Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti–dsDNA autoantibody production, a SLE–related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti–dsDNA autoantibody positive (anti–dsDNA +, n = 811) and anti–dsDNA autoantibody negative (anti–dsDNA –, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti–dsDNA + SLE. Far fewer and weaker associations were observed for anti–dsDNA – SLE. For example, rs7574865 in STAT4 had an OR for anti–dsDNA + SLE of 1.77 (95% CI 1.57–1.99, p = 2.0E-20) compared to an OR for anti–dsDNA – SLE of 1.26 (95% CI 1.12–1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti–dsDNA + SLE and were not associated with anti–dsDNA – SLE. In conclusion, we identified differential genetic associations with SLE based on anti–dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti–dsDNA – SLE.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can involve virtually any organ system. SLE patients produce antibodies that bind to their own cells and proteins (autoantibodies) which can cause irreversible organ damage. One particular SLE–related autoantibody directed at double-stranded DNA (anti–dsDNA) is associated with kidney involvement and more severe disease. Previous genome-wide association studies (GWAS) in SLE have studied SLE itself, not particular SLE manifestations. Therefore, we conducted this GWAS of anti–dsDNA autoantibody production to identify genetic associations with this clinically important autoantibody. We found that many previously identified SLE–associated genes are more strongly associated with anti–dsDNA autoantibody production than SLE itself, and they may be more accurately described as autoantibody propensity genes. No strong genetic associations were observed for SLE patients who do not produce anti–dsDNA autoantibodies, suggesting that other factors may have more influence in developing this type of SLE. Further investigation of these autoantibody propensity genes may lead to greater insight into the causes of autoantibody production and organ damage in SLE.
Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies associated with specific clinical manifestations. Previous studies have shown an association between differential DNA methylation and SLE susceptibility, but have not investigated SLE-related autoantibodies. Our goal was to determine whether DNA methylation is associated with production of clinically relevant SLE-related autoantibodies, with an emphasis on the anti-dsDNA autoantibody. In this study, we characterized the methylation status of 467,314 CpG sites in 326 women with SLE. Using a discovery and replication study design, we identified and replicated significant associations between anti-dsDNA autoantibody production and the methylation status of 16 CpG sites (pdiscovery<1.07E-07 and preplication<0.0029) in 11 genes. Associations were further investigated using multivariable regression to adjust for estimated leukocyte cell proportions and population substructure. The adjusted mean DNA methylation difference between anti-dsDNA positive and negative cases ranged from 1.2% to 19%, and the adjusted odds ratio for anti-dsDNA autoantibody production comparing the lowest and highest methylation tertiles ranged from 6.8 to 18.2. Differential methylation for these CpG sites was also associated with anti-SSA, anti-Sm, and anti-RNP autoantibody production. Overall, associated CpG sites were hypomethylated in autoantibody positive compared to autoantibody negative cases. Differential methylation of CpG sites within the major histocompatibility region was not strongly associated with autoantibody production. Genes with differentially methylated CpG sites represent multiple biologic pathways, and have not been associated with autoantibody production in genetic association studies. In conclusion, hypomethylation of CpG sites within genes from different pathways is associated with anti-dsDNA, anti-SSA, anti-Sm, and anti-RNP production in SLE, and these associations are not explained by genetic variation. Thus, studies of epigenetic mechanisms such as DNA methylation represent a complementary method to genetic association studies to identify biologic pathways that may contribute to the clinical heterogeneity of autoimmune diseases.
Systemic lupus erythematosus (SLE) patients exhibit immense heterogeneity which is challenging from the diagnostic perspective. Emerging high throughput sequencing technologies have been proved to be a useful platform to understand the complex and dynamic disease processes. SLE patients categorised based on autoantibody specificities are reported to have differential immuno-regulatory mechanisms. Therefore, we performed RNA-seq analysis to identify transcriptomics of SLE patients with distinguished autoantibody specificities. The SLE patients were segregated into three subsets based on the type of autoantibodies present in their sera (anti-dsDNA+ group with anti-dsDNA autoantibody alone; anti-ENA+ group having autoantibodies against extractable nuclear antigens (ENA) only, and anti-dsDNA+ENA+ group having autoantibodies to both dsDNA and ENA). Global transcriptome profiling for each SLE patients subsets was performed using Illumina® Hiseq-2000 platform. The biological relevance of dysregulated transcripts in each SLE subsets was assessed by ingenuity pathway analysis (IPA) software. We observed that dysregulation in the transcriptome expression pattern was clearly distinct in each SLE patients subsets. IPA analysis of transcripts uniquely expressed in different SLE groups revealed specific biological pathways to be affected in each SLE subsets. Multiple cytokine signaling pathways were specifically dysregulated in anti-dsDNA+ patients whereas Interferon signaling was predominantly dysregulated in anti-ENA+ patients. In anti-dsDNA+ENA+ patients regulation of actin based motility by Rho pathway was significantly affected. The granulocyte gene signature was a common feature to all SLE subsets; however, anti-dsDNA+ group showed relatively predominant expression of these genes. Dysregulation of Plasma cell related transcripts were higher in anti-dsDNA+ and anti-ENA+ patients as compared to anti-dsDNA+ ENA+. Association of specific canonical pathways with the uniquely expressed transcripts in each SLE subgroup indicates that specific immunological disease mechanisms are operative in distinct SLE patients’ subsets. This ‘sub-grouping’ approach could further be useful for clinical evaluation of SLE patients and devising targeted therapeutics.
