R.J.T. Smeenk

ANTI-NUCLEOSOME ANTIBODIES COMPLEXED TO NUCLEOSOMAL ANTIGENS SHOW ANTI-DNA REACTIVITY AND BIND TO RAT GLOMERULAR-BASEMENT-MEMBRANE IN-VIVO

Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis.

Anti-DNA antibodies and lupus nephritis: the complexity of crossreactivity

It has been suggested that crossreactivity of anti-DNA antibodies plays a central role in the development of lupus nephritis. Experiments with monoclonal anti-DNA antibodies initially seemed to sustain this intriguing hypothesis but such studies may easily lead to incorrect conclusions. In this short article, Kees Brinkman and colleagues discuss the validity of these studies and challenge the role of crossreactivity in the pathogenesis of lupus nephritis.

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.

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

SummaryAntibodies 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.

The detection of anti-Ro/SS-A and anti-La/SS-B antibodies: A comparison of counterimmunoelectrophoresis with immunoblot, ELISA, and RNA-precipitation assays

The presence in serum of anti-Ro/SS-A and/or anti-La/SS-B autoantibodies is a characteristic of autoimmune diseases such as Sjögrens syndrome and systemic lupus erythematosus. To evaluate different assays currently available for the detection of these antibodies 50 sera were tested using the four different assay methods: counterimmunoelectrophoresis (CIE), RNA precipitation assay, immunoblotting technique and ELISA. The RNA-precipitation assay showed the highest sensitivity and specificity. The CIE for the detection of anti-Ro/SS-A antibodies gave comparable results whereas the Ro/SS-A ELISA and Ro/SS-A or HeLa immunoblot showed lower sensitivities (96% and 80% respectively). Sensitivity was even lower (66%) when only reactivity towards the 60 kDa Ro/SS-A protein was considered. The ELISA for the detection of anti-La/SS-B antibodies showed a sensitivity of 98%, the immunoblotting technique of 86% and the CIE only 67%. The high sensitivity of the La/SS-B ELISA went together with a low specificity of 14%. We conclude from these data that for the detection of anti-Ro/SS-A and anti-La/SS-B antibodies the RNA precipitation assay shows the highest sensitivity and highest specificity. For routine screening purposes the CIE is the most convenient and reliable assay to detect anti-Ro/SS-A antibodies. For the detection of anti-La/SS-B antibodies the immunoblot corresponds most closely to the RNA precipitation assay.

The use of polyethylene glycol precipitation to detect low-avidity anti-DNA antibodies in systemic lupus erythematosus☆

With a recently introduced method for measurement of low-avidity anti-DNA, the polyethylene glycol (PEG) precipitation assay (Riley et al., 1979), high levels of DNA binding by normal human serum (NHS) were found when circular PM2-DNA was used as antigen. THe nature of this DNA binding was studied. The removal of low-density lipoproteins (LDL) from the serum, e.g., by Aerosil absorption, eliminated DNA binding by NHS. Purified LDL bound DNA to the same extent as NHS. Non-specific binding of NHS or LDL to DNA was prevented by adding dextran sulphate to the incubation mixture. Analysis on sucrose gradients showed that only large DNA-anti-DNA complexes were precipitated by 3.5% PEG. The PEG assay with dextran sulphate is a sensitive assay for low-avidity anti-DNA antibodies. It adds important information to results obtained with the Farr assay, which mainly detects antibodies of high avidity.

Significance of anti‐nuclear and anti‐extracellular matrix autoantibodies for albuminuria in murine lupus nephritis; a longitudinal study on plasma and glomerular eluates in MRL/l mice

The relationship between autoantibody reactivities and nephritis in systemic lupus erythematosus (SLE) is unclear. We studied MRL/l mice which developed a considerable albuminuria (either mice with short (< 1 week) or heavy and prolonged (3 weeks) albuminuria) and compared them with non‐albuminuric age‐matched controls, with young (12 weeks old) non‐albuminuric mice and with mice which were followed for 36 weeks and did not develop albuminuria. In a longitudinal prospective study on plasma samples we correlated a variety of anti‐nuclear reactivities and reactivities against extracellular matrix (ECM) components, with the onset of albuminuria. We found that at the onset of albuminuria, anti‐DNA was higher while anti‐nucleosome and anti‐H2A/H2B‐DNA subnucleosome reactivities were lower compared with age‐matched non‐albuminuric mice. We also studied glomerular eluates of these mice in ELISA and in indirect immunofluorescence (IF). In the eluates we found with IF that anti‐glomerular basement membrane (GBM)‐tubular basement membrane (TBM) antibodies were already present in 12‐week‐old non‐albuminuric mice. These eluates showed no anti‐nuclear antibodies. In eluates of albuminuric mice more immunoglobulin was deposited, and anti‐ECM, anti‐DNA and anti‐nucleosome reactivities were higher than in eluates of age‐matched non‐albuminuric mice. The deposition of anti‐nucleosome antibodies preceded the deposition of anti‐DNA antibodies since they were deposited to a greater extent in mice with a short albuminuria. We conclude that anti‐GBM‐TBM antibodies are the first autoantibodies that deposit in glomeruli of MRL/l mice at an early age. The onset of albuminuria is associated with additional deposition of both anti‐ECM and anti‐nuclear (anti‐nucleosome and anti‐DNA) antibodies, but the difference with non‐albuminuric mice seems to be more quantitative than qualitative.

