antibody, showing nuclear and cytoplasmic staining of hepatocytes and lymphocytes; J-L: PBC section incubated with homologous PBC serum and IgM secondary antibody, showing cytoplasmic staining of sinusoidal cells and lymphocytes. Magnification × 200. FITC: Fluorescein isothiocyanate.

Figure 2

Immunofluorescence of primary biliary cirrhosis, acute alcoholic hepatitis (AAH) or normal sections incubated with primary biliary cirrhosis, disease control or normal serum and fluorescein isothiocyanate -conjugated IgG secondary antibody. A-C: Primary biliary cirrhosis (PBC) section incubated with normal serum; D-F: Acute alcoholic hepatitis section incubated with normal serum; G-I: AAH section incubated with PBC serum; J-L: Normal section incubated with PBC serum, showing weak positive staining of cholangiocytes; M-O: PBC section incubated with steatohepatitis serum, showing positive staining of hepatocytes; P-R: Hepatitis B virus section incubated with steatohepatitis serum. Magnification × 200. FITC: Fluorescein isothiocyanate.

Figure 3

Immunofluorescence of sections from the primary biliary cirrhosis and disease control groups incubated with hepatitis B virus serum and fluorescein isothiocyanate conjugated IgG secondary antibody. A-C: Primary biliary cirrhosis (PBC) section with positive staining of intra-sinusoidal and portal lymphocytes; D-F: Hepatitis C virus (HBV) section with positive staining of cholangiocytes; G-I: AAH section with positive staining of hepatocytes; J- L: Hepatitis C virus section. Magnification × 200. FITC: Fluorescein isothiocyanate.