Autoimmune diseases other than lupus share common anti-DNA idiotypes

We examined the sera of 170 patients with various autoimmune diseases other than systemic lupus erythematosus (SLE) for the presence of an anti-DNA antibody idiotype termed 16/6 and known to occur with high frequency in sera of patients with SLE. The idiotype was found in 6/15 sera from patients with polymyositis (49%), 3/18 with multiple sclerosis (17%), 3/18 with primary Sj?gren's syndrome (18%), 9/40 with autoimmune thyroid diseases (23%), 2/35 with myasthenia gravis (6%), and 3/42 patients with rheumatoid arthritis (7%). The idiotype was not detected among 12 patients with scleroderma or 77 normal controls. The presence of the 16/6 idiotype was associated with the presence of another anti-DNA idiotype termed 134-Id. Serum samples were also tested for activity against DNA, various synthetic polynucleotides, and cardiolipin. The serum activity against these antigens was found to be polyspecific, though overlap in reaction against the various polynucleotides was not absolute. The 16/6 idiotype is thought to be coded by a germline gene. The presence of this idiotype in various autoimmune diseases points to a pathophysiologic link between the diseases.     

Cross-reactive idiotypes in anti-DNA antibodies of systemic lupus erythematosus patients

Cross-reactive idiotypes associated with the combining site of anti-double-stranded DNA antibodies from systemic lupus erythematosus patients were demonstrated by the ability of isologous lupus sera to block functionally the binding of target anti-DNA antibodies to DNA in vitro. A framework idiotype, denoted AM Id, was identified using xenogeneic anti-idiotype antibodies rendered specific by affinity absorptions. The AM Id was found in 85% of patients with systemic lupus erythematosus (n = 63) and correlated positively with anti-DNA antibodies. Analysis of the distribution of the AM Id among individuals showed that, while present in anti-DNA antibodies to varying degrees in individual patients, it was also found in variable amounts on non-DNA-binding immunoglobulins. These results indicate that the AM Id and anti-DNA antibodies represent overlapping populations of immunoglobulins.     

Shift of private and not of cross-reactive anti-DNA idiotypes in systemic lupus erythematosus.

The shift of private idiotype (Id) and cross-reactive Id (CRI) on anti-DNA antibodies in a lupus patient KE was investigated during a 7-year period. Anti-private Id and anti-CRI activities were separated by affinity chromatography from rabbit (R)-anti-Ids raised against KE anti-DNA antibodies during active (1/84) and inactive (4/90) stages of the disease. Anti-CRI isolated from the 84 R-anti-Id appeared to recognize binding site-related Ids that are shared with KE non-anti-DNA antibodies, unrelated lupus patients' sera, and certain normal sera. Id expression on serial serum samples of KE using these fractionated R-anti-Ids as probes showed that the 1/84 private Id expression declined while the 4/90 private Id expression gradually increased. Expression of the CRI showed a relatively stable pattern. These results suggest that anti-DNA populations detected by anti-private Id can shift, while populations expressing CRI may stay stable.     

Private idiotypes on human polyclonal IgG anti-DNA antibodies are not expressed on coexisting IgM anti-DNA antibodies in systemic lupus erythematosus

Sharing of private idiotypes (Id) on human polyclonal IgG anti-double-stranded DNA (dsDNA) with coexisting IgM anti-dsDNA was investigated using rabbit (R) anti-Id raised against IgG anti-dsDNA. The R-anti-Id showed specificity to private Id in or near the antigen-binding sites. The R-anti-Id poorly bound to the immobilized enriched IgM anti-dsDNA preparation but significantly bound to IgG anti-dsDNA preparation by a direct-binding ELISA (0.020 OD vs 0.295 OD, respectively). The R-anti-Id poorly inhibited the binding of IgM anti-dsDNA to immobilized dsDNA but significantly inhibited the binding of IgG anti-dsDNA to dsDNA (6% vs 55% inhibition, respectively). This was confirmed by poor inhibition of binding of the R-anti-Id to immobilized IgG anti-dsDNA by the enriched IgM anti-dsDNA preparation (maximum of 26% inhibition at 50 micrograms/ml). Nonsharing of private Id between IgG and coexisting IgM anti-dsDNA may represent the idiotypic diversity of human anti-DNA antibodies secondary to the frequent occurrence of somatic mutation on anti-DNA antibody during class switching.     

