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.

Caspase induction by IgA antimitochondrial antibody: IgA-mediated biliary injury in primary biliary cirrhosis

“…The development of recombinant IgA was not beneficial to this work due to the low yield of expression systems. 10 New technology using the Sap peptide for the isolation and purification of functionally intact IgA has enabled new studies concerning the role of this Ig isotype in mucosal disease. 11 For the assay system, we chose the pIgR expressing MDCK cell model because a significant body of data already exists on the use of IgA against HIV and Rota virus in infected cells.…”

“…Media consisted of DMEM supplemented with 10% fetal calf serum (FCS). 9,10 The inserts were composed of thin membranes of polyethylene terephthalate with 1.0 m pores, 1.6 ϫ 10 6 pores/cm 2 . The cell cultures were maintained at 37°C with 5% CO 2 in a humid incubator.…”

“…IgA AMAs are readily detected in the bile, saliva, and even the urine of patients with PBC. 6 -8 Previous work involving confocal microscopy studies of Madine-Darby canine kidney (MDCK) cells expressing pIgR 9,10 has demonstrated that AMA IgA co-localizes, and thus potentially interacts with, PDC-E2 in the cytoplasm of these cells. A limitation of the studies to date has been the absence of functional effects of IgA AMAs on pIgR positive cells, due in large part to the difficulty in obtaining enough polymeric IgA.…”

Experimental evidence on the immunopathogenesis of primary biliary cirrhosis

“…We cannot preclude the possibility that the apical staining obtained with anti-PDC-E2 monoclonal antibodies may be secondary to the presence of immune complexes formed by secreted IgA and AMA antigens, as one line of evidence seems to suggest. 100 Apoptosis of biliary epithelial cells in PBC warrants further discussion and may prove crucial for immune tolerance breakdown, 63,101 as illustrated under other conditions. Quite surprisingly, Odin et al first demonstrated that PDC-E2 remains intact and retains its immunogenicity during cholangiocyte apoptosis because of a cell-specific lack of glutathionylation of biliary epithelial cells.…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…Several immunoglobulin isotypes from many different species, including murine IgG, 2,3,[21][22][23][24][25] chimeric IgG, [26][27][28][29][30] human IgG, [31][32][33][34][35][36][37][38][39] IgA, [40][41][42] IgM 41 and IgE, [43][44][45] as well as pig Igs 30,45 have been successfully expressed in insect cells. These molecules are all correctly processed (signal peptide cleaved), glycosylated and assembled (H2L2) with a production rate varying from 0.75 to 100 mg/l, depending on the antibody ( …”