Pathogenic anti-DNA idiotype-reactive IgG in intravenous immunoglobulin preparations.

This study was addressed to explore the reactivity of natural anti-idiotypes from commercial lots of immunoglobulins to several idiotypes (Ids), usually expressed by anti-DNA molecules in lupus nephritis. Eleven intravenous immunoglobulin (IVIG) preparations and nine (three polyvalent and six hyper-immune) intramuscular IgG were investigated for specific content of anti-DNA, anti-F(ab')2 and antibodies reacting with several anti-DNA IgG Ids. Two samples (nos 6 and 11) showed high reactivity with allogeneic F(ab')2 and with F(ab')2 of myeloma proteins bearing the anti-DNA Id 3I+ and the 8.12+. Since both 3I and 8.12 Id markers are known to characterize pathogenic anti-DNA IgG in systemic lupus erythematosus (SLE), anti-Id antibodies to these markers were obtained by absorbing the IVIG samples nos 6 and 11 to Sepharose columns coupled with pooled F(ab')2 fragments of 3I(+)-F4(+)-8.12(+)-myeloma proteins. Inhibition experiments showed that anti-8.12 Id-eluted IgG induced a selective suppression of the DNA-reactive antibodies derived from patients with active lupus nephritis to their substrate, suggesting the involvement of 8.12+ molecules in the SLE glomerular damage. Since 8.12+ anti-DNA are nephritogenic antibodies, the occurrence of anti-8.12+ Id in commercial IVIG may be of potential therapeutic relevance in modulating the pathogenic SLE Id network. Previous variable results of IVIG treatment in SLE, such as resolution of proteinuria or worsening nephritis, could be related to variable enrichment of different lots of IVIG in suppressive anti-pathogenic Id antibodies.     

Family distribution of anti-F(ab'')2 antibodies in relatives of patients with systemic lupus erythematosus.

Recently we reported an inverse relationship between the levels of anti-F(ab')2 antibodies and disease activity in systemic lupus erythematosus (SLE). The present study focused on anti-F(ab')2 antibodies in unaffected relatives of SLE patients. Sixty sera from first degree family members from 11 SLE families and 49 sera from 8 control families were studied. Percentage of SLE family members with anti-DNA antibodies (15%) was higher than than control family sera (8%, P less than 0.05). Anti-F(ab')2 antibodies were measured using ELISA assays. The SLE family sera had higher amounts of anti-F(ab')2 antibodies than the normal control family group (P = 0.0051). In an effort to determine if anti-F(ab')2 antibodies found in high titres in the sera of some SLE family members had specificity for the F(ab')2 fragment of anti-DNA antibodies of the SLE relative patients, DNA-anti-DNA inhibition experiments were performed using anti-F(ab')2 prepared from the relative in parallel with anti-F(ab')2 prepared from normal controls with equivalent high titres of serum anti-F(ab')2. Inhibition exhibited by anti-F(ab')2 of first degree relatives was higher than that obtained from control normal donors (P less than 0.02). Such differences in inhibition were not recorded using a control tetanus toxoid-anti-tetanus toxoid assay. In direct binding ELISA experiments, peroxidase-conjugated anti-F(ab')2 antibodies from the same first degree relative showed high relative specificity against purified anti-DNA antibodies of his SLE proband when compared to those obtained against different anti-DNA antibodies isolated from unrelated SLE patients (P less than 0.001). Such a substantial difference was not observed in parallel experiments using peroxidase conjugated anti-F(ab')2 antibodies from normal controls unrelated to SLE subjects.     

