Anti-Ro antibodies and neonatal lupus

The presence of antibodies directed against the Ro (SS-A) antigen is the strongest risk factor for the neonatal lupus syndrome. In most instances, the pregnant woman is asymptomatic; however, the presence of subclinical connective tissue disease and the subsequent emergence of overt connective tissue disease have been reported. This article will review the molecular biology of the Ro antigen, assays for anti-Ro antibodies, prevalence of anti-Ro antibody in normal people and patients with systemic lupus erythematosus, and the clinical, immunogenetic and pathophysiologic features of the neonatal lupus syndrome. Finally, current concepts concerning treatment of pregnant women with anti-Ro antibodies will be reviewed.     

Lupus pneumonitis and anti-SSA(Ro) antibodies

Interstitial pneumonitis has previously been thought to be an uncommon feature in systemic lupus erythematosus (SLE). A review of 63 patients with SLE from 1984 to 1987 at Tulane University revealed a frequency of 25.4% (N = 16) of lupus pneumonitis. Serologic testing for antibodies to SSA(Ro) revealed that 81% (13 of the 16) of the patients with lupus pneumonitis have anti-SSA(Ro) antibodies, compared to a frequency of 38% (24 of the 63) for the entire group with SLE (p less 0.001). The association of lupus pneumonitis and anti-SSA(Ro) antibodies is discussed in light of a speculative immunopathogenic role for the antibody to SSA(Ro) antigen.     

Optimizing the clinical value of polyreactive autoantibodies to double-stranded DNA

The serological characteristic of systemic lupus erythematosus is the presence of antibodies to double stranded (ds) DNA. Raised levels of these antibodies have been found occasionally in patients with other inflammatory rheumatic diseases. Evaluating 67 patients with anti-dsDNA antibodies we searched for additionally immunological features to improve their clinical relevance for connective tissue diseases. We found out that coexistent antinuclear antibodies, antibodies to Sm, U1-nRNP, Ro and La, except in D-penicillamine induced lupus syndrome, were most specific for connective tissue diseases. Permanent antinuclear antibody negative systemic lupus erythematosus may not really exist.     

Antibodies to cytoplasmic antigens in lupus erythematosus

Seven patients with classic cutaneous lupus ery-thematosus are described. Three of these patients had features satisfying four of the American Rheumatism Association (ARA) preliminary criteria for the diagnosis of systemic lupus erythematosus (SLE). Their sera, however, lacked antinuclear antibodies but demonstrated precipitating antibodies reactive against cytoplasmic RNP (La) and non-nucleic acid (Ro) antigens. Four additional ANA-negative patients lacking significant skin disease but having a lupus-like multisystem disease were found to have antibodies to soluble cytoplasmic antigens. Thirty-three of 130 ANA-positive SLE patients, but none of 16 discoid lupus patients, possessed these anticytoplasmic antibodies. These findings suggest that antibodies to Ro and La may be a marker for systemic disease in ANA-negative patients with 1) cutaneous lupus and 2) a distinct sub-population of patients with a lupus-like syndrome without skin disease.     

Interstitial pneumonitis in antinuclear antibody–negative systemic lupus erythematosus: a new clinical manifestation and possible association with anti–ro (ss-a) antibodies

The clinical manifestations of patients with systemic lupus erythematosus (SLE) and negative fluorescent antinuclear antibody have been well documented. This report describes 2 cases of antinuclear antibody–negative SLE with antibodies only to Ro (SS-A) and the previously unreported clinical manifestation of lupus interstitial pneumonitis. A review of all SLE patients from 1984–1986 with interstitial pneumonitis revealed that 10 of the 12 patients had antibodies to Ro (SS-A). The frequency of anti–Ro (SS-A) with interstitial pneumonitis in SLE patients suggests a possible association between the two.     

Sera from patients with autoimmune disease recognize conformational determinants on the 60-kd Ro/SS-A protein.

