Anti-Ro/SSA and La/SSB antibodies

The Ro/La system is considered as an heterogeneous antigenic complex, constituted by three different proteins (52?kDa Ro, 60?kDa Ro and La) and four small RNAs particles. Anti-Ro/SSA are the most prevalent specificity among many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous LE (SCLE), neonatal lupus and primary biliary cirrhosis. In contrast, anti-La/SSB is more associated with Sjögren's syndrome (SS). The differences between 52?kDa, 60?kDa Ro and La could explain why different assays did not show equivalent performance in anti-Ro and anti-La autoantibodies detection. The RNA precipitation assay had the highest sensitivity and specificity, usually considered as the reference methods. CIE is considered the most reliable to detect anti-Ro/SSA antibodies in routine practice, performing better than immunoblotting (IB) and some ELISAs. It shows a high sensitivity (89%) and specificity (100%). ELISA is generally considered a safe, rapid, sensitive and specific tecnique. Therefore, its high sensitivity often corresponds to a very low clinical specificity and the assay can give false positive results. Therefore, it is very important to search anti-Ro and anti-La only in selected patients, using the assay with high specificity and good predictive value, in order to have clinically significant and true positive results.     

Ro (SSA) and La (SSB) antibodies

Summary This review traces the historical development of information regarding the Ro (SSA) and La (SSB) autoantibody systems over the past twenty years. Clinical and serologic findings are integrated with fundamental observations in this rapidly expanding area of research. Retrospective analysis of the physicochemical properties of the antigens and the cellular staining characteristics of antibodies to these antigens suggest that SjD and Ro and SSA, as well as SjT and La, SSB, and Ha antigens probably are similar macromolecules. The immunologic identity of Ro with SSA and La with SSB and Ha has been established previously. Antibodies to these antigens are directed against macromolecules containing small RNA nucleotides.Antibodies to the Ro (SSA)-La(SSB) antigen system commonly are detected in the sera of patients with systemic lupus erythematosus and Sjögren's syndrome and appear to be of diagnostic significance. These antibodies occur in up to one quarter of patients with systemic lupus erythematosus (SLE) without the sicca complex, but also in patients with ANA negative SLE who have a prominent photosensitive dermatitis and may have serious renal disease, subacute cutaneous SLE, and in infants and mothers of infants with neonatal SLE. Thus, these antibody systems form a serologic link between many unusual connective tissue diseases and systemic SLE.Antibodies to Ro (SSA)-La(SSB) are associated not only with Sjögren's syndrome occurring alone, but also with Sjögren's syndrome occurring in the setting of other connective tissue diseases including SLE and rheumatoid arthritis. Anemia, leukopenia, and thrombocytopenia, as well as hyperglobulinemia and the presence of rheumatoid factor, cryoglobulins, and antibodies to nuclear antigens are associated significantly with Ro positivity in Sjögren's syndrome patients. There is a striking association of vasculitis in the clinical setting of Sjögren's syndrome with the presence of antibodies to Ro (SSA). In addition to peripheral nerve involvement, unusual central nervous system manifestations as well as myositis occur in these Ro(SSA) positive Sjögren's syndrome patients. Deposition of immunoglobulin and complement within vessel walls of kidney and muscle from Ro positive patients with Sjögren's syndrome suggests a possible role for immune complex deposition in the pathogenesis of the vasculitis.Supported by National Institutes of Health grant 5ROI-AM-25650-03 and Research Career Development Award 5-KO-4-AM-00524-02     

B cell apotopes of the 60-kDa Ro/SSA and La/SSB autoantigens

Apoptosis has been proposed to influence the initiation and diversification of autoimmunity to the Ro (SSA)/La (SSB) ribonucleoprotein (RNP) particle and serve as a target for autoantibody-mediated tissue injury. We have developed a new approach to B cell epitope mapping which identifies "apotopes," defined as epitopes expressed on the surface of apoptotic cells. Preliminary studies support a role for apotopes as diagnostic markers in systemic lupus erythematosus (SLE) and primary Sj?gren's syndrome. For example, apotopes within the NH(2)-terminal and central regions of La react with the majority of sera from mothers of infants with congenital heart block. Furthermore, a Ro60 apotope is specific for a subset of SLE with isolated anti-Ro60 responses. The mapping of B cell apotopes may prove superior to standard epitope mapping by suggesting novel pathways of autoantibody production and identifying pathogenic species of autoantibodies.     