In vitro inhibition of anti-DNA producing cells from systemic lupus erythematosus patients by autologous anti-F(ab')2 antibodies

We have previously documented an inverse relationship between serum levels of anti-F(ab')2 antibodies and disease activity in systemic lupus erythematosus (SLE). The present study focused on the specific in vitro inhibition of anti-DNA producing cells from SLE patients by autologous anti-F(ab')2 antibodies. Peripheral blood lymphocytes (PBL) from eleven inactive SLE patients with no apparent disease activity were cultured in vitro to evaluate anti-DNA antibody secretion. Low levels of synthesis of anti-DNA antibody were detected in 3 of 11 SLE patients using unstimulated PBL; on the contrary, pokeweed mitogen stimulation of cultured cells increased production of anti-DNA in all SLE subjects. Parallel cultures were also performed in the presence of heterologous and autologous anti-F(ab')2 antibodies and results on production of anti-DNA evaluated. Lymphocytes from SLE patients in remission showed inhibition of synthesis of anti-DNA antibodies when autologous anti-F(ab')2 antibodies were added to the cultures, whereas production of anti-tetanus toxoid IgG by the same cells was not significantly altered under the same conditions. These data suggest that a functional anti-idiotypic role may be assigned to anti-F(ab')2 antibodies during clinical remission of SLE.     

Human anti-F(ab')2 antibodies show preferential reactivity for F(ab')2 molecules bearing lambda light chains.

In order to evaluate binding specificities of anti-F(ab')2 antibodies from patients with systemic lupus erythematosus (SLE) and from normal healthy controls, F(ab')2 fragments were prepared from 24 IgG myelomas with defined isoelectric points, DNA-associated idiotypes, and kappa/lambda light chain types. Using ELISA and hemagglutination assays, anti-F(ab')2 antibodies from 12 healthy controls and 29 SLE patients were observed to exhibit preferential binding (lambda > kappa) to myeloma F(ab')2 fragments composed of lambda light chains (P < 0.0001). No correlation of anti-F(ab')2 binding and presence of cationic, neutral, or anionic isoelectric points or for DNA-associated idiotypes on monoclonal F(ab')2 was detected. Anti-F(ab')2 antibodies, often elevated in SLE during remission, show preferential specificity for F(ab')2 fragments bearing lambda light chains.     

Lupus-specific kidney deposits of HSP90 are associated with altered IgG idiotypic interactions of anti-HSP90 autoantibodies

Previous studies have shown that autoantibodies to heat shock protein 90 (HSP90) are elevated in a significant proportion of patients with systemic lupus erythematosus (SLE) who are more likely to have renal disease and a low C3 level. Using samples from 24 patients, we searched for glomerular deposits of HSP90 in renal biopsy specimens from seven patients with lupus nephritis and 17 cases of glomerulonephritis from patients without SLE. Positive glomerular immunofluorescent staining for HSP90 was observed in six of seven cases of SLE and positive tubular staining in two of seven SLE patients. The staining for HSP90 was granular in nature and was located in subepithelial, subendothelial and mesangial areas. None of the non-SLE renal biopsies revealed positive staining for HSP90 deposition. Further we showed the presence of anti-HSP90 IgG autoantibodies in IgG from sera of patients with SLE as well as in normal human IgG (IVIg). In normal IgG this autoreactivity could be adsorbed almost completely on F(ab')2 fragments from the same IgG preparation, coupled to Sepharose and could be inhibited by the effluent obtained after subjecting normal IgG to HSP90 affinity column. These findings indicate that anti-HSP90 natural autoantibodies are blocked by idiotypic interactions within the IgG repertoire. Unlike natural autoantibodies, anti-HSP90 IgG from SLE patients' sera were only moderately adsorbed on F(ab')2 fragments of normal IgG. These results demonstrate that immunopathogenesis of lupus nephritis is associated with HSP90 (as an autoantigen) and that the pathology is associated with altered idiotypic regulation of the anti-HSP90 IgG autoantibodies.     

Monoclonal antibodies against human anti-F(ab')2 antibodies react with light chain epitopes.

