Exposure of chromatin and not high affinity for dsDNA determines the nephritogenic impact of anti-dsDNA antibodies in (NZB×NZW)F1 mice

Recent studies have demonstrated that the nephritogenicity of antibodies to dsDNA and nucleosomes confers to binding of glomerular membrane-associated nucleosomes, and not to cross-reacting glomerular antigens. There is no known parameter that determines antibody pathogenicity aside from specificity for dsDNA/nucleosomes, and systemic lupus erytheomatosus (SLE) patients may have high titer anti-dsDNA antibodies irrespective whether they have lupus nephritis or not. One parameter may be antibody affinity, as theoretically only high affinity antibodies may bind in vivo in a stable way. This was analyzed in (NZB × NZW)F1 mice with full-blown lupus nephritis. These mice had serum antibodies to dsDNA, and IgG autoantibodies bound in situ in glomerular membrane-associated electron dense structures as determined by immune electron microscopy (IEM). Intrinsic affinity of purified circulating and glomerular IgG anti-dsDNA antibodies was determined by surface plasmon resonance. The results demonstrate that affinity of glomerular-bound anti-dsDNA antibodies was higher than for those in circulation. However, affinity of glomerular in situ-bound antibodies from different mice varied considerably, from K_D in the range from 10^{? 8} to 10^{? 13}. These results indicate that antibody affinity is not a decisive pathogenic factor, but rather that availability of chromatin fragments may be the factor that determines whether an anti-dsDNA antibody binds in glomeruli or not.     

A quantitative approach to the determination of antigen in immune complexes

Immune complexes are present in the circulation of healthy individuals and the formation of such complexes is part of a normal immune process. During some pathological conditions, significant amounts of immune complexes are formed and deposited in the kidney and other tissues, causing severe injury. Since the levels of immune complexes can provide valuable prognostic information, dozens of methods have been developed to detect and quantify these complexes. However, many of these methods are non-specific, not quantitative, and give false-positive results. Methods based on detecting the antigen portion of immune complexes can yield more precise information about circulating immune complexes. We have used a quantitative dot-blot assay, which permits detection of antigen even if buried, to determine the levels of antigen in circulating immune complexes. In healthy donors, significant amounts of immune complexes containing DNA and beta(2)-glycoprotein I were detected (natural immune complexes). Natural immune complexes with Lewis X antigen were also observed in the circulation of healthy persons. In experimentally induced murine systemic lupus erythematosus (SLE) and SLE patients, there was a correlation between the clinical manifestations and the levels of DNA in the circulating immune complexes. At severe SLE flares, the level of DNA in circulating immune complexes decreased, probably due to tissue deposition of immune complexes. The low levels of DNA in immune complexes circulating in SLE patients correlated with low serum concentrations of the complement component C1q. No direct correlation was found between the levels of circulating anti-dsDNA antibodies and DNA in immune complexes. Thus, quantitation of antigen levels in circulating immune complexes can be used to determine the prognosis of autoimmune diseases.     

Pure anti-dsDNA mAbs need chromatin structures to promote glomerular mesangial deposits in BALB/c mice

The glomerular targets for nephritogenic antibodies have been identified as membrane-associated chromatin fragments. The processes responsible for their deposition are poorly understood. To determine early events in antibody-mediated nephritis, we injected highly pure anti-dsDNA mAbs into BALB/c mice. Mice receiving one dose of anti-dsDNA mAbs were sacrificed 6 or 24 h later. No direct binding of mAbs to glomerular membranes or to the mesangial matrix was observed by immune electron microscopy. In contrast, repeated injections of the same antibodies over 4 weeks resulted in deposition of electron dense structures predominantly in the mesangial matrix. These structures contained mAbs and chromatin fragments as determined by co-localization immune electron microscopy. Biotinylated anti-dsDNA mAbs, injected into nephritic (NZB × NZW)F1 or MRL^lpr/lpr mice were detected in newly formed electron dense structures within glomerular capillary membranes. There were no correlation between mAb affinity for DNA, as determined by surface plasmon resonance analyses, and ability to bind chromatin fragments in vivo. No direct binding of mAbs to inherent membrane antigens was observed. Quantification of DNA in sera before and after one single injection of antibodies revealed increased DNA levels at 6 h after injection of anti-dsDNA mAb, and lower levels after 24 h. Repeated injections of anti-dsDNA caused an increase in circulating DNA. These results indicate that availability of chromatin fragments, presumable in circulation, is important for glomerular mesangial matrix deposition of anti-dsDNA antibody-containing immune complexes in context of lupus nephritis.     