Clonal frequency analysis of B cells producing pathogenic anti-DNA antibody-associated idiotypes in systemic lupus erythematosus.

In order to identify the mechanism responsible for autoantibody production in systemic lupus erythematosus (SLE), B cell repertoires associated with anti-DNA idiotypes were explored by a limiting dilution analysis using Epstein-Barr virus (EBV) transformation methods and ELISA spot assays. The frequencies of B cell clones producing antibodies to DNA and to conventional antigens, tetanus toxoid, dinitrophenyl, or keyhole limpet hemocyanin were higher in active SLE compared to those in inactive SLE and in normal subjects. In addition, there was a disproportionate increase in anti-DNA antibody- and anti-DNA idiotype (Id)-producing clones at the precursor cell levels as well as at the mature cell level. On the other hand, numbers of anti-Id clones against anti-DNA-Id, termed 0-81 Id, were markedly increased at inactive stages of the disease but not at active stages. These were confirmed by serial studies in some patients with SLE. These results support a two-step mechanism for autoantibody production, in which initial polyclonal activation is followed by an antigen-driven process, and indicate an alteration of the precursor B cell repertoire in SLE, which may also associate with a preferential expansion of anti-DNA clones.     

Suppressor cell function and anti-DNA antibody idiotypes in the serum of SLE patients and their first-degree relatives

Sixteen-six (16/6) is a major cross-reactive idiotype of monoclonal anti-DNA antibodies, which was derived from the fusion of lymphocytes of a patient with systemic lupus erythematosus (SLE). Antibodies with the 16/6 idiotype (16/6 Id) are increased in the sera of patients with SLE and deposited in their gomeruli and skin. Since stimulated lymphocytes from healthy persons have the capacity to produce 16/6 Id, the mechanisms controlling its expression in health and their possible failure in SLE are of considerable interest. A defect in suppressor cell function was found in a high proportion of patients with SLE and in some of their first-degree relatives. Suppressor cell function in 15 SLE patients and in 53 relatives was compared with the level of 16/6 Id as well as with immunoglobulin levels and anti-DNA antibodies. Ten of 15 SLE patients and 26 of 53 first-degree relatives had increased serum 16/6 levels, which was found in only 1 of 35 healthy controls and household members. Of the 10 SLE patients with increased 16/6, six had a suppressor cell defect (P less than 0.1). Among the 26 first-degree relatives with elevated 16/6 Id levels, 12 had associated suppressor defect and in only two cases was a suppressor cell defect unaccompanied by increased 16/6 (P less than 0.005). For the group of 18 patients and relatives showing concomitant suppressor cell defect and increased 16/6, a correlation was found between the severity of the suppressor cell defect and the level of 16/6 Id in the serum. The increased 16/6 in the relatives was not associated with hypergammaglobulinemia or with measurable anti-DNA activity in the serum. We conclude that the suppressor cell defect in relatives of SLE patients is often associated with increased expression of antibodies with the 16/6 idiotype. However, additional mechanisms are involved in the regulation of 16/6 Id and the development of clinical SLE, since increased 16/6 was commonly found in the presence of a normal suppressor T-cell function.     

Platelet-associated DNA and anti-DNA antibody in systemic lupus erythematosus with nephritis.

We report DNA and specific anti-dsDNA antibody (dsDNAb) in excess of controls, associated with platelets from 35 patients with systemic lupus erythematosus (SLE). Deoxyribonuclease (DNase) treatment of intact platelets resulted not only in a drop in platelet-associated DNA, but also a dramatic fall in platelet-associated dsDNAb. This suggests that at least some DNA and virtually all the dsDNAb is bound to the platelet surface, probably in the form of an immune complex. The quantity of platelet-associated DNA did not correlate with amounts of platelet-associated anti-dsDNA antibody (PAdsDNAb); perhaps because platelets also have receptors for DNA itself. Total platelet-associated IgG was elevated in only six patients with normal PAdsDNab.     

