A novel monospecific IgG2/lambda-autoantibody with specificity for a mitochondrial antigen: evidence for an antigen-driven pathogenetic B-cell response in rheumatoid synovial tissue, induced by tissue alteration.
To elucidate the pathogenic role of synovial B cells in rheumatoid arthritis (RA), nine human IgG/lambda-secreting B-cell hybridomas from rheumatoid synovial tissue of a patient with definite RA were screened by enzyme-linked immunosorbent assay and indirect immunofluorescence on tissue cryosections for detection of antibodies against autoantigens. One IgG2/lambda monoclonal antibody (mAb) from the B-cell hybridoma ELG211/15/63 (= hybr63) exhibited intense immunofluorescence reactivity in the cytoplasm of chondrocytes and epithelial cells of the gastrointestinal tract, especially in parietal cells of gastric mucosa (human and mouse tissue), representing a mitochondrial pattern. This result was confirmed by morphometric analysis of immunoelectron microscopy data, exhibiting a significantly higher labeling density in mitochondria (p < or = 0.001) than in the cytoplasmic background, with predominant staining in the inner mitochondrial membrane and mitochondrial matrix (p < or = 0.05). Immunoblotting experiments carried out with gastric mucosa, and a mitochondrial protein preparation revealed two major proteins of 38 and 50 kd under reducing conditions. The analysis of the IgV(H) genes from this B-cell hybridoma showed highest homology to the human germline gene DP53 (96%). The IgV(L) region gave highest homology to the human germline gene DP5 (93%). In the complementarity-determining regions, residues of the H- and L-chain variable regions replacement mutations only indicated that this B-cell clone had been antigen-selected for its affinity (ratio of replacement to silent mutations: > or = 7). To analyze the in vivo expansion of the B-cell clone, primers specific for the V(H) to D to J(H) rearrangement of this B-cell hybridoma were used. Specific amplifications could be detected within part of the synovial tissue but not within the cells of the synovial fluid and peripheral blood of the patient. The ability of the IgG2/lambda mAb to induce an inflammatory reaction was tested by intraperitoneal application in severe combined immunodeficiency (SCID) mice, which resulted in an inflammatory, predominantly granulocytic infiltration of the peritoneum. Consequently, intrasynovial cell death or cartilage destruction seems to be a possible source of liberation of mitochondrial antigens, inducing a local, antigen-driven IgG2/lambda B-cell response with the ability to induce an inflammatory reaction. These data suggest that tissue destruction may serve as a source of arthritogenic antigens that perpetuate and amplify the local pernicious inflammatory process in RA synovialitis.
Krenn
V
V
Institute of Pathology, University of Würzburg, Germany.
Molitoris
R
R
Berek
C
C
Sack
U
U
König
A
A
Müller-Deubert
S
S
Kempf
V
V
Mosgoeller
W
W
Kerkau
T
T
Vollmers
H P
HP
Müller-Hermelink
H K
HK
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D017209:Apoptosis; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D005260:Female; D005803:Genes, Immunoglobulin; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D007150:Immunohistochemistry; D051379:Mice; D016513:Mice, SCID; D016253:Microscopy, Immunoelectron; D008875:Middle Aged; D008928:Mitochondria / Q000276:immunology*; D008969:Molecular Sequence Data; D013583:Synovial Membrane / Q000276:immunology*
D001323:Autoantibodies; D007074:Immunoglobulin G