ABSTRACT
Primary biliary cirrhosis (PBC) is a liver disease characterized by serum autoantibodies against the pyruvate dehydrogenase complex (PDC) located in the inner mitochondrial membrane. The predominant target in PDC has previously been localized to the inner lipoyl domain (ILD) of the E2 subunit. The etiology of PBC is unknown, although molecular mimicry with bacterial PDC has been proposed. Here, we have investigated the etiology of PBC and nature of the autoimmune response by analyzing the structure of a human monoclonal antibody with ILD specificity. Mutants of the monoclonal antibody, which was originally isolated from a patient with PBC, were expressed as Fab by phage display, and tested for reactivity against recombinant domains of the E2 subunit. Fab in which the V(H)-encoded portions were reverted to germline lost reactivity against the ILD alone, but recognized a different epitope in a didomain construct encompassing the ILD, hinge region and E1/E3 binding domain. The complete V(H) and V(L )germline revertant was unreactive with the human ILD and didomain, the Escherichia coli didomain, and whole PDC. We hypothesize that the IgM on the surface of the naïve B-cell first recognizes an as yet unidentified antigen, and that accumulation of somatic mutations results in an intermolecular epitope shift directed towards an epitope involving the E1/E3 binding domain. Further mutations result in the specificity being redirected to the ILD. These findings also suggest that bacterial molecular mimicry is not involved in initiating disease.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Epitopes/genetics , Gene Rearrangement, B-Lymphocyte/genetics , Liver Cirrhosis, Biliary/genetics , Liver Cirrhosis, Biliary/immunology , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , B-Lymphocytes/cytology , Cell Differentiation/genetics , Complementarity Determining Regions/genetics , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Gene Rearrangement, B-Lymphocyte, Heavy Chain/genetics , Genes, Immunoglobulin/genetics , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Lymphocyte Activation/genetics , Molecular Sequence Data , Mutation/genetics , Protein Structure, Tertiary , Protein Subunits , Pyruvate Dehydrogenase Complex/chemistry , Pyruvate Dehydrogenase Complex/immunology , Sequence AlignmentABSTRACT
Acute liver failure arises from an imbalance between liver cell death and regeneration. The severity of the insult determines whether liver cells die by apoptosis or necrosis, and this in turn affects the magnitude of the inflammatory reaction. Liver cell death occurs through complex cellular interactions and is mediated by immunological, inflammatory and chemical components. Regeneration of the liver cell mass also depends on cellular interactions, often involving those same mediators implicated in the injury itself. A greater understanding of these processes will lead ultimately to targeted rational therapy in acute liver failure.
Subject(s)
Liver Failure, Acute/physiopathology , Liver/cytology , Cell Death/immunology , Humans , Liver/immunology , Liver/pathology , Liver Failure, Acute/immunology , T-Lymphocytes, Cytotoxic/immunology , fas ReceptorABSTRACT
BACKGROUND/AIMS: Autoantibodies with specificity for the E2 component of the pyruvate dehydrogenase complex (PDC-E2) are commonly present in primary biliary cirrhosis. The aim of this study was to generate and characterise human anti-PDC-E2 monoclonal antibodies and analyse immunoglobulin gene usage and mutation for clues to pathogenesis. METHODS: Peripheral B-lymphocytes from two patients with primary biliary cirrhosis were used to generate heterohybridomas secreting PDC-E2 specific monoclonal antibodies. The antibodies were characterised by ELISA, immunoblotting, indirect immunofluorescence and enzyme inhibition techniques, and their encoding immunoglobulin genes were amplified, cloned and sequenced. RESULTS: Four IgGlambda and one IgMlambda monoclonal antibodies specific for PDC-E2 were generated: all gave bands at 74 kD and 52 kD on PDC immunoblots, two clones were specific for the lipoylated inner lipoyl domain, and all inhibited target enzyme function. Sequence analysis suggested unrestricted VH gene usage, but a strong preference for lambda light chains. The extent of somatic mutation was high (3-20%), with evidence for antigen selection in 3/5 VH sequences. CONCLUSIONS: These monoclonal antibodies closely resemble the hallmark autoantibodies of primary biliary cirrhosis. Their specificities demonstrate true cross reactivity between an epitope on PDC-E2 and Protein X, and the existence of a subset of B cells that recognise only the lipoylated form of the antigen. The pattern of immunoglobulin gene mutations suggests an antigen-driven selection of high affinity IgG autoantibodies, supporting a possible role for exogenous antigen in the pathogenesis of primary biliary cirrhosis.
