The constant region contributes to the antigenic specificity and renal pathogenicity of murine anti-DNA antibodies

Abstract

Affinity for DNA and cross-reactivity with renal antigens are associated with enhanced renal pathogenicity of lupus autoantibodies. In addition, certain IgG subclasses are enriched in nephritic kidneys, suggesting that isotype may determine the outcome of antibody binding to renal antigens. To investigate if the isotype of DNA antibodies affects renal pathogenicity by influencing antigen binding, we derived IgM, IgG1, IgG2b and IgG2a forms of the PL9–11 antibody (IgG3 anti-DNA) by in vitro class switching or PCR cloning. The affinity and specificity of PL9–11 antibodies for nuclear and renal antigens were analyzed using ELISA, Western blotting, surface plasmon resonance (SPR), binding to mesangial cells, and glomerular proteome arrays. Renal deposition and pathogenicity were assayed in mice injected with PL9–11 hybridomas. We found that PL9–11 and its isotype-switched variants had differential binding to DNA and chromatin (IgG3 > IgG2a > IgG1 > IgG2b > IgM) by direct and competition ELISA, and SPR. In contrast, in binding to laminin and collagen IV the IgG2a isotype actually had the highest affinity. Differences in affinity of PL9–11 antibodies for renal antigens were mirrored in analysis of specificity for glomeruli, and were associated with significant differences in renal pathogenicity in vivo and survival. Our novel findings indicate that the constant region plays an important role in the nephritogenicity of antibodies to DNA by affecting immunoglobulin affinity and specificity. Increased binding to multiple glomerular and/or nuclear antigens may contribute to the renal pathogenicity of anti-DNA antibodies of the IgG2a and IgG3 isotype. Finally, class switch recombination may be another mechanism by which B cell autoreactivity is generated.

Deficiency of Fibroblast Growth Factor-Inducible 14 (Fn14) Preserves the Filtration Barrier and Ameliorates Lupus Nephritis

“…Both anti-double-stranded DNA and antichromatin antibodies are closely associated with nephritogenicity. 2,39 Anticardiolipin IgG is commonly found in the MRL/lpr strain 40 and has been linked with proteinuria. 41 Antiribosomal P antibodies are highly specific for SLE 42 and share antigenic epitopes on the ribosome with anti-DNA antibodies.…”

“…The detection of glomerular IgG deposition was performed using biotinylated goat anti-mouse IgG (Vector Laboratories) and an NBT-BCIP solution (Roche Diagnostics) as described previously. 2 For Fn14 staining, rabbit anti-mouse Fn14 mAb (Epitomics) was used followed by applying horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and then DAB-chromogen (Dako). The WT1 staining protocol was similar to that for Fn14 expression but with a primary rabbit antibody to mouse WT1 (Santa Cruz).…”

“…16 Anti-double-stranded DNA, chromatin, and cardiolipin ELISAs were performed as previously described. 2,40 Antibodies against ribosomal P (P0, P1, and P2) were measured using a commercially available kit from Aesku Diagnostics (Wendelsheim, Germany). 38 Sera were diluted to 1:100, and OD values were normalized to the average absorbance of sera from two sick control MRL/lpr mice.…”

“…1 A better understanding of the pathogenesis of LN is important for identifying more targeted and less toxic therapeutic approaches. However, despite substantial progress, 2,3 several recent clinical trials testing novel therapies have failed to show improvement over the existing standard of care. Thus, mechanisms of renal damage in LN are not fully elucidated, which poses a real challenge for the development of improved treatment approaches.…”

Variable Region Identical IgA and IgE to Cryptococcus neoformans Capsular Polysaccharide Manifest Specificity Differences

“…3 and 4). In the past two decades, studies from at least seven independent laboratories have established that the C region can influence the specificity and affinity of certain antibodies (Abs) (5)(6)(7)(8)(9)(10)(11)(12). This effect has been reported for IgM, IgG, and IgA isotypes, but IgE has not yet been explored.…”