Objective. Although anti-double-stranded DNA (anti-dsDNA) antibodies are important in lupus nephritis, the question regarding which glomerular structures (␣-actinin, nucleosomes, or others) are recognized by nephritogenic anti-dsDNA antibodies is still controversial. In this study, we determined which glomerular structures are recognized by monoclonal and in vivobound nephritogenic antibodies.Methods.Western blotting was used to analyze the ability of nephritogenic anti-dsDNA antibodies to recognize glomerular and nucleosomal structures. Sera from patients with lupus nephritis, sera from random antinuclear antibody-positive patients, and paired antibodies from sera and kidney eluates from nephritic (NZB ؋ NZW)F 1 mice were analyzed for activity against proteins identified by monoclonal nephritogenic antibodies, and against ␣-actinin, dsDNA, nucleosomes, histone H1, heparan sulfate, DNase I, and type IV collagen. Immunoelectron microscopy was used to determine the glomerular localization of ␣-actinin and in vivo-bound autoantibodies in nephritic (NZB ؋ NZW)F 1 mouse kidneys.Results. Anti-␣-actinin antibodies were observed in human and murine lupus nephritis sera and in sera from patients without systemic lupus erythematosus and were not detected in kidney eluates from nephritic mice. Antibodies to dsDNA and histone H1 were detected in all eluates. Western blot analyses revealed that nephritogenic anti-dsDNA antibodies recognized a 32-kd band, identified as histone H1. Competitive enzyme-linked immunosorbent assay demonstrated that nephritogenic monoclonal antibodies, and dominant antibodies eluted from nephritic kidneys, cross-reacted with dsDNA and H1. This cross-reactive anti-H1 specificity was largely absent in sera from those mice. Immunoelectron microscopic analysis of nephritic (NZB ؋ NZW)F 1 mouse kidneys revealed that antibodies eluted from kidneys, but not anti-␣-actinin antibodies, bound to distinct nephritis-associated electrondense structures linked to glomerular basement membranes.
Conclusion.Cross-reactive anti-dsDNA/antihistone H1 antibodies, but not anti-␣-actinin antibodies, are central among those deposited in nephritic glomeruli.
Collectively, these results provide firm evidence that dominant target structures for nephritogenic autoantibodies are constituted by TUNEL-positive chromatin associated with glomerular capillary and mesangial matrix membranes at high affinity.
Cross-reactivity of anti-double stranded DNA (anti-dsDNA) antibodies with glomerular antigens has been postulated as a key factor in the development of lupus nephritis. Because no direct proof has been presented on anti-dsDNA antibodies binding in vivo to glomerular structures, we have analysed the binding of potentially nephritogenic anti-dsDNA antibodies to α-actinin and laminin. By enzyme-linked immunosorbent assay and surface plasmon resonance (SPR) analyses, we demonstrate that monoclonal antibodies (mAbs) bind both double-stranded DNA and α-actinin at high affinity. However, when added to nephritic kidney sections they did not bind to such structures, but rather to nucleosome-containing structures within the mesangial matrix or the glomerular basement membranes (GBMs). Nucleosomes, anti-nuclear antibodies and complexes of them were tested for their binding to glomerular components such as agrin, perlecan and laminin using SPR analysis. Nucleosomes bound to laminin, marginally to agrin, but not to perlecan or heparan sulphate-depleted agrin. Anti-histone H2B and anti-nucleosome antibodies in complex with nucleosomes slightly increased the binding of nucleosomes to agrin, while binding to laminin was slightly decreased compared to nucleosomes alone. In conclusion, the availability of nucleosomal antigens and the binding of these antigens to components of the mesangial matrix and GBM seem crucial for the glomerular deposition of immune complexes.
Recent studies have demonstrated that the nephritogenicity of antibodies to dsDNA and nucleosomes confers to binding of glomerular membrane-associated nucleosomes, and not to cross-reacting glomerular antigens. There is no known parameter that determines antibody pathogenicity aside from specificity for dsDNA/nucleosomes, and systemic lupus erytheomatosus (SLE) patients may have high titer anti-dsDNA antibodies irrespective whether they have lupus nephritis or not. One parameter may be antibody affinity, as theoretically only high affinity antibodies may bind in vivo in a stable way. This was analyzed in (NZB x NZW)F1 mice with full-blown lupus nephritis. These mice had serum antibodies to dsDNA, and IgG autoantibodies bound in situ in glomerular membrane-associated electron dense structures as determined by immune electron microscopy (IEM). Intrinsic affinity of purified circulating and glomerular IgG anti-dsDNA antibodies was determined by surface plasmon resonance. The results demonstrate that affinity of glomerular-bound anti-dsDNA antibodies was higher than for those in circulation. However, affinity of glomerular in situ-bound antibodies from different mice varied considerably, from K(D) in the range from 10(- 8) to 10(- 13). These results indicate that antibody affinity is not a decisive pathogenic factor, but rather that availability of chromatin fragments may be the factor that determines whether an anti-dsDNA antibody binds in glomeruli or not.
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