Our previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood.
The ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers.
These results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.
To compare the performance characteristics of cell-bound complement (C4d) activation products (CBCAPS) on erythrocyte (EC4d) and B cells (BC4d) with antibodies to double-stranded DNA (anti-dsDNA) and complement C3 and C4 in systemic lupus erythematosus (SLE).
The study enrolled 794 subjects consisting of 304 SLE and a control group consisting of 285 patients with other rheumatic diseases and 205 normal individuals. Anti-dsDNA and other autoantibodies were measured using solid-phase immunoassays while EC4d and BC4d were determined using flow cytometry. Complement proteins were determined using immunoturbidimetry. Disease activity in SLE was determined using a non-serological Systemic Lupus Erythematosus Disease Activity Index SELENA Modification. A two-tiered methodology combining CBCAPS with autoantibodies to cellular and citrullinated antigens was also developed. Statistical analyses used area under receiver operating characteristic curves and calculations of area under the curve (AUC), sensitivity and specificity.
AUC for EC4d (0.82±0.02) and BC4d (0.84±0.02) was higher than those yielded by C3 (0.73±0.02) and C4 (0.72±0.02) (p<0.01). AUC for CBCAPS was also higher than the AUC yielded by anti-dsDNA (0.79±0.02), but significance was only achieved for BC4d (p<0.01). The combination of EC4d and BC4d in multivariate testing methodology with anti-dsDNA and autoantibodies to cellular and citrullinated antigens yielded 80% sensitivity for SLE and specificity ranging from 70% (Sjogren's syndrome) to 92% (rheumatoid arthritis) (98% vs. normal). A higher proportion of patients with SLE with higher levels of disease activity tested positive for elevated CBCAPS, reduced complement and anti-dsDNA (p<0.03).
CBCAPS have higher sensitivity than standard complement and anti-dsDNA measurements, and may help with the differential diagnosis of SLE in combination with other autoantibodies.
Autoimmune Diseases; Systemic Lupus Erythematosus; Autoantibodies
Autoantibodies against a panoply of self-antigens are seen in systemic lupus erythematosus, but only a few (anti-Sm/RNP, anti-Ro/La, anti-dsDNA) are common. The common lupus autoantigens are nucleic acid complexes and levels of autoantibodies can be extraordinarily high. We explore why that is the case. Lupus is associated with impaired central or peripheral B-cell tolerance and increased circulating autoreactive B cells. However, terminal differentiation is necessary for autoantibody production. Nucleic acid components of the major lupus autoantigens are immunostimulatory ligands for toll-like receptor (TLR)7 or TLR9 that promote plasma cell differentiation. We show that the levels of autoantibodies against the U1A protein (part of a ribonucleoprotein) are markedly higher than autoantibodies against other antigens, including dsDNA and the non-nucleic acid-associated autoantigens insulin and thyroglobulin. In addition to driving autoantibody production, TLR7 engagement is likely to contribute to the pathogenesis of inflammatory disease in lupus.
autoantibodies; lupus erythematosus; systemic; innate immunity; TLR7; B cells; immune tolerance
Systemic lupus erythematosus (SLE) is a multi-systemic autoimmune
disease leading to immunological aberrations and excessive multiple autoantibody
production. The aim of this study was to investigate the prevalence of
multiple autoantibodies in SLE patients utilizing the multiplex system method.
We analyzed the presence of elevated titers of anti-Ro, anti-La, anti-RNP,
anti-Sm, anti-Jo1, anti-centromere, anti-Scl-70, anti-histone, and anti-dsDNA
antibodies in 199 serum samples (113 SLE patients, 86 healthy donors). We
compared the type, level and number of autoantibodies and the correlation
the autoantibody profile and disease severity utilizing the SLEDAI score.
Elevated titers of at least one autoantibody were detected in 48% of 42 SLE
patients. Elevated titers of anti-Ro antibodies were most commonly detected. The
distribution of specific autoantibodies was: anti-Ro- 23.8%, anti-dsDNA- 19%,
anti-histone- 19%, anti-RNP- 14.2%, anti-La antibodies- 11.9%, anti-Sm- 7.1%,
anti-Scl 70-4.7%, and anti-centromere- 2.4%. Utilizing ROC analysis, the sensitivity
and specificity of anti-DNA antibodies at a cutoff value of 34 IU/ml were 87.1% and
79.4% respectively. Elevated titers of anti-Jo1 antibody were not detected. There
was a correlation with the titer of anti-Ro antibodies and disease activity by the
SLEDAI score. Seven patients harbored one autoantibody only, 15 patients
harbored 2-3 autoantibodies, 3 patients harbored 4-5 autoantibodies, and one
patient harbored 6 autoantibodies. A correlation between the number of
autoantibodies per patient and disease severity was found. One patient with
a multitude of
autoantibodies had severe lupus and a myriad of clinical manifestations.
