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.     

Hidden high-avidity anti-DNA antibodies occur in normal human gammaglobulin preparations.

An ability to detect hidden high-avidity DNA-binding protein in human gammaglobulin samples was investigated. Using ion exchange chromatography on QAE-Sephadex A-50 a highly positive Farr assay DNA-binding fraction was reproducibly isolated from several commercial normal human gammaglobulin preparations. The estimated dissociation constant had a value of 1.04 x 10(-11) M thus confirming high avidity protein-DNA complex formation. Anti-DNA antibodies (Ab) ELISA data revealed that immunoglobulin (Ig) G, IgM and IgA participated in the protein-DNA interaction. Inhibitory experiments involving a number of polynucleotides, synthetic and natural polyanions demonstrated that both double-stranded DNA (dsDNA) and heat-denatured DNA but not RNA inhibited the protein-3H-DNA binding. Generally, the inhibiting effect was more pronounced when purine base-containing polynucleotides and polydeoxy- rather than polyribonucleotides were used. Synthetic polyanions and normal human sera (NHS) also markedly reduced the binding. The presence of hidden high-avidity DNA-binding antibodies in normal gammaglobulin preparations was suggested.     

Character of anti-DNA antibodies in systemic lupus erythematosus

Anti-DNA antibodies in twenty-one systemic lupus erythematosus (SLE) sera were analysed by precipitation in gel, complement fixation, and the Farr globulin precipitation technique. Fifteen sera precipitated in agarose with both native and denatured DNA (Group 1) and six precipitated solely with denatured DNA (Group 2). Prior incubation of the sera with RNA or DNA digest abolished the precipitin reactions of all Group 2 sera, but had no appreciable effect on the precipitin patterns of the Group 1 sera. Molecular class of anti-DNA antibodies did not correlate with precipitation pattern. The ability of sucrose density gradient fractions of serum to fix complement with denatured DNA at 4°C did not correspond with the presence of precipitating antibodies against denatured DNA.The Farr globulin binding assay probably allowed detection of all anti-DNA antibodies, and each of the sera bound most of the [¹⁴C]DNA. No binding was observed when ¹⁴C-labelled nucleotides were substituted for the [¹⁴C]DNA. Both native and denatured unlabelled DNA were able to significantly inhibit binding by all sera tested (Groups 1 and 2), while RNA and DNA digest gave minimal inhibition. Three types of antigenic sites on the DNA molecule are postulated. It is suggested that all anti-DNA sera, regardless of their behaviour in precipitin or complement fixation tests, contain antibodies reactive with all three determinants on the DNA molecule.     

Monoclonal human anti-DNA antibodies from EB virus-transformed lymphocytes of systemic lupus erythematosus (SLE) patients

Sixteen monoclonal human anti-DNA antibodies were obtained from Epstein-Barr virus-transformed lymphoblastoid cells of patients with systemic lupus erythematosus (SLE) and were studied in terms of antigenic specificity. All of the antibodies showed polyspecificity to polynucleotides. Among them, some antibodies had a specificity to single-stranded (ss) DNA. Especially, O-8 antibodies showed a preference for polynucleotides with pyrimidine bases. The binding specificity of the antibody was also studied using different sizes of dT oligomers in order to assess the size of the epitope. It was revealed that oligonucleotides with a size of more than 25–30 nucleotides are required for inhibition of the antibody to ss-DNA. Other studies also demonstrated that anti-ss-DNA (O-8) antibody and anti-double-stranded (ds) DNA (NE-28) antibody bound to different combining sites in the same polynucleotides, poly(dT). These results suggest that some anti-ss-DNA antibodies are directed to the conformational structure related to the base sequence and that nucleic acids, therefore, might be responsible for the possible immunogenic stimulus causing the anti-DNA immune response. We also indicate that this type of antibody would be popular among serum anti-DNA antibodies in SLE.     

