anti-DNA Antibodies Research Articles

Sex ratios among children of lupus pregnancies

A recent paper1 stated that maternal antibody to the N-methyl-D-aspartate receptor (NMDAR) causes death of female fetuses of BALB/c mice, leading to offspring male/female (m/f) ratios greater than the normal ratio of 1.0. Since about 40% of patients with systemic lupus erythematosus (SLE) have anti-NMDAR, if the mouse model is predictive, m/f ratios in human SLE pregnancies might exceed the United States normal ratio of 1.048. Our pregnancy studies offer an opportunity to test this prediction. PROMISSE (Predictors of Pregnancy Outcome: Biomarkers in Antiphospholipid Syndrome and Systemic Lupus Erythematosus) is a multicenter, prospective study of pregnancies of mothers with lupus;2 the Research Registry for Neonatal Lupus (RRNL) is a registry of families in which the mother has anti-SSA/Ro and/or SSB/La antibodies and at least one child with neonatal lupus. RRNL includes both affected and unaffected children.3 The table displays m/f ratios of liveborn children of RRNL and PROMISSE, stratified by maternal serology and maternal and fetal diagnosis, and compared to the PROMISSE control group of normal women and to the United States norm. (PROMISSE patients with antiphospholipid antibody were excluded). Ratios are similar if stillborn children are counted. The m/f ratios are 1.0 for children of mothers with negative tests for anti-NMDAR and <1.0 for those with positive tests (p = 0.36). Because anti-DNA antibodies often coexist with anti-NMDAR antibodies, we compared m/f ratios in anti-DNA positive and negative patients and found no difference (p = 0.30). M/f ratios of patients with and without pregnancy complications did not differ (p = 0.34). Thus our human data do not confirm the mouse experiments. Table Male/female ratios of liveborn children in RRNL and PROMISSE studies, stratified by maternal or fetal diagnosis, fetal sex, and maternal serology. Inclusion of stillborn children does not change the ratios. See text for abbreviations. Prior data on human SLE fetal sex ratios are weak: in a national birth registry containing 68,600 women, conclusions were based on the 42 women who had a diagnostic code identifying them has having lupus.4 A summary of published case series (diagnostic criteria unspecified) reported studies ranging in size between 14 and 249 children; the four largest series, constituting half the infants, had a combined m/f ratio of 1.07, 5 Our prospective studies, having rigorous definition of maternal diagnosis and fetal outcome, are more likely to be correct, and the mouse-based hypothesis of male predominance of liveborn children incorrect. Our data reemphasize that the mouse models do not invariably predict human outcomes.

Lupus nephritis and non-Hodgkin lymphoma simultaneously diagnosed in a patient on methimazole

A 78 year old white male on methimazole due to Grave's thyroiditis presented with acute renal failure after a short term history of progressive shortness of breath, malaise, myalgias, arthralgias, and bilateral lower limb swelling. The abdomen was remarkable for splenomegaly and lower extremities for erythema nodosum. No peripheral lymphadenopathy was detected. Serum albumin was 1.7 g/dl and very high erythrocyte sedimentation rate. Urine sediment was very active with dysmorphic red blood cells and casts and significant proteinuria (6.6 grams/day). Serum complements were abnormally low and antinuclear and anti-DNA antibodies were positive. Renal histopathology revealed membranoproliferative glomerulonephritis, along with a full house pattern on IFF consistent with lupus nephritis. Bone marrow aspiration revealed a 40% infiltration by a lymphocyte population of small cells consistent with a B cell non-Hodgkin lymphoma. The patient was treated with methylprednisolone, cyclophosphamide and rituximab and acute dialysis. Over the following weeks the patient became dialysis independent and returned to his baseline GFR.

Homozygous FCGR3A-158F mutation is associated with delayed B-cell depletion following rituximab but with preserved efficacy in a patient with refractory lupus nephritis.

Rituximab (RTX), an anti-CD20 monoclonal antibody, has shown promising results in a small group of systemic lupus erythematosus (SLE) patients treated for lupus nephritis (LN). However, such observations were not confirmed in the double-blind LUNAR study. Accordingly, the factors associated with the clinical response remain to be characterized. We report the case of a young woman with known LN successfully re-treated with RTX and steroids and homozygous for the low-affinity FCG3RA 158F genotype. Although B-cell depletion was delayed, complete remission with anti-DNA antibody negativity and proteinuria normalization were maintained for 5 years. The implications for disease pathogenesis and clinical monitoring are discussed.