Anti-alpha-actinin antibodies: a new marker of lupus nephritis

The exact role of anti-ds (double stranded) DNA antibodies in the pathogenesis of kidney injury in lupus nephritis remains a focus of continuing investigation. One theory explaining the pathogenicity of anti-dsDNA antibodies in lupus nephritis is direct cross-reactivity with renal antigens. Several years ago, alpha-actinin was identified as a major cross-reactive target for pathogenic anti-dsDNA antibodies in murine SLE. Indeed, binding of a nephritogenic murine anti-dsDNA antibody was stronger to the alpha-actinin derived from a lupus prone mouse mesangial cell line as compared to alpha-actinin in a non-autoimmune mouse mesangial cell line. Furthermore, we recently showed that immunization of non-autoimmune mice with alpha-actinin induces anti-chromatin antibodies, glomerular IgG deposition and proteinuria. In humans, anti-alpha-actinin autoantibodies (Ab) were associated with anti-dsDNA Ab in SLE. In those patients, anti-alpha-actinin rather than anti-dsDNA Ab were significantly associated with glomerulonephritis and disease activity. The anti-alpha-actinin reactivity was associated with high avidity anti-dsDNA Ab. Moreover, the anti-alpha-actinin response was related to the actin-binding site of alpha-actinin. Taken together, these studies indicate that detection of anti-alpha-actinin Ab, in association with anti-dsDNA Ab, may constitute a new marker in lupus nephritis.     

Binding to histone of an anomalous IgG from patients with SLE and drug-induced lupus

The mechanism of attachment of circulating immune complexes (CIC) to glomerular basement membranes (GBM) in systemic lupus erythematosus (SLE) has not yet been elucidated. One difficulty is that CIC must be strongly cationic for such deposition to occur, which is opposite to the anionic nature of putative DNA-anti-DNA immune complexes (DNA-IC). The strongly cationic histone has been proposed as a potential "planted antigen"; it would decorate the GBM to function as a ligand for DNA in the DNA-IC. However, DNA-IC, aggregated IgG and most of the IgG "anti-histone antibodies" in SLE patient sera bind to histone on a solid phase not through DNA, but through the Fcgamma. Here, we investigated the nature of the anti-histone "antibody" in sera of 18 patients with SLE and 57 with drug-induced lupus (DIL). The binding to nucleosomes of IgG from these patients was mainly pepsin-resistant and F(ab')(2)-dependent, whereas the binding to histone was mainly pepsin-sensitive and Fcgamma-dependent. Surprisingly, after molecular sieving of 12 of these sera, the pepsin-sensitive histone-binding IgG was located mainly in the 150-kDa monomeric IgG peak. The binding to nucleosomes was only in the 150-kDa peak. These findings are consistent with the existence of an anomalous IgG in SLE and DIL sera, capable, like aggregated IgG, DNA-IC and other CIC, of binding to histone-decorated structures. We propose that this anomalous IgG plays an essential role in the pathogenesis of lupus nephritis and other related inflammatory conditions. These observations also explain the large discrepancies in the reports on anti-histone autoantibodies in autoimmune conditions.     

