DNA In Systemic Lupus Erythematosus Research Articles

Whole-genome bisulfite sequencing identified the key role of the Src family tyrosine kinases and related genes in systemic lupus erythematosus.

As a multisystemic autoimmune illness, the basic mechanisms behind the pathophysiology of systemic lupus erythematosus (SLE) remain poorly understood. We aimed to investigate the possible significance of SLE's DNA methylation and gainfurtherinsightinto potential SLE-related biomarkers and therapeutic targets. We used whole genome bisulfite sequencing (WGBS) method to analyze DNA methylation in 4 SLE patients and 4 healthy people. 702 differentially methylated regions (DMRs) were identified, and 480 DMR-associated genes were annotated. We found the majority of the DMR-associated elements were enriched in repeat and gene bodies. The top 10 hub genes identified were LCK, FYB, PTK2B, LYN, CTNNB1, MAPK1, GNAQ, PRKCA, ABL1, and CD247. Compared to the control group, LCK and PTK2B had considerably decreased levels of mRNA expression in the SLE group. Receiver operating characteristic (ROC) curve suggested that LCK and PTK2B may be potential candidate biomarkers to predict SLE. Our study improved comprehension of the DNA methylation patterns of SLE and identified potential biomarkers and therapeutic targets for SLE.

The Role of Plasma Cell-Free Mitochondrial DNA and Nuclear DNA in Systemic Lupus Erythematosus.

The roles of plasma cell-free (pcf) mitochondrial DNA (mtDNApcf) and nuclear DNA (nDNApcf) in the pathogenesis of systemic lupus erythematosus (SLE) remain unclear. We analyzed the relative copies of mtDNApcf and nDNApcf and investigated their association with the levels of plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG), plasma malondialdehyde (MDA) and mRNA of leukocyte C-type lectin domain family 5 member A (CLEC5A) in SLE patients. A total of 80 SLE patients and 43 healthy controls (HCs) were enrolled. Their plasma samples were subjected to the measurements of mtDNApcf copies, nDNApcf copies, 8-OHdG and MDA, respectively. Their leukocytes were analyzed for CLEC5A mRNA expression. SLE patients had higher nDNApcf copies (2.84 ± 1.99 vs. 2.00 ± 0.88, p = 0.002), lower mtDNApcf copies (4.81 ± 6.33 vs. 9.83 ± 14.20, p = 0.032), higher plasma 8-OHdG (0.227 ± 0.085 vs. 0.199 ± 0.041 ng/mL, p = 0.016), lower plasma MDA (3.02 ± 2.20 vs. 4.37 ± 2.16 μM, p = 0.001) and similar leukocyte CLEC5A mRNA expression levels (1.21 ± 1.17 vs. 1.26 ± 1.05, p = 0.870), as compared with those of HCs. Among the HCs, SLE patients with SLE Disease Activity Index (SLEDAI) ≤8, and SLE patients with SLEDAI >8, their respective mtDNApcf copies decreased stepwisely (9.83 ± 14.20 vs. 6.28 ± 7.91 vs. 3.19 ± 3.35, p = 0.054). The nDNApcf copies of HCs, SLE patients without nephritis, and SLE patients with nephritis were increased stepwisely (2.00 ± 0.88 vs. 2.63 ± 1.74 vs. 3.16 ± 2.34, p = 0.043). Among SLE patients, higher nDNApcf copies were associated with higher levels of plasma 8-OHdG (p < 0.001) but lower plasma MDA (p = 0.019). Among HCs but not SLE patients, higher nDNApcf copies (p = 0.013) or lower mtDNApcf copies (p < 0.001) were related to higher levels of leukocyte CLEC5A mRNA expression. Higher nDNApcf, lower mtDNApcf, increased ROS-elicited oxidative DNA damage and dysregulated leukocyte CLEC5A expression might be implicated in the pathogenesis of SLE.

Contribution of Impaired DNASE1L3 Activity to Anti-DNA Autoantibody Production in Systemic Lupus Erythematosus.

