The anti-Ro/SSA and anti-La/SSB antigen-antibody systems

INTRODUCTION — Autoantibodies directed against Ro/SSA and La/SSB autoantigens were originally identified in patients with Sjögren's disease and systemic lupus erythematosus (SLE). Subsequent studies showed that anti-Ro/SSA antibodies may be present in patients with other autoimmune diseases, including systemic sclerosis, idiopathic inflammatory myopathies (IIM), interstitial lung disease (ILD), mixed connective tissue disease (MCTD), primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis), and rheumatoid arthritis (RA). Additionally, anti-Ro/SSA antibodies (with or without anti-La/SSB antibodies) identify pregnant women who are at increased risk of having a child with neonatal lupus syndrome (see "Neonatal lupus: Epidemiology, pathogenesis, clinical manifestations, and diagnosis"). Anti-Ro/SSA antibodies may also be the only autoantibodies present in a subset of patients with "antinuclear antibody (ANA)-negative" SLE.

The anti-Ro/SSA and anti-La/SSB antibody systems and the clinical significance of these antibodies are reviewed here. An overview of the ANAs important in SLE and in related autoimmune diseases and the clinical significance of autoantibodies directed against double-stranded deoxyribonucleic acid (dsDNA), U1 ribonucleoprotein (RNP), Sm, and ribosomal P proteins are reviewed separately. (See "Measurement and clinical significance of antinuclear antibodies" and "Antibodies to double-stranded (ds)DNA, Sm, and U1 RNP" and "Antiribosomal P protein antibodies".)

Ro/SSA ANTIGENS — Serum containing autoantibodies directed against the Ro/SSA antigens may recognize one or both of two cellular proteins with molecular weights of approximately 52 and 60 kD [1-4]. These autoantigens are referred to as "Ro52" and "Ro60," respectively. Ro52 and Ro60 do not have amino acid sequence homology, and autoantibodies directed against Ro52 do not cross-react with Ro60, and vice versa. The two autoantigens were originally thought to interact with each other, but subsequent studies have shown that the two proteins reside in distinct cellular compartments, with Ro60 localized to the nucleus and nucleolus and with Ro52 localized to the cytoplasm.

Ro60 (also known as TROVE2) — The amino acid sequence of the Ro60 autoantigen was described in the late 1980s [1,2]; Ro60 was shown to bind small, non-coding ribonucleic acids (RNAs) termed "Y RNAs." Although the function of Y RNAs is unknown, one report suggests that Y RNAs may be a source of micro (mi)RNA molecules, which have a role in the regulation of messenger (m)RNA stability and translation [5]. The crystal structure of Ro60 suggests that the protein can bind to both single- and double-stranded (ds)RNA. The protein may function as a "RNA chaperone" that binds to misfolded pre-5S ribosomal RNA and may hasten the degradation of the defective molecule. Ro60 may also bind to other cellular and viral RNAs in the cell, including Epstein-Barr virus (EBV) early RNA 1 (EBER1) [6]. Mice that lack the functional gene encoding Ro60 develop an autoimmune syndrome characterized by anti-ribosome and anti-chromatin antibodies, as well as glomerulonephritis [7], suggesting that Ro60 may have an important role in preventing systemic autoimmune disease.

Ro52 (also known as TRIM21) — The amino acid sequence of the Ro52 autoantigen was reported in 1991 [3]. Ro52 is an interferon-inducible protein that belongs to the "tripartite motif" family of proteins. Ro52 contains an amino (N)-terminal RING finger domain, followed by B-box and coiled-coil domains and by a carboxy (C)-terminal B30.2 or "PRYPRY" domain. The protein localizes to the cytoplasm and functions as an E3 ubiquitin ligase, an enzyme that adds ubiquitin molecules to target proteins [4].