Anti-F(ab')2 antibodies affinity isolated from sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or normal SLE relatives were used to produce monoclonal antibodies (mAbs) in Balb/c and NZB mice. Four of five mAbs showed only primary light chain specificity. Only one mAb produced in an NZB mouse against anti-F(ab')2 from a single SLE patient showed anti-mu-chain specificity. Parallel identical control immunizations with IgG or a single human IgG kappa myeloma produced mAbs with a predominant gamma-chain/Fc fragment specificity. Anti-light chain specificity of mAbs was demonstrated to involve epitopes requiring tertiary structure of the entire light chain instead of antigens confined to Ckappa/lambda or Vkappa/lambda fragments. Anti-kappa specificity of three mAbs was extremely similar but not identical to that defined by anti-Km1 allotyping systems. No evidence was obtained with any of the mAbs produced for antigens unique to SLE or RA anti-F(ab')2 antibodies. The light chain antigenic prominence of many anti-F(ab')2 antibodies may reflect structural features shared by this group of immunoglobulins somehow important for their biologic function.     

Anti-idiotypic antibody against anti-DNA in sera of laboratory personnel exposed to lupus sera or nucleic acids.

We tested for anti-DNA, anti-idiotypic, antinuclear, and lymphocytotoxic antibodies in the sera of three groups of normals: volunteers never exposed to lupus sera or nucleic acids (group I), research personnel handling nucleic acids (group II), and laboratory personnel handling lupus sera (group III). There was no significant differences among the groups with respect to levels of either single stranded or double stranded anti-DNA. Group I showed no significant differences in binding to F(ab')2 fragments of lupus anti-DNA, lupus non-anti-DNA or normal IgG. Compared to group I, groups II and III bound significantly higher to anti-DNA F(ab')2 fragments compared to non-anti-DNA F(ab')2 or normal F(ab')2 fragments. Sera from the three groups were negative for antibodies and all but one individual from group III had normal antinuclear antibody titres. These results indicate that sera of normals exposed to lupus sera or to nucleic acids contain an anti-idiotype directed against anti-DNA antibody. The possible role of these anti-idiotypes in regulating the anti-DNA antibody is discussed.     

SLE患者血清抗F(ab′)_2片段抗体的测定及其临床意义

用酶解法制备正常人 IgG-F(ab')_2片段及 SLE 患者血清抗 ds-DNA-F(ab')_2片段,ELISA 法检测同一组(46例)SLE 患者血清中抗 F(ab')_2片段抗体,两者检测结果呈高度相关性.同时发现,缓解期 SLE 患者血清中抗 F(ab')_2片段抗体水平明显高于活动期 SLE 患者,而抗 ds-DNA 抗体水平与之相反.提示,正常人 IgG-F(ab')_2片段与 SLE 患者血清抗 ds-DNA-F(ab')_2片段可能有共同的结构表位,其抗 F(ab')_2片段抗体测定可作为临床观察 SLE 活动性的一个指标.     

Insoluble DNA-like phosphorylated polystyrene: specific interactions with anti-DNA antibodies from systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is an autoimmune disease. Antibodies directed mainly against DNA and/or phospholipids are present in the serum of SLE patients. Therefore phosphorylated polystyrene derivatives acting as DNA-like polymers should be able to interact with the SLE anti-DNA antibodies. Such functional polymers were synthesized and subsequently their interactions with the anti-DNA antibodies studied. Adsorption experiments performed with both anti-DNA antibodies and normal immunoglobulins showed high affinity constants of the phosphorylated polymer for anti-DNA antibodies (4 x 10(9) M-1) whereas for normal IgG the affinity was low (2 x 10(5) M-1). Moreover, the interaction was specific involving the idiotypic moiety of the anti-DNA antibodies and an array of phosphoester groups at the surface of this biomaterial.     

