Higher anti-heparan sulphate reactivity during systemic lupus erythematosus (SLE) disease exacerbations with renal manifestations; a long term prospective analysis.

Cross-reactive antibodies against heparan sulphate (HS) have been suggested to play a role in initiating renal disease in SLE. Recently, we found that HS-reactivity is mediated by anti-DNA antibodies complexed with DNA and histones. To evaluate the clinical significance of anti-HS reactivity, we studied prospectively a cohort of 72 consecutive SLE patients, of whom 22 experienced 40 exacerbations. In 20 of these exacerbations renal symptoms were present. In these 20 exacerbations significantly higher anti-DNA (median 1:160) and anti-HS (median 1:30) titres were detected compared with exacerbations without renal manifestations (median 1:60 for anti-DNA and negative for anti-HS). There were no correlations with other symptoms of SLE. Anti-HS titres showed a significant correlation with anti-DNA antibody titres (rs = 0.57, P < 0.05). Anti-HS without anti-DNA reactivity was never detected. Some SLE patients showed a high anti-DNA titre without anti-HS reactivity, suggesting that not all anti-DNA antibodies are able to bind to histone/DNA complexes and thus to exhibit anti-HS reactivity. Our findings indicate that anti-HS reactivity is correlated with renal disease in SLE.

Antigen-specificity of antibodies bound to glomeruli of mice with systemic lupus erythematosus-like syndromes.

BACKGROUND: MRL/l mice with diffuse proliferative glomerulonephritis, and graft-versus-host (GVH) mice with membranous glomerulonephritis, are both regarded as models for human systemic lupus erythematosus. In these two models, the specificity of the nephritogenic antibodies was analyzed. EXPERIMENTAL DESIGN: The nephritogenic antibodies were eluted from isolated glomeruli with an acid buffer. The antibodies were purified from these eluates on protein-A sepharose under high salt conditions to exclude the presence of antigens potentially bound to the antibodies. The specificities of the antibodies were analyzed towards components of the glomerular basement membrane (GBM), nuclear antigens and brush border of proximal epithelial cells in the indirect immunofluorescence (IF), enzyme linked immunosorbent assay, and Crithidia luciliae assay. Furthermore, we studied the glomerular binding by IF and immunoelectron microscopy after intravenous injection of the eluted antibodies into control mice. RESULTS: Glomerular eluates of both MRL/l and GVH mice showed nuclear staining and linear to homogeneous binding to GBM and tubular basement membrane in IF on normal mouse kidney. In enzyme linked immunosorbent assay, both eluates reacted not only with DNA, but also with histones and laminin, an important component of the GBM, however not with brush border. Reactivity with dsDNA was also found in the Crithidia luciliae assay for both eluates. In vivo binding was determined by injection of eluates into control MRL/n and F1 hybrid mice. The GVH eluate showed linear to homogeneous binding to the GBM 1.5 hour after injection which changed into a granular, membraneous pattern after 5 days, as determined by IF and immunoelectron microscopy. The MRL/l eluate also demonstrated linear to homogeneous binding to the GBM 1.5 hour after intravenous injection, that had decreased after 5 days, but did not change into a granular pattern. CONCLUSIONS: In these two models of systemic lupus erythematosus nephritis not only antibodies to DNA and histones, but also antibodies to laminin are involved in the induction of murine systemic lupus erythematosus nephritis. The latter antibody specificity has not been identified before in renal or glomerular eluates obtained from diseased MRL/l mice.

Anti-DNA antibodies can bind to the glomerulus via two distinct mechanisms.