Anti-DNA autoantibodies are pathogenic in systemic lupus erythematosus (SLE). Cell-free chromatin associated long DNA fragments are antigens for anti-DNA antibodies. In health state, released by cell death and actively secreted by live cells, these cell-free DNA are cleared by deoxyribonucleases (DNASES). In SLE, cell-free DNA are accumulated. The defective clearance of long fragments of cell-free DNA in SLE is largely attributed to impaired deoxyribonuclease 1 like 3 (DNASE1L3). DNASE1L3 null mutation results in monogenic SLE. The SLE risk single-nucleotide polymorphism (rs35677470) encodes R260C variant DNASE1L3, which is defective in secretion, leading to reduced levels of DNASE1L3. In addition, neutralizing autoantibodies to DNASE1L3 are produced in SLE to inhibit its enzymatic activity.

The aberrant expression of CD45 isoforms and levels of sex hormones in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a common autoimmune disease with significant gender bias in women, and sex hormones are considered to play an important role in the regulation of immune activity. The CD45 isoforms generated through alternative splicing of mRNA identify different functional status of lymphocytes and also are suggested as a biomarker for assessing the progression of SLE, while the modulation of CD45 expression in SLE patients is not clear. In this study, the peripheral blood sera of 46 SLE patients and 15 health individuals were collected for detecting the levels of sex hormones and immune associated factors. The expression of CD45 isoforms and the status of CD45 DNA methylation of the peripheral mononuclear blood cells were detected by flow cytometry and bisulfite sequencing PCR, respectively. The levels of complement C3 and IgA decreased, especially decline of the serum IgA to the level of selective immunoglobulin A deficiency, and the C-reactive protein increased in SLE patients when compared with healthy controls, which manifested the abnormal immune activity of the SLE patients. Sex hormones detection showed a decreased testosterone and increased prolactin in SLE. An accelerated expression of CD45RO, reduced CD45RA and CD45RB, and a relative hypermethylation of CD45 DNA in SLE were also identified that provided a clue to explain the possible regulatory mechanism for the immune function in SLE. The results indicated that the aberrant CD45 isoforms, DNA methylation and hormone levels might be correlated with the imbalanced immune activity of SLE patients. Key Points • Selective immunoglobulin A deficiency was significantly higher in SLE than in healthy individuals. • SLE patients had decreased testosterone and increased prolactin in the sera. • An aberrant expression of CD45 isoforms and CD45 DNA methylation were identified in SLE.

DNA as a self-antigen: nature and regulation.

High-affinity antibodies to double-stranded DNA are a hallmark of systemic lupus erythematosus (SLE) and are thought to contribute to disease flares and tissue inflammation such as nephritis. Notwithstanding their clinical importance, major questions remain about the development and regulation of these pathogenic anti-DNA responses. These include the mechanisms that prevent anti-DNA responses in healthy subjects, despite the constant generation of self-DNA and the abundance of DNA-reactive B cells; the nature and physical form of antigenic DNA in SLE; the regulation of DNA availability as an antigen; and potential therapeutic strategies targeting the pathogenic DNA in SLE. This review summarizes current progress in these directions, focusing on the role of secreted DNases in the regulation of antigenic extracellular DNA.

A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination.

NETosis, an antimicrobial form of neutrophil cell death, is considered a primary source of citrullinated autoantigens in rheumatoid arthritis (RA) and immunogenic DNA in systemic lupus erythematosus (SLE). Activation of the citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is believed to be essential for neutrophil extracellular trap (NET) formation and NETosis. PAD4 is therefore viewed as a promising therapeutic target to inhibit the formation of NETs in both diseases. In this review, we examine the evidence for PAD4 activation during NETosis and provide experimental data to suggest that protein citrullination is not a universal feature of NETs. We delineate two distinct biological processes, leukotoxic hypercitrullination (LTH) and defective mitophagy, which have been erroneously classified as “NETosis.” While these NETosis mimics share morphological similarities with NETosis (i.e., extracellular DNA release), they are biologically distinct. As such, these processes can be readily classified by their stimuli, activation of distinct biochemical pathways, the presence of hypercitrullination, and antimicrobial effector function. NETosis is an antimicrobial form of cell death that is NADPH oxidase-dependent and not associated with hypercitrullination. In contrast, LTH is NADPH oxidase-independent and not bactericidal. Rather, LTH represents a bacterial strategy to achieve immune evasion. It is triggered by pore-forming pathways and equivalent signals that cumulate in calcium-dependent hyperactivation of PADs, protein hypercitrullination, and neutrophil death. The generation of citrullinated autoantigens in RA is likely driven by LTH, but not NETosis. Mitochondrial DNA (mtDNA) expulsion, the result of a constitutive defect in mitophagy, represents a second NETosis mimic. In the presence of interferon-α and immune complexes, this process can generate highly interferogenic oxidized mtDNA, which has previously been mistaken for NETosis in SLE. Distinguishing NETosis from LTH and defective mitophagy is paramount to understanding the role of neutrophil damage in immunity and the pathogenesis of human diseases. This provides a framework to design specific inhibitors of these distinct biological processes in human disease.