Epstein-Barr virus-transformed B cells bearing idiotypes of anti-DNA autoantibodies

Epstein-Barr virus (EBV)-transformed B cells obtained from healthy subjects had the same idiotypes of anti-DNA autoantibodies on their surface as those obtained from patients suffering from systemic lupus erythematosus. These clones secreted anti-single-stranded or antidouble-stranded DNA antibodies. Among them, some produced anti-DNA idiotype-positive antibodies but failed to bind DNA. This was confirmed by a competitive inhibition radioimmunoassay. It was then considered whether or not the expression of anti-DNA idiotype on B-cell clones related to the anti-DNA antibody activityin vivo. The amounts of anti-DNA antibodies were not associated with the incidence of idiotype-positive B cells in the EBV-transformed cell lines from normals. The results indicate that the clones committed to the synthesis of anti-DNA idiotype-positive antibodies commonly exist at a resting state in the circulation of healthy subjects, probably through the self-tolerance regulatory system.     

Accessory cell activity of monocytes in anti-DNA antibody production in systemic lupus erythematosus.

Depletion of monocytes from peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE) resulted in a decreased anti-DNA antibody synthesis in vitro. The addition of monocytes restored the response by lymphocytes and the maximum response in the presence of 2.5-8% of monocytes, whereas greater than 15% of monocytes caused rather decreased antibody responses. In order to further evaluate the role of monocytes on an anti-DNA antibody synthesis, we designated an accessory cell index, based on reconstitution experiments using isologous SLE lymphocytes, and compared it in active or inactive SLE and controls. The studies revealed that active SLE monocytes enhanced spontaneously occurring anti-DNA antibody synthesis by SLE lymphocytes. These results indicate that SLE monocytes actively participate in spontaneously occurring anti-DNA autoantibody synthesis in humans.     

Specific detection of circulating DNA:anti-DNA immune complexes in human systemic lupus erythematosus sera using murine monoclonal anti-DNA antibody.

The presence of DNA-anti-DNA immune complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by a new solid phase radioimmunoassay (RIA). This assay used murine monoclonal anti-double stranded DNA (dsDNA) antibody to recognize DNA present in the complexes and 125I-rabbit anti-human gamma globulin as a tracer. DNA-anti-DNA immune complexes were found in certain SLE sera but not in sera from patients with other immune complex diseases and from healthy blood donors. The presence of circulating DNA-anti-DNA complexes was associated with low C4 levels. It was not related to the presence of immune complexes detected by the polyethylene glycol assay suggesting either that the assay did not detect all DNA-anti-DNA complexes or that other antigen-antibody systems constitute the major immune complex components in SLE sera. The clinical significance of circulating DNA-anti-DNA complexes in SLE sera as well as the potential use of this solid phase RIA using various monoclonal antibodies to detect specific antigen-antibody systems is discussed.     

Shared idiotypes are expressed on mouse and human anti-DNA autoantibodies.

The expression of common idiotypes on human and mouse anti-DNA monoclonal autoantibodies made by hybridomas was examined by their competitive binding to anti-idiotype antibodies. Some murine autoantibodies inhibited the binding of a human anti-DNA autoantibody 16/6 to monoclonal or polyclonal anti-idiotypic antibodies. Another human antibody (134) was not inhibited in its binding to homologous anti-idiotypic antibodies. The expression of the human 16/6 idiotype on mouse antibodies was restricted to those that had a specificity similar to the 16/6 antibody itself, their major properties being that they reacted more strongly with single stranded DNA (ssDNA) than double stranded DNA (dsDNA). One mouse antibody expressing the 16/6 idiotype also bound weakly to RNA. The results imply structural similarities between the binding sites of the antibodies in the two species, and are consistent with evolutionary conservation of V genes coding for primitive ancestral antibodies that react with DNA and become diversified through somatic mutation.     