Pregnancy Outcomes in Patients with Autoimmune Diseases and Anti-Ro/SSA Antibodies

Anti-Ro/SSA antibodies are associated with neonatal lupus (congenital heart block (CHB), neonatal transient skin rash, hematological and hepatic abnormalities), but do not negatively affects other gestational outcomes, and the general outcome of these pregnancies is now good, when followed by experienced multidisciplinary teams. The prevalence of CHB, defined as an atrioventricular block diagnosed in utero, at birth, or within the neonatal period (0–27 days after birth), in the offspring of an anti-Ro/SSA-positive women is 1–2%, of neonatal lupus rash around 10–20%, while laboratory abnormalities in asymptomatic babies can be detected in up to 27% of cases. The risk of recurrence of CHB is ten times higher. Most of the mothers are asymptomatic at delivery and are identified only by the birth of an affected child. Half of these asymptomatic women develop symptoms of a rheumatic disease, most commonly arthralgias and xerophtalmia, but few develop lupus nephritis. A standard therapy for CHB is still matter of investigation, although fluorinated corticosteroids have been reported to be effective for associated cardiomyopathy. Serial echocardiograms and obstetric sonograms, performed at least every 1–2 weeks starting from the 16th week of gestational age, are recommended in anti-Ro/SSA-positive pregnant women to detect early fetal abnormalities that might be a target of preventive therapy.     

Congenital heart block associated with maternal anti SSA/SSB antibodies :a report of four cases

Congenital heart block (CHB) associated with maternal anti-SSA/SSB antibodies: a report of four cases. CHB detected in utero is strongly associated with maternal antibodies to SSA (Ro) and SSB (La). Their pathogenic role in the development of CHB has been established in several studies. The mothers of affected infants frequently had autoimmune disease (systemic lupus erythematosus, Sj?gren's syndrome) or were entirely asymptomatic. It is very difficult to identify pregnant asymptomatic mothers carrying anti-SSA/SSB antibodies. We report four cases of infants born to asymptomatic mothers with anti-SSA/SSB antibodies, three of them developed isolated congenital cardiac heart block and one with no evidence of CHB. All three CHB are detected during pregnancy between 16 and 24 weeks of gestation. All maternal sera contained antibodies to SSA alone or the both SSA and SSB. Three of four subsequent pregnancies were complicated by heart block. One child affected died in utero. While the two other newborns with CHB required pacemaker insertion during the first 3 months of life. Although the association of anti-SSA/SSB with CHB is widely accepted, the precise mechanism by which these antibodies cause cardiac conduction abnormalities remains to be defined. Antibodies to SSA/SSB have been proposed to be a serologic marker for neonatal lupus syndrome and CHB. Fetal and neonatal diseases are presumed to be due to the transplacental passage of these IgG autoantibodies from the mother into the fetal circulation. Since these antibodies may have a pathogenic role in CHB, screening of infants with isolated CHB or neonatal lupus and their mothers for the presence of anti-SSA and anti-SSB is strongly recommended.     

IgM and IgG Subclass Distribution of Human Anti-Ro/SSA 60 kDa Autoantibodies

The Ro/SSA and La/SSB antigens are common targets for autoantibodies found in the sera of patients with Sjögren's syndrome and SLE. The anti-Ro/SSA and anti-La/SSB antibodies often appear together but are not cross-reactive. This paper describes the humoral autoimmune response to the Ro/SSA 60 kDa protein moiety with respect to the presence of IgM and IgG1-4 antibodies. IgM antibodies to the Ro 60 kDa protein coexisted with IgG anti-Ro 60 kDa antibodies in nearly half of the sera. A similar fraction also contained IgM anti-La/SSB antibodies. The frequency of sera with IgM antibodies of both specificities was that expected from random overlap.
A predominating igGl anti-Ro 60 kDa response was found in all patients, but anti-Ro 60 kDa antibodies of the other IgG subclasses were present also in a high number of sera. This is in contrast to the reported IgG subclass distribution of anti-La/SSB antibodies.
Mapping of IgM and IgG 1–4 antibody recognition of different parts of the Ro 60 kDa protein was also performed. IgM and IgGl-4 antibodies of all sera reacted with the central part of the Ro 60 kDa protein, encompassing amino acid residues 181–320.     