DNA levels in immune complexes circulating in mice with induced systemic lupus erythematosus

The levels of DNA in IgG immune complexes, which appeared in the circulation of mice after the induction of systemic lupus erythematosus (SLE), were measured by an immunochemical quantitative assay using monoclonal anti-dsDNA antibodies. The amount of DNA in immune complexes was already high at 10-12 days following the injection of a human monoclonal anti-DNA antibody bearing the major idiotype designated 16/6 in complete Freund's adjuvant, i.e. long before the appearance of clinical manifestations. The injections of these antibodies in the alum-precipitated form did not induce the formation of DNA:anti-DNA complexes as well as SLE itself. The levels of DNA in circulating immune complexes were in general high throughout the whole experimental period (up to 7 months) decreasing gradually before the first clinical manifestations appeared and thereafter, when the disease was fully developed. Such a decrease could be explained by the retention of immune complexes in kidneys. The levels of DNA in immune complexes circulating in normal mice or in mice receiving injections of complete Freund's adjuvant was very low. Treatment of experimental SLE that affected the clinical manifestations prevented the formation of high levels of DNA containing immune complexes.     

Renal expression of polyomavirus large T antigen is associated with nephritis in human systemic lupus erythematosus

We have demonstrated that glomerular expression of polyomavirus large T antigen (T-ag) in a binary tetracycline-regulated T-ag transgenic mouse model (i) terminated tolerance for nucleosomes, (ii) released complexes of nucleosomes and T-ag to the microenvironment from dead cells, and (iii) that these complexes bound induced anti-nucleosome antibodies and finally (iv) that they associated with glomerular membranes as immune complexes. This process may be relevant for human lupus nephritis, since productive polyomavirus infection is associated with this organ manifestation. Here, we compare nephritis in the T-ag transgenic mouse with nephritis in human SLE. Glomerular sections were analysed by transmission electron microscopy, immune electron microscopy (IEM) and by co-localization IEM and TUNEL IEM assays to compare morphological changes, composition of immune complexes and formation of nucleosome-T-ag complexes. Affinity of nucleosome-T-ag complexes for glomerular collagen IV and laminin was determined by surface plasmon resonance (SPR). Analyses revealed electron dense structures in both human and murine kidney samples. These EDS were shown to contain T-ag, DNA and histones, indicating that extra-cellular chromatin may originate from polyomavirus infected cells in human kidneys. SPR analyses demonstrated high affinity of nucleosomes and nucleosome-T-ag complexes for collagen IV and laminin. Complexes of nucleosomes, T-ag and anti-T-ag and anti-dsDNA antibodies bind glomerular membranes and contribute to the evolution of lupus nephritis in human SLE.     

Glomerular expression of large polyomavirus T antigen in binary tet-off regulated transgenic mice induces apoptosis, release of chromatin and initiates a lupus-like nephritis

Binary tetracycline-regulated polyomavirus large T antigen transgenic mice were generated to study immunological tolerance for nucleosomes. Expression of T antigen resulted in binding of the protein to chromatin, and released T antigen-nucleosome complexes from dying cells maintained anti-dsDNA and anti-nucleosome antibody-production by activating autoimmune nucleosome-specific B cells and CD4+ and CD8+ T antigen specific T cells. Glomerular T antigen expression was observed in these mice. Here, we demonstrate that this expression was linked to glomerular cell apoptosis, release of nucleosomes and association of nucleosomes with glomerulus basement membranes, detected as electron dense structures. Immune electron microscopy (IEM) revealed that these structures were glomerular targets for induced anti-dsDNA and anti-T antigen antibodies. Co-localization IEM demonstrated that in vivo-bound auto-antibodies co-localized with experimental monoclonal antibodies to dsDNA and to T antigen. A comparative analysis of glomeruli from nephritic (NZWxNZB)F1 and T antigen expressing transgenic mice revealed deposition of nucleosomes in glomerular capillary and mesangial matrix membranes and binding of anti-nucleosome antibodies in both mice strains. A controlled experimental model that may elucidate the initial events accounting for nucleosome-mediated nephritis has not been available. The transgenic mouse may be important to describe early immunological and cellular events accounting for the enigmatic lupus nephritis.     

