Ro52, Ro60 and La IgG autoantibody levels and Ro52 IgG subclass profiles longitudinally throughout pregnancy in congenital heart block risk pregnancies

Congenital heart block occurs in fetuses of Ro/SSA and La/SSB positive women. To investigate the stability of maternal autoantibody levels during pregnancy, we followed Ro52, Ro60 and La autoantibody IgG level variation and Ro52 subclass profiles longitudinally in selected congenital heart block risk pregnancies. Serum samples were obtained from 12 Ro/La positive women diagnosed with a systemic rheumatic disease and followed on average 60 months (range two to 84) which included 13 pregnancies. Seven children were affected by neonatal lupus, whereof four developed complete congenital heart block. Serum was also collected from the babies at birth. Ro52, Ro60 and La IgG as well as subclass antibodies were analysed by ELISA using recombinant antigens. Six Ro/La negative rheumatic patients were included as controls for antibody levels during pregnancy. Ro52, Ro60 and La IgG levels decreased progressively from early to late pregnancy, significantly for Ro52 and Ro60 (P < 0.01). No peaks or persistent elevation of antibody levels were noted in any of the CHB risk pregnancies. Ro52 IgG1 antibody levels were significantly higher than IgG2 (P < 0.01), IgG3 (P < 0.01) and IgG4 (P < 0.05) levels in the mothers during pregnancy. Ro52 IgG1 and IgG4 levels decreased significantly from early to late pregnancy (P = 0.02), while levels of IgG2 and IgG3 were low and the decrease was not significant. All IgG subclasses were transferred to the children. We conclude that maternal levels of Ro52, Ro60 and La autoantibodies tended rather to decrease than to increase during pregnancy.     

Can B cell epitopes of 60 kDa Ro distinguish systemic lupus erythematosus from Sj?gren's syndrome?

Antibodies binding components of the Ro/La (or SSA/SSB) ribonucleoprotein particle are found in the sera of patients with systemic lupus erythematosus (SLE) and Sj?gren's syndrome (SS) as well as mothers who give birth to babies with neonatal lupus. Anti-La occurs in a subset of sera that contain anti-Ro, and anti-La is found more commonly in sera of patients with SS than in sera from SLE patients. The fine specificity of autoantibodies binding 60 kDa has been studied extensively. Recent data have suggested that there are disease-specific epitopes which identify patients with either SLE or SS. Alternatively, other data suggest that the B cell epitopes of 60kDa Ro vary according to the presence of anti-La. The present study was undertaken to determine whether binding of putative disease-specific 60 kDa Ro epitopes is associated with the diagnosis of SLE vs SS, or instead associated with the presence of anti-La. Anti-60 kDa Ro positive sera from 24 SLE patients and 44 SS patients were studied for antibodies binding two epitopes of 60 kDa Ro. We find the epitope defined by residues 171-190 is associated with anti-60 kDa Ro without anti-La, regardless of diagnosis. Meanwhile, binding of the epitope defined by residues 215-232 is not commonly found in anti-60 kDa Ro sera, especially in those sera with both anti-60 kDa Ro and anti-La. Thus, the fine specificity of antibody binding to 60 kDa Ro varies according to the presence of anti-La, not to the diagnosis of either SLE or SS.     

A B cell apotope of Ro 60 in systemic lupus erythematosus

OBJECTIVE: Previous studies have attempted to segregate anti-60-kd Ro/SSA (anti-Ro 60) responses in systemic lupus erythematosus (SLE) and primary Sj?gren's syndrome (SS) but have shown limited disease preference. The aim of the present study was to determine whether the presence of autoantibodies against an Ro 60 apotope (an epitope expressed on apoptotic cells) distinguishes anti-Ro 60 responses in SLE and primary SS. METHODS: Multiparameter flow cytometry was used to select early apoptotic cells and measure the simultaneous binding of annexin V, propidium iodide, and anti-Ro 60-positive IgG from SLE patients (n=21) and patients with primary SS (n=19). The specificity of the Ro 60 apotope was determined by inhibition experiments with recombinant and native Ro 60. RESULTS: Autoantibodies against the Ro 60 apotope were prevalent in SLE patients (13 of 21, 62%) and were rarely observed in patients with primary SS (1 of 19, 5%) (P=0.0002). Further, within SLE patients, autoantibodies to the Ro 60 apotope strongly distinguished patients with anti-Ro 60 alone (12 of 13, 92%) from those with both anti-Ro 60 and anti-La (1 of 8, 13%) (P=0.0005). When we considered all patients with anti-Ro 60 alone, the presence of autoantibodies to the Ro 60 apotope had both high sensitivity (92.3%) and high specificity (85.7%) for SLE compared with primary SS (P=0.0012). The presence of autoantibodies to the Ro 60 apotope may therefore be of diagnostic value in patients with isolated anti-Ro 60 responses. CONCLUSION: The preferential targeting of an Ro 60 apotope exposed on early apoptotic cells in a subset of SLE patients implies disease-specific pathways for the induction of anti-Ro 60 autoimmunity.     