自身抗原SSB/La抗原优势表位分析

探讨自身抗原Ｌａ的抗原优势表位，为疾病机制的研究提供依据。方法：根据计算机软件进行的蛋白质序列结构分析，采用ＰＣＲ法克隆自身抗原Ｌａ多肽片段的ｃＤＮＡ，定向插入表达载体ＰＧＥＸ－２Ｔ，并且导入大肠杆菌中表达重组事蛋白，用ＧＳＴ亲人事层析柱进行纯化，经免疫印迹法与患者阳性血清进行反应，结果：ＳＳＢ／Ｌａ的抗原优势表位主要存在于１－２８、８０－１０７，１０８１８８，２８３－３３８位。不同病人在不同病程     

New coupled-particle light-scattering assay for detection of Ro/SSA (52 and 60 kilodaltons) and La/SSB autoantibodies in connective tissue diseases

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sj?gren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.     

New Coupled-Particle Light-Scattering Assay for Detection of Ro/SSA (52 and 60 Kilodaltons) and La/SSB Autoantibodies in Connective Tissue Diseases

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sjögren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.     

Effective separation of the 52 kDa SSA/Ro polypeptide from the 48 kDa SSB/La polypeptide by altering conditions of polyacrylamide gel electrophoresis

A method is described for the separation of the 52 kDa SSA/Ro polypeptide from the 48 kDa SSB/La polypeptide. These two proteins anomalously co-migrate with the same relative mass under conditions of conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis according to Laemmli using a stock solution of acrylamide: bisacrylamide 30:0.8, ratio = 37.5. A higher ratio of monomer to cross-linker, ratio = 172.4 used in a 15% acrylamide solution, readily separates the two peptide chains. This method facilitates the detection of the 52 kDa SSA/Ro component which otherwise might have been incorrectly assigned as a 48 kDa SSB/La polypeptide.     

Nuclear-Targeting Autoantibodies Induced Nuclear PARP Cleavage Accompanied by More Pronounced Decrease of Peripheral White Blood Cells Than Ro/SSA and La/SSB Antigen-Targeting Autoantibodies

Autoantibody production and leukocytopenia may be linked in patients with lupus erythematosus (LE). Unclear is the ability of different autoantibody species to induce apoptosis and cell loss. Laboratory routine analyses (white blood cell counts, autoantibody detection), and flow cytometry (annexin V, CD3, CD4, CD8) have been performed in 126 consecutive LE-patients. Nuclei of PBMC were investigated flow cytometrically for the presence of the 85 kDa poly-(ADP-ribose)-polymerase (PARP) fragment. Peripheral total white blood cells (WBC), lymphocytes, T-cells, CD3+ CD4+, and CD3+ CD8+ cells were significantly decreased in patients with LE (P from 1.2 × 10^–14 to P < .0008). In the presence of either antinuclear (P from 1.2 × 10^–14 to P < .0008) or anti-dsDNA antibodies (P from 2.9 × 10^–12 to P < .007) were significantly diminished. Differences in cell numbers in LE patients with versus without anti-Ro/SSA were less pronounced: significant differences could be only obtained in lymphocytes and T-cells (P < .02). Anti-La/SSB antibodies were accompanied by significant increased leukocytes (P < .02). PARP cleavage (85 kDa) in nuclei was preferentially observed in cases with nuclear targeting autoantibodies. These results indicate that nuclear targeting autoantibodies are associated to lower peripheral blood cells counts than Ro/SSA, and La/SSB cytoplasmic targeting autoantibodies. This provides an explanation for the pathogenesis of cytopenias associated with SLE.     

La/SSB ribonucleoprotein levels increased in transformed cells.

Autoantibodies to the La/SSB ribonucleoprotein are commonly found in patients with Sjögren''s syndrome. Previous studies have shown that La/SSB accumulates in cells shortly after viral infection. We have extended these studies by investigating levels of the La/SSB antigen in virally and spontaneously transformed cell lines (contact-insensitive and tumourigenic) relative to their non-transformed counterpart cell lines (contact-sensitive and non-tumourigenic). Transformed BALB/3T12-3 and KNRK fibroblasts were visibly brighter by immunofluorescence assay than non-transformed BALB/3T3 and NRK fibroblasts respectively, when reacted with anti-La/SSB specific sera. This was confirmed by flow cytometry, as La/SSB levels were elevated in the transformed counterparts of the same cell lines. An anti-Sm monoclonal antibody and normal human serum reacted with these cell lines failed to show a significant increase by flow cytometry. Finally, a two-fold increase in the La/SSB antigen was demonstrated in cell lysates of these cell lines by a capture ELISA. These data show that La/SSB is elevated in transformed cell lines compared with non-transformed counterpart cell lines and suggest that this increase is not restricted to viral transformation.     

