A common anti-cardiolipin antibody idiotype in autoimmune disease: identification using a mouse monoclonal antibody directed against a naturally-occurring anti-phospholipid antibody

We have recently produced a series of human monoclonal antibodies reacting with cardiolipin. One of these, H3, a polyspecific IgM/k derived from a normal individual, was used to raise mouse monoclonal antibody to its idiotype. Two anti-idiotypic antibodies, S2.9 (IgG2b) and S2.10 (IgM) were selected for their specific reaction with H3.S2.9 did not react with five other human monoclonal antibodies of IgM/k class despite the fact that these shared some antigen-binding characteristics with H3.S2.9 was able to block the binding of H3 to all of its cross-reactive antigens including cardiolipin, while S2.10 was not. S2.9 was equally efficient in blocking the binding of H3 to three of its cross-reactive antigens, cardiolipin, diphtheria and tetanus toxoids; greater than 90% inhibition could be achieved at an equimolar ratio of H3 to S2.9. The anti-idiotype S2.9 was used to demonstrate the presence of the H3 idiotype in serum. This idiotype was found in amounts greater than that seen in 42 normal individuals, in 30 of 36 patients with systemic lupus erythematosus (SLE), eight of 20 patients with rheumatoid arthritis (RA), 8 of 20 patients with Felty's syndrome as well as 10 of 23 patients with syphilis. Not one of nine patients with drug-induced lupus syndrome had abnormal levels. In patients with SLE and Felty's syndrome there was a good correlation between the amount of anti-cardiolipin antibodies and the amount of H3 idiotype (rs = 0.70 and 0.69 respectively). No such correlation was found in syphilitics or in patients with RA. In patients with SLE the H3 idiotype was present on IgM and IgG anti-cardiolipin antibodies. In 15 of 16 SLE sera with high levels of cardiolipin antibody, S2.9 blocked binding of serum antibodies to cardiolipin by 13-72%, with a mean value of 49%. One patient had a high level of anti-cardiolipin antibody which could not be blocked by S2.9. These results indicate that a mouse monoclonal antibody which reacts with an idiotope in the antigen-binding region of a naturally-occurring phospholipid antibody also defines a common idiotype of anti-cardiolipin antibodies in patients with autoimmune disease.     

IgG class anti-cardiolipin antibody as a possible marker for evaluating fetal risk in patients with systemic lupus erythematosus

To identify the correlation between incidence of anti-phospholipid antibodies and fetal prognosis in pregnant SLE patients, we measured the amount of anti-cardiolipin antibody in their sera, using solid-phase enzyme immunoassay (EIA) methods. Findings in the group having poor obstetric results (fetal loss group) and in those with a history of full-term births (live birth group) were compared with regard to other anti-phospholipid antibodies. The incidence of IgG class anti-cardiolipin antibody was 60% in the fetal loss group and 19% in the live birth group, (P less than 0.05). The incidence of the other anti-phospholipid antibodies, including lupus anticoagulant and biological false-positive serological test for syphilis (BFP-STS), did not differ significantly between the two groups. Therefore, the presence of IgG class anti-cardiolipin antibody may prove to be a useful marker for evaluating fetal risk in SLE patients.     

Use of monoclonal antibodies to identify shared idiotypes on anticardiolipin and anti-DNA antibodies in human sera.

Using hybridoma technology we produced monoclonal antibodies (MoAb) to idiotypic determinants on human anti-cardiolipin antibodies purified from a patient with SLE. Hybridomas were screened by inhibition of cardiolipin binding activity of sera from patients with SLE. Seven hybridomas were selected, two of which were studied extensively. Sera from a number of patients with SLE were found to share idiotypic determinants. This cross-reacting idiotype was not detectable on anti-cardiolipin antibodies in syphilis sera. The cross-reacting idiotype was present in sera with anti-ssDNA antibodies even though some of these sera had no anti-cardiolipin antibodies. We propose that these MoAb may recognize a regulatory idiotype.     

Induction of experimental anti-phospholipid syndrome associated with SLE following immunization with human monoclonal pathogenic anti-DNA idiotype.