The genetic basis of Ro and La antibody formation in systemic lupus erythematosus

Summary Antibodies against Ro and La, including recombinant La and recombinant 60 kD-Ro, were determined by counter immunoelectrophoresis and ELISA in over 300 central European systemic lupus erythematosus (SLE) patients. The presence of both Ro and La antibodies was strongly associated with the MHC haplotype B8-C4AQ0-DR3-DQ2, the association being stronges for DR3. After exclusion of all B8-DR3 positive patients only DR3 positive patients still showed an increased incidence of Ro and La antibodies, suggesting DR3 as the primary association factor. High titers of La antibody, but not of 60 kD-Ro antibody, were also significantly associated with the presence of DR3. Other DR and DQ antigens or heterozygous DQ combinations were not significantly associated with Ro and La antibodies.E. Albert, M. Baur, A. Corvetta, J. Frey, J. R. Kalden, G. G. de Lange, H. H. Peter, K. Pirner, R. Röther, P. Schneider, S. Seuchter, P. Späth, C. Specker, W. Stangel, S. Stannat-Kießling     

Dissociation of immune responses to the SS-A (Ro) 52-kd and 60-kd polypeptides in systemic lupus erythematosus and Sj?gren's syndrome

The cellular RNA particle SS-A (Ro) is a target of autoimmune response in many patients with Sj?gren's syndrome (SS) and systemic lupus erythematosus (SLE). Recent immunologic, biochemical, and DNA cloning studies have shown that the SS-A particle consists of at least 2 polypeptide components, of 52 kd and 60 kd. Immunodiffusion analysis of 60 sera from patients with primary SS revealed 47 (78%) to be SS-A precipitin positive. Western blotting studies of the sera showed 3 groups of reactivities: 22 (47%) possessed autoantibodies against both the 60-kd and the 52-kd polypeptides, 19 (40%) reacted only with the 52-kd protein, and 6 (13%) were nonreactive in Western blots although positive in immunodiffusion. Fifty-one of 90 SLE sera (57%) were SS-A precipitin positive by immunodiffusion. In Western blots, 24 (47%) possessed antibodies against both the 60-kd and the 52-kd antigens, while 9 (18%) reacted only with the 60-kd protein. Eighteen (35%) were nonreactive by Western blot, although positive by immunodiffusion. Antibody to the 52-kd antigen without concomitant antibody to the 60-kd antigen was seen only in patients with primary SS, whereas antibody to the 60-kd antigen without concomitant antibody to the 52-kd antigen was seen only in SLE patients. Although antibodies to SS-A are detected in both SS and SLE, our findings show that there is dissociation of immune responses to the 2 component antigens of the particle, which may be evidence of different events initiating the autoimmune process in these diseases.     

A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein,a major lupus autoantigen

Antibodies against a conserved RNA-binding protein, the Ro 60-kDa autoantigen, occur in 24-60% of all patients with systemic lupus erythematosus. Anti-Ro antibodies are correlated with photosensitivity and cutaneous lesions in these patients and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with complete congenital heart block and photosensitive skin lesions. In higher eukaryotes, the Ro protein binds small RNAs of unknown function known as Y RNAs. Because the Ro protein also binds misfolded 5S rRNA precursors, it is proposed to function in a quality-control pathway for ribosome biogenesis. Consistent with a role in the recognition or repair of intracellular damage, an orthologue of Ro in the radiation-resistant eubacterium Deinococcus radiodurans contributes to survival of this bacterium after UV irradiation. Here, we show that mice lacking the Ro protein develop an autoimmune syndrome characterized by anti-ribosome antibodies, anti-chromatin antibodies, and glomerulonephritis. Moreover, in one strain background, Ro-/- mice display increased sensitivity to irradiation with UV light. Thus, one function of this major human autoantigen may be to protect against autoantibody development, possibly by sequestering defective ribonucleoproteins from immune surveillance. Furthermore, the finding that mice lacking the Ro protein are photosensitive suggests that loss of Ro function could contribute to the photosensitivity associated with anti-Ro antibodies in humans.     