Ro52 interacts with a broad range of substrates and may either enhance or inhibit the function of target proteins. It interacts with the Fc portion of immunoglobulin G (IgG) molecules, via its C-terminal B30.2 domain, and catalyzes the polyubiquitination of misfolded IgG, thereby hastening its degradation in proteasomes [8,9]. The presence of a domain that can bind the Fc portion of antibody molecules raises the possibility that Ro52 has a role in "intracellular antibody immunity." Antibody-coated viral particles might be carried inside the cell during infection. Interaction between Ro52 and the antibody-pathogen complex may result in ubiquitination and subsequent degradation of the pathogen [10].

Ro52 may also have important roles in the regulation of inflammation. Ro52 adds an ubiquitin molecule to activated inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB) and downregulates proinflammatory nuclear factor kappa-B (NFKB) signaling [11]. Ro52 also inhibits inflammation by targeting interferon regulatory factors (IRF) 3 and 7 for ubiquitin-mediated degradation [12,13]. Consistent with the effects of Ro52 on NFKB signaling and IRF stability, mice that lack Ro52 develop an autoimmune disease, which is characterized by hypergammaglobulinemia and renal disease [14,15].

Ro52, like Ro60 and La (see 'La/SSB antigen' below), may also have a role in the regulation of mRNA stability. Ro52 interacts with and enhances the activity of decapping protein (DCP) 2 [16], a protein that removes the 5' cap from mRNA molecules. Removal of the 5' cap exposes mRNA molecules to degradation by exoribonuclease I.

La/SSB ANTIGEN — Autoantibodies directed against the La/SSB autoantigen (La) interact with a 47-kD protein, which shuttles between the nucleus and cytoplasm but which is predominantly found in the nucleus [17]. A complementary DNA (cDNA) encoding La was identified in 1988 [18]. The N-terminus of the protein contains a "La" RNA-binding domain and an adjacent RNA recognition motif (RRM), which cooperate to bind RNA. The C-terminus of the protein contains a second RRM followed by a short basic motif ("SBF" domain) and a nuclear localization sequence [19]. The N-terminal portion of La mediates the interaction with the 3' end of RNA polymerase III transcripts [20]. La participates in the processing of small, noncoding RNAs such as ribosomal 5S RNA.

La also binds cellular transfer (t)RNAs, as well as viral RNA molecules, including those produced by Epstein-Barr virus (EBV), Hepatitis A and C viruses, respiratory syncytial virus, adenovirus, and human immunodeficiency virus (HIV) [17,21-25]. La may regulate the translation of viral RNAs by enhancing internal ribosomal entry site function. A report suggests that La has a critical role in regulating RNA interference (RNAi) by enhancing RNAi turnover in the RNA-induced silencing complex (RISC) [26]. If confirmed, this result would suggest that La has a role in the regulation of messenger (m)RNA stability and translation. A mouse model that lacks the La protein has not been reported.

DETECTION OF ANTI-Ro AND ANTI-La ANTIBODIES

Solid phase assays — In the past, antibodies directed against the Ro and La antigens were detected by immunodiffusion or by counter-immunoelectrophoresis. Most laboratories have adopted solid phase assays, including enzyme-linked immunosorbent assays (ELISA) or antigen-coated fluorescent microsphere and flow cytometry-based assays. Solid phase assays use either native or recombinant protein as substrate. These assays have advantages over other techniques including decreased cost, increased sensitivity for the detection of the corresponding autoantibodies, and the ability to quantify the amount of antibody. The increased sensitivity of solid phase assays comes at the cost of decreased specificity for the diagnosis of autoimmune diseases. The reported prevalence of anti-Ro and anti-La autoantibodies in patients with autoimmune diseases depends to a great extent on the method used to detect the autoantibodies.

Immunofluorescence staining on HEp-2 substrates — Patients in whom the only autoantibodies present are anti-Ro antibodies may have a falsely negative antinuclear antibody (ANA) test using indirect immunofluorescence and the traditional human epithelial cell line-2 (HEp-2) cell substrate; Ro60 immunoreactivity may be lost during the preparation of the cells and Ro52 is a cytoplasmic, rather than nuclear, autoantigen.