Psychiatric symptoms before systemic lupus erythematosus is diagnosed

Psychiatric symptoms are rarely reported as an initial feature of systemic lupus erythematosus (SLE). Nevertheless, many patients have the feeling that psychiatric symptoms occurred before they were diagnosed as having SLE. This feeling was confirmed by an enquiry among members of the Dutch Lupus Patients Society: half of them had experienced psychiatric complaints before SLE was diagnosed. Two-thirds of these patients searched for professional help for these complaints. This motivated us to study whether SLE patients were admitted into psychiatric hospitals without being diagnosed as having SLE. Sera from 2121 patients admitted to a psychiatric hospital and from 500 controls matched for sex and age were tested for the presence of antinuclear antibodies (ANA) and antibodies to DNA. ANA were found in 3% of patients, as well as controls. Anti-DNA antibodies were found in 1% of both patients and controls. Two out of 114 patients psychiatric patients with ANA and/or anti-DNA antibodies had SLE and/or Sjögrens syndrome. We concluded that SLE is not an important cause of admission to psychiatric hospitals. Routine tests for the determination of antinuclear and anti-DNA antibodies on admissions in these hospitals thus would not seem useful. To study whether patients with another chronic disease also had psychiatric complaints before being diagnosed, we performed the same enquiry among members of the Dutch Sarcoidosis Patients Society. The results were almost equal to those of the enquiry of the members of the Dutch Lupus Patients Society. Why members of both societies so often report psychiatric symptoms before their disease is diagnosed should be a subject of further studies.

Characterization of murine monoclonal antibodies against 60-kD Ro/SS-A and La/SS-B autoantigens

Small cytoplasmic ribonucleoproteins (scRNPs) are important autoantigens in patients with systemic lupus erythematosus and Sjögrens syndrome. MoAbs against these proteins were made by immunization of BALB/c mice with purified human recombinant 60‐kD Ro/SS‐A or 50‐kD La/SS‐B proteins. Five stable hybridoma cell lines were obtained, of which four secreted anti‐Ro/SS‐A antibodies (clones 1D8, 1D11, 2G10 and 6G8) and one produced anti‐La/SS‐B antibodies (clone 7F6). The MoAbs were further characterized using four different immunoassays: immunofluorescence, immunoblotting, RNA precipitation combined with Northern blotting, and recombinant protein precipitation. All lour MoAbs against Ro/SS‐A recognized the native protein and one of them (2G10) recognized also intact scRNP particles. Interestingly, hY3‐RNA was reproducibly not efficiently precipitated by MoAb 2G10. Epitope mapping using deletion mutants of the 60‐kD Ro/SS‐A antigen showed that MoAb ID8 recognized the C‐terminal part of this protein, while 1D11 and 2G10 recognized distinct epitopes in the region between the RNP motif and the putative zinc finger domain. The epitopes recognized by these MoAbs lire highly conserved among species, and the epitope recognized by MoAb 2G10 may be identical to an autoepitope recognized by sera of patients. This is the first report describing the isolation and characterization of MoAbs of the IgG class against the 60‐kD Ro/SS‐A and La/SS‐B autoantigens obtained by immunization with purified human recombinant proteins. These MoAbs can be of great use in studying the cellular processes in which scRNPs are involved, and may help to determine why these scRNPs become autoantigenic in autoimmune diseases.

CLINICAL-EVALUATION OF A MODIFIED ELISA, USING PHOTOBIOTINYLATED DNA, FOR THE DETECTION OF ANTI-DNA ANTIBODIES

The measurement of anti-dsDNA antibodies is important for the diagnosis and the follow-up of patients with systemic lupus erythematosus (SLE). For routine detection of anti-dsDNA, the Farr assay and the immunofluorescence technique (IFT) on Crithidia luciliae proved to be very useful. The anti-dsDNA ELISA is not used for routine purposes in our institute since it is flawed by false-positive results due to binding of negatively charged (immune) complexes to the employed precoat (protamine sulphate). Recently, a new anti-dsDNA ELISA has been described in which photobiotinylated dsDNA is coated to streptavidin coated plates. To investigate whether this modified ELISA is more specific than the classical anti-dsDNA ELISA, we tested sera of patients with SLE (n = 51), myasthenia gravis (MG, n = 25), rheumatoid arthritis (RA, n = 25) and Sjögrens syndrome (SS, n = 23) and sera of healthy blood bank donors (BBD, n = 25). In both assays the sera of the SLE patients gave significantly higher values than the sera of healthy blood bank donors. In the classical ELISA, 84% of the sera from patients with RA and 28% of sera of patients with MG were found positive. For the modified assay the figures were 8% and 24%, respectively. This modified ELISA was further studied and clinically evaluated by comparing it with the classical anti-DNA ELISA and two other anti-DNA assays (Farr assay and IFT), using 500 sera sent to our institute for routine anti-DNA determination and sera of an additional 75 healthy blood bank donors. Quantitatively, both ELISAs showed the same high degree of correlation with the IFT. The modified ELISA gave a better correlation with the Farr assay than the classical anti-DNA ELISA. From our data we conclude that the ELISA using photobiotinylated DNA is a more reliable assay than the classical anti-DNA ELISA.