It is generally assumed that antibodies to double stranded DNA (anti-DNA) play a pivotal role in the pathogenesis of SLE nephritis. Recently, we reported that anti-DNA antibodies can bind to heparan sulphate proteoglycan (HSPG), a constituent of the glomerular basement membrane (GBM), via histones and DNA. We postulated that these histone/DNA/anti-DNA complexes can bind via their histone part to the glomerulus in vivo. To test this hypothesis we performed in vitro binding studies with isolated GBM loops and renal perfusion studies in the rat using histones, DNA and an anti-DNA monoclonal antibody (mAb) with high avidity for dsDNA. A strong granular binding of anti-DNA mAb to isolated GBM loops occurred via histones and DNA and a moderate granular binding was found via DNA alone. Anti-DNA mAb alone did not bind to the GBM loops. After perfusion of histones, DNA and immediately thereafter anti-DNA, we found with immunoelectron microscopy (IEM) a strong binding to endothelial cells in the glomerulus and to a lesser extent in the GBM. When the anti-DNA mAb was injected i.v. one hour after perfusion of histones and DNA, we observed a strong fine granular binding to the capillary wall by immunofluorescence (IF) in a membranous pattern along with some minor mesangial deposits. After perfusion of DNA alone followed by anti-DNA mAb, binding in the glomerulus was less than with histones and DNA, and was more restricted to the mesangium. No direct binding to the glomerulus was observed after perfusion with anti-DNA mAb alone, histones and anti-DNA mAb, or histones, DNA and a control mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-dsDNA: choice of assay in relation to clinical value.

Antibodies to DNA are quite specific for systemic lupus erythematosus (SLE) and occur in the majority of SLE patients. Therefore, their detection is an important diagnostic aid to the clinician. Detection of anti-dsDNA may precede the diagnosis of SLE by more than a year. Fluctuations in the level of anti-dsDNA in an individual patient may give important information on the clinical status of the patient. Four of the most important methods developed for the measurement of anti-dsDNA antibodies will be discussed in this paper: the Farr assay, the PEG assay, the indirect immunofluorescence test on Crithidia luciliae and the ELISA. They will also be compared with one commercially available (Farr) assay, the Amersham anti-dsDNA kit. Each method, detects a part of the spectrum of anti-dsDNA antibodies produced by a patient. The Farr assay is the most specific for SLE; however, milder forms of the disease in which patients have only low avidity anti-dsDNA may easily be missed by this technique. Clinically, high avidity anti-dsDNA is related more frequently to the occurrence of nephritis, whereas low avidity anti-dsDNA antibodies are found more often in patients with central nervous system involvement. Traditionally, SLE is considered an immune-complex disease, in which inflammatory processes are initiated by local deposition of DNA/anti-dsDNA complexes. More recently, a major role was thought to be played by crossreactions of anti-dsDNA with tissue constituents. Our current view, however, is that such a crossreactivity plays only a minor role; we postulate that binding to glomerular constituents is caused by anti-dsDNA antibodies complexed with DNA and histones.

Anti-heparan sulphate reactivity in sera from patients with systemic lupus erythematosus with renal or non-renal manifestations.

Previously, we have shown that anti-DNA can bind to heparan sulphate (HS), a constituent of the glomerular basement membrane (GBM). We hypothesized that binding of anti-DNA to HS in the GBM plays a role in the onset of systemic lupus erythematosus (SLE) nephritis. To test this hypothesis we measured the anti-HS reactivity in cross-sectional and longitudinal studies of SLE patients with or without nephritis. In the transverse serum study single serum samples from 26 SLE patients were studied. We found no correlation between anti-HS reactivity and previously development of nephritis (anti-HS positive: seven out of 16 with history of nephritis, two out of 10 without nephritis). However, six of the seven anti-HS positive sera in the nephritis group were obtained within 1 month of the onset of nephritis, suggesting a temporal relationship between anti-HS reactivity and onset of nephritis. In the longitudinal serum study between six and 16 serum samples were studied from each of 10 SLE-patients. In five out of five episodes of nephritis we found anti-HS reactivity before the onset or exacerbation of the nephritis. In four non-renal manifestations anti-HS reactivity was found in only one episode; in none of the three patients who remained clinically stable did serum samples show anti-HS reactivity. Anti-HS reactivity was only found in sera positive for anti-DNA by Farr assay but the anti-HS titre was not a mere reflection of the reactivity measured in the Farr assay. This indicates that only a subpopulation of anti-DNA can bind to HS. We found a high correlation (r = 0.99) between anti-HS reactivities in plasma and serum and we conclude that anti-HS reactivity in serum samples from SLE patients is not due to in vitro complex formation during clotting. Although further prospective analysis is necessary, our data suggest that measurement of anti-HS reactivity in SLE patients might identify patients at risk for the development of nephritis.

Cross-reactivity of monoclonal anti-DNA antibodies with heparan sulfate is mediated via bound DNA/histone complexes.