Anti-DNA antibody subpopulations and lupus nephritis

As a consequence of increased insight into the cellular and molecular mechanisms responsible for induction of B cell and T cell autoimmunity to DNA and nucleosomes, there is an obvious need to reconsider the dogma stating that anti-dsDNA antibodies serve as marker antibodies for SLE and also that anti-dsDNA antibodies per se are responsible for the initiation of lupus nephritis. Given that the potential to produce anti-dsDNA antibodies is an inherent property of the normal immune system and that few anti-DNA antibodies have nephritogenic potential, we must try to solve the problem whether it is avidity for DNA, specificity for unique DNA structures or cross-reactivity with non-DNA molecules, that make such antibodies pathogenic and thus potential markers for SLE and lupus nephritis. In this review, we will summarize contemporary problems related to these questions; (1) try to focus on phenotypic differences with respect to the ability to produce anti-dsDNA antibodies between individuals suffering from SLE and those not belonging to this diagnostic group, and (2) to describe differences between pathogenic and non-pathogenic anti-dsDNA antibodies.     

Histone-containing immune complexes are to a large extent responsible for anti-dsDNA reactivity in the Farr assay of active SLE patients

Increased titres of anti-dsDNA antibodies, especially if of high avidity, are associated with renal exacerbations in patients with systemic lupus erythematosus (SLE). One of the most reliable assays to measure anti-dsDNA antibodies, the Farr assay, is believed to detect preferentially high avidity antibodies. Purified non-complexed monoclonal antibodies (mAbs) against nucleosomes, obtained from mice with SLE, are not reactive in the Farr assay, but can become so once complexed to nucleosomes. These Farr-positive, nucleosome containing, immune complexes were also able to bind in vivo to the glomerular basement membrane (GBM), predominantly via heparan sulphate (HS). To evaluate whether in SLE patients the same kind of immune complexes are responsible for Farr reactivity, IgG from serum or plasma was isolated under dissociating and physiological conditions. We observed that after purification under dissociating conditions, Farr reactivity was significantly decreased (P<0.0001) in contrast to reactivity with histones and two 'control' antigens: Epstein Barr Virus (EBV) and Ro/SS-A. Reactivity with nucleosomes also decreased after purification, although to a lesser extent. Plasma purified under physiological conditions showed no decrease in Farr reactivity. The importance of histones for the generation of immune complexes is supported by the two following observations. Firstly, the presence of histones could be demonstrated in serum and plasma of SLE patients but not in serum of healthy controls or in IgG preparations purified under dissociating conditions. Secondly, Farr reactivity of purified IgG preparations could be restored by addition of purified histones. From these studies we conclude that histones containing immune complexes are responsible for a large part of the Farr reactivity in active SLE, and are therefore indirectly implicated in the pathogenesis of lupus nephritis.     

Concomitant early appearance of anti-ribonucleoprotein and anti-nucleosome antibodies in lupus prone mice

OBJECTIVE: To gain insights on initial stages of the autoimmune response in lupus prone mice taking advantage of new sensitive and quantitative techniques for the detection of autoantibodies specific for RNA- (ribonucleoproteins) and DNA-protein (chromatin) complexes. METHODS: DNA and nucleosome antibodies were detected by ELISA, antibodies to SmB, U1A-RNP, Ro52, Ro60 and La by a new radioligand assay, using de novo synthesized radio-labeled antigens. RESULTS: Analysis of anti-chromatin (including anti-nucleosome, anti-dsDNA and anti-histone antibodies) and of anti-snRNP antibodies (including anti-U1A-RNP, anti-SmB, anti-Ro52, anti-Ro60, anti-La antibodies) was performed in sequential sera from B/W, MRL+/+, MRL Yaa and MRL lpr/lpr mice. In a cohort of 105 MRL+/+ mice of different ages, 59, 51, and 57 mice were positive for anti-nucleosome, anti-SmB and anti-U1A-RNP, respectively. None of them was positive for anti-dsDNA. Importantly, antibody positivities were not randomly distributed but were significantly clustered in individual mice. Appearance of DNA- and RNA-protein complex antibodies started at approximately 18-20 weeks of age, preceding that of the anti-dsDNA (or anti-histone) antibodies that only started at 30-32 weeks. Anti-nucleosome, anti-SmB and anti-U1A-RNP antibody responses did not display any cross-reactivity as demonstrated by inhibition and adsorption experiments. CONCLUSION: These data indicate that anti-nucleosome and anti-snRNP antibodies appear early and concomitantly in lupus prone mice even though they do not share any cross-reactivity. These results fit with the assumption that their production is triggered by tightly physically associated nucleosomes and snRNP autoantigens contained in the same apoptotic bodies.     