In conclusion, the multiplex system is specific and sensitive, provides
an autoantibody profile in a single test, and may be useful as a diagnostic
test for SLE. Elevated anti-Ro antibodies are associated with
severe disease. An autoantibody load may be indicative of more severe disease.
Sjogren's syndrome type B (SSB)/La antibody is an autoantibody generally observed in connective tissue diseases whereas double-stranded deoxyribonucleic acid (dsDNA) antibodies are the most characteristic autoantibodies found in systemic lupus erythematosus (SLE) patients. The relationship of these autoantibodies remains unclear. The aim of the study was to determine the profile of antibody production in rabbits immunized with synthetic SSB peptides alone or with dsDNA. For this purpose, 214–225aa peptide of SSB antigen was synthesized based on the organic chemistry solid-phase peptide synthesis. Rabbits were immunized with the following antigens: i) synthetic SSB peptides linked with keyhole limpet hemocyanin (KLH); ii) dsDNA; iii) SSB plus dsDNA; iv) KLH; and v) phosphate-buffered saline. SSB peptide antibody was measured using the enzyme-linked immunosorbent assay while extractable nuclear antigens (ENA) antibody and dsDNA antibody were measured by immunoblotting and immunofluorescence, respectively. The results showed that a specific anti-SSB peptide antibody was produced following immunization with SSB epitope alone or with dsDNA. The SSB peptide antibody titer in the coimmunization group was higher than that of the SSB alone group. In addition, antibodies against ribonucleoprotein (RNP), Smith and/or dsDNA were detected in rabbits of the coimmunization group. The presence of anti-dsDNA antibodies in the rabbits immunized with SSB peptide suggested the induction of epitope spreading. In conclusions, SSB antibodies were produced in rabbits immunized with SSB peptide or SSB+dsDNA, whereas SSB antibody titers were higher in the coimmunization group. Furthermore, coimmunization was associated with epitope spreading.
Sjogren's syndrome type B antigen peptides; double-stranded deoxyribonucleic acid; co-immunization; epitope spreading
Systemic lupus erythematosus is characterized by production of autoantibodies to RNA or DNA–protein complexes such as small nuclear ribonucleoproteins (snRNPs). A role of Epstein–Barr virus in the pathogenesis has been suggested. Similar to Epstein–Barr virus, cytomegalovirus (CMV) infects the majority of individuals at a young age and establishes latency with a potential for reactivation. Homology of CMV glycoprotein B (UL55) with the U1snRNP-70 kDa protein (U1–70 k) has been described; however, the role of CMV infection in production of anti-snRNPs is controversial. We investigated the association of CMV serology and autoantibodies in systemic lupus erythematosus.
Sixty-one Mexican patients with systemic lupus erythematosus were tested for CMV and Epstein–Barr virus serology (viral capsid antigen, IgG, IgM) and autoantibodies by immunoprecipitation and ELISA (IgG and IgM class, U1RNP/Sm, U1–70 k, P peptide, rheumatoid factor, dsDNA, β2-glycoprotein I).
IgG anti-CMV and IgM anti-CMV were positive in 95% (58/61) and 33% (20/61), respectively, and two cases were negative for both. Clinical manifestation and autoantibodies in the IgM anti-CMV(+) group (n = 20) versus the IgM anti-CMV(-)IgG (+) (n = 39) group were compared. Most (19/20) of the IgM anti-CMV(+) cases were IgG anti-CMV(+), consistent with reactivation or reinfection. IgM anti-CMV was unrelated to rheumatoid factor or IgM class autoantibodies and none was positive for IgM anti-Epstein–Barr virus–viral capsid antigen, indicating that this is not simply due to false positive results caused by rheumatoid factor or nonspecific binding by certain IgM. The IgM anti-CMV(+) group has significantly lower levels of IgG anti-U1RNP/Sm and IgG anti-U1–70 k (P = 0.0004 and P = 0.0046, respectively). This finding was also confirmed by immunoprecipitation. Among the IgM anti-CMV(-) subset, anti-Su was associated with anti-U1RNP and anti-Ro (P < 0.05). High levels of IgG anti-CMV were associated with production of lupus-related autoantibodies to RNA or DNA–protein complex (P = 0.0077).
Our findings suggest a potential role of CMV in regulation of autoantibodies to snRNPs and may provide a unique insight to understand the pathogenesis.