The ability to detect anti-Ro60 antibodies by indirect immunofluorescence has been enhanced by the use of a modified HEp-2 cell substrate ("HEp-2000"), in which transfection of a complementary DNA (cDNA) encoding Ro-60 was used to over-express the protein in 10 to 20 percent of the cells [27,28]. Anti-Ro60 antibodies produce nucleolar and nuclear speckled staining patterns in transfected cells on the HEp-2000 substrate. Only 10 to 20 percent of the cells will produce this staining pattern if anti-Ro60 antibodies are the only autoantibodies present in a patient's serum (picture 1). A limitation of the HEp-2000 substrate for the detection of anti-Ro60 antibodies occurs when other autoantibodies are present in addition to anti-Ro60 antibodies. For example, antibodies directed against DNA will produce a homogeneous nuclear staining pattern in all cells and may obscure the characteristic anti-Ro60 pattern (which would otherwise be detected in a subset of the HEp-2000 cells). As of 2012, only approximately 25 percent of clinical and commercial laboratories in the United States use the HEp-2000 substrate, while in Europe and Australia, HEp-2000 is used as the ANA substrate in approximately 75 percent of such laboratories. In a 2016 survey of national and international clinical laboratories that participated in the College of American Pathologists' Proficiency Testing Program, 80 percent of 644 responding laboratories reported using the HEp-2 cell substrate, while 18 percent used the HEp-2000 cell substrate [29]. In laboratories using the traditional HEp-2 cell substrate, solid phase assay, instead of indirect immunofluorescence, is the more reliable approach to detecting anti-Ro60 antibodies.

Antibodies directed against Ro52, like those directed against Ro60, are difficult to detect by indirect immunofluorescence using the traditional HEp-2 cell substrate. In addition, when overexpressed in HEp-2 cells, Ro52 localizes to the cell cytoplasm (picture 2). Because Ro52 is not a nuclear autoantigen and because anti-Ro52 autoantibodies are difficult to detect using the HEp-2 cell substrate, a patient who has only anti-Ro52 antibodies may have a negative test for ANA.

Although La has been shown to shuttle between the nucleus and cytoplasm, the protein is predominantly found in the nucleus of HEp-2 cells. Autoantibodies directed against La produce a speckled nuclear staining pattern (picture 3).

CLINICAL SIGNIFICANCE — For the most part, the solid-phase assays used to detect anti-Ro antibodies detect both anti-Ro60 and anti-Ro52 antibodies. Anti-Ro antibodies may be present in patients with a range of autoimmune disorders, including systemic lupus erythematosus (SLE; 32 percent), Sjögren's disease (59 percent), idiopathic inflammatory myopathies (IIM; 19 percent), systemic sclerosis (21 percent), mixed connective tissue disease (MCTD; 29 percent [30]), and rheumatoid arthritis (RA; 15 percent), as well as primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis), interstitial lung disease (ILD) [31], and undefined connective tissue disease (UCTD) [32]. Women with anti-Ro antibodies (with or without anti-La antibodies and with or without autoimmune disease) are at increased risk for having a child with neonatal lupus syndrome. Anti-Ro antibodies may be the only autoantibodies present in more than half of the patients with "antinuclear antibody (ANA)-negative" SLE. Anti-Ro antibodies may also be the first detectable autoantibodies that precede the development of SLE and Sjögren's disease in asymptomatic individuals. (See 'Sjögren's disease' below and 'Anti-Ro antibodies and 'ANA-negative' SLE' below and 'Neonatal lupus syndrome' below and 'Anti-Ro antibodies and other autoimmune diseases' below.)

In contrast to anti-Ro antibodies, which may be present in a variety of autoimmune diseases, anti-La antibodies are specific for the diagnosis of Sjögren's disease and SLE [33]. In addition, as with anti-Ro antibodies, anti-La antibodies may be detected in the mothers of children who are born with neonatal lupus syndrome [34,35]. These women may or may not have evidence of systemic autoimmune disease.