MIV-7 is a human monoclonal antibody that binds to DNA and carries a pathogenic anti-DNA idiotype 16/6. The antibody was generated by fusing peripheral blood lymphocytes of a healthy donor which were stimulated with an anti-idiotypic antibody to B11 (a human mAb anti-mouse mammary tumor virus-MMTV). The MIV-7, in addition to being an anti-DNA antibody, also binds to MMTV glycoproteins. Following immunization into the footpad of naive BALB/c mice with MIV-7, the mice developed anti-phospholipid syndrome (APLS) and SLE. The APLS was characterized by thrombocytopenia, the presence of anticardiolipin antibodies, lupus anticoagulant (prolonged APTT), high resorption rate of fetuses and lower mean weights of the placentae and fetuses. The SLE was characterized by serological markers (e.g. anti-DNA), laboratory (increased sedimentation rate and proteinuria) and histological findings (deposition of immune complexes in the glomeruli). Active immunization of mice with mouse monoclonal anti-cardiolipin antibodies led to the induction of primary APLS without SLE. The results add to our previous passive transfer model in which mouse monoclonal anti-cardiolipin antibody generated from immunized mice (CAM) was infused into the tail vein and also resulted in induction of pure APLS [11]. Our results demonstrate the ability to induce secondary APLS to SLE following immunization with a pathogenic idiotype of anti-DNA antibodies and to induce primary APLS with anti-cardiolipin mAb. The existence of these experimental models may permit controlled studies of novel therapeutic models.     

Heterogeneity of binding reactivity to different phospholipids of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis.

The binding specificities were investigated of anti-phospholipid antibodies derived from sera from 55 patients with SLE and related diseases, and from 33 patients with syphilis. Antibodies from both these groups of patients bind strongly to cardiolipin in solid-phase immunoassays, but only antiphospholipid antibodies from patients with autoimmune diseases are associated with thrombotic complications and recurrent spontaneous abortions. IgG anti-phospholipid antibodies from both groups of patients cross-reacted with a range of negatively charged phospholipids, but binding to neutral phospholipids was largely restricted to sera from patients with syphilis. A monoclonal IgM lambda anti-cardiolipin antibody, derived from a patient with autoimmunity, was used to inhibit binding of anti-phospholipid antibodies to cardiolipin and to phosphatidic acid. This antibody inhibited the binding of autoimmune sera to cardiolipin more strongly than sera from syphilis patients, but the converse pattern of inhibition of binding to phosphatidic acid was observed. The VDRL titre correlated with anti-phospholipid antibody activity in sera from syphilis patients, but not from those with autoimmunity. Lupus anti-coagulant activity correlated weakly with IgG antibody levels to each of the negatively charged phospholipids among the patients with autoimmunity. Lupus anticoagulant activity did not correlate uniquely with any anti-phospholipid antibody specificity. These results provide further documentation of the great heterogeneity of anti-phospholipid antibodies associated with autoimmune disease and syphilis.     

Anti-DNA idiotype- and anti-idiotype-specific T cell responses in patients with systemic lupus erythematosus and their first-degree relatives.

We examined the proliferative responses of T cells of patients with systemic lupus erythematosus (SLE), their first-degree relatives, and healthy donors, to a human monoclonal antibody that bears a common anti-DNA idiotype, 16/6 Id, and to a murine, 16/6 Id-specific, monoclonal antibody. Both 16/6 Id+ and 16/6 Id-specific antibodies were previously shown to be involved in the induction of experimental SLE in mice. Here we show that T cells of fewer SLE patients, as compared with healthy donors, could proliferate to both antibodies. The difference between T cell responses of patients and controls to the 16/6 was found to be significant. The proliferative responses of T cells of first degree relatives of SLE patients to the anti-16/6 Id were found to be significantly lower compared with the responses detected in healthy donors and in SLE patients. The responses of T cells of SLE relatives to the 16/6 Id were found to be lower than those of healthy donors, but this difference was not significant. The present study suggests a possible involvement of T cells, and specifically of idiotype and anti-idiotype specific T cells, in SLE.     

Anti-mitochondrial type M5 and anti-cardiolipin antibodies in autoimmune disorders: studies on their association and cross-reactivity.

In a series of 42 positive sera, anti-mitochondrial type M5 antibodies (AMA-M5) were found most frequently in patients with SLE (24) and SLE-like syndromes. Patients with AMA-M5 displayed a higher prevalence of thrombocytopenia, thrombosis, biological false positive seroreactions for syphilis, lupus-like anticoagulant activity and anti-cardiolipin antibodies in comparison with a group of 43 SLE AMA-M5 negative patients. The strong association between anti-phospholipid and AMA-M5 antibodies cannot be explained entirely by cross-reactivity between these two groups of antibodies, as indicated by absorption experiments and studies using affinity purified antibody preparations. However, cardiolipin liposomes were able to reduce partially the titres of AMA-M5 sera, suggesting that a small population of AMA-M5 antibodies exists that cross-reacts with cardiolipin. The existence of this population was further substantiated by our demonstration that an IgM monoclonal antibody, from a patient with Waldenström''s macroglobulinaemia, displayed both anti-cardiolipin and AMA-M5 activity, and AMA-M5 activity was completely inhibited by cardiolipin.     