IL‐21 promotes the production of anti‐DNA IgG but is dispensable for kidney damage in lyn−/− mice

The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear Ags and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of systemic lupus erythematosus, lack an inhibitor of B-cell and myeloid cell activation. This results in B-cell hyper-responsiveness, plasma cell accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn(-/-) mice. We find that IL-21 mRNA is reduced in the spleens of lyn(-/-) IL-6(-/-) and lyn(-/-) Btk(lo) mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for plasma cell accumulation and IgM autoantibodies in lyn(-/-) mice, it is required for anti-DNA IgG antibodies and some aspects of T-cell activation. Surprisingly, GN still develops in lyn(-/-) IL-21(-/-) mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA Ags. These studies identify a specific role for IL-21 in the class switching of anti-DNA B cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn(-/-) mice.

Erratum to: Simultaneous Positivity for Anti-DNA, Anti-Nucleosome and Anti-Histone Antibodies is a Marker for More Severe Lupus Nephritis

Erratum to: Simultaneous Positivity for Anti-DNA, Anti-Nucleosome and Anti-Histone Antibodies is a Marker for More Severe Lupus Nephritis

Role of peroxynitrite-modified biomolecules in the etiopathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by autoantibodies directed against various biomolecules. The initial immunogens that drive the development of SLE are unknown, but characteristics of the immune response in SLE suggest that it is an antigen-driven response, and a chromatin antigen could be one of the immunogens for the production of antinuclear antibodies (ANA) in SLE. Other factors implicated in the pathogenesis of SLE include nitrogen-free radicals such as nitric oxide and peroxynitrite. The free radical-mediated damage to proteins results in the modification of amino acid residues, cross-linking of side chains and fragmentation. The tyrosine residues in proteins are susceptible to attack by various reactive nitrogen intermediates, including peroxynitrite to form 3-nitrotyrosine (3-NT). The presence of nitrated proteins in vivo indicates that peptides derived from the proteolytic degradation of modified proteins could serve as neoantigens. Histones are highly conserved proteins that are rich in basic amino acids lysine and arginine. Autoantibodies against histones and anti-DNA antibodies are present in SLE. The anti-DNA autoantibodies coexist with anti-histone autoantibodies and may react with chromatin-associated histones and histone complexes. Elevated levels of reactive nitrogen species (RNS) in SLE patients suggest a possible role in the pathogenesis of the disease. The alteration of proteins resulting from photomodification or peroxynitrite could lead to the development of antibodies. Therefore, the modified proteins or photoadducts could have important implications in autoimmunity, and understanding the pathophysiology of peroxynitrite-modified biomolecules could lead to a better understanding of autoimmune phenomenon in SLE.

Generation of gene-engineered chimeric DNA molecules for specific therapy of autoimmune diseases.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of self-reactive B and T cells and autoantibody production. In particular, double-stranded DNA-specific B cells play an important role in lupus progression, and their selective elimination is a reasonable approach for effective therapy of SLE. DNA-based vaccines aim at the induction of immune response against the vector-encoded antigen. Here, we are exploring, as a new DNA-based therapy of SLE, a chimeric DNA molecule encoding a DNA-mimotope peptide, and the Fv but not the immunogenic Fc fragment of an FcγRIIb-specific monoclonal antibody. This DNA construct was inserted in the expression vector pNut and used as a naked DNA vaccine in a mouse model of lupus. The chimeric DNA molecule can be expressed in eukaryotic cells and cross-links cell surface receptors on DNA-specific B cells, delivering an inhibitory intracellular signal. Intramuscular administration of the recombinant DNA molecule to lupus-prone MRL/lpr mice prevented increase in IgG anti-DNA antibodies and was associated with a low degree of proteinuria, modulation of cytokine profile, and suppression of lupus nephritis.