Parvovirus B19 viremia in Egyptian adults with systemic lupus erythematosus

Objective This study was undertaken to investigate the seroprevalence of parvovirus B19 DNA in systemic lupus erythematosus (SLE) patients and its correlation with disease activity. Background Infection with parvovirus B19 (B19) has been suggested to contribute to the pathogenesis of SLE. B19 infection may simulate both clinical and laboratory features of SLE, presenting either as a potential first-time diagnosis of SLE or as an exacerbation of previously established disease. Patients and methods Sera from 30 adult patients with SLE and from 15 normal controls were examined for parvovirus B19 infection by means of nested PCRs to detect B19 DNA. Results B19 DNA was detected in two of 15 (13.5%) controls and in 12 (40%) of 30 SLE patients, with no significantly positive rates observed in SLE patients compared with healthy controls ( P > 0.05). B19 was positive in seven (58.5%) patients with severe activity compared with 8.3% without activity, 16.7% patients with mild activity, and 16.7% patients with moderate activity. No significant differences were observed between patients with virological positivity and those with virological negativity for B19 infection as regards the activity of SLE or any clinical manifestations of SLE. Conclusion Parvovirus B19 infection is not associated with SLE.

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.

The Role of Extracellular DNA in Autoimmunity in SLE

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the expression of antibodies to DNA. These antibodies form immune complexes that can stimulate cytokine production as well as deposit in the tissues to incite inflammation and damage. For the formation of immune complexes, the availability of extracellular DNA in an immunologically relevant form is essential. While apoptosis has been implicated as the source of this nuclear material in SLE, as shown with in vitro or in vivo systems, extracellular DNA can originate from apoptotic as well as necrotic cells. In experimental models, the release of DNA occurs with the administration of cells induced to die, in vitro as well as the administration of agents to induce cell death in situ. This release can be influenced by the presence of inflammatory cells such as macrophages that can interact with dead and dying cells to modulate the translocation of DNA from the inside to the outside of cells. In vivo, both glucocorticoids and oestrogens can modify the extent of DNA release from the administration of dead and dying cells. Together, these findings indicate that the generation of extracellular DNA in SLE can result from cell death and that steps in this process represent potential targets for new therapies.

Haemodialysis as a model for studying endogenous plasma DNA: oligonucleosome-like structure and clearance.

The rate of clearance of extracellular plasma DNA in man has important implications for pathogenetic mechanisms in systemic lupus erythematosus (SLE), as well as for certain other clinical states. Present knowledge of this parameter is derived exclusively from studies of injected, naked DNA in animals. Recent information indicates that the physiologic form of plasma DNA in SLE is that of oligonucleosome-like molecules rather than of naked DNA and consists of multimeric complexes of DNA bound to histone, probably arising from an apoptotic process. In order to study the rate at which these oligonucleosome-like complexes are removed from plasma and to do so in man rather than experimental animals, we exploited the observation that during haemodialysis large amounts of DNA are released, apparently within the dialysis coil, into the patient's plasma. Since this release appears to cease promptly with termination of the procedure, it offered the potential for estimating the rate of removal of such DNA from human plasma. Moreover, if that DNA, as postulated, were shown to possess an oligonucleosome-like structure resembling that found endogenously in human SLE, the relevance of such information to the human disease state would be further enhanced. The present results support the conclusion that DNA released into plasma during haemodialysis possesses such an oligonucleosome-like structure. The plasma half-life of that DNA in man was found not to exceed 4 min. The highly dynamic state thus implied for extracellular endogenous plasma DNA in man has important implications for pathogenetic mechanisms dependent on dsDNA in SLE. Moreover, individuals undergoing chronic haemodialysis, who are thereby exposed to a very large cumulative amount of such DNA, might serve as models for studying its long-term sequelae.