To study in more detail the cross-reactive binding of anti-DNA antibodies to heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG) purified from glomerular basement membranes (GBM), the binding pattern of 31 murine IgG anti-DNA MoAbs, derived from MRL/lpr, NZB/W and graft-versus-host diseased mice, was analysed. In ELISA we found binding of 10 anti-DNA MoAbs to HS. Seven of the 10 anti-HS positive clones bound to HSPG but not to the HSPG core protein in ELISA and/or on Western blots. However, DNase-I treatment partly reduced this binding, whereas after purification of MoAb by protein-A sepharose chromatography under dissociative conditions, all clones completely lost their binding capacity to HS and HSPG. Culturing of hybridoma cells in the presence of 3H-thymidine revealed DNA bound to the MoAb. Although the binding to HS and HSPG could be reconstituted by the addition of the protein-A column effluent, this was not possible by the addition of DNA alone. Therefore, we performed immunoprecipitation of the effluent with purified MoAb and subsequent SDS-PAGE which showed that the complex also contained histones. However, histones alone were also not able to reconstitute the binding to HS and HSPG. It is concluded that binding of anti-DNA MoAb to HS and GMB-HSPG is mediated via bound complexes containing both DNA and histones. A comparable reaction with polyclonal anti-DNA Ab might play a role in the pathogenesis of SLE nephritis, since histones have a very high affinity for HS, the major glycosaminoglycan of the GBM.

Murine monoclonal antibodies to DNA. A comparison of MRL/lpr NZB/W and chronically graft-versus-host-diseased mice.

Hybridomas producing monoclonal antibodies to DNA were prepared from NZB/W F1 (n = 20), MRL/lpr (n = 13), mice with a chronical graft versus-host-disease (GVHD) (n = 8) and polyclonally stimulated mice (n = 9). Screening was performed by means of an anti-DNA ELISA. Reaction patterns in four different anti-DNA assays (anti-DNA ELISA, indirect immunofluorescence on Crithidia luciliae, PEG assay and Farr assay) as well as avidity and cross-reactivity of these monoclonals were studied in relation to anti-DNA (sub)class and murine origin of the clones. It was found that monoclonal anti-DNA derived from mice with chronic GVHD did not differ from monoclonal anti-DNA derived from NZB/W F1 or MRL/lpr mice, with respect to isotype distribution, avidity towards DNA, cross-reactivity and assay behaviour in the anti-DNA assays mentioned before. In contrast, monoclonal anti-DNA obtained from polyclonally stimulated mice were all of the IgM isotype and displayed a stronger cross-reactive behaviour than the other three models. Altogether, these results exclude the possibility that anti-DNA in the GVHD mice originates from the non-specific pool of natural autoantibodies and further emphasize the relevance of chronic GVHD as a murine model of systemic lupus erythematosus.

Antibodies to DNA in patients with systemic lupus erythematosus. Their role in the diagnosis, the follow-up and the pathogenesis of the disease.

In this paper an overview of the present knowledge on antibodies against DNA will be presented. Diagnostic, prognostic and pathogenic aspects of anti-DNA will be highlighted. Detection of antibodies to DNA in the circulation of a patient by an assay selective for high avidity anti-DNA is highly diagnostic for SLE. Anti-DNA of low avidity occurs in rheumatic diseases other than SLE as well, making detection of such antibodies of less diagnostic value. Furthermore, data will be presented that show that the anti-DNA ELISA in its present form is not suited as a diagnostic tool. Not only disease features of SLE vary from patient to patient, anti-DNA avidity does so too. A relationship between anti-DNA avidity and clinical features can be found: high avidity anti-DNA is more abundant in patients with nephritis, low avidity anti-DNA in patients with CNS involvement. Prognostically, a steady increase of the level of high avidity anti-DNA generally heralds an upcoming exacerbation in a patient. Furthermore, 85% of the patients who do not have SLE at the time (high avidity) anti-DNA is detected in their serum, will develop the disease within the next few years. It is noteworthy, that patients with only low avidity anti-DNA in their circulation develop a more mild form of SLE; the (low) avidity of their anti-DNA seldomly increases during the course of their disease. The relevance of anti-DNA to the pathogenesis of SLE still is a matter of debate. On the one hand, the association of parameters of anti-DNA that determine the size of the complexes formed with DNA is in favour of the classical hypothesis, which states that SLE is primarily an immune complex disease. On the other hand, recent data on crossreactions of anti-DNA with phospholipids, glycosaminoglycans and other (poly-negative) structures plead for a role of such crossreactivities in the pathogenesis of SLE.