The pathogenic hallmark of systemic lupus erythematosus (SLE or lupus) is the autoimmune response against self nuclear antigens, including dsDNA. The increased expression of the pro-inflammatory cytokine IL-1β has been found in the cutaneous lesion and peripheral blood mononuclear cells from lupus patients, suggesting a potential involvement of this cytokine in the pathogenesis of lupus. IL-1β is produced primarily by innate immune cells like monocytes and can promote Th17 cell response, which is increased in lupus. IL-1β production requires cleaving pro-IL-β into IL-1β by the caspase-1-associated multiprotein complex called inflammasomes. Here we show that self dsDNA induces IL-1β production from human monocytes dependently of serum or purified IgG containing anti-dsDNA antibodies by activating the NLRP3 inflammasome. Reactive oxygen species (ROS) and K+ efflux were involved in this activation. Knocking down the NLRP3 or inhibiting caspase-1, ROS and K+ efflux decreased IL-1β production. Supernatants from monocytes treated with a combination of self dsDNA and anti-dsDNA antibody-positive serum promoted IL-17 production from CD4+ T cells in an IL-1β dependent manner. These findings provide new insights in lupus pathogenesis by demonstrating that self dsDNA together with its autoantibodies induces IL-1β production from human monocytes by activating the NLRP3 inflammasome through inducing ROS synthesis and K+ efflux, leading to the increased Th17 cell response.
Interferon-α (IFNα) is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variation near IRF7 is implicated in SLE susceptibility. SLE-associated autoantibodies can stimulate IFNα production through the Toll-like receptor/IRF7 pathway. This study was undertaken to determine whether variants of IRF7 act as risk factors for SLE by increasing IFNα production and whether autoantibodies are important to this phenomenon.
We studied 492 patients with SLE (236 African American, 162 European American, and 94 Hispanic American subjects). Serum levels of IFNα were measured using a reporter cell assay, and single-nucleotide polymorphisms (SNPs) in the IRF7/PHRF1 locus were genotyped.
In a joint analysis of European American and Hispanic American subjects, the rs702966 C allele was associated with the presence of anti–double-stranded DNA (anti-dsDNA) antibodies (odds ratio [OR] 1.83, P = 0.0069). The rs702966 CC genotype was only associated with higher serum levels of IFNα in European American and Hispanic American patients with anti-dsDNA antibodies (joint analysis P = 4.1 × 10−5 in anti-dsDNA–positive patients and P = 0.99 in anti-dsDNA–negative patients). In African American subjects, anti-Sm antibodies were associated with the rs4963128 SNP near IRF7 (OR 1.95, P = 0.0017). The rs4963128 CT and TT genotypes were associated with higher serum levels of IFNα only in African American patients with anti-Sm antibodies (P = 0.0012). In African American patients lacking anti-Sm antibodies, an effect of anti-dsDNA–rs702966 C allele interaction on serum levels of IFNα was observed, similar to the other patient groups (overall joint analysis P = 1.0 × 10−6). In European American and Hispanic American patients, the IRF5 SLE risk haplotype showed an additive effect with the rs702966 C allele on IFNα level in anti-dsDNA–positive patients.
Our findings indicate that IRF7/PHRF1 variants in combination with SLE-associated autoantibodies result in higher serum levels of IFNα, providing a biologic relevance for this locus at the protein level in human SLE in vivo.
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies, which plays an important role in disease development and progression. Lupus preferentially affects women during their reproductive years. The pathogenesis of lupus is contributed by both genetic factors and epigenetic modifications that arise from exposure to the environment. Epigenetic marks, including DNA methylation, histone post-translational modifications and microRNAs (miRNAs), interact with genetic programs to regulate immune responses. Epigenetic modifications influence gene expression and modulate B cell functions, such as class switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation, thereby informing the antibody response. Epigenetic dysregulation can result in aberrant antibody responses to exogenous antigens or self-antigens, such as chromatin, histones and dsDNA in lupus. miRNAs play key roles in the post-transcriptional regulation of most gene-regulatory pathways and regulate both the innate and the adaptive immune responses. In mice, dysregulation of miRNAs leads to aberrant immune responses and development of systemic autoimmunity. Altered miRNA expression has been reported in human autoimmune diseases, including lupus. The dysregulation of miRNAs in lupus could be the result of multiple environmental factors, such as sex hormones and viral or bacterial infection. Modulation of miRNA is a potential therapeutic strategy for lupus.
activation-induced cytidine deaminase (AID); antibody; autoantibody; autoimmunity; B cell; class switch DNA recombination (CSR); epigenetics; microRNA; somatic hypermutation (SHM); systemic lupus erythematosus (SLE)
To evaluate the role of serum IgG, IgM and IgA anti-dsDNA antibody isotypes in the diagnosis of systemic lupus erythematosus (SLE), and their association with clinical features and disease activity, in a large cohort of SLE patients.
Sera of 200 SLE patients (mean age 34±10.3 years; 26 male and 174 female; median duration of disease 115 months, range 7–378), and of 206 controls, including 19 Sjögren's syndrome, 27 rheumatoid arthritis, 26 psoriatic arthritis, 15 idiopathic inflammatory myopathies (IIM), 13 systemic sclerosis, 49 infectious diseases and 57 healthy subjects, were tested for anti-dsDNA IgG, IgM and IgA isotypes.
