Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC

Hepatology. 2002 Dec;36(6):1383-92. doi: 10.1053/jhep.2002.37140.

Abstract

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pyruvate dehydrogenase complex (PDC-E2)-specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti-PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Calpha constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further support of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity
  • Autoantibodies / immunology
  • Baculoviridae / genetics
  • Cell Line
  • Cloning, Molecular
  • Dihydrolipoyllysine-Residue Acetyltransferase
  • Epithelial Cells / cytology
  • Epithelial Cells / immunology
  • Humans
  • Immunoglobulin A / genetics
  • Immunoglobulin A / immunology*
  • Immunohistochemistry
  • Liver Cirrhosis, Biliary / immunology*
  • Pyruvate Dehydrogenase Complex / immunology*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology

Substances

  • Antibodies, Monoclonal
  • Autoantibodies
  • Immunoglobulin A
  • Pyruvate Dehydrogenase Complex
  • Recombinant Proteins
  • Dihydrolipoyllysine-Residue Acetyltransferase