Evaluation of the cross-reaction between anti-DNA and anti-cardiolipin antibodies in SLE and experimental animals.

The cross-reaction between anti-DNA and anti-cardiolipin IgG antibodies and its relation to the standard test for syphilis was studied with sera and monoclonal antibodies derived from human patients and mice with systemic lupus erythematosus (SLE). Syphilitic sera of humans and rabbits infected with the spirochete Treponema pallidum were also tested in this study. In addition, rabbits were immunized with ssDNA and cardiolipin and the cross-reactions of the induced antibodies were studied in two different assay systems. The results of these experiments suggest: that the anti-DNA and anti-cardiolipin IgG autoantibodies in SLE sera constitute separate antibody populations and, therefore, cardiolipin cannot play a role in the induction of immune response to DNA in SLE; that in immunized experimental animals there is a significant level of cross-reaction between anti-DNA and anti-cardiolipin-the detection of this cross-reaction depends on highly amplified solid phase assay systems which measure low affinity antibodies and that there is no correlation between the activity of syphilitic sera in the serologic test for syphilis and their binding to pure cardiolipin-this implies that cardiolipin may not be the dominant ingredient in this test as previously proposed.     

A common anti-DNA antibody idiotype and anti-phospholipid antibodies in sera from patients with schistosomiasis and filariasis with and without nephritis

The serum concentration of a public idiotype (16/6 ID), originally identified on a human hybridoma-derived monoclonal anti-DNA antibody, was raised in patients with S. mansoni and filariasis with or without nephritis, compared to patients with S. haematobium (in which nephritis does not occur) or idiopathic nephritis not associated with infection. There was no significant difference in 16/6 ID levels between patients with or without renal disease, whether associated with S. mansoni or filarial infection. The 16/6 ID-positive antibodies did not bind to native or denatured DNA, using competitive fluid-phase inhibition studies. Anti-cardiolipin antibody (ACA) concentration was increased in patients with either S. mansoni or filarial infection. In filariasis, both IgG and IgM ACA isotypes were elevated, irrespective of the presence of nephritis. In S. mansoni infection, IgA ACA were limited to patients with renal disease, and IgG ACA isotype to patients without overt nephritis. Both isotypes of anti-cardiolipin antibody correlated with levels of the 16/6 ID in patients with S. mansoni or filariasis. The 16/6 idiotype was therefore a feature of chronic helminthic infection, but was not related to anti-DNA antibody levels or specificity. The occurrence of nephritis as a complication of these infections was not related to the prevalence of the 16/6 idiotype.     

Binding affinity of serum immunoglobulin G to cardiolipin and other phospholipids in patients with systemic lupus erythematosus and syphilis.

Qualitative and quantitative assays for human antibodies to cardiolipin and other phospholipids were used in tests for these reactions in sera from patients with systemic lupus erythematosus (SLE) and syphilis. Of 22 SLE serum samples tested by the qualitative assay, 8 showed positive staining to cardiolipin, phosphatidic acid, and/or phosphatidylserine. All 47 syphilitic sera reacted with these three phospholipids. The apparent affinity of anticardiolipin binding was estimated by normalizing absolute binding levels as a function of serum concentration to the maximum percent bound. It was evident that antibody affinity was four- to fivefold lower in the SLE sera than in the syphilitic sera. Twelve serum samples from patients with one or more features of the anti-cardiolipin syndrome demonstrated mean binding values which were not distinguishable from binding in other SLE sera. In sera from patients with active SLE, binding affinity for cardiolipin was somewhat greater than that in samples from patients with inactive disease, but the differences were not statistically significant. The low anticardiolipin binding affinity which was observed in patients with SLE compared with that in patients with syphilis casts doubt on a pathogenic role for these reactions.     

Anti-idiotype immunomodulation of experimental anti-phospholipid syndrome via effect on Th1/Th2 expression.