Simultaneous Positivity for Anti-DNA, Anti-Nucleosome and Anti-Histone Antibodies is a Marker for More Severe Lupus Nephritis

The purpose of this study is to examine autoantibody profile of systemic lupus erythematosus (SLE) patients with lupus nephritis (LN) and to establish the correlation between the antibody reactivity and disease activity of LN. Autoantibodies and serological parameters were measured and analyzed in 589 SLE patients. The associations of the co-positivity of anti-dsDNA, -nucleosome and -histone antibodies (3-pos) with clinical, serological and outcome parameters were analyzed. At the study entry, the prevalence for anti-dsDNA (61.52 % vs. 34.11 %, P < 0.0001), anti-nucleosome (56.09 % vs. 37.21 %, P = 0.0002) and anti-histone (49.35 % vs. 33.33 %, P = 0.0013) antibodies in patients with LN were significantly higher than that in patients without LN. Patients with 3-pos had a higher proportion of proliferative renal lesions (class III + IV). The incidence of a poor renal outcome (7.14 % vs. 2.52 %, P = 0.0174) in LN patients with 3-pos was significantly higher than those without 3-pos. Moreover, the rate of remission (73.63 % vs. 82.37 %, P = 0.0245) was significantly reduced and recurrence increased (58.90 % vs. 23.44 %, P < 0.0001) in 3-pos patients as compared to that in non 3-pos within the LN group. Our data indicate a strong association between the 3-pos and renal disease activities, especially proliferative glomerulonephritis. The ability of 3-pos to predict renal flares may lead to major additional benefits in the follow-up of these patients.

A Rare Cell-penetrating Anti-DNA Antibody Inhibits DNA Repair, Sensitizes Tumors To DNA-damaging Therapy, and is Synthetically Lethal to BRCA2-deficient Cancer Cells

A Rare Cell-penetrating Anti-DNA Antibody Inhibits DNA Repair, Sensitizes Tumors To DNA-damaging Therapy, and is Synthetically Lethal to BRCA2-deficient Cancer Cells

Targeting Cancer with a Lupus Autoantibody

Systemic lupus erythematosus (SLE) is distinct among autoimmune diseases because of its association with circulating autoantibodies reactive against host DNA. The precise role that anti-DNA antibodies play in SLE pathophysiology remains to be elucidated, and potential applications of lupus autoantibodies in cancer therapy have not previously been explored. We report the unexpected finding that a cell-penetrating lupus autoantibody, 3E10, has potential as a targeted therapy for DNA repair-deficient malignancies. We find that 3E10 preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes cultured tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we demonstrate that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low-dose doxorubicin. Our results establish an approach to cancer therapy that we expect will be particularly applicable to BRCA2-related malignancies such as breast, ovarian, and prostate cancers. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the intrinsic deficiencies in DNA repair and the unexpectedly low rates of breast, ovarian, and prostate cancers observed in SLE patients. In summary, this study provides the basis for the potential use of a lupus anti-DNA antibody in cancer therapy and identifies lupus autoantibodies as a potentially rich source of therapeutic agents.

Lupus Antibody Tops Cancer Cells

A lupus causing anti-DNA antibody penetrates living cells and targets DNA repair for therapeutic advantage in human cancer cells.

Nephritogenic-antinephritogenic antibody network in lupus glomerulonephritis

Lupus glomerulonephritis (LGN) is one of the most threatening manifestations of systemic lupus erythematosus (SLE) and a major predictor of poor prognosis. The mechanisms leading to kidney inflammation are not completely clear; however, autoantibodies seem to play a pivotal role. Apoptosis dysregulation in SLE is likely to trigger generation of autoantibodies, the released nucleosomes being the driving autoantigen for further epitope amplification and selection of DNA or nucleosome-specific B cells. Growing evidence supports a multistep path to LGN involving initial autoantibody binding to chromatin fragments in the mesangial matrix, where they can induce mesangial inflammation leading to a shut-down of the renal DNase gene, generation and deposition of secondary necrotic chromatin on the glomerular basement membrane favouring antibody binding, complement activation and development of membrano-proliferative glomerular lesions. Anti-DNA IgG antibodies display the major pathogenetic potential in LGN initiation; however, other isotypes (IgA or IgE) as well as autoantibodies targeting other molecules (e.g. anti-C1q, anti-C reactive protein) can perpetuate renal injury. Conversely, protective autoantibodies are also likely in SLE which can contain renal damage targeting either DNA (i.e. IgM anti-DNA) or other molecules (e.g. pentraxin 3). Thus, lupus nephritogenic-antinephritogenic antibodies orchestrate the balance between harm and defence of renal tissue.