An immunodominant La/SSB autoantibody proteome derives from public clonotypes

The La/SSB autoantigen is a major target of long‐term humoral autoimmunity in primary Sjögren's Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti‐Ro60/Ro52/La responses target an NH2‐terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high‐resolution Orbitrap mass spectrometry to determine the clonality, isotype and V‐region sequences of LaA‐specific autoantibodies in seven patients with primary SS. Anti‐LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope‐specific affinity chromatography were analysed by combined database and de‐novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa‐restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3‐30 heavy chain paired with IGKV3‐15 light chain and the second by an IGHV3‐43/IGKV3‐20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity‐determining regions, consistent with a common breach of B cell tolerance followed by antigen‐driven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein–RNA complexes are mediated by public sets of autoreactive B cell clonotypes.     

Different temporal expression of immunodominant Ro60/60 kDa-SSA and La/SSB apotopes

Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH₂-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.     

A radioimmunoassay for antibodies to the Ro/SSA particle

A radioimmunoassay (RIA) for antibodies to the Ro/SSA particle is described using Iodogen to radiolabel the antigenic protein moiety of Ro/SSA with 125I. RIA methods utilizing either 33% saturated ammonium sulfate (NH4)2SO4 or 3.5% polyethylene glycol to separate bound from free antigen are comparable. Either method is similar to ELISA in sensitivity and specificity. This RIA provides a fluid phase assay for the Ro/SSA-anti-Ro/SSA system not previously available.     

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization

The B cell epitope mapping of La/SSB was performed using 20 mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145–164, 289–308, 301–320 and 349–368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes:¹⁴⁷HKAFKGSI¹⁵⁴, ²⁹¹NGNLQLRNKEVT³⁰², ³⁰¹VTWEVLEGEVEKEALKKI³¹⁸ and ³⁴⁹GSGKGKVQFQGKKTKF³⁶⁴. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presented 83.3% similarity with the ¹³⁹HKGFKGVD¹⁴⁶ region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope ¹⁴⁷HKAFKGSI¹⁵⁴ to 100% to epitope ³⁴⁹GSGKGKVQGKKTKF³⁶⁴. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope ¹⁴⁷HKAFKGSI¹⁵⁴ presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.     

Cross-recognition between histones and La/SSB may account for anti-DNA reactivity in SLE patients

Antibodies to La/SSB are detected in sera of patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE). The vast majority of anti-La/SSB positive sera contain antibodies directed towards a linear B-cell epitope of La/SSB spanning the sequence 349-364aa (pep349-364). The aim of this study was to evaluate the fluctuation of antibody levels to major B-cell epitopes of La/SSB over time and investigate for their possible crossreactions. Sequential sera from 15 SLE and 15 pSS patients, followed from 3 to 10 years were obtained. All patients with SLE were positive for anti-Ro/SSA, anti-La/SSB and anti-dsDNA antibodies and patients with pSS were positive for anti-Ro/SSA and anti-La/SSB antibodies. Sera from 30 patients with SLE without anti-La/SSB antibodies and 30 healthy individuals served as disease and negative control respectivelly. All sera tested for the presence of anti-pep349-364 antibodies, using a specific ELISA. Specific anti-pep349-364 IgG was purified from sera of SLE patients and evaluated for cross reactivity against dsDNA and histones. In all SLE sera the levels of anti-pep349-364 antibodies varied in time and fluctuated in parallel with anti dsDNA antibodies. Anti-pep349-364 IgG purified from 7 SLE patients. Five out of 7 were found to react with calf thymus DNA in ELISA. All purified (7/7) anti-pep349-364 IgG preparations reacted with histone H1 and failed to produce a positive immunofluorescence pattern in Crithidia luciliae anti-dsDNA assay which lacks histones. Competative inhibition experiments demonstrated that histone H1 could inhibit completely the binding of anti-pep349-364 IgG to pep349-364 while pep349-364 inhibited by 70% the binding of anti-pep349-364 IgG to histone H1. These findings indicate that a subgroup of SLE patients possess cross-reacting anti-histone H1 antibodies and anti-pep349-364 antibodies, which can be faulty considered as anti-dsDNA reactivity in regular ELISA techniques.