Mice with experimental anti-phospholipid syndrome (APS), induced by active immunization with a human anti-cardiolipin MoAb (H-3), were treated with mouse anti-idiotypic MoAb (anti-H3, named S2.9) and with an irrelevant anti-idiotype. The immunized mice produced high titres of mouse anti-cardiolipin antibodies along with clinical manifestations of experimental APS: prolonged activated partial thromboplastin time (aPTT), thrombocytopenia and high rate of fetal loss. Treatment with the specific anti-Id (S2.9) as a whole molecule or F(ab)2 fraction, resulted in a decrease in serum levels of the anti-cardiolipin antibodies, rise in platelet count, shortened aPTT and reduced rate of fetal loss. The anti-Id effect was associated with a rise in the number of IL-2 and interferon-gamma (IFN-gamma)-secreting cells (Th1) and reduction in IL-4- and IL-6-secreting cells (Th2). The beneficial effect of the anti-Id treatment in mice with experimental APS induced by active immunization with an idiotype further supports the idiotypic aetiology of experimental APS and points to the role of Th1 cytokines in suppression of its manifestations.     

Expression of an interspecies idiotype in sera of SLE patients and their first-degree relatives.

Sera from 29 SLE patients and 81 first-degree healthy family members were tested for quantitative expression of a cross-reactive idiotype present on a murine monoclonal anti-Sm autoantibody (Y2). Forty-one percent of SLE patients and 27% of all relatives showed increased serum levels of the Y2 idiotype compared to 6% in a normal, unrelated control group. In addition, female relatives of SLE patients showed slightly increased levels of anti-Sm antibodies compared to male relatives (15% vs 3%). In one of the 28 families and three unrelated SLE patients studied, there was a significant correlation between the Y2 idiotype expression and expression of another idiotype present on anti-DNA antibodies (1341d). Affinity column absorption studies showed that these two idiotypes were present on different antibody molecules. This study demonstrates: (1) a genetic predisposition for an anti-Sm antibody idiotype expression in humans; and (2) that two different idiotypes may be under parallel or coordinate regulation.     

In vitro T-cell functions specific to an anti-DNA idiotype and serological markers in patients with systemic lupus erythematosus (SLE)

The human monoclonal autoantibody 16/6 is a common anti-DNA idiotype found to have clinical relevance in patients with systemic lupus erythematosus (SLE). Therefore the ability of peripheral blood T cells of SLE patients and healthy controls to proliferate and to produce helper T-cell factors following stimulation with this idiotype was tested. It was found that T cells of 75% of healthy donors proliferated to the 16/6 idiotype, whereas only 22% of SLE patients responded to this idiotype by proliferation. On the other hand, the capability to produce T-cell helper factors specific to the 16/6 idiotype was found in a higher percentage of SLE patients (48%) as compared to healthy controls (31%). The low frequency of proliferative responses in SLE patients might be due either to the chronic exposure to the 16/6 idiotype or to the production of antiidiotype antibodies against the 16/6 idiotype, which interfere with the response to the latter stimulator.     

Binding profiles of anticardiolipin antibodies in sera from patients with SLE and infectious diseases.

Inhibition experiments were performed to study the specificity of IgG-class antibody, binding to cardiolipin immobilized onto a polystyrene surface, in sera from patients with systemic lupus erythematosus (SLE) or infection. Six different phospholipids (three anionic: cardiolipin, phosphatidylserine and phosphatidic acid, and three neutral: phosphatidylcholine, phosphatidylethanolamine and platelet activating factor), lipopolysaccharide from Salmonella Minnesota (ReLPS), strain Re595 and lipoteichoic acid from Streptococcus pyogenes were used as inhibitors, in the form of liposomes. Eight of fifteen SLE sera exhibited strong reactivity to phosphatidylserine liposomes; other anionic phospholipids, cardiolipin and phosphatidic acid, were less effective inhibitors. The binding was inhibited effectively only by cardiolipin in three of the SLE sera, and by none of the anionic phospholipids tested in the remaining four SLE sera. In most sera from patients with bacterial infections (including syphilis), anti-cardiolipin antibodies (ACA) were inhibited only by cardiolipin, but in some cases also by phosphatidic acid. In Gram-negative infections, ACA were inhibited by ReLPS more effectively than by cardiolipin. ReLPS also inhibited ACA in two of five chlamydial sera. Appreciable inhibition of ACA by phosphatidylserine did not occur in infections. Thus, in contrast to previous studies, broad reactivity to anionic phospholipids occurred in only about half of SLE sera. This pattern of polyreactivity was not seen in infections.     

Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-beta2-glycoprotein I (beta2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to 'pure' phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific 'pure' anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein-Barr virus infection. However, anti-beta2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.     

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.     