Effect of anti-idiotypic antibody on production of anti-DNA antibodies by splenocytes in lupus prone mice

It has been suggested that production of autoantibodies is regulated by idiotype-antiidiotype network. In this study, we examined modulatory effect of the antiidiotypic antibody on the synthesis of anti-DNA antibodies by New Zealand black/New Zealand white F1 mice (B/W F1) splenocytes. The antiidiotypic antibodies were prepared by immunization of a monoclonal anti-DNA antibody derived from B/W F1 to rabbits. The prepared antiidiotypic antibody had specificity to the antigen binding site of anti-DNA antibodies. B/W F1 splenocytes were adjusted to 1 X 10(6) cells/ml and cultured in 1.0 ml aliquots in the presence of varying concentrations of the antiidiotypic antibody for 48 hours. The cells were then washed three times, resuspended in RPMI1640 containing 10% fetal calf serum and cultured again. On days 3 and 7 of the culture, the supernatants were harvested and secretion of anti-DNA antibodies was measured by ELISA. Production of anti-DNA antibodies by B/W F1 splenocytes was suppressed by pretreatment with the antiidiotypic antibody. When the concentration of antiidiotypic antibody was 1 microgram/ml, anti-DNA activity of the supernatants decreased 50%, compared with control on day 3, but the effect was reduced on day 7. The treatment of antiidiotypic antibody did not affect the proliferation and viability of B/W F1 splenocytes. The results indicated that anti-DNA antibodies synthesis were regulated by idiotype-antiidiotype network and could be manipulated by the antiidiotypic antibody.     

Detection of DNA-bound immunoglobulins in patients with lupus nephritis, using monoclonal anti-DNA antibody.

In order to investigate the relationship between renal histopathology and the characteristics of circulating immune complexes (CICs) in patients with lupus nephritis (LN), we measured the sizes of CICs, DNA-bound immunoglobulins in patients with systemic lupus erythematosus (SLE) and different histopathological forms of nephritis. Sera were obtained from nine patients: four with diffuse proliferative LN (DPLN), four with membranous LN (MLN), and one with mesangial LN, who fulfilled the criteria of the American Rheumatism Association for SLE. The DNA-bound immunoglobulins were measured by ELISA, in which ELISA plates were coated with mouse monoclonal anti-DNA antibodies. The sizes of CICs were analysed by sucrose density gradient ultracentrifugation. Large (larger than 19S), intermediate (19-7S) and small (nearly 7S) sized DNA-bound immunoglobulins (high peaks of IgG and IgA, but low IgM peaks) were found in the patients with DPLN. By contrast, in patients with MLN, the sizes of ICs; DNA-bound IgG, IgA were in general slightly larger than 7S. In one patient with DPLN, at the onset, various sized DNA-bound IgG, IgA and IgM were found. After the methylprednisolone pulse therapy, CICs became smaller and gradually disappeared. We conclude that the characteristics of DNA-anti-DNA IgG, IgA complexes may determine the localization of ICs in the glomeruli and suggest that CICs play an important role in the pathogenesis of LN.     

Frequency of anti-DNA antibody producing cells from normals and patients with systemic lupus erythematosus

The frequency of anti-DNA antibody producing cells from normals and patients with systemic lupus erythematosus (SLE) was determined. Peripheral blood lymphocytes (PBL) from normals and patients with SLE were cultured for 8 and 15 days with and without transformation by Epstein-Barr virus (EBV). Culture supernatants were examined for the presence of anti-DNA antibody using an enzyme-linked immunosorbent assay. We found that PBL from patients with SLE spontaneously produce anti-DNA antibodies whereas PBL from normals do not. After EBV transformation, anti-DNA antibody producing cells were detected in both cultures from patients with SLE as well as from normals. These data suggest that the high levels of anti-DNA antibody observed in patients with SLE represent activation of B cells committed to anti-DNA antibody production and that such cells are present but are not activated in normal individuals.     