Anti-Ro antibodies are found in a large proportion of patients with systemic lupus erythematosus and primary Sj?gren's syndrome. These antibodies also characterize neonatal lupus, subacute cutaneous lupus erythematosus, and vasculitis associated with Sj?gren's syndrome and rheumatoid arthritis. Anti-Ro-positive sera may contain either or both of 2 sets of antibodies, recognizing either a 60-kd or a 52-kd polypeptide component of the Ro particle. We found in this study that the immune response to the 60-kd Ro antigen is heterogeneous. Some sera specifically recognize the native Ro antigen but fail to bind the corresponding denatured polypeptides. In addition, after immunodepletion using the denatured 60-kd Ro polypeptide, all anti-Ro-positive sera tested still contained high titers of antibodies recognizing conformational determinants on the Ro antigen. The frequent immunodominance of anti-Ro antibodies targeted to conformational determinants suggests that native autoantigens may directly drive the autoimmune response.     

Interstitial pneumonitis in antinuclear antibody-negative systemic lupus erythematosus: a new clinical manifestation and possible association with anti-Ro (SS-A) antibodies

The clinical manifestations of patients with systemic lupus erythematosus (SLE) and negative fluorescent antinuclear antibody have been well documented. This report describes 2 cases of antinuclear antibody-negative SLE with antibodies only to Ro (SS-A) and the previously unreported clinical manifestation of lupus interstitial pneumonitis. A review of all SLE patients from 1984-1986 with interstitial pneumonitis revealed that 10 of the 12 patients had antibodies to Ro (SS-A). The frequency of anti-Ro (SS-A) with interstitial pneumonitis in SLE patients suggests a possible association between the two.     

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.     

THE MOLECULAR BASIS OF THE SSA/Ro ANTIGENS AND THE CLINICAL SIGNIFICANCE OF THEIR AUTOANTIBODIES

The SSA/Ro antigens are nuclear and cytoplasmic polypeptideswhich serve as autoantigens in systemic lupus erythemato-sus(SLE) and Sjögren's syndrome (SS). They contain two majorisoforms of 60 and 52 kD. The former is the native antigen whilethe latter is a major autoantigen in its denatured form. A thirdprotein of 46 kD termed ‘calreticulin-Ro’ is anautoantigen found in the sera of some patients with SLE. However,it is probably unrelated to the SSA/Ro system. The clinicalrelevance of anti SSA/Ro antibodies in rheumatic diseases hasalso been considered. Initially these antibodies were thoughtto be an epiphenomenon of autoimmune diseases. Recent studieshave shown that they are associated with specific clinical manifestationsand disease subsets. Furthermore, animal models have demonstratedthat they may enhance tissue damage. It seems that anti-SSA/Roantibodies may play a role in the pathogenicity of SLE and SS. KEY WORDS: SSA/Ro antigen, Autoantibodies, Systemic lupus erythematosus, Sjögren's syndrome     

Anti-SSA/Ro and anti-SSB/La antibodies. What's new?

