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Pathogenic profiles and molecular signatures of antinuclear autoantibodies rescued from NZM2410 lupus mice.

Liang Z, Xie C, Chen C, Kreska D, Hsu K, Li L, Zhou XJ, Mohan C - J. Exp. Med. (2004)

Bottom Line: Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99.To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies.The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.

View Article: PubMed Central - PubMed

Affiliation: Simmons Arthritis Research Center, University of Texas Southwestern Medical School, Dallas 75390, USA.

ABSTRACT
Two outstanding questions concerning antinuclear antibodies (ANAs) in lupus involve their pathogenic potential and their molecular signatures. To address these questions, a panel of 56 antinuclear and 47 nonnuclear binding monoclonal antibodies was rescued from four seropositive NZM2410 lupus mice. The monoclonals varied in their reactivity to nucleosomes, ssDNA, dsDNA, and glomerular substrate. A large fraction of the antibodies demonstrated apparent polyreactivity (to DNA, histones, and glomerular antigens) due to bound, DNase-1 sensitive nuclear antigenic bridges. Although nephrophilic immunoglobulin (Ig) M and IgG antibodies were the most pathogenic, the dsDNA-binding antibodies were modestly so; in contrast, antinucleosome antibodies were clearly not pathogenic. Compared with the nonnuclear antigen-binding monoclonal antibodies rescued from the same mice, ANAs exhibited increased utilization of VH5/7183 genes and highly cationic heavy chain (HC) CDR3 regions. Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99. To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies. The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.

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Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).
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fig1: Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).

Mentions: With respect to the latter specificity, potential dual reactivity to both histones and DNA seemed somewhat unlikely given the diametrically opposite polarities of these two antigens. One possibility was that the apparent reactivity to histones and/or DNA was mediated by nucleosomal Ags (originating, for example, from apoptotic hybridoma cells in culture) that were bound to the Ag-binding grooves of these mAbs; these moieties may have served as antigenic bridges facilitating contact with the DNA and/or histones coated on the ELISA plates. Thus, it was certainly conceivable that an anti-DNA or antinucleosome Ab might be masquerading as an antihistone Ab (or vice versa) through this mechanism. To ascertain if this phenomenon was indeed responsible for the observed dsDNA/histone dual reactivity, six representative dual-reactive ANAs were subjected to DNase-I treatment to digest away any bound nucleosomal material before reassaying their reactivity to dsDNA and/or histones. DNase-1 treatment of the Ab abrogated the ability of most of these “dual-reactive” Abs to bind histones (Fig. 1), but not to dsDNA (not depicted), suggesting that a substantial fraction of the apparent antihistone reactivity in the sera of these mice may be attributed to dsDNA-specific Abs. Interestingly, ∼10–20% of the mAbs appeared to retain a certain degree of histone reactivity despite DNase-1 treatment of both the Ab and the antigenic substrate used for ELISA. It is currently unclear if these few clones represented true histone/DNA dual-reactive Abs, or whether the observed reactivity pattern was due to residual, DNase-1–resistant, nucleosomal fragments tightly bound within the Ag-binding pockets of these Abs.


Pathogenic profiles and molecular signatures of antinuclear autoantibodies rescued from NZM2410 lupus mice.

Liang Z, Xie C, Chen C, Kreska D, Hsu K, Li L, Zhou XJ, Mohan C - J. Exp. Med. (2004)

Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211797&req=5

fig1: Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).
Mentions: With respect to the latter specificity, potential dual reactivity to both histones and DNA seemed somewhat unlikely given the diametrically opposite polarities of these two antigens. One possibility was that the apparent reactivity to histones and/or DNA was mediated by nucleosomal Ags (originating, for example, from apoptotic hybridoma cells in culture) that were bound to the Ag-binding grooves of these mAbs; these moieties may have served as antigenic bridges facilitating contact with the DNA and/or histones coated on the ELISA plates. Thus, it was certainly conceivable that an anti-DNA or antinucleosome Ab might be masquerading as an antihistone Ab (or vice versa) through this mechanism. To ascertain if this phenomenon was indeed responsible for the observed dsDNA/histone dual reactivity, six representative dual-reactive ANAs were subjected to DNase-I treatment to digest away any bound nucleosomal material before reassaying their reactivity to dsDNA and/or histones. DNase-1 treatment of the Ab abrogated the ability of most of these “dual-reactive” Abs to bind histones (Fig. 1), but not to dsDNA (not depicted), suggesting that a substantial fraction of the apparent antihistone reactivity in the sera of these mice may be attributed to dsDNA-specific Abs. Interestingly, ∼10–20% of the mAbs appeared to retain a certain degree of histone reactivity despite DNase-1 treatment of both the Ab and the antigenic substrate used for ELISA. It is currently unclear if these few clones represented true histone/DNA dual-reactive Abs, or whether the observed reactivity pattern was due to residual, DNase-1–resistant, nucleosomal fragments tightly bound within the Ag-binding pockets of these Abs.

Bottom Line: Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99.To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies.The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.

View Article: PubMed Central - PubMed

Affiliation: Simmons Arthritis Research Center, University of Texas Southwestern Medical School, Dallas 75390, USA.

ABSTRACT
Two outstanding questions concerning antinuclear antibodies (ANAs) in lupus involve their pathogenic potential and their molecular signatures. To address these questions, a panel of 56 antinuclear and 47 nonnuclear binding monoclonal antibodies was rescued from four seropositive NZM2410 lupus mice. The monoclonals varied in their reactivity to nucleosomes, ssDNA, dsDNA, and glomerular substrate. A large fraction of the antibodies demonstrated apparent polyreactivity (to DNA, histones, and glomerular antigens) due to bound, DNase-1 sensitive nuclear antigenic bridges. Although nephrophilic immunoglobulin (Ig) M and IgG antibodies were the most pathogenic, the dsDNA-binding antibodies were modestly so; in contrast, antinucleosome antibodies were clearly not pathogenic. Compared with the nonnuclear antigen-binding monoclonal antibodies rescued from the same mice, ANAs exhibited increased utilization of VH5/7183 genes and highly cationic heavy chain (HC) CDR3 regions. Most intriguingly, the CDR3 regions of the ANAs exhibited alternating arginine/lysine peaks at H96, H98, and H100, with neutral troughs at H95, H97, and H99. To summarize, glomerular-binding anti-dsDNA antibodies appear to be the most pathogenic variety of lupus autoantibodies. The presence of an alternating charge pattern in their HC CDR3 regions appears to be a prominent hallmark of ANAs.

Show MeSH
Related in: MedlinePlus