The Generation of Extracellular DNA in SLE: the Role of Death and Sex

DNA is a large macromolecule that plays a central role in the pathogenesis of systemic lupus erythematosus (SLE), serving as a target antigen of autoantibodies as well as a major component of immune complexes. These complexes can both promote immune disturbances as well as deposit in the kidney to incite inflammation. While the origin of anti-DNA autoantibodies in SLE has received intense investigation, the mechanisms by which DNA exits cells to form immune complexes in the circulation is not well understood. To determine the origin of DNA circulating in the blood in SLE, our laboratory has been using a murine model system to track the in vivo fate of DNA from Jurkat T cells that have been made apoptotic or necrotic in vitro and then administered to mice. Results of these studies indicate that DNA from apoptotic and necrotic cells appears in the blood in a time- and dose-dependent manner. Irrespective of origin, this DNA has properties of nucleosomes as shown by its molecular weight. The process of release requires the presence of macrophages and can be modified by glucocorticoids as well as inflammation. In addition, sex may play a role in the generation of extracellular DNA from dead cells as male and female mice differ in their responses in this model. Together, these studies clarify the origin of extracellular DNA circulating in the blood in SLE and suggest steps in this process that can be interdicted by novel therapy.

Peptide mimicking antigenic and immunogenic epitope of double-stranded DNA in systemic lupus erythematosus.

Autoantibodies to double-stranded (ds) DNA are an important diagnostic marker and pathogenic factor for systemic lupus erythematosus (SLE). Identifying dsDNA mimotopes is a way to discover diagnostic and therapeutic candidates for SLE. 'Mono-specific' SLE anti-dsDNA antibodies were obtained by affinity purification using dsDNA-coupled Sepharose column. Using the anti-dsDNA antibodies to screen a phage peptide library, we were able to identify a mimotope that has a motif peptide sequence of RLTSSLRYNP. This chemically synthesized peptide could be recognized by 88% (37 out of 42) of anti-dsDNA antibody-positive SLE sera with a cut-off point at mean + 3 SD of the negative control sera at OD(492). The reaction of the peptide with SLE sera in ELISA was highly correlated with that of dsDNA (r = 0.809, P < 0.0001). Of particular interest, not only dsDNA but also single-stranded (ss) DNA and native RNA could inhibit the binding of the peptide with SLE sera, suggesting that the mimotope is shared by ds and ssDNAs as well as native RNA, whereas denatured RNA was not observed to inhibit the binding. The peptide was also able to elicit an immune response in rabbits and the anti-peptide rabbit serum was observed to cross-react with the peptide, ss and dsDNAs, and ss and dsDNAs could inhibit the binding of the anti-peptide serum and the peptide. However, the inhibition was not obtained with RNA. Our findings demonstrate the potential of the peptide mimic in diagnostic tests of SLE, and in the investigation of anti-DNA antibody origin and of DNA-anti-DNA antibody interaction.

Cross-reactivity of antiidiotypic antibodies with DNA in systemic lupus erythematosus.

To assess the functional relationship between antibodies reactive with DNA and antibodies reactive with the idiotypes (idiopeptides) of anti-DNA antibodies that are associated with systemic lupus erythematosus (SLE) in mice. Antiidiotypic antibodies that appeared spontaneously in lupus mice, and others that were induced by immunization of normal, non-lupus mice, were analyzed for their reactivity by a range of direct binding, competition enzyme-linked immunosorbent assay (ELISA), and surface plasmon resonance (SPR) methods. Their reactions were assessed against synthetic peptides representing sequences of the V(H) region of anti-DNA monoclonal antibody (mAb) V-88, against the native mAb itself, and against mammalian DNA. In lupus mice, only sera with the highest reactivity against double-stranded DNA (dsDNA) also reacted with idiopeptides in ELISA, and this showed a strong statistical correlation. However, there was no significant relationship between antiidiotypic antibodies and anti-single-stranded DNA antibodies. Immunization of (BALB/c x NZW)F1 mice with idiopeptides p64 (V(H) residues 64-80) or p92 (V(H) residues 92-105) induced antibodies that reacted not only against the respective peptides, but also against the native parent anti-DNA mAb V-88. Furthermore, the immune antiidiopeptide antibodies cross-reacted with dsDNA. Competition SPR experiments with the BIAcore system supported this observation. The binding reaction of V(H) peptide p64 (representing the CDR-H2/FR-H3 region of V-88) with antiidiopeptide antibodies was inhibited by dsDNA. This study identified a unique set of autoantibodies in SLE. They react with both autoantibody idiotopes and with dsDNA, thus having a dual specificity for 2 autoantigens. Because these antiidiotope antibodies arise naturally during the development of lupus disease, and because they bind also to dsDNA, this provides a mechanism whereby the production of anti-dsDNA antibodies is stimulated. These idiotopes on autoantibodies in lupus act as natural mimotopes for inducing anti-dsDNA antibodies, which, due to their dual specificity, may significantly contribute to the pathology of nephritis in SLE.