Monoclonal anti-la antibody derived from a mouse with experimental SLE is similar to human anti-la antibodies

The La antigen is a highly conserved protein, originally defined by sera of patients with Sj?gren's syndrome or systemic lupus erythematosus (SLE). In the present study, we have produced and characterized a monoclonal anti-la antibody derived from mice with experimental SLE. The induction of SLE in these mice was achieved by their immunization with a murine monoclonal anti-idiotypic antibody against a common idiotype (16/6 Id) found in SLE patients. The monoclonal anti-La antibody derived from these mice was found to be virtually identical to the anti-La antibodies found in human autoimmune sera. First, its binding to different nuclear extracts, as well as to protease-digested HeLa nuclear protein extracts, was found to be identical to that of human anti-La antibodies. Second, an inhibition study on blotted proteins demonstrated a very close relationship between the epitopes recognized by the murine monoclonal anti-La antibody and the human anti-La serum. Third, the monoclonal anti-La antibody was found by immunofluorescence to be directed against a nuclear antigen that gave a speckled pattern. Finally, the monoclonal anti-La antibody immunoprecipitated the La-associated small RNAs. This report provides evidence for the similarity of murine anti-La antibody produced in experimental SLE and human anti-La antibodies formed in autoimmunity.     

Anti-phospholipid antibodies in syphilis and a thrombotic subset of SLE: distinct profiles of epitope specificity.

In a study of connective tissue and infectious disease sera, we have demonstrated IgM and IgG anti-cardiolipin activity, in a solid phase radioimmunoassay, in systemic lupus erythematosus (SLE), rheumatoid arthritis, syphilis and in acute malaria caused by four different species of Plasmodium. The highest values were noted in SLE (IgM anti-cardiolipin P less than 0.005, IgG anti-cardiolipin P less than 0.01), but there was no correlation with anti-dsDNA, rheumatoid factor or VDRL titres in any disease group. Anti-cardiolipin binding was significantly associated with the lupus anticoagulant, thrombocytopenia, spontaneous abortions and thromboses in the SLE patients. Ten SLE sera from this thrombotic subset and 10 syphilitic sera with similar anti-cardiolipin activity, were tested against four phospholipid antigens and showed significantly different anti-phosphatidyl ethanolamine/anti-phosphatidyl serine binding ratios (P less than 0.001). These differences in phospholipid epitope specificity could explain the specificity of the VDRL antigen in syphilis serology, and we discuss a putative role for anti-phosphatidyl serine in the thrombotic diathesis of SLE.     

Lymphocyte autoantibodies and alloantibodies in HIV-positive haemophilia patients.

In order to elucidate the fine specificity of anti-cardiolipin antibodies (ACA) in patients with SLE compared to patients with syphilis (SY) various inhibition experiments were performed. Seven SLE sera and eight SY sera positive for ACA were diluted and preincubated with either cardiolipin VDRL-antigen, mitochondial particles, dsDNA, ssDNA or dilution buffer. The sera were subsequently assayed for residual ACA activity of IgG or IgM class using a sensitive ELISA technique. Significant inhibition of IgM ACA activity in SLE sera was found with cardiolipin, VDRL-antigen and mitochondrial particles. Cardiolipin inhibited binding to a significantly higher extent than the other antigens. In SY sera significant inhibition of the IgM ACA activity was found with all antigens used. The strongest inhibition was seen using VDRL-antigen. Inhibition of IgG ACA activity could only be clearly estimated in SY sera where VDRL-antigen was found to be a much stronger inhibitor than the rest, purified cardiolipin being the weakest. Only two out of seven SLE sera were IgG ACA positive which made a clear conclusion impossible but a strong inhibitory capability of pure cardiolipin and a weaker inhibition with VDRL-antigen was found. This study disclosed a difference between SLE and SY sera showing strong reactivity of ACA in SLE sera with purified cardiolipin, contrasting to ACA in SY sera which predominantly reacted with cardiolipin in the liposome environment, as found in the VDRL-antigen and in mitochondrial particles.     

DNA Antibody Idiotypes: An Analysis of Their Clinical Connections and Origins

Approximately thirty common DNA antibody idiotypes have been described on hybridoma derived or affinity purified DNA-binding antibodies. There are associations between some idiotypes and the clinical manifestations of systemic lupus erythematosus although none are sufficiently firm to be clinically useful in identifying subsets of SLE or in assessing disease activity in individual patients. The expression of these idiotypes is not confined to DNA antibodies in SLE. They may be found in the serum from patients with a range of autoimmune rheumatic disorders, infectious diseases and blood dyscrasias. In most cases the antigen binding specificity of the antibody bearing the idiotype is unknown. The precise relationship between the various idiotypes is becoming better understood with increasing availability of genetic and structural data. DNA antibody idiotype manipulation may provide a potential new therapeutic modality in SLE.