title: Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC.
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.
authors.affiliation: Division of Rheumatology/Allergy and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Fukushima
authors.forename: Greg
authors.initials: G
authors.lastname: Nalbandian
authors.forename: Judy
authors.initials: J
authors.lastname: Van De Water
authors.forename: Kandra
authors.initials: K
authors.lastname: White
authors.forename: Aftab A
authors.initials: AA
authors.lastname: Ansari
authors.forename: Patrick
authors.initials: P
authors.lastname: Leung
authors.forename: Thomas
authors.initials: T
authors.lastname: Kenny
authors.forename: Shizuo G
authors.initials: SG
authors.lastname: Kamita
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
authors.forename: Ross L
authors.initials: RL
authors.lastname: Coppel
authors.forename: Freida
authors.initials: F
authors.lastname: Stevenson
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ishibashi
authors.forename: M Eric
authors.initials: ME
authors.lastname: Gershwin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004847:Epithelial Cells / Q000166:cytology / Q000276:immunology; D006801:Humans; D007070:Immunoglobulin A / Q000235:genetics / Q000276:immunology*; D007150:Immunohistochemistry; D008105:Liver Cirrhosis, Biliary / Q000276:immunology*; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007070:Immunoglobulin A; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D051047:Dihydrolipoyllysine-Residue Acetyltransferase