488 The Deficit in Immunoglobulin A, Autoimmunity Risk

Introduction and objectivesThe immunodeficiencies (ID), by the subsequent impairing of the immunoregulation, may be at the origin of certain autoimmune diseases (AID).The deficit in immunoglobulin A is one of the...

Microparticles as autoantigens in human and murine lupus

Systemic lupus erythematosus is a systemic inflammatory disease characterized by anti-DNA production in association with immune complex deposition. These complexes can deposit in the tissue to incite inflammation as well as stimulate cytokine production by plasmacytoid dendritic cells. While the properties of anti-DNA antibodies have been extensively characterized, little is known about the nature of the DNA in the immune complexes although its origin is generally considered to be cell death. Among sources of extracellular DNA, microparticles (MPs) are small membrane-bound vesicles released from activated and dying cells by a blebbing process. As shown by flow cytometry, these particles display DNA as well as other nuclear molecules in an antigenically accessible form as indicated by the binding of monoclonal anti-DNA antibodies as well as plasma from patients with lupus. Furthermore, plasma from patients with lupus contains increased number of particles with bound IgG, suggesting that MPs can form immune complexes found in lupus. To assess whether MPs play a similar role in murine lupus, we used flow cytometry to measure the presence of MPs with bound IgG in the blood of MRL-lpr/lpr and NZB/W mice; in addition, we tested the binding of plasma of these mice to MPs from apoptotic cells. The results of these studies showed important differences in the serological findings of the two strains as reflected by the much higher numbers of MPs with bound IgG in the blood of MRL-lpr/lpr compared with NZB/W mice. These studies also showed that antibodies from MRL-lpr/lpr mice bound much better to MPs from apoptotic cells than those from NZB/W mice. Since particles in NZB/W blood bound to monoclonal anti-nuclear antibodies as well as MRL-lpr/lpr plasma, these findings indicate antigenic activity despite the lack of particle IgG complexes in NZB/W blood. Together, these studies indicate important differences in the serological features of the two strains as reflected by the capacity of antibodies to bind to MPs. These differences may impact on the process of immune complex formation and its consequences as well as the respective role of anti-DNA and other autoantibodies in nephritis in NZB/W mice.

Epigenetics and lupus

Lupus develops when genetically predisposed people encounter environmental agents that initiate flares. Current evidence indicates that the environmental contribution is mediated by T-cell DNA demethylation. DNA methylation patterns are established during differentiation, and silence inappropriate or unnecessary genes by promoting a condensed chromatin configuration that is inaccessible to transcription factors. The methylation patterns are then replicated each time a cell divides by DNA methyltransferase 1 (Dnmt1). Dnmt1 is upregulated during mitosis, binds the replication fork, and catalyzes transfer of the methyl group from S-adenosylmethionine (SAM) to dC bases in the daughter DNA strand only where the parent strand is methylated. Environmental agents that block ERK pathway signaling prevent Dnmt1 upregulation, and low Dnmt1 levels synergize with dietary micronutrient deficiencies that decrease SAM pools to impair methylation of the daughter strand. This activates genes silenced only by DNA methylation. Inhibiting T-cell DNA methylation converts helper CD4+ T cells into autoreactive, cytotoxic, proinflammatory cells that cause lupus-like autoimmunity in mice. Similar changes in CD4+ T-cell DNA methylation and gene expression are found in patients with active lupus. Procainamide and hydralazine, which cause ANAs in a majority of patients and lupus in a genetically predisposed subset, also inhibit T-cell DNA methylation. The lupus T-cell DNA methylation defect has been traced to low Dnmt1 levels caused by decreased ERK pathway signaling, and the signaling defect has now been traced to PKCδ inactivation caused by oxidative damage. The importance of decreased ERK pathway signaling was confirmed by generating a transgenic mouse with an inducible dominant negative MEK. Inducing the signaling defect selectively in T cells decreases Dnmt1, causing anti-DNA antibodies in mice without lupus genes, and higher anti-DNA antibody levels and an immune complex glomerulonephritis in mice with lupus genes. Autoantibody levels and kidney disease are suppressed by dietary transmethylation micronutrient supplementation in these mice. Epigenetic mechanisms also contribute to the gender dimorphism in lupus. Immune genes on the normally silenced X chromosome demethylate in women with active lupus, contributing to flare severity. In contrast, men with only one X chromosome require a greater genetic predisposition and/or greater degree of DNA demethylation to develop a lupus flare equal in severity to women. Together, these studies indicate that environmental agents including oxidative stress and diet combine to inhibit T-cell DNA methylation, and that the epigenetically modified cells cause lupus-like autoimmunity in genetically predisposed people and mice.