Sjögren's disease — Anti-Ro antibodies detected by a solid phase immunoassay that includes a mixture of Ro60 and Ro52 are frequently detected in the serum of patients with Sjögren's disease. Because these autoantibodies are relatively specific for Sjögren's disease, an American-European Consensus Group included the presence of anti-Ro with or without anti-La antibodies as one of six classification criteria for this disease [36]. A subsequent American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR; formerly known as European League Against Rheumatism) classification system for primary Sjögren's disease gave even greater weight to the presence of anti-Ro antibodies. (See "Diagnosis and classification of Sjögren’s disease", section on 'Antibodies to Ro/SSA and La/SSB' and "Diagnosis and classification of Sjögren’s disease", section on 'Classification criteria'.)

Anti-Ro60 versus anti-Ro52 antibodies — With recognition that the autoantigen designated "Ro" consists of both 60-kD and 52-kD proteins, solid phase immunoassays have been developed for each [37]. The prevalence of anti-Ro60 and anti-Ro52 antibodies in patients with SLE was reported to be 49 and 43 percent, respectively. In patients with Sjögren's disease, these antibodies were detected in 67 and 75 percent of affected individuals. The presence of anti-Ro52 antibodies may be associated with more severe disease [38], and there is an increased risk of developing ILD in Sjögren's disease patients with anti-Ro52 antibodies [39]. The prevalence of the two types of anti-Ro antibodies diverge in patients with other autoimmune diseases [37]. Anti-Ro52 antibodies but not anti-Ro60 antibodies were detected in patients with IIM (35 versus 0 percent), and anti-Ro52 was more common than anti-Ro60 in patients with systemic sclerosis (19 versus 6 percent) and MCTD (29 versus 19 percent [30]).

In a study of anti-Jo-1 antibody-positive patients with IIM, 36 of 89 consecutive patients (40 percent) had coexisting anti-Ro52 antibodies [40]. The presence of anti-Ro52 antibodies appeared to identify a subset of anti-Jo-1 antibody-positive patients with more severe muscle and joint disease. As in patients with Sjögren's disease, the presence of anti-Ro52 antibodies may identify patients with MCTD or systemic sclerosis who are at increased risk of lung fibrosis [30,41].

Most commercial laboratories use a single "anti-Ro" antibody test for both anti-Ro60 and anti-Ro52 antibodies using a combination of the two antigens, despite the different clinical information provided by the individual detection of anti-Ro60 and anti-Ro52 autoantibodies. The practice of combining Ro60 and Ro52 in solid phase immunoassays provides less useful clinical information than do separately performed assays for Ro60 and Ro52 [42-44]. In addition, assays that combine Ro52 and Ro60 antigens may fail to detect up to 20 percent of patients who have antibodies against either Ro52 or Ro60 [37].

Anti-Ro antibodies and 'ANA-negative' SLE — As described above, sera that have only anti-Ro60 and/or anti-Ro52 autoantibodies may be reported to be "ANA-negative." When evaluating patients who are thought to have SLE or Sjögren's disease but who are ANA-negative by indirect immunofluorescence, it is important to request testing for anti-Ro antibodies using a solid phase assay [45]. (See 'Immunofluorescence staining on HEp-2 substrates' above and "Clinical manifestations and diagnosis of systemic lupus erythematosus in adults", section on 'ANA-negative lupus'.)

Neonatal lupus syndrome — Neonatal lupus is a passively transferred autoimmune disease that occurs in some babies born to mothers with anti-Ro with or without anti-La antibodies. The most serious complication in the neonate is complete heart block, which occurs in approximately 1 to 2 percent of such pregnancies. Neonatal lupus accounts for 90 to 95 percent of cases of congenital heart block occurring in utero or in the neonatal period. The pathogenesis and clinical features of the neonatal lupus syndrome, as well as the management of congenital complete heart block, are presented separately. (See "Neonatal lupus: Epidemiology, pathogenesis, clinical manifestations, and diagnosis" and "Congenital third-degree (complete) atrioventricular block".)