Antiidiotypic antibodies against anti-DNA antibodies in sera of families of lupus patients

We searched for antiidiotypes directed against anti-DNA in sera of healthy family members of lupus patients. Controls were healthy individuals without a personal or family history of lupus. No significant differences were noted between the family members' and the control group's sera with respect to binding to DNA or to non-anti-DNA F(ab)₂ fragments. Family members' sera had higher binding to anti-DNA F(ab)₂ and to normal IgG F(ab)₂ fragments (P<0.01). Sera of the family members had significantly higher binding to anti-DNA F(ab)₂ than to normal IgG F(ab)₂ fragments (P<0.0036). Inhibition experiments have shown that the antiidiotype is directed against the framework determinants and not against the antigen binding sites of the idiotype. The anti-idiotypic antibodies were directed against cross-reactive anti-DNA idiotypes and were not restricted to the idiotypes of the lupus proband. Age, sex, and blood relationship to the lupus patient did not influence the presence of antiidiotypes in the family members. The possible role of environmental factors in the induction of antiidiotypes and the role of the latter in regulating anti-DNA antibodies are discussed.     

Relation between lymphocytotoxic antibodies, anti-DNA antibodies and a common anti-DNA antibody idiotype PR4 in patients with systemic lupus erythematosus, their relatives and spouses.

Forty-two patients with systemic lupus erythematosus (SLE), 65 of their healthy relatives and 20 spouses were studied for the presence of lymphocytotoxic antibodies (LCA), anti-lymphocyte antibodies (ALA), antibodies to DNA and a common idiotype (Id) PR4. Seventy-one per cent of the patients had positive levels of LCA, and in 34% the PR4 Id was detected; normal levels were found in their families. Anti-PR4, an anti-Id, failed to block the lymphocytotoxic activity in those nine patients who both carried the Id and had LCA. This indicates that the Id was not present on LCA. There was no correlation between anti-DNA antibodies and LCA, suggesting that different mechanisms are involved in their expression.     

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.     

Molecular and structural analysis of nuclear localizing anti-DNA lupus antibodies

To determine the structure of three nuclear localizing lupus anti-DNA immunoglobulins (Igs) and to search for clues to mechanisms of cellular and/or nuclear access, their H- and L-chain variable region sequences were determined and subjected to three-dimensional modeling. Although the results indicate heterogeneity in their primary structures, the H chains are encoded by 3 members of the J558 V_H gene family with a common tertiary conformation that is not shared by a J558-encoded nonnuclear localizing anti-DNA control Ig. Furthermore, at least two of the Igs share a conformational motif in the H-chain CDR3, and all three Igs contain multiple positively charged amino acids in their CDRs, resembling nuclear localization signals that direct protein nuclear import. Notably, each V_H and V_K gene is also found recurrently among previously described autoantibodies. Molecular analysis further indicates that both germline-encoded and significantly mutated V genes can generate nuclear localizing anti-DNA Ig.     

Role of anti-DNA antibodies in the pathogenesis of lupus nephritis

Despite a good association between anti-DNA autoantibodies and lupus nephritis, it is difficult to determine the pathogenic potential of an anti-DNA autoantibody response. It is proposed that anti-DNA antibodies can exert their pathogenic effects through deposition as immune complexes in the kidney or through the recognition of cross-reactive antigens in the kidneys. Several studies in literature demonstrate that cross-reactivity of anti-DNA antibodies with kidney antigens is critical for their pathogenic potential. This raises the question whether DNA is responsible for the activation and selection of B cells generating cross-reactive anti-DNA antibodies. Recent studies suggest that antigens other than DNA can initiate an antibody response that is cross-reactive with dsDNA. Moreover, we and other have demonstrated that lupus nephritis can occur in the absence of anti-DNA antibodies. Thus, reactivity to dsDNA should be considered as one of the characteristic of polyreactive autoantibodies and not a primary requisite for the pathogenesis of lupus nephritis.     

Specificity and binding kinetics of murine lupus anti-DNA monoclonal antibodies implicate different stimuli for their production

The origin and relative biological importance of the many different DNA-reactive antibodies that appear in systemic lupus erythematosus are not well understood. A detailed analysis of their fine specificity and binding characteristics with DNA is a necessary step in understanding their biology. We have examined here two monoclonal antibodies (mAb) IV-228 and V-88 that are, respectively, characteristic of antibodies, which bind exclusively to single-stranded (ss) DNA and to both double-stranded (ds) DNA and ssDNA. By surface plasmon resonance (SPR) on BIAcore, we characterized the kinetics of binding of each antibody to synthetic ss and ds oligonucleotides. Antibody V-88 and IV-228 showed different patterns of reactivity for both ss and ds oligonucleotides, characterized by distinctly different kinetic parameters. Analysis of their binding kinetics indicates the importance of base composition in defining DNA epitopes, and shows that some epitopes, such as that recognized by mAb V-88, are expressed on dsDNA and ssDNA, whereas others, as recognized by IV-228, are not. The base preferences of V-88 for ds GC-rich structures over AT-rich, and of IV-228 for ss T-rich structures, also reveal distinct differences between these antibodies. We conclude that the different binding properties of the antibodies will relate to their biological activities. The base preferences of the antibodies suggest that they might be induced by different immunological stimuli, such as those that could be provided by the various DNA fragments and structures released during programmed cell death.     