Physicochemical and immunological studies on 4-hydroxynonenal modified HSA: Implications of protein damage by lipid peroxidation products in the etiopathogenesis of SLE

4-Hydroxynonenal (HNE) is the most abundant and toxic aldehyde generated by the oxidation of plasma membrane polyunsaturated fatty acids. Systemic lupus erythematosus (SLE), a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. HNE has been extensively used as a biomarker of oxidative stress. It can form adduct with proteins, making them highly immunogenic. Increased levels of such aldehyde-protein adducts have been reported in various pathological states, including autoimmune disorders like SLE and arthritis. In the present study, HNE-mediated structural changes in human serum albumin (HSA) were characterized by UV, fluorescence, CD and FT-IR spectroscopy as well as by polyacrylamide gel electrophoresis. Furthermore, immunogenicity of native and HNE-modified HSA was probed in female rabbits. The HNE-modified HSA was highly immunogenic eliciting high titre immunogen specific antibodies. Binding of SLE anti-DNA antibodies was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to HNE-modified HSA as compared to native HSA or native DNA. Our results suggest that HNE modification generates neoepitopes on HSA causing enhanced autoantibodies production. The results point towards the possible role of HNE-modified HSA in SLE etiopathogenesis.

Human complement receptor 2 (CR2/CD21) as a receptor for DNA: Implications for its roles in the immune response and the pathogenesis of systemic lupus erythematosus (SLE)

Human CR2 is a B cell membrane glycoprotein that plays a central role in autoimmunity. Systemic lupus erythematosus (SLE) patients show reduced CR2 levels, and complete deficiency of CR2 and CR1 promotes the development of anti-DNA antibodies in mouse models of SLE. Here we show that multiple forms of DNA, including bacterial, viral and mammalian DNA, bind to human CR2 with moderately high affinity. Surface plasmon resonance studies showed that methylated DNA bound with high affinity with CR2 at a maximal KD of 6nM. DNA was bound to the first two domains of CR2 and this binding was blocked by using a specific inhibitory anti-CR2 mAb. DNA immunization in Cr2−/− mice revealed a specific defect in immune responses to bacterial DNA. CR2 can act as a receptor for DNA in the absence of complement C3 fixation to this ligand. These results suggest that CR2 plays a role in the recognition of foreign DNA during host-immune responses. This recognition function of CR2 may be a mechanism that influences the development of autoimmunity to DNA in SLE.

Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)‐bisphosphate regulates B‐cell activation and autoantibody production

TAPP1 and TAPP2 (where TAPP is tandem PH domain containing protein) are dual PH domain adaptors that selectively bind PI(3,4)P2 (phosphatidylinositol (3,4)-bisphosphate). PI(3,4)P2 is a lipid messenger generated by phosphoinositide 3-kinase (PI3K) and SHIP, both of which are critical regulators of B-cell activation. To determine the functional role of TAPP-PI(3,4)P2 interactions, we utilized a double knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPP1 and TAPP2. TAPP KI mice show evidence of altered B-cell development, but generate phenotypically normal mature B-cell populations. Total serum immunoglobulin IgM and IgG levels were found to be markedly elevated in TAPP KI mice. B cells purified from TAPP KI mice were hyper-responsive to antigen receptor cross-linking, showing increased proliferation, CD86 expression, and Akt phosphorylation on Ser473 and Thr308. Female TAPP KI mice developed elevated levels of anti-DNA and antinuclear antibodies with age, associated with IgG deposition in kidneys and significant glomerulonephritis pathology. Together our results indicate that interaction of TAPPs with PI(3,4)P2 mediates feedback inhibition impacting on BCR signaling, with functional significance for control of autoreactive B cells.

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

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.

The Inhibition of Anti-DNA Binding to DNA by Nucleic Acid Binding Polymers

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs) on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3), hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.