For mothers who have given birth to a child with neonatal lupus syndrome, the risk of congenital heart block in subsequent pregnancies increases to 15 percent [46]. Most mothers of babies with neonatal lupus do not have SLE or another autoimmune disease, although nearly half will develop an autoimmune disease (usually Sjögren's disease) over time [47].

Prenatal surveillance for heart block in the setting of maternal anti-Ro/SSA antibodies is discussed in detail elsewhere. (See "Neonatal lupus: Epidemiology, pathogenesis, clinical manifestations, and diagnosis", section on 'Fetal surveillance for heart block'.)

Anti-Ro antibodies preceding autoimmune disease — Autoantibodies may precede the diagnosis of SLE by years, if not decades. A study using serum samples of 130 patients with known SLE from the Department of Defense Serum Repository to test for the presence of autoantibodies (by enzyme-linked immunosorbent assays [ELISA]) prior to the diagnosis detected anti-Ro antibodies in 48 percent of the patients [48]. The mean interval between the dates of the stored serum sample in which anti-Ro antibodies were present and the diagnosis of SLE was longer than 3.6 years. Relative to all other autoantibodies tested, anti-Ro antibodies were the first to develop in patients who eventually were diagnosed with SLE. In a study of 175 serum samples obtained prior to a diagnosis of Sjögren's disease in 117 individuals, anti-Ro antibodies were detected a median of four years prior to diagnosis [49]. The number of normal individuals who are anti-Ro antibody positive, but who never develop an autoimmune disease, is unknown.

Anti-Ro antibodies in patients with cutaneous lupus erythematosus — Anti-Ro antibodies are frequently detected in patients with dermatologic manifestations of lupus erythematosus. In one study, anti-Ro antibodies were detected in 173 of 235 (74 percent) of patients with subacute cutaneous lupus erythematosus (SCLE) [50]. The relationship between anti-Ro antibodies and cutaneous photosensitivity is uncertain. One report suggests that the presence of anti-Sm antibodies, rather than anti-Ro antibodies, is associated with photosensitivity in patients with SLE [51].

Anti-Ro antibodies and other autoimmune diseases — Anti-Ro antibodies may be present in conditions other than SLE and Sjögren's disease, including PBC, IIM, and RA. Anti-Ro52 antibodies were detected in 28 to 32 percent of patients with PBC and the presence of these antibodies provide an important clue to the diagnosis of this treatable, but sometimes asymptomatic, autoimmune liver disease [52-54]. (See "Clinical manifestations, diagnosis, and prognosis of primary biliary cholangitis".)

Anti-Ro antibodies have also been detected in 6 to 12 percent of patients with RA [55,56]. No difference was seen between the clinical presentations of anti-Ro antibody-positive versus anti-Ro antibody-negative patients. However, one study suggests that RA patients with anti-Ro antibodies may have a poorer clinical response to treatment with infliximab, a tumor necrosis factor (TNF) inhibitor, compared with patients lacking such autoantibodies [57].

Anti-Ro antibody-negative, anti-La antibody-positive patients — Anti-La antibodies are rarely detected in the absence of anti-Ro antibodies. In a study of 201 mothers of children with neonatal lupus syndrome, 113 had antibodies directed against both Ro and La, and 85 had antibodies directed against Ro alone. Only three patients were found to have anti-La antibodies in the absence of anti-Ro antibodies [34]. Among 335 patients with Sjögren's disease, 74 had both anti-Ro and anti-La antibodies, and 85 had only anti-Ro antibodies. Only four patients were anti-Ro antibody-negative and anti-La antibody-positive [58]. A study involving the Sjögren's Syndrome International Clinical Alliance (SICCA) cohort concluded that patients who were anti-La antibody positive but anti-Ro antibody negative were unlikely to have Sjögren's disease [59]. Among 165 PBC patients with sicca syndrome-associated autoantibodies, 58 were anti-Ro and anti-La antibody-positive, and 61 had only anti-Ro antibodies. Two patients had only anti-La antibodies [60]. These studies demonstrate that anti-Ro antibody-negative, anti-La antibody-positive patients are uncommon; the clinical significance of this combination of serological results is uncertain.