Cross-reactive binding patterns of monoclonal antibodies to DNA are often caused by DNA/anti-DNA immune complexes.

Recently, the role of antibodies to DNA in the pathogenesis of systemic lupus erythematosus (SLE) has been reevaluated, since observed cross-reactive binding of anti-DNA to tissue-related antigens might substantially contribute to the inflammatory process of the disease. Evidence of this cross-reactivity has, in part, been obtained from studies with monoclonal anti-DNA. However, we now report that the presence of DNA/anti-DNA immune complexes in monoclonal antibody preparations may be the cause of the observed cross-reactive binding patterns. Studying a panel of anti-DNA producing hybridomas (n = 63), we detected such immune complexes in 76% of the obtained culture supernatants by using an anti-protamine sulphate (PS) ELISA; complexes formed with 1 microgram/ml DNA or more were traced in this assay. In cultures of anti-DNA-producing hybridomas, complexes were detected from day 3 on. Treatment of supernatants with DNase reduced the anti-PS reactivity to an average of only 20% of the original reactivity. Contaminating DNA/anti-DNA immune complexes were found to play no role in the cross-reactivity of anti-DNA antibodies with cardiolipin, a minor role in cross-reactivity with dextran sulphate, but a substantial role in the cross-reactivity with heparan sulphate, histones and other nuclear or cytoplasmic antigens. Our results clearly demonstrate that exclusion of the presence of immune complexes in antibody preparations is a prerequisite when cross-reactivity patterns of anti-DNA antibodies are studied.

Binding of anti-DNA antibodies to glomerular heparan sulfate: a new clue for the pathogenesis of SLE nephritis?

Antibodies against heparan sulfate (HS) were detected in sera from patients with active systemic lupus erythematosus (SLE) and in sera from MRL/l mice with a spontaneous SLE-like disease. By inhibition studies it was shown that this reactivity towards HS was due to cross-reactivity of anti-DNA antibodies. Immunoglobulin eluted from human and mouse kidneys with diffuse proliferative SLE glomerulonephritis also bound to HS. This crossreaction of anti-DNA antibodies was further substantiated by the finding that 17 out of 42 anti-DNA monoclonal antibodies (mAb) also bound to HS, 11 out of 17 HS positive mAb bound to heparan sulfate proteoglycan (HSPG) purified from human glomerular basement membranes (GBM) and 7 out of 42 bound directly to isolated human GBM-loops. The binding to HS, HSPG, and GBM could be inhibited in a dose-dependent manner by DNA. In a retrospective analysis of sera from 10 SLE patients, in which 6-16 serum samples per patient were studied, we found in all 5 episodes of onset or exacerbation of a SLE nephritis an anti-HS activity in the serum, prior to onset of the nephritis. In 4 episodes of onset or exacerbation of non-renal manifestations, anti-HS activity was only found in the serum during one episode. Based on these studies we postulate that binding of anti-DNA antibodies to HS within the GBM may be an important immunopathological event in the development of SLE nephritis.

Plasmodium berghei: reduction of the mouse's specific lymphoproliferative response in relation to corticosterone and pregnancy.

Spleen cells from mice immune to Plasmodium berghei exhibited a significantly increased in vitro proliferative response to parasitized reticulocytes compared to spleen cells from normal mice. The specific response to malaria antigen was decreased in spleen cells from pregnant immune mice in contrast to the nonspecific response to the mitogen phytohemagglutinin. Addition of mouse serum to spleen cell cultures of immune mice depressed both the phytohemagglutinin and the specific proliferative response, whereas serum of pregnant mice exerted an even stronger inhibition than serum of nonpregnant mice. Charcoal adsorption of mouse sera for the elimination of steroid hormones removed the serum dependent immunosuppression from normal as well as pregnant serum. Corticosterone added to the spleen cell cultures depressed also the proliferative response. These findings demonstrate that the response to malaria antigen is decreased in immune mice during pregnancy. The possible effect of serum corticosterone on the depression of the immune response is discussed.