Anti-DNA autoantibody activity and idiotypic relationships of human monoclonal proteins

Previous studies showed that polyclonal anti-DNA antibodies from patients with systemic lupus erythematosus (SLE) share cross-reactive idiotypes (CRI). In this report, we used human myeloma proteins (HMP), isolated from the serum of patients with multiple myeloma or Waldenstr?m macroglobulinemia, as probes to further explore this idiotypic cross-reactivity. Fourty-four HMP were tested for DNA-binding capacity and for expression of CRI associated with lupus anti-DNA antibodies. Anti-DNA IgG were immunoaffinity purified from the serum of patient TOF with severe SLE. A xenogeneic anti-idiotype antibody to this IgG was raised in rabbit. This anti-idiotype recognized CRI associated with the combining site of anti-DNA IgG from unrelated SLE patients. Using inhibition competitive-immunoassays, we found that these CRI were present on all but one of the DNA-binding HMP. Furthermore, we observed that these CRI were detectable on an IgG2 lambda, a HMP devoid of anti-DNA activity. These findings are in agreement with those previously obtained in similar studies using murine monoclonal anti-DNA antibodies. These converging results suggest that antibodies expressing anti-DNA-related CRI and antibodies exhibiting anti-DNA-binding affinity constitute overlapping molecular subpopulations.     

Binding to histone of an anomalous IgG from patients with SLE and drug-induced lupus

The mechanism of attachment of circulating immune complexes (CIC) to glomerular basement membranes (GBM) in systemic lupus erythematosus (SLE) has not yet been elucidated. One difficulty is that CIC must be strongly cationic for such deposition to occur, which is opposite to the anionic nature of putative DNA-anti-DNA immune complexes (DNA-IC). The strongly cationic histone has been proposed as a potential "planted antigen"; it would decorate the GBM to function as a ligand for DNA in the DNA-IC. However, DNA-IC, aggregated IgG and most of the IgG "anti-histone antibodies" in SLE patient sera bind to histone on a solid phase not through DNA, but through the Fcgamma. Here, we investigated the nature of the anti-histone "antibody" in sera of 18 patients with SLE and 57 with drug-induced lupus (DIL). The binding to nucleosomes of IgG from these patients was mainly pepsin-resistant and F(ab')(2)-dependent, whereas the binding to histone was mainly pepsin-sensitive and Fcgamma-dependent. Surprisingly, after molecular sieving of 12 of these sera, the pepsin-sensitive histone-binding IgG was located mainly in the 150-kDa monomeric IgG peak. The binding to nucleosomes was only in the 150-kDa peak. These findings are consistent with the existence of an anomalous IgG in SLE and DIL sera, capable, like aggregated IgG, DNA-IC and other CIC, of binding to histone-decorated structures. We propose that this anomalous IgG plays an essential role in the pathogenesis of lupus nephritis and other related inflammatory conditions. These observations also explain the large discrepancies in the reports on anti-histone autoantibodies in autoimmune conditions.     

Specificity of IgM Antibodies to Pooled Human F(ab')2 Fragments

An isotope specific immunoassay which minimizes interference by endogenous rheumatoid factors was used to determine the specificity of IgM anti-F(ab')₂ antibodies in human serum. We underscore the heterogeneity of these antibodies. While one subset of IgM anti-F(ab')₂ antibodies reacts only with intact F(ab')₂, another recognizes determinants present following reduction and alkylation of F(ab')₂ and separation of Fd' fragments from light chains. IgM anti-F(ab')₂ antibodies in sera from rheumatoid arthritis patients do not react significantly with intact pooled IgG and, therefore, probably are not anti-idiotypic antibodies. Some sera, but not all, contain elevated levels of antibodies that are crossreactive with rabbit F(ab')₂. Such crossreactive antibodies may interfere with assays which utilize F(ab')₂ fragments of rabbit antibodies specific for antigens of clinical relevance.     

Evidence in patients with systemic lupus erythematosus of the presence of antibodies against RNA-dependent DNA polymerase of baboon endogenous virus.