Antinuclear autoantibodies in flaky skin (fsn) mutant mice

Mice homozygous.for the flaky skin (fsn) single gene mutation have a severe hyperproliferative disease resulting complex phenotype, which includes widespread inflammation and autoimmunity. Flaky skin mice have several serological and pathological features that share similarities with the human systemic autoimmune disease systemic lupus erythematosus (SLE). Analyses of the antinuclear and anti-dsDNA autoantibodies in fsn/fsn mice indicate that they are low titer IgG antibodies. These low titer anti-dsDNA autoantibodies can ultimately form immune complex deposition in the glomeruli associated kidney damage. IgE antibodies were identified in the immune complex deposition, however their role in the pathology is not determined. It is hypothesized that the mechanism of autoantibody production and autoimmune disease pathogenesis in mice homozygous for the fsn mutation is initiated by non-specific polyclonal activation of B-lymphocytes resulting in the synthesis of low affinity autoantibodies.     

Antinuclear Autoantibodies in Flaky Skin ( fsn ) Mutant Mice

Mice homozygous for the flaky skin ( fsn ) single gene mutation have a severe hyperproliferative disease resulting complex phenotype, which includes widespread inflammation and autoimmunity. Flaky skin mice have several serological and pathological features that share similarities with the human systemic autoimmune disease systemic lupus erythematosus (SLE). Analyses of the antinuclear and anti-dsDNA autoantibodies in fsn / fsn mice indicate that they are low titer IgG antibodies. These low titer anti-dsDNA autoantibodies can ultimately form immune complex deposition in the glomeruli associated kidney damage. IgE antibodies were identified in the immune complex deposition, however their role in the pathology is not determined. It is hypothesized that the mechanism of autoantibody production and autoimmune disease pathogenesis in mice homozygous for the fsn mutation is initiated by non-specific polyclonal activation of B-lymphocytes resulting in the synthesis of low affinity autoantibodies.     

Elevated levels of soluble CD40 ligand (sCD40L) in serum of patients with systemic autoimmune diseases

The CD40–CD40L costimulatory pathway is involved in the evolution of many autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Increased levels of sCD40L in the serum have been associated with disease activity in SLE. The aim of this study was to investigate the role of sCD40L in the development of lupus nephritis and examine its possible association with cryoglobulinemia in Sjögren's syndrome. We used a 2-site sandwich ELISA to measure the levels of sCD40L in sera, from 64 patients with SLE, RA and SS and 17 healthy blood donors. Biological specimens from the affected tissues such as urine from patients with lupus nephritis and saliva from patients with SS were also tested. In this regard, paired sera and first morning urine samples from 6 SLE patients (3 with active lupus nephritis and 3 with inactive lupus nephritis) were tested with the sCD40L ELISA protocol as well as paired sera and salivary samples from 5 patients with SS and cryoglobulinemia, 5 patients with SS and anti-Ro or anti-La autoantibodies and 5 age-matched healthy control donors. We also examined possible correlations of sCD40L levels with several laboratory and clinical parameters in SS and SLE. We found that sera from SLE and SS patients had significantly higher levels of sCD40L compared to sera from healthy control donors. No sCD40L was detected, in urine samples of patients with either active or inactive nephritis and in salivary samples from SS patients or normal subjects. Soluble CD40L is elevated in sera of SS and SLE patients but further investigation is needed to determine its possible role in SLE nephritis and Sjögren's syndrome.     