Polyhydroxyethylmethacrylate-based magnetic DNA-affinity beads for anti-DNA antibody removal from systemic lupus erythematosus patient plasma

The aim of this study is to prepare magnetic poly(2-hydroxyethylmethacrylate) (mPHEMA) beads and to investigate their utility for the removal of anti-DNA antibodies from systemic lupus erythematosus (SLE) patient plasma. mPHEMA beads, in the size range of 80-120 microm, were produced by a modified suspension technique. Then, DNA was coupled onto mPHEMA beads by carbodiimide activation. The amount of ligand coupled was changed by changing the initial concentrations of carbodiimide and DNA. Human immunoglobulin G (HIgG) and anti-DNA antibody adsorption from aqueous solutions and human plasma were examined in a batch system. mPHEMA beads were characterized by swelling tests, electron spin resonance (ESR) and scanning electron microscopy. Important results obtained in this study are as follows: the swelling ratio of mPHEMA beads was 34%. The presence of magnetite particles in the polymeric structure was confirmed by ESR. The mPHEMA beads have a spherical shape and porous structure. Maximum DNA coupling of carbodiimide activated mPHEMA beads was 4.4 mg/g. Maximum HIgG adsorption from an aqueous solution was 47.5 mg/g. Anti-DNA antibody adsorption from SLE plasma was observed as 87.6 mg/g. Non-specific HIgG adsorption was 0.1 mg/g. More than 90% of the adsorbed HIgG molecules and anti-DNA antibodies were desorbed succesfully by using NaSCN solution. It was possible to reuse these DNA-affinity beads without significant losses in the antibody adsorption capacities.     

Standardization of anti-DNA antibody assays

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA–anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.     

Somatically mutated IgG anti-DNA antibody clonally related to germ-line encoded IgM anti-DNA antibody.

Among 15 anti-DNA antibody-producing hybridomas derived from a single NZB X NZW F1 mouse, an IgM and an IgG were shown to use the same VH gene of the Q52 family. Using a combination of two primers both consisting of a mixture of oligonucleotides (one complementary to the 5' end of VH segment and one to the 3' end of VH segment of Q52 family) we determined the sequences of several members of germ-line VH genes in the Q52 family derived from NZB and NZW strains. Comparison of the sequences with those of cloned VH cDNA obtained from the hybridomas revealed that the VH sequence of the IgM anti-DNA antibody was identical to that of a cloned NZW germ-line VH gene, except for the priming sites. In contrast, the VH sequence of the IgG counterpart contained somatically mutated nucleotides. Because the IgG anti-DNA antibody showed a higher DNA binding activity than did the IgM antibody, we conclude that these changes in nucleotide sequences were induced and selected through an antigen-driven mechanism as is the case in a normal immune response. It is tempting to speculate that the germ-line encoded, low-affinity IgM autoantibody undergoes somatic mutations and isotype switching, resulting in generation of pathogenic, high-affinity autoantibodies in autoimmune diseases.     

A hypothesis about anti-DNA antibody

"Anti-DNA antibody" is the antibody which can bind DNA molecules. It is well accepted that this antibody, especially in anti-double strand DNA antibody, has a high disease specificity for SLE. Usually, the antibody is detected by using DNA molecule as a binding target in the clinical laboratory test performed in vitro. However, it is quite unknown what the antibody is actually attacking in vivo. This review will introduce many findings known to date and try to solve the mystery of anti-DNA antibody with a hypothesis that the antibody nonspecifically crossreacts to negatively charged substances(e.g., polysaccharides) in vivo. Probably, a type of charge interaction is dominantly involved in this binding.     

Anti-acetylcholine receptor antibody related idiotypes in myasthenia gravis

Anti-acetylcholine receptor antibody associated idiotypes were defined by six murine monoclonal antibodies raised against purified receptor antibodies. Four of the monoclonal antibodies bound to idiotopes located within or close to the antigen binding site of the anti-receptor antibodies; the other two monoclonal antibodies were directed against framework determinants. These monoclonal antibodies recognized idiotopes present on immunoglobulins in 14-60% of patients presenting myasthenia gravis, indicating substantial idiotype sharing. These idiotopes were also found in patients with no detectable anti-receptor antibody activity in their serum. In all patients studied, the pattern of idiotypes fluctuated considerably during the course of the disease regardless of clinical symptoms. This suggests continuous modulation of the autoimmune process in myasthenia gravis.