The role of antibodies to DNA in systemic lupus erythematosus--a review and introduction to an international workshop on DNA antibodies held in London, May 1996.

The role of antibodies to DNA in systemic lupus erythematosus--a review and introduction to an international workshop on DNA antibodies held in London, May 1996.

The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus.

To gain insight into the mechanisms of autoantibody induction, sera from 40 patients with systemic lupus erythematosus (SLE) were tested by ELISAs for antibody binding to denatured individual histones, native histone-histone complexes, histone-DNA subnucleosome complexes, three forms of chromatin, and DNA. Whole chromatin was the most reactive substrate, with 88% of the patients positive. By chi-square analysis, only the presence of anti-(H2A-H2B), anti-[(H2A-H2B)-DNA], and antichromatin were correlated with kidney disease measured by proteinuria > 0.5 g/d. SLE patients could be divided into two groups based on their antibody-binding pattern to the above substrates. Antibodies from about half of the patients reacted with chromatin and the (H2A-H2B)-DNA subnucleosome complex but displayed very low or no reactivity with native DNA or the (H3-H4)2-DNA subnucleosome complex. An additional third of the patients had antibody reactivity to chromatin, as well as to both subnucleosome structures and DNA. Strikingly, all sera that bound to any of the components of chromatin also bound to whole chromatin, and adsorption with chromatin removed 85-100% of reactivity to (H2A-H2B)-DNA, (H3-H4)2-DNA, and native DNA. Individual sera often bound to several different epitopes on chromatin, with some epitopes requiring quaternary protein-DNA interactions. These results are consistent with chromatin being a potent immunogenic stimulus in SLE. Taken together with previous studies, we suggest that antibody activity to the (H2A-H2B)-DNA component signals the initial breakdown of immune tolerance whereas responses to (H3-H4)2-DNA and native DNA reflect subsequent global loss of tolerance to chromatin.

Endogenous circulating DNA in systemic lupus erythematosus. Occurrence as multimeric complexes bound to histone.

Little is known about endogenous systemic lupus erythematosus (SLE) plasma DNA even though it is the presumed precursor of DNA-containing immune complexes, thought to play a central role in lupus glomerulonephritis. DNA purified from SLE plasma formed discrete bands, corresponding to sizes of about 150-200, 400, 600, and 800 bp, closely resembling the characteristic 200 bp "ladder" found with oligonucleosomal (ON) DNA. By radiolabeling DNA while in whole plasma, the very small amounts present could be further characterized. All of 24 such specimens formed two or more discrete bands on 6% PAGE. Detergent treatment of plasma resulted in a DNA migration pattern similar to that of purified DNA, suggesting disruption of DNA-protein complexes. DNA purified from authentic ON and detergent-treated ON behaved similarly. A significant portion of DNA, labeled in SLE plasma could be specifically immunoprecipitated with monoclonal antihistone antibody as was the case with ON. These immunoprecipitates, when redissolved, exhibited the expected size distribution upon PAGE. It is concluded that DNA in SLE plasma occurs as a series of multimeric complexes, at least a portion of which is noncovalently bound to histone. These results are consistent with an ON-like structure for SLE plasma DNA as had been suggested by theoretical considerations and may have important implications for its immunologic behavior in SLE and perhaps other disorders.

Low avidity antibodies to double stranded DNA in systemic lupus erythematosus: a longitudinal study of their clinical significance.

In a longitudinal study the relevance of the detection of low avidity antibodies to double stranded DNA (dsDNA) as measured by the polyethylene glycol (PEG) assay in patients with systemic lupus erythematosus (SLE) was evaluated. It was found that 35 patients positive in the PEG assay only--that is, having only low avidity anti-dsDNA in their circulation, had a mild course of their SLE with absence of renal involvement. The PEG assay had little predictive value but a high specificity (90.2%) for clinical exacerbations; furthermore, a change in avidity of anti-dsDNA in such patients was seldom observed. In 14 patients positive in both Farr and PEG assays--that is, with a relative preponderance of high avidity anti-dsDNA, there was a clear correlation between rises in Farr assay and major exacerbations, while the PEG assay on its own was not helpful in predicting disease manifestations; disease manifestations were often heralded by a change in the Farr/PEG ratio, with renal and cerebral exacerbations associated with the greatest increase in the Farr/PEG ratio (more than 10 times the previous value).