Autoantibodies to C-reactive protein is a common finding in SLE,but not in primary Sjögren's syndrome,rheumatoid arthritis or inflammatory bowel disease

The occurrence of antibodies to human C-reactive protein (CRP) was analysed by enzyme-linked immunosorbent assay (ELISA) in 56 patient sera known to contain antibodies to double-stranded DNA (dsDNA) and in 16 sera from patients with primary Sj?gren's syndrome (SS), 15 rheumatoid arthritis, 31 Crohn's disease, and 37 ulcerative colitis. Eighty-seven per cent of the patients with anti-dsDNA antibodies had systemic lupus erythematosus (SLE) and the remaining had autoimmune hepatitis. The cut-off for positive anti-CRP test was set at the 95th percentile of 100 healthy blood donors. Twenty of 56 anti-dsDNA sera (36%) and two of 16 SS sera (13%) had antibodies reactive with human CRP, whereas all other samples were negative. Thirteen of 27 SLE patients (48%) were positive on at least one occasion. The sera containing anti-CRP antibodies only reacted with surface-bound antigen, but not with native CRP in solution. In conclusion, we found that autoantibodies to CRP are common in sera from patients with anti-dsDNA antibodies. It is not likely that this explains the relative failure of CRP response in patients with active SLE. However, it cannot be excluded that anti-CRP autoantibodies have other biological potentials of pathophysiological interest in SLE, for instance by binding to CRP deposited on cell and tissue surfaces.     

Microbes and Autoimmunity

Cross-reactivity of anti-double stranded DNA (anti-dsDNA) antibodies with glomerular antigens has been postulated as a key factor in the development of lupus nephritis. Because no direct proof has been presented on anti-dsDNA antibodies binding in vivo to glomerular structures, we have analysed the binding of potentially nephritogenic anti-dsDNA antibodies to α-actinin and laminin. By enzyme-linked immunosorbent assay and surface plasmon resonance (SPR) analyses, we demonstrate that monoclonal antibodies (mAbs) bind both double-stranded DNA and α-actinin at high affinity. However, when added to nephritic kidney sections they did not bind to such structures, but rather to nucleosome-containing structures within the mesangial matrix or the glomerular basement membranes (GBMs). Nucleosomes, anti-nuclear antibodies and complexes of them were tested for their binding to glomerular components such as agrin, perlecan and laminin using SPR analysis. Nucleosomes bound to laminin, marginally to agrin, but not to perlecan or heparan sulphate-depleted agrin. Anti-histone H2B and anti-nucleosome antibodies in complex with nucleosomes slightly increased the binding of nucleosomes to agrin, while binding to laminin was slightly decreased compared to nucleosomes alone. In conclusion, the availability of nucleosomal antigens and the binding of these antigens to components of the mesangial matrix and GBM seem crucial for the glomerular deposition of immune complexes.     

Nephritogenic antibodies bind in glomeruli through interaction with exposed chromatin fragments and not with renal cross-reactive antigens

Cross-reactivity of anti-double stranded DNA (anti-dsDNA) antibodies with glomerular antigens has been postulated as a key factor in the development of lupus nephritis. Because no direct proof has been presented on anti-dsDNA antibodies binding in vivo to glomerular structures, we have analysed the binding of potentially nephritogenic anti-dsDNA antibodies to α-actinin and laminin. By enzyme-linked immunosorbent assay and surface plasmon resonance (SPR) analyses, we demonstrate that monoclonal antibodies (mAbs) bind both double-stranded DNA and α-actinin at high affinity. However, when added to nephritic kidney sections they did not bind to such structures, but rather to nucleosome-containing structures within the mesangial matrix or the glomerular basement membranes (GBMs). Nucleosomes, anti-nuclear antibodies and complexes of them were tested for their binding to glomerular components such as agrin, perlecan and laminin using SPR analysis. Nucleosomes bound to laminin, marginally to agrin, but not to perlecan or heparan sulphate-depleted agrin. Anti-histone H2B and anti-nucleosome antibodies in complex with nucleosomes slightly increased the binding of nucleosomes to agrin, while binding to laminin was slightly decreased compared to nucleosomes alone. In conclusion, the availability of nucleosomal antigens and the binding of these antigens to components of the mesangial matrix and GBM seem crucial for the glomerular deposition of immune complexes.     