Anti-SSA/Ro and anti-SSB/La autoantibodies recognize different epitopes on polypeptides associated with small RNAs called scYRNA situated mostly in the cytoplasmic compartment (70%) and few in the nuclear compartment (30%). These hYRNPs (h=human) can be found on the cytoplasmic membrane or in small blebs during apoptosis after various stimuli such as UVB, 17-beta-estradiol, viral infection, TNF alpha and other cellular apoptosis inducing molecules. At least two major different proteins are called SSA/Ro: a 52 kDa Ro (with two subtypes alpha and beta) and a 60 kDa Ro. There is only one SSB/La protein of 48 kDa. In some circumstances, other proteins such as calreticuline (MW 57 kDa) join Ro/SSA proteins on some YRNAs. Anti-SSA/Ro antibodies are detected in the sera of 30% of patients with SLE, even during preclinical setting; anti-Ro/SSA are strongly associated (90%) with some subtypes of SLE such as old-onset (>50 y) SLE, subacute lupus erythematosus, drug-induced subacute lupus erythematosus and in patients with hereditary C2 or C4 or C1q deficiency with lupus or lupus-like disease. Anti-SSA/Ro are also associated with primary Sj?gren syndrome (50% to 60%) and with undifferenciated connective tissue disease (UCTD). Anti-SSA/Ro antibodies are almost always present in sera of mothers with babies with neonatal lupus syndrome (NNL) and with complete congenital heart block (CCHB). This last event is very unusual in pregnant patients with anti-Ro/SSA antibodies (1% to 2% of primigeste women). Some good evidences such as experimental models in vitro or ex-vivo, argue for the responsibility of maternal anti-Ro/SSA 52 kDa and/or anti-La/SSB antibodies (or associated IgG antibodies) as major etiologic factor of CCHB and NNL. IgG anti-Ro 52 beta kDa has been shown able to interrupt the atrioventricular conduction as well as the L calcium channel influx of fetal cardiocytes. Other factors must be taken into account to explain discordant twins (with and without CCHB). More recently anti-Ro/SSA antibodies were associated with QT interval prolongation in newborns without CCHB.     

Identification of mothers at risk for congenital heart block and other neonatal lupus syndromes in their children. comparison of enzyme-linked immunosorbent assay and immunoblot for measurement of anti–ss-a/ro and anti–ss-b/la antibodies

Objective. To identify the fine specificity patterns of maternal anti–SS-A/Ro and anti–SS-B/La antibodies that are associated with the birth of a child with transient or permanent manifestations of neonatal lupus syndromes, and to suggest a predictor algorithm for use in counseling. Methods. Sera were obtained from 4 groups of mothers: 57 whose children had congenital heart block, 12 whose children had transient dermatologic or hepatic manifestations of neonatal lupus but no detectable cardiac involvement, 152 with systemic lupus erythematosus and related autoimmune diseases, who gave birth to healthy infants, and 30 with autoimmune diseases whose pregnancy resulted in miscarriage, fetal death, or early postpartum death unrelated to neonatal lupus. Antibodies to SS-A/Ro and SS-B/La were assessed by enzymelinked immunosorbent assay (ELISA) and by sodium dodecyl sulfate (SDS)-immunoblot. Results. Anti–SS-A/Ro antibodies were identified by ELISA in 100%, 91%, 47%, and 43% of the mothers of infants with heart block, with transient neonatal lupus, healthy infants, and fetal death, respectively. High titers of anti–SS-A/Ro antibodies were present more often in mothers of children with cardiac disease or transient neonatal lupus than in either of the other 2 groups. Maternal antibodies to SS-B/La were detected by ELISA in 76% of the heart block group, 73% of the cutaneous neonatal lupus group, 15% of the group with healthy children, and 7% of the fetal death group. On SDS-immunoblot, sera from 91% of the heart block group mothers who had antibodies to SS-A/Ro but not to SS-B/La recognized at least 1 SS-A/Ro antigen, with significantly greater reactivity against the 52-kd component. In contrast, only 62% of the anti–SS-A/Ro positive, anti–SS-B/La negative responders in the healthy group recognized the 52-kd and/or the 60-kd component. Although there was no profile of anti–SS-A/Ro response unique to the mothers of children with heart block or cutaneous manifestations of neonatal lupus, only 1% of the healthy infants were born to mothers with antibodies directed to both the 52-kd SS-A/Ro and 48-kd SS-B/La antigens and not to the 60-kd SS-A/Ro antigen. Conclusion. Women with antibodies to both SS-A/Ro and SS-B/La have an increased risk of giving birth to children with neonatal lupus, especially if the anti-SS-A/ Ro response identifies the 52-kd component on SDS-immunoblot. Women whose sera contain only anti-SS-A/Ro antibodies in low titer and only recognize determinants that are altered by conditions of SDS-immunoblot have a low risk for giving birth to a child with neonatal lupus. Specific antibody profiles do not distinguish among the manifestations of the neonatal lupus syndromes.     