Plasma dna in systemic lupus erythematosus. Characterization of cloned base sequences

Little is known about plasma DNA in patients with systemic lupus erythematosus (SLE). Previous studies have suggested that it may, in fact, not be derived from the random nucleolysis that might be expected to accompany cell death, which might have been the simplest explanation of its origin. To extend the results of other studies, plasma DNA, obtained from a subgroup of SLE patients who had large amounts of immunoprecipitable plasma DNA, was cloned into a plasmid vector, and the nucleotide base sequences were studied by nucleic acid hybridization. One possible explanation for the anomalous renaturation kinetics of plasma DNA in SLE--namely, that it contained non-human genomic base sequences--was rendered untenable by the fact that 51 consecutive DNA clones from 2 SLE patients were each shown to contain human genomic DNA sequences. Plasma DNA from SLE patients also differed from human genomic control DNA in that those sequences derived from the highly repetitive fraction of human DNA were specifically enriched in one such sequence, Alu, which constituted 55% of the repeat sequences in the plasma DNA clones as compared with 13% in control DNA clones. There was a decrease in the frequency of non-Alu repeat sequences (9% for plasma DNA compared with 23% in control DNA). These differences were statistically significant, and they indicate that SLE plasma DNA contains a nonrandom selection of human genomic base sequences. Several explanations for these findings are considered.

Complement activation by antibodies to DNA in systemic lupus erythematosus measured by enzyme immunoassay

An enzyme immunoassay to detect complement-fixing antibodies to DNA (CF-antiDNA) was developed. Of SLE sera, 64% had these antibodies as did 6% of 50 rheumatoid arthritis and 3.2% of 93 normal human sera. The mean CF-antiDNA level was higher in the sera of SLE patients with renal disease than those SLE patients who had no renal disease ( P < 0.0001), and higher in those SLE patients with active rather than inactive renal disease ( P = 0.006). CF-antiDNA was more closely associated with renal activity than total IgG-antiDNA or CH50. These observations suggest that both the quality and quantity of anti-DNA antibodies play a role in the pathogenesis of renal disease, and that modern enzyme immunoassays help distinguish the relative importance of complement-fixing antibodies to anti-DNA from that of total anti-DNA.

Circulating DNA in systemic lupus erythematosus. Isolation and characterization.

Immunoprecipitable double-stranded (dsDNA) was previously shown to persist in the circulation of a clinically recognizable subgroup of patients with systemic lupus erythematosus (SLE). Plasma from 10 such patients was subjected to a DNA isolation procedure that used a combination of proteolysis, phenol extraction, and hydroxylapatite adsorption and elution in the presence of urea. The isolated dsDNA was radiolabeled by nick translation and then characterized by isopyknic ultracentrifugation in CsCl under both neutral and alkaline conditions, as well as after digestion with S1-endonuclease. These experiments demonstrated essential identity in nucleotide base composition between the plasma-derived DNA and human genomic DNA. The presence of specific human base sequences in the plasma DNA was demonstrated by finding that authentic human genomic DNA accelerated the renaturation of plasma DNA when compared with the effect of nonhuman, control DNA. The proportion of such sequences in plasma DNA was estimated by attempting to renature the plasma DNA in the presence of human DNA under conditions shown to result in complete renaturation of human DNA in model experiments. In this way, a minimum of 47% of plasma DNA base sequences could be shown also to be present in human genomic DNA. However, an average of 10-20% of the plasma-derived DNA failed to renature under these conditions, a result that was further confirmed by comparing the renaturation of the tritium-labeled plasma DNA specimens, in double-label experiments, with internal controls consisting of 14C-labeled authentic human DNA. Attempts to drive the reaction to completion with human DNA led to a similar conclusion. The relative nonrenaturability of this fraction of plasma DNA did not appear to be attributable to extensive chain breakage, although adequate analysis of this DNA subfraction was limited by reagent availability. It was therefore concluded that, in this group of SLE patients, persistently circulating DNA consisted largely of base sequences also found in human genomic DNA. The additional presence in plasma of a DNA subfraction that differed in its renaturation behavior from human genomic DNA was recognized, although its significance could not be established with certainty.