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.

Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis

“…Future studies should focus on the antigen specificity of the IgM and IgG produced in these coronal arrangement-comprising plasma cells. Although IgA transcytosis has been considered one of the contributing factors toward bile duct lesions in PBC, 33,34 it is unlikely that the coronal arrangement we observed in this study includes IgA þ plasma cells. However, we observed that IgA staining was often seen in the degenerated cholangioepithelium of CNSDC or at the apical margin of damaged bile duct cells (data not shown).…”

Low specificity of anti‐tissue transglutaminase antibodies in patients with primary biliary cirrhosis

“…Furthermore, although in PBC subjects the serum AMA is mostly of the IgM and IgG type (39), the biliary AMA is mostly of the IgA isotype (40,41) due to the presence in the ductal epithelium of IgA specific surface receptors that perform the IgA transcytosis (42). These data led to the hypothesis that PBC might develop after a locally AMA IgA driven response in the mucosa (43). We have found that AMA of the IgA isotype are present in the serum of 24% of PBC patients, and that there is a considerable correlation (82%) between the results of the AMA IgA and the anti-tTG IgA tests.…”

The causes of primary biliary cirrhosis: Convenient and inconvenient truths

“…The fact that such apical staining is seen only with selected and not all mAbs that react with PDC‐E2 and that distinct epitopes can be identified with these apically staining mAbs led to the initial hypothesis that a target molecule cross‐reactive with PDC‐E2 could be located at the apical surface of BECs in PBC 57–59. However, subsequent research demonstrated that the apical staining was due to a new complex between AMA of immunoglobulin A (IgA) isotype and PDC‐E2, and this raised the potential role of IgA as a participant in the immune‐mediated destruction of BECs 60, 61…”