A new autoantibody, anti-210kDa microtubule associated protein antibody, detected in the serum of patients with various liver diseases and SLE

A new autoantibody to 210kDa microtubule associated protein (MAP) obtained from rabbit liver excrude was detected in the serum with various liver diseases and SLE. As there were no reports concerning autoantibodies to MAPs in animals and human, it may say that anti-210kDa MAP autoantibody we detected was a new cytoskeleton antibody. Using western blotting method, the anti-210kDa MAP autoantibody was frequently found in the patients with alcoholic liver diseases (52.5%), PBC (55.6%), autoimmune hepatitis (83.3%), SLE (71.4%) but rarely in the patients with viral liver diseases (26.4%) and none in normal controls at a serum dilution of 1:10. In addition, at a serum dilution of 1:100, the anti-210kDa MAP autoantibody was found in the patients with alcoholic liver diseases (22.5%), PBC (44.4%), autoimmune hepatitis (66.7%), SLE (71.4%), viral liver diseases (17.0%) and none in normal controls. It was confirmed that the anti-210kDa MAP autoantibody was frequently detected in cases of alcoholic liver diseases, PBC, autoimmune hepatitis and SLE than in those of viral liver diseases and normal control.     

Umbilical cord blood levels of maternal antibodies reactive with p200 and full‐length Ro 52 in the assessment of risk for cardiac manifestations of neonatal lupus

Objective

Maternal anti‐Ro autoantibodies are associated with cardiac manifestations of neonatal lupus (cardiac NL), yet only 2% of women with this reactivity have an affected child. Identification of a more specific marker would channel intense monitoring to fetuses at greater risk. This study aimed to determine whether autoantibodies against Ro 52 amino acids 200–239 (p200) confer added risk over autoantibodies to full‐length Ro 52, Ro 60, or La.

Methods

Anti‐Ro–exposed pregnancies resulting in cardiac NL or no cardiac manifestations were identified from the Research Registry for Neonatal Lupus and the PR Interval and Dexamethasone Evaluation study. Umbilical cord (n = 123) and maternal (n = 115) samples were evaluated by enzyme‐linked immunosorbent assay.

Results

The frequencies of p200, Ro 52, Ro 60, and La autoantibodies were not significantly different between affected and unaffected children. However, neonatal anti–Ro 52 and Ro 60 titers were highest in cardiac NL and their unaffected siblings compared to unaffected neonates without a cardiac NL sibling. Although both maternal anti–Ro 52 and p200 autoantibodies were less than 50% specific for cardiac NL, anti‐p200 was the least likely of the Ro autoantibodies to be false‐positive in mothers who have never had an affected child. Titers of anti–Ro 52 and p200 did not differ during a cardiac NL or unaffected pregnancy from the same mother.

Conclusion

Maternal reactivity to p200 does not confer an added risk to fetal conduction defects over full‐length Ro 52 or Ro 60 autoantibodies. Mothers who may never be at risk for having an affected child have lower anti–Ro 60 titers and may require less stringent echocardiographic monitoring compared to women with high‐titer autoantibodies.     

Prevalence and clinical relevance of 52-kDa and 60-kDa Ro/SS-A autoantibodies in Japanese patients with systemic sclerosis

OBJECTIVE—To determine the prevalence of 52-Kda and 60-Kda Ro/SS-A autoantibodies in serum samples from Japanese patients with systemic sclerosis (SSc).
METHODS—Serum samples from 263 Japanese patients with SSc were examined by double immunodiffusion and enzyme linked immunosorbent assay (ELISA).
RESULTS—By double immunodiffusion, 29 serum samples from patients with SSc were found to possess anti-Ro/SS-A antibodies. By ELISA, 31 serum samples were positive for anti-Ro52 and/or anti-Ro60. Of 27 serum samples that were positive by both methods, 15 reacted with both Ro52 and Ro60, five with Ro52 alone, and seven with Ro60 alone. Eleven of 12 patients with both anti-Ro52 and anti-Ro60 and all five patients with anti-Ro52 alone had Sjögren's syndrome, while only one of six patients with anti-Ro60 alone had this disorder.
CONCLUSIONS—Anti-Ro52 may be a serological marker for the presence of Sjögren's syndrome in anti-Ro/SS-A-positive patients with SSc.