The Lupus-derived Anti-double-stranded DNA IgG Contributes to Myofibroblast-like Phenotype in Mesangial Cells

Anti–double-stranded DNA (dsDNA) antibodies have been indicated to play a major role in the pathogenesis of lupus nephritis (LN), which is characterized by mesangial alterations, including phenotypic changes. To explore the effects of anti-dsDNA antibodies on the phenotype of mesangial cells (MCs), the anti-dsDNA IgG in sera and histological features of glomeruli were analyzed in the mice models of immune-complex glomerulonephritis. The MCs were cultured in vitro with the addition of anti-dsDNA or non-anti-dsDNA IgG. Compared to the anti-dsDNA-negative controls, the serum positive mice had increased extracellular matrix accumulation and higher alpha-smooth muscle actin expression in the mesangial region. The anti-dsDNA IgG enhanced the synthesis of transforming growth factor beta, alpha-smooth muscle actin, and fibronectin, and even induced the myofibroblast-like morphological features in cultured MCs. Our results indicated that anti-dsDNA antibodies contribute to the phenotypic changes in MCs, which suggests another mechanism of renal injuries in LN induced by anti-dsDNA antibodies.     

DNA-anti-DNA circulating complexes in the nephritis of systemic lupus erythematosus.

In order to determine whether circulating antigen-antibody complexes in systemic lupus erythematosus (SLE) consist of DNA and anti-DNA, cryoglobulins were isolated from the sera of 38 patients with SLE nephritis and analysed for DNA and anti-DNA. Cryoglobulins were detected in 36 of the 38 sera, and DNA was found in 30 of 33 examined by either fluorescence of ethidium bromide or a radioimmunoassay. Anti-DNA activity was not detectable in any of the whole cryoglobulins but anti-IgG activity was found in 17. Twenty cryoglobulins were therefore treated by acid dissociation and ultracentrifugation to obtain isolated immunoglobulins; IgG was isolated from all, and IgM from eight. Using a modified Farr assay to detect anti-dsDNA and an enzyme-linked immunoadsorption assay to detect anti-ssDNA, anti-dsDNA activity alone was found in five IgG fractions, anti-ssDNA activity alone in five, and both anti-ds- and anti-ssDNA activity in four. Anti-dsDNA activity was found in three of the IgM fractions. In all, anti-dsDNA activity was found in nine of these 20 cryoglobulins, and anti-DNA in 14. Analysis of these 14 cryoglobulins with anti-DNA Ig fractions showed that there was enrichment of the IgG anti-DNA activity in the cryoglobulin compared to the patient's serum in all but two cases. In six of the 20 cryoglobulins studied there was no detectable anti-DNA activity in isolated IgG or IgM fractions. We therefore concluded that DNA-anti-DNA complexes were present in most of our patients with SLE nephritis, but there was clearly a substantial minority in whom they were undetectable. In some of the latter who also had active disease the cryoglobulins had anti-IgG activity. Thus it would seem that SLE can occur in the absence of DNA-anti-DNA complexes, but with other complexes present. DNA was also found in cryoglobulins isolated from patients with idiopathic glomerulonephritis, but, in contrast to recent reports, anti-DNA activity was not detectable in immunoglobulins isolated from their cryoglobulins.     

Binding of circulating SLE autoantibodies to oxygen free radical damaged chromatin

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by various immunologic disorders, including production of autoantibodies, formation of immune complexes, decreased serum complement levels, and lymphocytopenia. One of the hallmarks of this disease is the loss of tolerance to nuclear antigens. The dominant presence of antibodies against the exposed conformational epitopes on chromatin strongly suggests that the pathogenic immune response in lupus is driven by chromatin. In the present study, the binding of SLE autoantibodies with native chromatin and oxygen free radical damaged chromatin was studied. As assessed by direct binding and inhibition ELISA, circulating SLE autoantibodies exhibited a high degree of specificity towards the reactive oxygen species (ROS)-modified chromatin in comparison to native chromatin and this binding specificity was reiterated visually by gel retardation assay. The data suggested possible role of modified chromatin in the induction of SLE autoantibodies and higher recognition of oxidatively damaged chromatin by antibodies in sera of SLE patients. It is indicated that free radical modified chromatin or nucleosomes might be the antigen for the production of circulating autoantibodies in SLE.