Immunoglobulin G (IgG) in six out of 30 patients with systemic lupus erythematosus (SLE) strongly inhibited the activity of RNA-dependent DNA polymerase (RDPase) of baboon endogenous virus, M7, while IgG obtained from scleroderma patients, rheumatoid arthritis patients and normal subjects was less reactive. Experiments with anti-human IgG and with IgG F (ab')2-bound immunoaffinity columns indicated that the inhibition of RDPase was antibody-mediated. The RDPase inhibiting activity of SLE IgG was considered not to be due to cross-reactions of anti-nuclear antibodies including anti-DNA, anti-ribonucleoprotein, anti-Sm and anti-SS.B antibodies. SLE IgG preferably inhibited the RDPase activity of baboon endogenous virus and a feline endogenous virus, RD114. These findings support the hypothesis that retrovirus(es) might be involved in SLE.     

Analysis of anti-idiotypic antibodies against anti-microsomal antibodies in patients with thyroid autoimmunity.

Anti-microsomal antibody (AMA) activity was inhibited in 14 of 16 sera and in all 12 IgG preparations from patients with postpartum thyroiditis following incubation with F(ab')2 fragments from normal polyspecific immunoglobulin for therapeutic use (ivIg). Similar results were observed with sera from seven of seven patients with Graves' disease and five of six patients with autoimmune hypothyroidism. Results of these competitive binding assays and affinity chromatography of AMA IgG on Sepharose-bound F(ab'), fragments from ivIg indicated that AMA antibodies reacted with ivIg through idiotypic-anti-idiotypic interactions. Eight out of 10 IgG preparations from patients with autoimmune thyroid disease also showed inhibition of AMA activity when coincubated with autologous IgM at various IgG:IgM molar ratios. These observations suggest that ivIg can inhibit anti-microsomal antibodies through idiotype-anti-idiotype interactions and that such interactions occur with IgM anti-idiotype antibodies in vivo, providing evidence of a role for idiotypic network regulation in the control of thyroid autoimmunity.     

Quantitative immunoassay for IgA class circulating immune complexes using solid phase Facb fragment of anti-C3

A quantitative assay of IgA class circulating immune complexes (IgA-CIC) by a solid phase anti-C3 enzyme immunoassay (anti-C3 EIA) is described. A stable and reproducible standard for determination of IgA-CIC was prepared successfully by chemical binding of complement C3 to human serum IgA. Two of 27 sera from patients with systemic lupus erythematosus (SLE), however, contained high concentrations of IgA class anti-F(ab')2 antibodies that caused false positive results when the F(ab')2 of anti-C3 was used for EIA. Solid phase Facb of anti-C3 was found to eliminate the false positive results caused by IgA class anti-F(ab')2 and IgA class rheumatoid factor. Good reproducibility and recovery were observed with this Facb anti-C3 EIA using the IgA-C3, a stable standard material, and so this method should be useful clinically in elucidating the role of IgA-CIC.     

Noncognate binding to histones of IgG from patients with idiopathic systemic lupus erythematosus

The mechanism of attachment of circulating immune complexes (CIC) to glomerular basement membranes (GBM) has not yet been elucidated. Since it has been proposed that histone may be the ligand between GBM and DNA/anti-DNA CIC, we explored by ELISA and Western blots the nature of the interaction of IgG with histone on solid phase. Cognate binding of IgG anti-histone antibody was characteristically dependent on in its F(ab')(2) fragment and was inhibited by free histone. On the other hand, heat-aggregated IgG, a model for CIC, and IgG from most patients with idiopathic SLE had a characteristic noncognate binding behavior to histone: it was dependent on Fcgamma rather than on the F(ab')(2) fragment and was not effectively inhibited by free histones. Also, binding to histone of in vitro generated DNA/anti-DNA immune complexes was not dependent on DNA as a histone ligand, but on Fcgamma. Finally, there was good agreement between the binding of this IgG to histone and to C1q. We concluded that: (1) altered IgG and/or CIC bind to solid-phase-attached histone primarily through their Fcgamma and (2) CIC may mimic IgG antihistone antibodies in solid-phase immunoassays.     