     

Auto-antibodies to anti-ENA SSA/RO (52 and 60 kDa): an auto-immunity laboratory's experience

PURPOSE: Anti-SSA/Ro and anti-SSB/La autoantibodies are frequently encountered in SLE or SGS where anti-SSA subtypes 52 and 60 kDa seems to be differently found in connection with the disease type: anti-SSA/Ro 60 kDa more frequently found in SLE and anti-SSA/Ro 52 kDa in SGS. We try to find if it was interesting in identifying these specificities for all anti-ENA screening. METHOD: The study included 162 patients' sera found anti-SSA 52 and/or anti-SSA 60 and/or anti-SSB positive among 1600 screening tests from the different hospital's services. We used two assays: first, dotblot (Innolia-Ana Update INGEN) as a screening test and second, an Elisa (ENA-LISA BMD) as confirmation. Thirty-eight control sera were found negative with dotblot. RESULTS: Only one subtype of anti-SSA (52 or 60 kD) or anti-SSB was found for 55 sera (44 anti-SSA 52, 10 anti-SSA 60, 1 anti-SSB) and 107 sera were found positive for two or more (73 anti-SSA 52 + 60 and 34 anti-SSA 52 or 60 with another anti-ENA). While anti-SSA 60 kDa alone or not was always positive with the Elisa test, neither anti-SSA 52 alone was anti-SSA Elisa's positive. Diseases associations results show a greater linking of anti-SSA 60 kDa with SLE, a frequent linking of combined reactivity anti-SSA 52/60 in SLE and SGS and a greater spreading of anti-SSA 52 kDa alone among pathological groups, showing an autoimmune disease's linking in 68%. Among SGS, 29% had only anti-SSA 52 kDa. CONCLUSION: We suggest screening specific tests for identifying anti-SSA/Ro 52 kDa reactivity which are missed by routine testing (tests using animal's antigens) and could represent an additional serum marker in Connective Tissue Diseases.     

Cardiac involvement in patients with systemic lupus erythematosus and correlation of valvular lesions with anti-Ro/SS-A and anti-La/SS-B antibody levels

The aim of this study was to evaluate the incidence of morphologic and functional cardiac abnormalities in patients with systemic lupus erythematosus (SLE) and to correlate the findings with levels of anti-Ro/SS-A, anti-La/SS-B, and anti-cardiolipin antibody (aCL). Sixty-two patients with SLE were enrolled in this study. All patients underwent complete history taking, clinical assessment, and standard two-dimensional and Doppler echocardiography. Anti-Ro/SS-A, anti-La/SS-B, and aCL levels were measured using a standardized ELISA test. The patients were subdivided into two subgroups based on the presence or absence of valvular involvement. The two subgroups were then compared. Valvular involvement was present in 19 patients (30.6%), pericardial effusion in 12 patients (19.4%), impaired left ventricular relaxation abnormalities in 2 patients (3.2%), and pulmonary hypertension in 3 patients (4.8%). More patients in the valvular involvement group had positive anti-Ro/SS-A antibodies than in the valvular noninvolvement group (7/19 vs. 4/43). The difference was significant, with P 0.01. Serum levels of anti-Ro/SS-A levels were significantly higher in the valvular involvement group (33.7 ± 36.0 vs. 13.7 ± 25.1; P 0.01), as were the serum anti-La/SS-B levels (21.9 ± 23.5 vs. 10.7 ± 17.8; P 0.05). The results suggest a causative correlation between anti-Ro/SS-A and anti-La/SS-B antibodies and the pathogenesis of the valvular lesions in SLE patients.     

Antiphospholipid antibody profiles and their clinical associations in Chinese patients with systemic lupus erythematosus

OBJECTIVE: Different prevalences of antiphospholipid antibodies (aPL) have been reported in different populations of patients with systemic lupus erythematosus (SLE). Chinese are generally believed to have lower risk of vascular thrombosis. We examined the prevalence of aPL including lupus anticoagulant (LAC), anticardiolipin (aCL) and anti-beta2-glycoprotein I (anti-beta2-GPI) antibodies, the level of thrombotic risk, and the association of aPL with thrombotic and pregnancy outcomes in a Chinese cohort with SLE at the university lupus clinic during the period 1986-2003. METHODS: aPL were measured in 272 SLE patients, and medical records were reviewed for vascular thrombosis and pregnancy outcomes. RESULTS: The prevalence of LAC, IgG aCL, and IgG anti-beta2-GPI antibodies was 22.4%, 29.0%, and 7.7%, respectively. There were 38 episodes of thrombosis after a mean duration of followup of 11.0 +/- 6.8 SD years, giving a thrombotic rate of 1.26/100 patient-years. All aPL were shown to be associated with vascular thrombosis. IgG anti-beta2-GPI antibodies were found to be associated with recurrent thrombosis [8.0/100 patient-years or 25.0% (7/28)]. Patients taking hydroxychloroquine were found to have fewer thrombotic complications than those who were not (OR 0.17, 95% CI 0.07-0.44; p < 0.0001). LAC was the strongest factor associated with recurrent miscarriages [relative risk 12.3, 95% CI 1.22-123.31; p = 0.03). The diagnosis of secondary antiphospholipid syndrome was satisfied in 8.9% of patients. CONCLUSION: The lifetime and recurrent thrombotic rates in our patients with aPL were not particularly different from those in the literature. However, the lower prevalence of aPL in our cohort may suggest a role of other prothrombotic factors in predisposition to thrombosis.     