Selecting a cutoff corresponding to 95% specificity, the sensitivity of IgG, IgM and IgA anti-dsDNA antibodies in SLE was 55%, 30% and 49%, respectively; 12.5%, 1% and 7.5% of SLE patients had positive IgG, IgM or IgA isotype alone, respectively. SLE patients with glomerulonephritis showed higher levels of IgA anti-dsDNA (p = 0.0002), anti-dsDNA IgG/IgM (p = 0.001) and IgA/IgM (p<0.0001) ratios than patients without renal disease. No significant associations have been found between anti-dsDNA isotypes and other clinical features. IgA anti-dsDNA (p = 0.01) (but not IgG or IgM) and IgG/IgM ratio (p = 0.005) were significantly higher in patients with more active disease (ECLAM score >4).
The detection of IgA anti-dsDNA autoantibodies seems to improve our ability to diagnose SLE and to define lupus nephritis phenotype and active disease. By contrast, IgM anti-dsDNA antibodies might be protective for renal involvement. These data support the hypothesis that anti-dsDNA antibody class clustering may help to refine SLE diagnosis and prognosis.
Despite the uncertainty in the diagnosis of neuropsychiatric involvement in systemic lupus erythematosus (SLE), attempts have been made to record the association of certain antibodies in serum with neuropsychiatric (NP) manifestations. We aimed to assess the behaviour and the association of serum and cerebrospinal fluid (CSF) autoantibodies with NP manifestations in SLE patients (NPSLE).
Forty-seven SLE patients, hospitalized because of NP manifestations were included. They were evaluated at hospitalization and six months later, and serum and CSF samples were obtained at each evaluation. As controls, serum samples were taken from 49 non-NPSLE patients at hospitalization and six months later; serum and CSF samples were also obtained from 6 SLE patients with septic meningitis, 16 surgical SLE patients and 25 patients without autoimmune diseases. Antinuclear, anti-dsDNA, anti-ribosomal P, Anti-N-Methyl-D-Aspartate receptor (NMDAR), anti-cardiolipin, and anti-β2 glycoprotein-I antibodies were measured. In serum, anti-ribosomal P, anti-NMDAR, and other antibodies did not differentiate among SLE groups, and the levels of all antibodies were similar among the SLE groups. Six-months later, this scenario remained unchanged and the decrease in the levels of some autoantibodies reflected a decline in disease activity, rather than a change in NPSLE. In CSF, only the presence and the levels of anti-NMDAR antibodies showed a characteristic distribution in central NPSLE and septic meningitis patients. Six months later the prevalence of most antibodies in CSF did not change, however the levels of anti-dsDNA, anti-ribosomal P, and anti-NMDAR decreased.
In NPSLE, autoantibodies in serum do not reflect their behaviour in CSF. All autoantibodies were elevated in septic meningitis reflecting the global penetration of serum antibodies into the CSF in this condition. Anti-NMDAR antibodies in CSF identified patients with central NPSLE; their continued presence in CSF 6 months after neurologic symptoms raise questions regarding the conditions under which they are pathogenic.
Systemic lupus erythematosus (SLE) is characterized by production of a variety of autoantibodies. Although anti-double-stranded DNA (anti-dsDNA) antibodies contribute to the pathogenesis of lupus nephritis (LN), they are not sufficient for diagnosis and evaluation of disease activity. To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system. We screened 17 proteins that showed higher positive signals in the active phase than in the inactive phase of SLE, and higher positive signals in the serum of SLE patient with nephritis than in that of patient without nephritis. Of these, two LN-associated autoantigens, ribosomal RNA-processing protein 8 (RRP8) and spermatid nuclear transition protein 1 (TNP1) were identified by immunoprecipitation and immunofluorescence of renal tissues. Circulating anti-RRP8 and anti-TNP1 autoantibodies were recognized and deposited as an immune complex (IC) in glomeruli. IC was deposited preferentially in glomeruli rather than in other organs in C57BL/6 mice injected with RRP8 or TNP1. ELISA analysis of sera from patients with various rheumatic diseases demonstrated reactivity for RRP8 and TNP1 in 20% and 14.7% of SLE patients, respectively, whereas there was little or no reactivity in patients with other rheumatic diseases. Among SLE patients, 63.6% and 45.5% of those with LN were positive for anti-RRP8 and anti-TNP1 antibodies, compared with 12.5% and 9.4% of SLE patients without nephritis, respectively. Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA. These proteins released from apoptotic cells form ICs with each autoantibody, and their ICs may become trapped at anionic sites in the glomerular basement membrane, leading to deposition in glomeruli. These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.
An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.
Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.
B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.
The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.