Detection and purification of antiidiotypic antibody against anti-DNA in intravenous immune globulin

Pooled normal human IgG for therapeutic use, following depletion of anti-DNA, anti-Fc, and anti-F(ab)₂ of normal IgG, expressed antiidiotypic activity against anti-DNA derived from lupus sera. The antiidiotype enriched by elution from anti-DNA affinity columns bound directly to anti-DNA IgG and inhibited the binding of lupus sera to DNA but did not bind to normal IgG or inhibit the binding of anti-tetanus toxoid to tetanus toxoid. Antiidiotypes in pooled normal sera may have a role in the clinical improvement seen in patients with autoimmune diseases receiving intravenous immune globulin.     

The network theory in autoimmunity: In vitro modulation of DNA-binding cells by antiidiotypic antibody present in inactive lupus sera

Lupus or normal controls' sera, depleted of anti-DNA antibody and of DNA, were tested for their capacity to suppress the binding of³H-DNA to DNA-binding cells. Sera from lupus patients with inactive disease were effective in suppressing the binding (P<0.01) and, in the presence of complement, were more efficient in suppression (P<0.001). The same sera were capable of suppressing the in vitro secretion of DNA antibodies but not polyclonal IgG by autologous cells. The suppressor factor in the inactive lupus serum was shown to reside in the F(ab)'₂ fragments of IgG and has a specificity for the F(ab')₂ fragments but not the Fc fragments of anti-DNA antibody. Sera from active lupus patients and human serum albumin were incapable of suppression. Normal sera from donors who had contact with lupus blood components were efficient in suppressing DNA-binding cells (P<0.01).     

Isolation of human anti-idiotypes broadly cross reactive with anti-dsDNA antibodies from patients with Systemic lupus erythematosus

Antibodies to double stranded (ds)DNA play a central role in clinical diagnosis and disease expression in Systemic lupus erythematosus (SLE). This paper describes the isolation of anti-idiotype reagents (anti/antidsDNA) from four SLE sera and the demonstration of broad and quantitatively similar cross reactivity to both polyclonal and monoclonal anti-dsDNA antibodies isolated from SLE patients. Seven affinity-purified polyclonal and three monoclonal human anti-dsDNA preparations reacted preferentially with anti-idiotype F(ab')(2) coated plates compared to normal immunoglobulin (Ig)G F(ab')(2) coated plates in ELISA. In contrast, autoantibodies of other specificities (anti-Ro/SSA, anti-La/SSB, and anti-U(1)RNP) reacted equally with anti/anti-dsDNA F(ab')(2) and normal IgG F(ab')(2) coated plates. Such anti-idiotypic antibodies could play a significant role in the regulation of anti-dsDNA antibody levels in SLE.     

Discrepancy in the expression of autoantibodies in healthy aged individuals

Sera from 50 healthy old subjects and from 51 young controls were tested by ELISA assays for a panel of autoantibodies, including IgM RF, anti-DNA, anti-F(ab')2, antithyroglobulin, anti-human albumin, anti-human hemoglobin, anti-secretory component from human IgA, and anti-gliadin. In vitro production of anti-DNA as well as anti-F(ab')2 antibodies were measured after stimulation of PBMC by pokeweed mitogen (PWM) in 12 healthy elderly subjects and 11 young controls. Sera from elderly donors contained threefold higher amounts of IgM RF than young controls (P less than 0.001). On the contrary, the levels of anti-DNA as well as anti-F(ab')2 antibodies were similar in both groups (P less than 0.3 for the two determinations). Anti-DNA and anti-F(ab')2 antibodies were also measured in supernates of PWM-stimulated glass nonadherent PBMC cultures from both old and young healthy donors without finding any significant difference between the two groups. Additional ELISA tests were also performed in both elderly and young control sera to detect antibodies against six other different antigens. No significant difference was found in the percentages of positive sera between the two groups. This discrepancy in production of IgM RF compared to other autoantibodies in healthy elderly subjects does not provide support for a general increase of autoantibodies with aging. The biological significance of an increase in IgM RF with aging remains to be determined.