NZB/NZW F1-Derived Monoclonal Anti-Dna Autoantibodies: Dual-Specificity and Immune Complex Formation

172 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.The glomerulonephritis associated with the autoimmune disease Systemic Lupus Erythematosus (SLE) is mediated by immune complexes composed of DNA, complement components and anti-DNA autoantibodies, with the primary pathogenic antibodies being dsDNA specific. Recent studies had identified a subpopulation of anti-DNA autoantibodies that possessed dual-specificity and appeared to form very avid immune complexes. The dual-specific autoantibodies were derived from the NZB/NZW F1 murine model of lupus. Further characterization of four anti-DNA (two anti-ssDNA and two anti-dsDNA) monoclonal autoantibodies revealed that only the two anti-dsDNA autoantibodies, BV 17-45 and BV 16-13, exhibited dual-specificity (anti-dsDNA and anti-IgG) under native conditions. Temperature-dependent denaturation studies and SDS denaturation/CD analysis identified the IgG epitope recognized by both BV 17-45 and BV 16-13 as non-linear. Enzymatic digests and native Western blot analysis localized the anti-IgG epitope to the hinge region of IgG2a. By utilizing synthetic hinge peptides and a 12 amino acid phage display library, the location of the anti-hinge autoepitope was further defined to the upper hinge region of IgG2a murine immunoglobulin. To examine the role of each specificity (anti-dsDNA and anti-IgG2a hinge) in the formation of immune complexes, affinities and Krel values were determined for both BV 17-45 and BV 16-13 employing various lengths of dsDNA fragments (21bp, 43bp, 84bp, and 114bp) and a synthetic nine amino acid hinge peptide dimer. The results showed that the affinities for both BV 17-45 and BV 16-13 for dsDNA was higher than for the synthetic nine amino acid hinge peptide dimer. It was also observed that the affinities of the two monoclonal antibodies for dsDNA was directly dependent upon the length of dsDNA. This trend was a direct consequence of dual-specificity. The larger dsDNA fragments localized several autoantibody active sites, in close proximity, thereby causing binding to both self (via the hinge specificity) and dsDNA resulting in the formation of avid immune complexes. The overall effect was a significant increase in functional affinity of ∼ 4000 to 8000-fold. A dual-specificity autoimmune complex model was proposed and described to explain the role of the anti-dsDNA and anti-IgG2a specificity in the formation of avid immune complexes.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD