Experimental immune mediated arthritis in rhesus monkeys. A model for human rheumatoid arthritis?

The induction of experimental arthritis in rhesus monkeys was studied by intradermal immunization of bovine type II collagen and antigens derived from Mycobacterium tuberculosis, Streptococcus pyogenes, and Eubacterium aerofaciens. The tested bacterial antigens proved to be not arthrogenic. Bovine type II collagen induced clinical arthritis in 50% of the rhesus monkeys. Type II collagen induced arthritis in rhesus monkeys proved to be a potential model to study clinical, serological, histological, genetic, and immunologic features associated with human RA.

Immune complex formation between IgM rheumatoid factor and IgG generated by hyaluronic acid.

Immunoglobulin inclusions are regularly detected in granulocytes obtained from the synovial fluid of rheumatoid arthritis (RA) patients. In contrast, granulocytes isolated from the blood of the same patients usually contain no immunoglobulin inclusions. Using the indirect granulocyte phagocytosis test (IGPT), we obtained evidence that this discrepancy can be explained by the finding that hyaluronic acid (HA), a component of synovial fluid, increases the avidity of rheumatoid factor (RF), resulting in the formation of IgG-IgM-RF complexes. The addition of HA to RA sera and subsequent testing by IGPT revealed an increased uptake of the induced immune complexes by these cells, which was dependent on the HA dose. Furthermore, supplementation of normal human serum with purified IgM-RF generated a positive IGPT result in a dose-dependent manner in the presence of HA. We conclude that HA, a component of synovial fluid, might facilitate immune complex formation in the joint cavity, resulting in the inflammatory reaction in the joints of RA patients.

Circulating immune complexes and rheumatoid arthritis: the induction of immune complex formation between rheumatoid factor and IgG by polyethylene glycol.

Circulating immune complexes (CIC) as detected by the C1q binding assay (C1qBA) in sera from patients with rheumatoid arthritis (RA) were not demonstrable in these sera with the indirect granulocyte phagocytosis test (IGPT). This discrepancy could be explained by the finding that polyethylene glycol (PEG), used in the C1qBA, enhanced the binding of rheumatoid factor IgM (RFIgM) and IgG resulting in immune complex (IC) formation. The addition of PEG to RA sera and subsequent testing of these sera in the IGPT revealed increased uptake of IC by these cells, dependent on the PEG dose. Addition of purified RFIgM to a normal human serum generated a positive IGPT in a dose dependent way. We conclude that in RA sera PEG induces IC between RFIgM and IgG. Therefore, assays devised to measure CIC in RA sera which are based on PEG (like the C1qBA) overestimate the amounts of IC present in the circulation in vivo.

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.

Effects of cyclosporin A on autoimmune disease in MRL/1 and BXSB mice.

MRL/1 and BXSB mice were treated daily with cyclosporin A (CyA) in an oral dose of 25 mg/kg body weight. With this dose, blood levels within the therapeutic range were obtained. In normal mice CyA in this dose significantly prolonged the survival of an H-2 incompatible skin graft, and suppressed delayed-type hypersensitivity (DTH). It had no influence on the magnitude of a primary antibody response. Autoimmune mice were treated from 6 to 22 weeks of age. CyA treatment did not alter significantly the anti-DNA and anti-IgG autoantibody levels in either strain compared with control mice, who received olive oil. There was a slight but significant increase in serum IgG levels in CyA-treated MRL/1 mice. Clinical signs of glomerulonephritis (decreased kidney function and albuminuria), and glomerular proliferation were not altered by CyA treatment in either strain. The amount of mesangial IgG deposits was reduced in CyA-treated MRL/1 mice, and remained unchanged in BXSB mice. The extent of the interstitial and perivascular infiltrates and the frequency and severity of necrotizing arteritis in the kidneys of MRL/1 mice were reduced by CyA treatment. The most prominent effect of CyA was an evident reduction in lymphoproliferation in MRL/1 mice. Mortality was not reduced by CyA treatment in MRL/1 and BXSB mice.

Cross-reactivity of human and murine anti-DNA antibodies with heparan sulfate. The major glycosaminoglycan in glomerular basement membranes.

In 30 of 33 human systemic lupus erythematosus (SLE) sera and in 10 sera from MRL/l mice with spontaneous SLE, antibodies against heparan sulfate were detected. The anti-heparan sulfate titers showed a significant correlation with the anti-DNA antibody titers. By inhibition studies it was demonstrated that heparan sulfate could inhibit the binding of anti-DNA antibodies to DNA, whereas DNA could block the binding to heparan sulfate. That this reaction is due to crossreactivity of anti-DNA antibodies was further substantiated by the finding that two monoclonal anti-DNA antibodies also bound to heparan sulfate. Antibodies eluted from human and mouse kidneys with diffuse SLE glomerulonephritis showed a similar binding to DNA and heparan sulfate when these eluted antibodies were tested in vitro. Heparan sulfate is the major glycosaminoglycan constituent of the glomerular basement membrane. Our findings suggest that heparan sulfate might serve as a target antigen in vivo for cross-reactive anti-DNA antibodies.