Anti-dsDNA antibodies are the most studied antibodies of the lupus-related autoantibodies. The dogma is that these are the most important autoantibodies in systemic lupus erythematosus. In this review, evidence is presented to show that these antibodies (as measured by modern clinical laboratories) are not the most important autoantibodies in the diagnosis of systemic lupus erythematosus, and are of limited value in clinical correlation and in predicting disease flares. In addition, they are not likely to be the initiating autoantibodies in lupus nephritis. Thus, several pervasively held beliefs on anti-dsDNA antibodies are not valid. We suggest that anti-dsDNA antibodies should be considered as just one of the many autoantibodies associated with systemic lupus erythematosus.
Systemic lupus erythematosus; anti-dsDNA antibodies; autoantibodies; lupus nephritis
For the development of rabbit models of Systemic Lupus Erythematosus (SLE), immunoglobulin allotype-defined pedigreed rabbits from the National Institute of Allergy and Infectious Diseases rabbit resource more closely approximate human populations due to their non-inbred pedigreed structure. In an initial study from this laboratory, peptides (SM and GR) from the spliceosomal Smith (Sm) and the NMDA glutamate receptor NR2b, on branched polylysine backbones (BB) elicited antinuclear and anti-dsDNA autoantibodies typical of SLE, as well as seizures and nystagmus sometimes observed as neurological manifestations in SLE patients. This suggested the feasibility of further selective breeding to develop a more reproducible rabbit model for investigations of SLE. Here we report the results of GR-MAP-8 and control BB immunization on autoantibody responses in a group of 24 rabbits specifically bred and developed from parents and ancestors tested for autoantibody responses. The changes in hematological profile and blood chemistry in the experimental rabbits were evaluated along with autoantibody responses. Elevations of total white blood cell (WBC), monocyte, eosinophil and basophil counts that developed following immunizations were moderately influenced by litter and presence of the antibody heavy chain allotype VH1a1. Autoantibody development followed a sequential pattern with anti-nuclear antibodies (ANA) followed by anti-dsDNA and subsequently anti-Sm and anti-RNP similar to SLE patients. High autoantibody levels to one autoantigen were not always associated with antibody response to another. Female rabbits had higher prevalence and levels of autoantibodies similar to human SLE. Higher autoantibody levels of anti-dsDNA and -ANA were observed among some full sibs and the presence of high responder ancestors in the pedigree was associated the augmented responses. We observed significant association between highest antibody responses to GR-MAP-8 and highest anti-dsDNA levels. Naturally occurring autoantibodies were found in some pre-immune sera and some unique ANA fluorescent staining patterns within the experimental group were observed. Background immunofluorescence in pre-immune sera, distinct patterns of programmed autoantibody responses unique among individual rabbits may have been modulated by genetic constitution, gender and environmental factors including exposure to antigens. The high incidence and intensity of autoantibody responses among descendants of high responders suggest that there may be an additive mode of inheritance with high heritability. It is conceivable that further rigorous pedigree selection for autoantibody responses could lead to development of rabbit models with spontaneous occurrence of SLE like serology and disease phenotypes.
Rabbits; Autoantibodies; Antibody heavy chain allotypes; Genetics; Lupus
Autoimmune diseases are considered the 3rd leading cause of morbidity and mortality in the industrialized countries. Autoimmune thyroid diseases (ATDs) are associated with high prevalence of nonorgan-specific autoantibodies, such as antinuclear antibodies (ANA), antidouble-stranded deoxyribonucleic acid (anti-dsDNA), antiextractable-nuclear antigens (anti-ENAs), rheumatoid factor (RF), and anticyclic-citrullinated peptides (anti-CCP) whose clinical significance is unknown.
We aimed to assess the prevalence of various nonorgan-specific autoantibodies in patients with ATD, and to investigate the possible association between these autoantibodies and occurrence of rheumatic diseases and, if these autoantibodies could be considered as predictor markers for autoimmune rheumatic diseases in the future.
This study had 2 phases: phase 1; in which 61 ATD patients free from rheumatic manifestations were assessed for the presence of these nonorgan-specific autoantibodies against healthy 61 control group, followed by 2nd phase longitudinal clinical follow-up in which cases are monitored systematically to establish occurrence and progression of any rheumatic disease in association to these autoantibodies with its influences and prognosis.
Regarding ATD patients, ANA, anti-dsDNA, Anti-ENA, and RF were present in a percentage of (50.8%), (18%), (21.3%), and (34.4%), respectively, with statistically significance difference (P < 0.5) rather than controls. Nearly one third of the studied group (32.8%) developed the rheumatic diseases, over 2 years follow-up. It was obvious that those with positive anti-dsDNA had higher risk (2.45 times) to develop rheumatic diseases than those without. There was a statistically significant positive linear relationship between occurrence of disease in months and (age, anti-dsDNA, anti-CCP, RF, and duration of thyroiditis). Anti-dsDNA and RF are the most significant predictors (P < 0.0001).