INDICATIONS FOR ANTI-RO AND ANTI-LA TESTING — The decision to obtain testing for anti-Ro and anti-La antibodies, using solid phase assays, depends upon the clinical setting. Patients in whom testing for anti-Ro and anti-La antibodies is indicated include:

●Patients with symptoms that suggest Sjögren's disease, such as xerostomia, keratoconjunctivitis sicca, and/or salivary and lacrimal gland enlargement, regardless of antinuclear antibody (ANA) status. (See 'Sjögren's disease' above and "Clinical manifestations of Sjögren's disease: Exocrine gland disease" and "Diagnosis and classification of Sjögren’s disease".)

●Women with systemic lupus erythematosus (SLE) or Sjögren's disease who wish to become pregnant. (See "Pregnancy in women with systemic lupus erythematosus", section on 'Presence of anti-Ro and anti-La antibodies'.)

●Women (with or without a systemic autoimmune disease) who have a history of giving birth to a child with congenital heart block. (See 'Neonatal lupus syndrome' above.)

●Women planning to become pregnant who are known to be ANA-positive. Note that the cost-effectiveness of such testing has not been studied.

●Patients suspected of having SLE in whom the screening indirect immunofluorescence test for ANA is negative. Such testing should employ a solid phase assay because anti-Ro antibodies may not be detected by indirect immunofluorescence using a traditional human epithelial cell line-2 (HEp-2) cell substrate. (See 'Anti-Ro antibodies and 'ANA-negative' SLE' above.)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Antinuclear antibodies".)

SUMMARY AND RECOMMENDATIONS

●Serum containing autoantibodies directed against the Ro/SSA autoantigens may recognize one or both of two cellular proteins with molecular weights of approximately 52 and 60 kD; the autoantigens are referred to as "Ro52" and "Ro60," respectively. Ro60 is localized to the nucleus and nucleolus, and Ro52 is localized to the cytoplasm. (See 'Ro/SSA antigens' above.)

●Autoantibodies directed against the La/SSB autoantigen (La) interact with a 47-kD protein, which shuttles between the nucleus and cytoplasm but which is predominantly found in the nucleus. (See 'La/SSB antigen' above.)

●Solid phase assays, including enzyme-linked immunosorbent assays (ELISA) and flow cytometry-based assays, with either native or recombinant protein as substrate, are generally used to detect antibodies directed against the Ro and La antigens. Anti-Ro antibodies may not be detected by indirect immunofluorescence using a traditional human epithelial cell line-2 (HEp-2) cell substrate. (See 'Immunofluorescence staining on HEp-2 substrates' above and 'Detection of anti-Ro and anti-La antibodies' above.)

●Anti-Ro antibodies are not specific for Sjögren's disease and systemic lupus erythematosus (SLE) and may also be present in patients with a range of autoimmune disorders, including dermatomyositis, idiopathic inflammatory myositis, systemic sclerosis, mixed connective tissue disease (MCTD), and primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis). Women with anti-Ro (with or without co-occurring anti-La) autoantibodies are at increased risk for having a child with neonatal lupus syndrome. Anti-Ro antibodies may be the only autoantibodies present in more than half of the patients with "antinuclear antibody (ANA)-negative" SLE. The combination of anti-Ro and anti-La antibodies is relatively specific for the diagnoses of SLE and Sjögren's disease. (See 'Clinical significance' above.)

●Patients for whom testing for anti-Ro and anti-La antibodies is indicated include (see 'Indications for anti-Ro and anti-La testing' above):

•Patients with findings that suggest Sjögren's disease

•Women with SLE or Sjögren's disease who wish to become pregnant

•Women with a history of giving birth to a child with congenital heart block

•Women planning to become pregnant who are known to be ANA-positive, although the cost-effectiveness of such testing has not been studied

•Patients with symptoms and findings that suggest the diagnosis of SLE in whom the indirect immunofluorescence test for ANA is negative

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Morris Reichlin, MD, who contributed to an earlier version of this topic review.