Antigen specificity of anti-nuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo.

Monoclonal anti-nuclear antibodies which are complexed to nucleosomes are able to bind to the glomerular basement membrane (GBM) in vivo, whereas purified antibodies do not bind. The positively charged histone moieties in the nucleosome are-responsible for the binding to anionic determinants in the GBM. We tested the hypothesis that the specificity of the autoantibodies complexed to the nucleosome influences the glomerular binding of the antibody-nucleosome complex. We induced the formation of these immune complexes in vivo, by intraperitoneal inoculation of hybridomas producing monoclonal anti-nuclear antibodies (four anti-histone, three anti-double stranded (ds)DNA and three anti-nucleosome antibodies) into nude BALB/c mice. In ascites and plasma from the mice inoculated with these hybridomas, nucleosome/autoantibody complexes were detected in comparable amounts. Immunofluorescence of kidney sections revealed that about 60% of the mice inoculated with anti-nucleosome or anti-dsDNA hybridomas had immunoglobulin deposits in the GBM, whereas only 15% of the mice with anti-histone hybridomas showed these deposits (p < or = 0.04). In the Matrigel-ELISA (used as a GBM surrogate) ascites from anti-nucleosome or anti-DNA hybridomas displayed significantly higher titers (p < or = 0.002) than ascites from anti-histone hybridomas. In conclusion, nucleosome/immunoglobulin complexes comprising anti-nucleosome or anti-dsDNA auto-antibodies do bind more frequently to the GBM in vivo than nucleosome/immunoglobulin complexes containing anti-histone antibodies. It therefore appears that the specificity of the antibody bound to the nucleosome is a critical determinant for the nephritogenic potential of the nucleosome-autoantibody complex.

Specificity of monoclonal anti-nucleosome auto-antibodies derived from lupus mice.

Recently, anti-nucleosome antibodies, which do not bind to DNA or to individual histones, have been identified in longitudinal studies in lupus mice. These anti-nucleosome antibodies occur early in spontaneous SLE and are formed prior to other anti-nuclear specificities. However, nucleosomal epitopes are yet to be fully characterized. We selected a panel of six monoclonal anti-nucleosome antibodies (mAbs) (#2, #32, #34, PL2-6, LG8-1 and LG10-1) derived from lupus mice. These mAbs were tested in ELISA on subnucleosome structures and on a panel of 53 histone peptides, covering the entire sequence of the five histones. Two mAbs reacted with one of these peptides, but the reactivity hardly exceeded the background reactivity. Based on the nucleosome and subnucleosome ELISA we identified different recognition patterns. Three mAbs showed the highest reactivity towards the intact nucleosome. For two of them (#32 and LG8-1) the nucleosomal epitope was primarily located on H2A-H2B/DNA, whereas for mAb #34 this primary epitope was located on H3/H4/DNA. Two mAbs (#2 and PL2-6) showed the highest reactivity with H2A-H2B/DNA and one mAb (LG10-1) recognized H3-H4/DNA. In the subnucleosome ELISA all but one (mAb #32) recognized more than one epitope, including DNA complexed to a variety of cationic molecules. Comparing these reactivities we identified for all mAbs one specific nucleosomal epitope, whereas reactivity with other subnucleosomes was comparable to the reactivity towards DNA complexed with cationic molecules. In inhibition experiments both in ELISA and in immunofluorescence it was found that only one of the mAbs (i.e. PL2-6), recognizing an epitope on H2A-H2B/DNA as primary epitope, could be inhibited by H2A-H2B/DNA in fluid phase. The two mAbs recognizing an epitope on H3-H4/DNA as primary epitope could be inhibited by H3-H4/DNA in fluid phase. From these analyses, we conclude first that for these nucleosome specific mAbs linear histone peptides are not very important. Second, that these mAbs all recognize different epitopes on both H2A/H2B-DNA and H3/H4-DNA and third that some solid phase H2A/H2B-DNA epitopes are not expressed on fluid phase H2A/H2B-DNA. Our findings suggest that in SLE the nucleosome can act as auto-antigen and that there is no immunodominant beta cell epitope within the nucleosome.

Heparin and heparinoids prevent the binding of immune complexes containing nucleosomal antigens to the GBM and delay nephritis in MRL/lpr mice.