Heart involvement in systemic lupus erythematosus, anti-phospholipid syndrome and neonatal lupus

Cardiac involvement is one of the main complications substantially contributing to the morbidity and mortality of patients suffering from systemic autoimmune diseases. All the anatomical heart structures can be affected, and multiple pathogenic mechanisms have been reported. Non-organ-specific autoantibodies have been implicated in immune complex formation and deposition as the initial triggers for inflammatory processes responsible for Libman-Sacks verrucous endocarditis, myocarditis and pericarditis. Anti-phospholipid antibodies have been associated with thrombotic events in coronary arteries, heart valve involvement and intra-myocardial vasculopathy in the context of primary and secondary anti-phospholipid syndrome. Antibodies-SSA/Ro and anti-SSB/La antigens play a major pathogenic role in affecting the heart conduction tissue leading to the electrocardiographic abnormalities of the neonatal lupus syndrome and have been closely associated with endocardial fibroelastosis.     

Antinuclear antibody profiles in relation to specific disease manifestations of systemic lupus erythematosus

Clinical Rheumatology - The aim of the study was to investigate the prevalence of antinuclear and associated antibodies (anti-dsDNA, anticardiolipin, anti-RNP, anti-Sm, anti-SSA and anti-SSB)...     

Reactivity with dichotomous determinants of Ro 60 stratifies autoantibody responses in lupus and primary Sjögren's syndrome

Objective

Analysis of B cell determinants of Ro 60 exposed on the surface of apoptotic cells (apotopes) or intracellular epitopes provides insight into the structural forms of the autoantigen that break immune tolerance. This study was initiated to compare anti–Ro 60 responses in systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS) against membrane‐bound and intracellular forms of Ro 60.

Methods

The reactivity of autoantibodies from patients with SLE and primary SS to Ro 60 apotopes and epitopes was assessed by multiparameter flow cytometry and solid‐phase immunoassay. Anti–Ro 60 IgG was eluted from early apoptotic cells or recombinant Ro 60 immobilized on nitrocellulose, and binding to membrane‐bound and intracellular forms of Ro 60 was quantitated by flow cytometry.

Results

An immunodominant apotope, which was recognized by IgG from a subset of SLE patients with anti‐Ro, but not anti‐La, autoantibodies, was mapped to a region forming a helix‐loop‐helix at the apical tip of the Ro 60 molecule. Immobilization of this region to the solid phase exposed an epitope that was recognized by IgG from primary SS and SLE patients whose sera had both anti‐Ro and anti‐La autoantibodies. Autoantibodies eluted from either the surface of apoptotic cells or the Ro 60 epitope on the solid phase were non–cross‐reactive and specifically recognized membrane‐bound or cytoplasmic forms of Ro 60.

Conclusion

This is the first example of a dichotomy of human autoantibody responses against mutually exclusive determinants linked to a single domain of a systemic autoantigen and supports a model in which tolerance is broken by different immunogenic forms of Ro 60.

     

Anti-SS-A (Ro) antibody is positively associated with steroid-induced psychiatric events in systemic lupus erythematosus patients

Forty-five systemic lupus erythematosus (SLE) patients who had steroid at a prednisolone dose of 0.8 mg/kg/day or more were retrospectively studied for psychiatric events that developed after the therapy. Simple insomnia was excluded. Their age, sex, dose, and duration of steroid, autoantibodies, and prophylactic heparin use were examined. Seventeen patients (female/male: 16/1, 36.7 ± 10.7 years old) developed psychiatric events 18.2 ± 10.2 days after steroid start, whereas 28 patients (27/1, 37.9 ± 13.1) did not. Anti-SS-A (Ro) antibody was more prevalent in patients with the events (15/17 vs. 14/28, p = 0.009). When heparin was administered concurrently with steroid, psychiatric events developed less frequently either in all of the patients (2/17 vs. 11/28, p = 0.048) or in patients positive for the anti-SS-A antibody (2/15 vs. 7/14, p = 0.041). SLE patients positive for the anti-SS-A (Ro) antibody would much more likely develop psychiatric events after a substantial-dose steroid therapy, and a concurrent prophylactic heparin administration might reduce the risk.