C. Kramers

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.

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 (<u20031 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.

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.

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.

A NEW ELISA FOR THE DETECTION OF ANTI-HEPARAN SULFATE REACTIVITY, USING PHOTOBIOTINYLATED ANTIGEN

Autoantibodies reacting with a great variety of autoantigens are characteristic for the autoimmune disease systemic lupus erythematosus (SLE). Although reactivity with heparan sulfate (HS) in sera of patients with SLE is found in association with the occurrence of nephritis, the aetiological significance of this association is not clear. The assay which is generally used to measure anti-HS reactivity is subject to false-positive results, as a consequence of the binding of negatively charged moieties within immune complexes to the precoat employed (protamine sulfate). Therefore, we have developed a new ELISA in which photobiotinylated HS is efficiently and reproducibly bound to streptavidin-coated wells. We compared the new ELISA with the classical anti-HS ELISA by testing culture supernatants of 20 murine monoclonal antibodies (mAb) to DNA (containing free anti-DNA and anti-DNA/nucleosome immune complexes) and preparations of these mAb (containing only free anti-DNA), purified under dissociating conditions. In the classical anti-HS ELISA, 14 out of 20 of the culture supernatants reacted positively with HS; after purification no reactivity remained. The discrepancy must be due to anti-DNA/nucleosome immune complexes present in the culture supernatants. In the new ELISA only four out of 20 culture supernatants and one of the purified preparations reacted with HS. This latter reactivity is probably not specific, since this mAb also reacted with streptavidin alone. To find out whether there is a correlation between the occurrence of nephritis and anti-HS reactivity, measured in this new anti-HS ELISA, we tested sera of patients with a renal- or non-renal exacerbation of SLE in the newly developed anti-HS ELISA. We observed a correlation between anti-HS reactivity and nephritis.

No evidence for an independent role of anti-heparan sulphate reactivity apart from anti-DNA in lupus nephritis

The presence of anti‐heparan sulphate (HS) reactivity in serum is closely related to the occurrence of nephritis in patients with systemic lupus erythematosus (SLE). Since patients with lupus nephritis in general also have high titres of anti‐DNA antibodies, we wanted to clarify the relationship between anti‐HS and anti‐DNA reactivity in serum. Therefore, we studied longitudinally six patients with lupus nephritis who experienced 12 exacerbations of their disease, and five SLE patients without nephritis experiencing 10 periods of non‐renal disease exacerbations. In addition, we tested single serum samples of another 24 patients obtained during a renal disease exacerbation and 22 sera of patients without nephritis. The sera of all patients were tested for anti‐DNA (Farr assay) and anti‐HS reactivity (ELISA). We confirmed that SLE patients during renal exacerbations have a significantly higher anti‐HS reactivity than patients without nephritis (P < 0·003). In addition, patients with nephritis also had higher titres of anti‐DNA antibodies during renal exacerbations than during non‐renal exacerbations (P < 0·01). A correlation between anti‐DNA and anti‐HS reactivity was observed (r= 0·40, P < 0·02), which in itself explains the correlation between nephritis and anti‐HS reactivity. Comparing sera from nephritis and non‐nephritis patients matched for anti‐DNA titre, we found no difference in anti‐HS reactivity, and therefore must conclude that the anti‐HS reactivity is a direct reflection of anti‐DNA reactivity.