Monoclonal anti-nucleosome antibodies (mAbs) complexed to nucleosomal antigens can bind to DNA and to heparan sulfate (HS) in ELISA and to the GBM in vivo in a rat renal perfusion system, whereas non-complexed mAbs do not bind [1]. In this study, we analyzed whether heparin (HEP) or N-desulfated/acetylated heparins (DSA-HEP), structurally and functionally strongly related to HS, are able to prevent the binding of these complexed mAbs to DNA and to HS in vitro and to rat GBM in vivo. In ELISA the binding of nucleosome complexed antinucleosome antibodies to DNA and HS was inhibited dose-dependently by HEP, DSA-HEP and low molecular weight (LMW) DSA-HEP. Intravenous injection of nucleosome/anti-nucleosome immune complexes without heparin/heparinoids in BALB/c mice led to GBM binding, while simultaneous injection of heparin/heparinoids with complexed antibodies or pretreatment with heparin subcutaneously prior to injection of complexes prevented this binding. Subsequently, we tested the preventive effect of HEP, DSA-HEP and LMW-DSA-HEP on progression of renal disease in MRL/lpr mice. Treatment was started at an age of eight weeks in a dose of 50 micrograms daily. With all three drugs albuminuria was significantly delayed compared to PBS treated controls (cumulative incidence of proteinuria at 20 weeks in controls 60% vs. 13%, 14% and 6% respectively for HEP, DSA-HEP and LMW-DSA-HEP; P < 0.05). At week 21 the glomerulonephritis was histologically less severe in heparin/heparinoid treated animals (P = 0.02). In immunofluorescence the amount of immunoglobulin and C3 deposits in the glomerular capillary wall tended to be less in heparin/heparinoid treated mice compared to PBS treated controls (P = 0.07). Furthermore, at 20 weeks anti-HS levels in plasma of heparin/heparinoid treated mice were significantly lower (P < 0.05). We conclude that interaction of heparin or heparin analogs with HS reactive immune complexes containing nucleosomal antigens prevents the binding of these immune complexes to the GBM and delays nephritis in MRL/lpr mice.

Dual reactivity of several monoclonal anti-nucleosome autoantibodies for double-stranded DNA and a short segment of histone H3.

We have shown previously that four IgG monoclonal autoantibodies (mAbs) reacted in ELISA with both double-stranded (ds) DNA and peptide 83-100 of histone H3. The peptide 83-100 contains a cysteine residue at position 96 and readily dimerizes at pH 7-8. We describe here that only the 83-100 dimers, and not the 83-100 monomers, are recognized by the four antibodies and inhibit in ELISA the binding of mAbs to dsDNA. The equilibrium affinity constants (Ka) and kinetic rate constants of two of these mAbs were measured in a biosensor system. Ka values were significantly higher when these mAbs were tested with dsDNA as compared with the 83-100 dimer. Further higher Ka values were measured with mononucleosomes containing DNA and histones. It is proposed that these four mAbs are directed against a topographic determinant formed by DNA and the region 83-100 of H3 present as a dimer at the surface of nucleosome, and that they react, although significantly less well, with DNA and peptide dimer tested separately. This study provides a quantitative and kinetic basis to interaction between several antibodies and distinct antigenic structures and allows us to better understand the structural basis of apparent autoantibody cross-reactivity.

In vivo ANA is a fixation artifact: nucleosome-complexed antinucleosome autoantibodies bind to the cell surface and are internalized.

It has been suggested that binding of anti-double-standed DNA antibodies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tissue damage in systemic lupus erythematosus. We have shown before that pathogenic antinuclear antibodies complexed to nucleosomal antigens can bind to heparan sulfate in the glomerular basement membrane in vivo. Because nucleosomes are also reported to bind to the cell surface, we hypothesized that in vivo ANA is a property of antinuclear antibodies bound to nucleosomal antigens. Therefore, we studied three antinucleosome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by immunofluorescence in mice inoculated intraperitoneally with the hybridoma producing the mAb. The same mAb complexed to nucleosomal antigens after intravenous injection into mice induced in vivo ANA, in contrast to purified noncomplexed mAb. To study this in more detail, we incubated complexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. However, no binding to the nucleus was observed by immunoelectron microscopy (IEM) and confocal laser microscopy. Noncomplexed mAb did not bind to the cell surface. Next, from mice bearing the hybridomas producing the mAb intraperitoneally, a small part of the kidney was snap frozen in liquid N2, fixed with acetone, and studied in immunofluorescence, whereas the remaining part of the kidney was fixed in vivo by renal perfusion with a mixture of 0.01 M sodium periodate, 0.075 M lysine HCl, 0.0375 M Na2HPO4, and 2% paraformaldehyde (PLP) and studied in both immunofluorescence and IEM. In the acetone-fixed kidney sections obtained without in vivo fixation we again observed in vivo ANA. However, after in vivo PLP perfusion fixation, no nuclear binding was found. In IEM, localization in cytoplasmic vesicles was seen. In conclusion, antinucleosome antibodies complexed to nucleosomal antigens can bind to the cell surface and are transported into the cytoplasm, but do not bind to the nucleus. The reported nuclear localization of antinuclear antibodies is caused by a fixation artifact.

