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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.

Objective. The autoantibody specificities that dominate the deposits in lupus kidneys remain unclear. Reasoning that previously utilized elution buffers such as acidic glycine and ammonium thiocyanate may not have been maximally effective in eluting all Ig deposits from the kidneys, this study was conducted to experiment with a stronger dissociating agent, urea-glycine.Methods. Seven antinuclear antibody-positive, nephritic female (SWR ؋ NZB)F 1 (SNF1) lupus mice were selected for the elution study. Deposited Ig was eluted from their kidneys using 3 different elution buffers: 0.15M glycine-HCl buffer, 1.3M ammonia thiocyanate/0.15M glycine-HCl buffer, and 5M urea/ 0.15M glycine-HCl buffer. All eluates were tested for specificity against a variety of nuclear and glomerular antigens.Results. Compared with conventional elution buffers, the urea-based regimen eluted severalfold more IgG and IgM antinuclear antibodies from the kidneys of nephritic SNF1 lupus mice. IgG anti-double-stranded DNA (anti-dsDNA) antibodies were not only the most prevalent species in these renal deposits, they were also heavily enriched in the kidneys, relative to the corresponding serum levels. A substantial fraction of the anti-single-stranded DNA and antihistone/DNA (but not antihistone) reactivity in these eluates was due to cross-reactive anti-dsDNA antibodies. No reactivity with SSA, SSB, Sm, RNP, Jo-1, Scl-70, or ribosomal P antigens could be demonstrated in these eluates. Importantly, the urea-glycine eluates differed from the conventional eluates in having significantly greater reactivity to glomerular substrate and laminin.Conclusion. This novel urea-based elution provides further support for the dominance of antibodies in lupus kidneys, with strong polyreactivity to DNA and glomerular substrate.Antibody-mediated end organ pathology is a cardinal feature of systemic lupus erythematosus. In particular, antinuclear antibodies (ANAs) have been accorded a dominant role in mediating this disease (1-3). Their temporal relationship to the clinical course of the disease and the findings of direct antibody transfer studies in murine models have together indicated their effector role in end organ disease. Although recent genetic studies have shed light on the potential origins of nephrophilic antibodies (4), the antibody specificities that prevail within the end organs, and are responsible for the pathology, remain unclear.One traditional approach to this question has been to simply examine the specificities of the autoantibodies deposited within the kidneys of mice that spontaneously develop lupus. In the MRL/lpr murine lupus model, antibodies specific for double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), nucleosomes, and histones have been eluted from diseased kidneys (5-7). In addition, van Bruggen et al have noted that eluates from MRL/lpr mouse kidneys demonstrated specificity for glomerular and tubular basement membrane antigens (7). Working with the same mouse model, others have observed that the renal eluates from these kidne...

The Sle1ab genomic interval on murine chromosome 1 mediates the loss of immune tolerance to chromatin resulting in antinuclear Abs (ANA) production in the lupus-prone NZM2410 mouse. Global gene expression analysis was used to identify the molecular pathways that are dysregulated at the initiation of B lymphocyte autoimmunity in B6.Sle1ab mice. This analysis identified that STAT3 and ras-ERK signaling pathways are aberrantly activated in Sle1ab B lymphocytes, consistent with increased production of IL-6 by splenic B lymphocytes and monocytes in B6.Sle1ab mice. In vitro treatment of splenic mononuclear cells isolated from ANA-positive Sle1ab mice with anti-IL-6 Ab or AG490, an inhibitor of STAT3 signaling pathway, suppressed ANA production in short-term culture, indicating that this pathway was essential to the production of autoantibodies. In vivo treatment of ANA-positive B6.Sle1ab mice with the ras pathway inhibitor, perillyl alcohol, suppressed the increase of ANA. These findings identify IL-6 as a early key cytokine in Sle1ab-mediated disease development and indicate that the STAT3 and ras-ERK signaling pathways are potential therapeutic targets for treating systemic lupus erythematosus.

Although the Ig H chains of anti-nuclear Abs (ANA) have been described to possess certain shared molecular signatures, it remains unclear whether the L chains of these Abs also possess distinctive molecular features. The present study examines this by generating and analyzing two comprehensive murine Ig L chain databases, one consisting of 264 monoclonal ANAs and the other consisting of 145 non-ANAs, drawn from previously published work. Importantly, clonal replicates were represented only once each, so as to minimize bias. ANAs and non-ANAs did not differ in Vκ family or Jκ gene usage, nor in their mutation frequencies. Interestingly, the L chains of ANAs exhibited differential usage of certain complementarity-determining region residues, arising almost entirely from the increased usage of certain Vκ germline genes, notably, Vκ ai4 among anti-dsDNA ANAs, Vκ23–45 among anti-ssDNA ANAs, and Vκ21–12 among non-ANAs. Finally, prompted by the increased prevalence of a particular Vκ1 family sequence among ANAs, we proceeded to clone a novel New Zealand Black Vκ1 germline gene, named bb1.1, which appears to be frequently used to encoded anti-ssDNA Abs. Collectively, these studies underline the potential contribution of particular Vκ germline genes in promoting or thwarting DNA binding.