ATD is more associated with rheumatic diseases than previously thought. Anti-dsDNA, RF, and anti-CCP antibodies may be used as predictive screening markers of systemic lupus erythematosus and RA, with early referral to rheumatologists for close follow-up and early diagnoses for appropriate disease management of the disease, as early disease control will allow better quality of life.
antidouble-stranded DNA; antiextractable-nuclear antigens; antinuclear antibodies; autoimmune thyroid diseases; rheumatoid factor; anticyclic-citrullinated peptides
Systemic lupus erythematosus (SLE) is characterized by impaired efferocytosis and aberrant activation of innate immunity. We asked if shedding of MER receptor tyrosine kinase (MerTK) and AXL into soluble (s) ectodomains was related to immunological and clinical aspects of SLE.
Levels of sMER and sAXL in the plasma of 107 SLE patients and 45 matched controls were measured by ELISA. In 40 consecutive SLE patients, we examined potential correlations between either sMER or sAXL and plasma levels of sCD163, a marker of M2 activation. All three soluble receptors were measured in supernatants of monocytes/macrophages cultured in various immunological conditions. Membrane expression of MerTK, AXL and CD163 was assessed by flow cytometry.
Both sMER and sAXL were associated with anti-chromatin and anti-phospholipid autoantibodies, and with hematological and renal involvement. However, sMER and sAXL did not significantly correlate with each other; sAXL correlated with growth arrest-specific 6 (Gas6), whereas sMER correlated with reduced free protein S (PROS) levels. Only sMER showed significant associations with lupus-specific anti-dsDNA, anti-Sm, anti-ribonucleoprotein (anti-RNP) and anti-Ro60 autoantibodies. Strong correlations with disease activity indices (Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), complement reduction, titer of circulating anti-dsDNA) were found for sMER, not for sAXL. Patients with active SLEDAI, nephritis, anti-dsDNA and anti-Ro60 positivity showed higher levels of sMER compared to controls. Levels of sMER, not sAXL, correlated with sCD163 levels, and these correlated with SLEDAI. Production of sMER and sCD163 occurred under “M2c” polarizing conditions, whereas sAXL was released upon type-I IFN exposure.
Alterations in homeostasis of anti-inflammatory and efferocytic “M2c” monocytes/macrophages may have a role in immunopathogenesis of SLE.
Immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) play important roles in the generation of autoantibodies in systemic lupus erythematosus. Systemic lupus is characterized by the production of an array of pathogenic high-affinity mutated and class-switched, mainly IgG, antibodies to a variety of self-antigens, including nuclear components, such as dsDNA, histones and chromatin. We previously found that MRL/Faslpr/lpr mice, which develop a systemic autoimmune syndrome sharing many features with human lupus, display greatly upregulated CSR, particularly to IgG2a, in B cells of the spleen, lymph nodes and Peyer’s patches. In MRL/Faslpr/lpr mice, the significant upregulation of CSR is associated with increased expression of activation-induced cytidine deaminase (AID), which is critical for CSR and SHM. We also found that HoxC4 directly activates the promoter of the AID gene to induce AID expression, CSR and SHM. Here, we show that in both lupus patients and lupus-prone MRL/Faslpr/lpr mice, the expression of HoxC4 and AID is significantly upregulated. To further analyze the role of HoxC4 in lupus, we generated HoxC4−/− MRL/Faslpr/lpr mice. In these mice, HoxC4-deficiency resulted in reduced AID expression, impaired CSR and decreased serum anti-dsDNA IgG, particularly IgG2a, autoantibodies, which were associated with a reduction in IgG deposition in kidney glomeruli. In addition, consistent with our previous findings that in MRL/Faslpr/lpr mice, upregulated AID expression is associated with extensive DNA lesions, comprising deletions and insertions in the IgH locus, we found c-Myc to IgH(c-Myc/IgH) translocations to occur frequently in B cells of MRL/Faslpr/lpr mice. The frequency of such translocations was significantly reduced in HoxC4−/− MRL/Faslpr/lpr mice. These findings suggest that in lupus B cells, upregulation of HoxC4 plays a major role in dysregulation of AID expression, thereby increasing CSR and autoantibody production, and promoting c-Myc/IgH translocations.
Activation-induced cytidine deaminase (AID); B cell lymphoma; cancer; class switch DNA recombination (CSR); c-Myc/IgH translocation; HoxC4; MRL/Faslpr/lpr mice; systemic lupus erythematosus (SLE)
The diagnostic significance of anti-double-stranded deoxyribonucleic acid (anti-dsDNA) determination was evaluated in a prospective manner from 1974 to 1982 in a group of 441 patients without systemic lupus erythematosus whose sera were found to contain antibodies to dsDNA on routine screening (Farr assay). Within one year 69% (304) of these patients fulfilled the preliminary American Rheumatism Association (ARA) criteria for systemic lupus erythematosus (SLE). Eighty-two of the remaining 137 patients were followed up for several years. At the end of the study 52% of these patients had also developed systemic lupus erythematosus. Patients who developed systemic lupus erythematosus were characterised by the occurrence of relatively high avidity anti-dsDNA in the circulation compared with patients who did not develop systemic lupus erythematosus. It can be concluded that about 85% of patients without systemic lupus erythematosus with anti-dsDNA in the circulation will develop SLE within a few years. Taking into account the relative avidity of anti-dsDNA, as determined by calculation of Farr/polyethylene glycol (PEG) ratios, we conclude that patients with relatively high avidity anti-dsDNA are more prone to develop systemic lupus erythematosus than patients with relatively low avidity anti-dsDNA.