Heparan sulfate staining of the glomerular basement membrane in relation to circulating anti-DNA and anti-heparan sulfate reactivity: a longitudinal study in NZB/W F1 mice.

Reactivity of serum antibodies with heparan sulfate (HS) has been associated with human and murine lupus nephritis, although the aetiological significance of this association is not clear. Recent work from our laboratories showed that binding of these antibodies to HS could be mediated by histone containing immune complexes. In human lupus nephritis we found a strong decrease in HS staining in the glomerular basement membrane (GBM). The aim of this study was to elucidate the correlation in experimental systemic lupus erythematosus (SLE) between albuminuria, staining of HS in the GBM and anti-DNA and anti-HS reactivity in plasma. We therefore studied NZB/W F1 mice during different stages of glomerular disease and compared them with age matched control NZB/W F1 mice without albuminuria. Anti-DNA and anti-HS reactivity were measured in longitudinally collected plasma samples and correlated with the onset of albuminuria, staining of HS in the glomerular basement membrane and deposition of immunoglobulins (Ig). HS staining was significantly decreased in the glomerular capillary loops of mice with prolonged proteinuria in comparison with age matched control mice (P = 0.0013). This decreased HS staining was correlated with increased Ig deposition in the capillary loops (tau = -0.42, P < 0.001), albuminuria (tau = -0.508, P < 0.001) and a decreased in anti-DNA levels measured in plasma (tau = 0.758, P < 0.005). Altered anti-HS reactivity in plasma did correlate with increased Ig deposition in the kidney (tau = 0.33, P < 0.05) but was not correlated with decreased staining of HS in the kidney. In conclusion, our study demonstrates that disappearance of staining of HS in the glomerular capillary loops is associated with albuminuria, increased Ig deposition in the glomerulus and decreased anti-DNA reactivity in plasma. Our findings are compatible with a model in which interaction ('masking') of HS with immune complexes consisting of anti-DNA antibodies and nucleosomes takes place.

Decrease of heparan sulfate staining in the glomerular basement membrane in murine lupus nephritis.

Recently we found in biopsies of human lupus nephritis a nearly complete loss of heparan sulfate (HS) staining in the glomerular basement membrane (GMB). To clarify the relationship between HS staining and albuminuria in lupus nephritis, we studied MRL/lpr mice with short (< 7 days) or prolonged duration of albuminuria (14-21 days) and compared these with mice of different ages without albuminuria. Kidney sections were stained for mouse immunoglobulin (Ig), HS, heparan sulfate proteoglycan (HSPG)-core protein and laminin in immunofluorescence. In mice with prolonged albuminuria HS staining in the glomerular capillary loops had almost completely disappeared, whereas staining was unaltered in non-albuminuric mice (P = 0.001). In mice with short duration of albuminuria, there was a tendency toward a decrease of HS staining (P = 0.06). The expression of HSPG-core protein and other extra cellular matrix (ECM) components was unaltered in all groups. HS staining correlated inversely with albuminuria (rs = -0.55; P < 0.001) and with staining of Ig deposits in the capillary loops (rs = -0.74; P < 0.001). Despite the nearly complete loss of HS staining in the GBM in mice with prolonged albuminuria, there was no change in glomerular HS content as assessed by agarose electrophoresis and HS inhibition ELISA. We conclude that the development of albuminuria in MRL/lpr mice is accompanied by a loss of HS staining in the GBM, probably due to the masking of HS by deposits of Ig. In vitro studies revealed that autoantibodies complexed to nucleosomal antigens can inhibit the binding of the anti-HS monoclonal antibody to HS. Whether this also occurs in vivo remains to be determined.