B cell repertoire for anti-DNA antibody in normal and lupus mice: differential expression of precursor cells for high and low affinity anti-DNA antibodies.

The precursor frequency for anti-DNA antibody producing cells and the affinity of antibodies secreted by these cells in both immature prereceptor B cell populations and mature B cell populations were compared between 8-week-old C57BL/6 female mice and 9-month-old B/WF1 female mice by producing a large collection of IgM secreting hybridomas from LPS-stimulated B cells. The data indicate that precursor cells for high affinity anti-DNA antibody are eliminated as they mature in C57BL/6 mice, while a sizable number of such clones are present in mature splenic B cells of aged B/WF1 mice. These results suggest that the emergence of precursors for high affinity anti-DNA producing cells in mature B cell population is an important factor in the pathogenesis of SLE.     

IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus

We describe the production of six mouse-human heterohybridomas secreting human IgG anti-dsDNA antibodies derived from patients with systemic lupus erythematosus (SLE). Peripheral blood cells used for fusion experiments were from patients who were shown to have high numbers of anti-DNA secreting B cells in the peripheral blood. All monoclonal antibodies bind to dsDNA in ELISA systems, five are reactive with Crithidia lucilae kinetoplasts and three precipitate dsDNA in the Farr assay. Inhibition studies revealed a remarkable specificity for certain polynucleotide structures. To our knowledge these are the first hybridomas described in the human system that secrete anti-dsDNA antibodies of the IgG class.     

Disease specificity of antibodies to poly (ADP-ribose); their relationships to anti-DNA antibodies and to disease activity in lupus.

In this study we have measured the level of anti poly (ADP-ribose) antibodies in the sera of a number of patients with SLE and their relatives, patients with a wide variety of other autoimmune and infectious diseases, and a group of normal healthy controls. It was found that these antibodies were not disease specific but were present in nine out of thirteen groups tested in significant numbers. The levels of anti poly (ADP-ribose) antibodies and anti DNA antibodies in SLE patients bled serially were also measured. The level of these antibodies fluctuated in parallel in many of these patients, although the anti poly (ADP-ribose) antibodies reflected disease activity more accurately in some.     

The effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.     

单克隆抗DNA自身抗体及其抗血清独特型交叉反应

由严重存在SLE,并无需外源免疫能自发产生抗DNA自身抗体的小鼠MRL-Lpr/Lpr脾细胞与SP2/0-Ag-14骨髓瘤细胞杂交,获得一系列杂交瘤细胞株,它们分别分泌单克隆抗DNA自身抗体。我们用其中的细胞株H241的单抗免疫家兔,从免疫血清中收获到抗H241独特型扰体。通过放射免疫分析法(RIA),对H241进行了敏感度分析,并研究了H241与抗H211血清反应中,H241与另24个抗DNA单抗独特型交叉反应。结果表明,H241是高敏感度的单抗,6ng非标记H241能使~(125)I-H241与抗H241独特型抗体的结合达50%抑制。实验还证明,24个抗DNA单抗中有15个与H241不同程度地存在独特型交叉反应。实验提示,虽然抗DNA自身抗体独特型往往结构分离,从而表现抗体多样性,但有些抗体由于独特型结构类似,多样性受到限制;全身性红斑狠疮小鼠控制免疫球蛋白合成的胚系基因含有某些抗DNA抗体的相关位点。     

Immunogenicity of singlet oxygen modified human DNA: implications for anti-DNA antibodies in systemic lupus erythematosus