Glomerulonephritis is a major cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). Deposition of autoantibodies in the glomeruli plays a key role in the development of lupus nephritis (LN). Different groups have proposed that either anti-nucleosome antibodies or antibodies that bind the intrinsic renal antigen, α-actinin, are central to the pathogenesis of LN. These theories have been based mainly on cross-sectional studies in patients and on experiments in animal models. No previous longitudinal studies have compared the relationships between levels of these antibodies and markers of renal function. We assessed how well anti-α-actinin, anti-nucleosome and anti-double-stranded DNA (anti-dsDNA) antibodies reflected renal outcome measures in patients with new-onset LN followed for up to 2 years.
Renal disease activity was monitored by measuring urine protein/creatinine ratio (PCR), serum albumin and a composite outcome of renal remission. At each time point, anti-nucleosome and anti-α-actinin antibodies were measured by enzyme-linked immunosorbent assay. High-avidity anti-dsDNA antibodies were measured using the Farrzyme assay. We analysed relationships between levels of the three antibodies and between antibody levels and renal outcome measures over time.
Levels of anti-nucleosome and anti-dsDNA were positively correlated with each other (r = 0.6, P = 0.0001) but neither correlated with anti-α-actinin level. At baseline, mean anti-nucleosome levels were higher in patients with LN than in healthy controls (0.32 versus 0.01, P < 0.001). The same was true for anti-dsDNA antibodies (0.50 versus 0.07, P < 0.001) but not for anti-α-actinin (0.33 versus 0.29). Over the follow-up period, anti-nucleosome and anti-dsDNA levels associated positively with urine PCR (P = 0.041 and 0.051, respectively) and negatively with serum albumin (P = 0.027 and 0.032, respectively). Both anti-nucleosome and anti-dsDNA levels were significantly lower during renal remission than when renal disease was active (P = 0.002 and 0.003, respectively). However, there was no relationship between anti-α-actinin levels and urine PCR, serum albumin or remission status.
This prospective longitudinal clinical study is the first to compare levels of anti-nucleosome, anti-dsDNA and anti-α-actinin antibodies in the same patients with SLE. Our results support the concept that, in the majority of patients, anti-nucleosome antibodies play a major role in pathogenesis of LN, in contrast to anti-α-actinin antibodies.
Systemic lupus erythematosus is a severe autoimmune disease that affects multiple organ systems resulting in diverse symptoms and outcomes. It is characterized by antibody production to a variety of self-antigens, but it is specifically associated with those against anti-dsDNA. Anti-dsDNA antibodies are present before the onset of clinical disease and are associated with severe manifestations of lupus such as glomerulonephritis. Their levels fluctuate with changes in disease activity and, in combination with the levels of complement proteins C3 and C4, are strong indicators of disease flare and treatment response in patients with lupus. The decreased complement levels that are noted during flares of lupus activity are believed to be secondary to increased autoantibody production and immune complex formation that results in tissue damage; however, recent data suggest that complement activation can also drive development of these pathogenic autoantibodies. This review will explore the various roles of complement in the development and pathogenesis of anti-dsDNA antibodies.
SLE; Autoantibodies; Anti-dsDNA antibodies; Complement; Clearance
Systemic lupus erythematosus (SLE) is characterized by over production of autoantibodies. C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein that participates in the systemic response to inflammation. Anti-CRP antibodies might have biological functions of pathogenetic interest in SLE. We evaluated anti-CRP antibodies in Indian SLE patients and their association with anti-dsDNA antibodies and complement levels (C3 and C4). One hundred SLE patients diagnosed according to the American College of Rheumatology criteria were included. Disease activity was assessed using SLE disease activity index (SLEDAI). Anti-CRP autoantibodies were detected by enzyme linked immunosorbent assay. Anti-dsDNA antibodies were detected by indirect immunofluroscence test (Euroimmun Lubeck, Germany). High sensitivity CRP and complement levels (C3, C4) were detected using a Nephelometer. (BN ProSpec, Dade Behring, Germany). Anti-CRP antibodies were detected in 26% of SLE patients. Mean age of disease onset among anti-CRP positives was 22.4 ± 7.5, and 26.6 ± 9.3 years among anti-CRP negatives (P > 0.05). Anti-dsDNA positivity was significantly higher among anti-CRP positives (32.7%) as compared to anti-CRP negatives (16%) (P = 0.00519). No statistically significant difference was observed in SLEDAI scores of anti-CRP positive group and anti-CRP negative group (P > 0.05). We observed a positive correlation between anti-CRP antibodies and anti-dsDNA antibodies.
Anti-C reactive protein antibodies; anti-dsDNA antibodies; high sensitivity C-reactive protein; systemic lupus erythematosus