Direct binding of monomeric anti-DNA antibodies to Raji cells.

The Raji-cell test is one of the most widely used methods for the detection and quantitation of immune complexes. Immune complexes and not 7 S IgG bind via C3 to complement receptors on the cell membrane of the Raji cell. During sucrose gradient fractionation of human and murine systemic lupus erythematosus sera, with a high Raji cell-binding activity, we could not demonstrate immune complexes in these sera. Subsequent analysis showed that the major part of the Raji cell binding was used by 7 S IgG with an anti-DNA specificity. Blocking experiments with complement-bearing aggregated IgG revealed that complement and Fc receptors were not involved in the binding of these anti-DNA antibodies to Raji cells. We conclude that the Raji cell test is not suitable for the detection and quantitation of immune complexes in sera containing anti-DNA antibodies.

Circulating immune complexes and rheumatoid arthritis.

Circulating immune complexes (CIC), as detected by the Clq binding assay (ClqBA) in sera from patients with rheumatoid arthritis (RA) were not demonstrable on analysis by ultracentrifugation on sucrose gradients. This discrepancy could be explained by the finding that polyethylene glycol 6000(PEG), used in the ClqBA to separate free radiolabelled Clq from complex bound Clq, increased the avidity of rheumatoid factor (RF), resulting in the formation of Clq binding RF IgM IgG complexes. Addition of purified RF IgM to normal human serum generated a positive ClqBA in a dose dependent way. The increased complex formation between RF IgM and IgG by PEG was also demonstrated in an enzyme linked immunoabsorbent assay and with sucrose gradients, where complexes became detectable when PEG was present. On the other hand RF IgM IgG Clq complexes obtained from the ClqBA dissociated upon removal of PEG. We conclude that high amounts of immune complexes, detected in RA sera by the ClqBA, are at least partly the result of in vitro complex formation between RF IgM and IgG. Therefore the results of this assay do not reflect the situation in the circulation in vivo.

Therapeutic efficacy of alpha-adrenoceptor blockade in primary and secondary Raynaud's syndrome.

Twenty patients with primary and 11 with secondary Raynaud's syndrome were treated with the alpha-adrenoceptor blocker phenoxybenzamine (10-20 mg daily). In the secondary group mean age and mean duration of symptoms as well as presence of positive ANA and positive Clq binding test were significantly higher than in the primary group. In both groups a beneficial effect of the medication on finger temperature 12 min after finger cooling and on clinical symptoms was established. Secondary Raynaud's syndrome reacted at least as well as primary syndrome.

Cross-reactivity of anti-DNA antibodies with proteoglycans.

Ten sera of patients with systemic lupus erythematosus (SLE) were tested in an enzyme linked immunosorbent assay for their ability to react with glycosaminoglycans, constituents of proteoglycans, in relation to their anti-DNA reactivity. The SLE sera reacted with hyaluronic acid and chondroitin sulphate and this reactivity correlated with the anti-DNA activity of these sera. By contrast, sera obtained from patients with other autoimmune diseases or normal sera lacked any of these reactivities. Anti-DNA antibodies purified by affinity chromatography with either oligo dT cellulose or Cibracon blue F3Ga Sepharose reacted with DNA as well as with hyaluronic acid. The cross-reactivity of anti-DNA antibodies could be confirmed by the reaction of a mouse monoclonal anti-DNA antibody with DNA, hyaluronic acid, and chondroitin sulphate. This pattern of cross-reactivities of anti-DNA antibodies suggests that several compounds can function as antigenic targets for these antibodies provided that their structures contain repeating negatively charged groups.

The interference of fibrinogen and heparin with the determination of circulating immune complexes in the C1q-binding assay.

When citrate plasma and serum of the same individual were tested simultaneously in the C1q-binding assay (C1qBA), binding levels in plasma were found to be 90-400% higher than in serum. The difference in 125I-Clq binding was due to the presence of fibrinogen in plasma. It was shown that complex formation between fibrinogen and 125I-Clq occurs and that this complex precipitates in the presence of polyethylene glycol, leading to the false positive results in the ClqBA. When heparin plasma was used to the assay, heparin itself also induced an increase in 125I-C1q binding that was not based on the presence of immune complexes. The effect of both fibrinogen and heparin could be inhibited by addition of protamine sulphate. Therefore, pretreatment of plasma with protamine sulphate makes it possible to use plasma samples for a reliable determination of C1q-binding levels. However, serum that is well clotted should be used preferentially.