Reactive oxygen species (ROS)-modified DNA has been shown to be a better and more discriminating immunogen than native DNA (nDNA) for the production of anti-DNA autoantibodies in SLE (systemic lupus erythematosus). Among ROS, the role of hydroxyl radical (.OH) in the induction of damage and modification of nDNA has been extensively studied while such documentation implicating singlet oxygen ((1)O(2)) in inducing immunogenicity in nDNA leading to the production of anti-double-stranded (ds) DNA autoantibodies in SLE is not yet available. This prospective study was undertaken to evaluate the immunogenicity of healthy human dsDNA modified with (1)O(2) generated by methylene blue plus radiant light. Female rabbits were immunized with (1)O(2)-modified human dsDNA to raise anti-dsDNA antibodies. (1)O(2)-modified anti-dsDNA rabbit immune sera and the (1)O(2)-modified anti-dsDNA rabbit purified immunoglobulin G (IgG) were tested against a variety of dsDNA antigenic substrates through direct enzyme-linked immunosorbent assay (ELISA). The immunogenicity of (1)O(2)-modified human dsDNA was further evaluated by studying its immunoreactivity with SLE patients' sera and SLE patients' purified anti-dsDNA IgG. As compared to healthy human sera, (1)O(2)-modified anti-dsDNA rabbit immune sera as well as the (1)O(2)-modified anti-dsDNA rabbit purified IgG demonstrated a strong affinity towards (1)O(2)-modified human dsDNA(.)(1)O(2)-modified human dsDNA proved to be potentially more immunogenic against SLE patients' whole sera and SLE patients' purified IgG as compared to healthy human sera. Our findings suggest that (1)O(2) may also be inducing immunogenicity in native dsDNA resulting in the production of anti-dsDNA autoantibodies as seen in SLE patients.     

Differences between human hybridoma platelet-binding antibodies derived from systemic lupus erythematosus patients and normal individuals

The binding and functional activities of platelet-binding hybridoma autoantibodies from SLE patients were compared with those derived from normal individuals. Twenty-nine SLE-derived hybridoma antibodies and 20 normal-derived hybridoma antibodies were analyzed for binding to glutaraldehyde fixed platelets, dDNA and phospholipids, and for lupus anticoagulant activity. Twenty-four of the 29 SLE-derived antibodies and 9 of the 20 normal-derived antibodies showed one or more activities in these assays. Of the 24 SLE-derived antibodies, 8 (33.3%) were reactive in only one assay (monospecific), while the other 16 (66.7%) had more than one of these activities (polyspecific). In contrast, none (0%) of the 9 normal-derived antibodies with known activities were monospecific, while all 9 (100%) showed polyspecificity. Statistical analyses demonstrated that there was no correlation of anti-DNA activity with anti-platelet and most anti-phospholipid activities for the SLE-derived antibodies, and strong positive correlations between these reactivities for the normal-derived antibodies. Similarly, differences were observed in Western blotting analyses; SLE-derived antibodies bound more specifically to individual platelet proteins than normal-derived antibodies. Moreover, in chromium-51 release assays, all of the SLE-derived platelet-binding antibodies were cytotoxic to platelets, while none of the normal-derived platelet-binding antibodies showed significant cytotoxicity. Our results suggest that hybridoma platelet-binding autoantibodies derived from SLE patients exhibit greater antigen specificity and functional activity than similar antibodies derived from normal individuals.     

Somatic diversification and affinity maturation of IgM and IgG anti-DNA antibodies in murine lupus

Molecular events occurring during the process of generation of pathogenic immunoglobulin (Ig)G anti-DNA antibodies in systemic lupus erythematosus (SLE) were studied using a newly established method. We analyzed the Ig variable (V) region gene sequence and DNA-binding activity of IgM and IgG anti-DNA monoclonal antibodies (mAb) from individual SLE-prone (NZB × NZW) F₁ mice. The first event appeared to be clonal selection and expansion of IgM anti-DNA clones, in which several clones had intraclonal V gene mutations. Although the number of mutations was small, the mutated IgM clones were associated with an increase in DNA-binding activity. The somatic mutations located in complementarity-determining regions (CDR) and in framework regions (FR) of V genes were apparently related to changes in DNA-binding activity. IgG anti-DNA clones that progressively increased in number with aging had numerous somatic mutations in the V region genes and there was a pair of clones which showed an intraclonal accumulation of mutations, in association with increase in the DNA-binding activity. All these findings show that somatic mutations associated with affinity maturation of the V region begin immediately before isotype-switching from IgM to IgG of the clones that have been selected and expanded, in an antigen-driven manner and/or by other forces. We propose that further accumulations of intraclonal somatic hypermutation, in association with selection and expansion of high affinity IgG clones, may lead to formation of highly pathogenic anti-DNA antibodies.