Deposition of antibodies to a soluble cytoplasmic antigen in the kidneys of patients with systemic lupus erythematosus.

Two patients with systemic lupus erythematosus were studied whose sera contained precipitating antibodies to a soluble cytoplasmic antigen, termed Ro. A reduction in the amount of these antibodies in each case was accompanied by a deterioration in the clinical status with the development of nephritis leading to death. Acid elution of gamma globulin was performed from homogenates of the renal cortex, and in both instances antibodies to Ro were demonstrated in the eluates by double immunodiffusion. The titer of these antibodies was measured in both sera and eluates, and specific enrichment of anti-Ro in the eluates was demonstrated in both patients. This strongly suggests the direct participation of Ro-anti-Ro immune complexes in the progressive renal disease and may underlie the association seen here between the decreasing serum titers of antibodies to Ro and the clinical deterioration in these two cases.     

ANTI La(SSB) IDENTIFIES A DISTINCTIVE SUBGROUP OF SYSTEMIC LUPUS ERYTHEMATOSUS

The relevance of antibodies for La(SSB) as a marker of a distinctivesystemic lupus erythematosus (SLE) subset was studied in 185lupus patients. Anti La was detected in 39 (21%) and was accompaniedinvariably by anti Ro. Clinically, anti La-positive patientswere distinguished by a later age of disease onset, and a lowfrequency of lupus nephritis. In common with other anti Ro-positivepatients they showed a high frequency of keratoconjunctivitissicca. Anti La identified patients with higher titres of antiRo antibodies and was significantly associated with HLA-DR3.Serological markers such as these are useful for identifyingmore homogeneous populations of SLE patients for studies ofaetiology and pathogenesis. KEY WORDS: SLE, anti La(SSB), anti Ro(SSA), Elderly lupus     

The frequency of autoantibodies in Turkish patients with lupus nephritis

We analysed the frequency of autoantibodies in 44 patients with lupus nephritis. Antinuclear antibody (ANA) was found in 91% of patients by an indirect immunofluorescence technique on HEp-2 cells at the time of study. Anti-dsDNA antibodies were found at high levels in most of the patients using the SynELISA test, which reveals both high and low avidity antibodies, but only in 64% of patients using the DAKO assay, which reveals high avidity antibodies. Anti-histone antibodies were found in 52% of the patients. Anti-SSA/Ro antibodies were positive in 27% of all patients; these patients also had prominent cutaneous involvement and photosensitivity. The frequency of anti-SSB/La and Anti-Sm antibodies was 7%. Immunoblotting experiments confirmed the findings shown by the techniques described above, and anti-ribosomal antibodies were found to be positive in more cases by the latter assay. IgA rheumatoid factor (RF) was more frequent (20.4%) than IgM RF (9.9%). In conclusion, Turkish patients suffering from lupus nephritis seem to express a total autoantibody profile similar to that reported for systemic lupus erythematosus from other parts of Europe. Received: 12 March 1996 / Accepted: 19 November 1996     

Long-term follow-up of autoantibody profiles in black female lupus patients and clinical comparison with Caucasian and Asian patients

The aim was to determine whether the autoantibody profile in Black female lupus patients is associated with clinical subsets, fluctuates over time and/or reflects disease activity. A clinical comparison with Caucasian and Asian patients matched for age of onset and disease duration was also undertaken. Up to seven serial bleeds from Black female lupus patients who had been followed up for periods of 3.15 yr were tested for antibodies to Ro/SSA, La SSB. Sm, RNP and ribosomal P using ELISA research assays. Significant differences in both clinical and serological profiles between the ethnic groups were found. Varying aspects of disease activity were linked to anti-DNA (renal, cardiovascular, global score), anti-ribosomal P (musculoskeletal, haematology) and anti-Sm (general) antibodies. There are differences in clinical and serological profiles amongst systemic lupus erythematosus patients of different ethnic origin. However, using the BILAG system, relatively few antibodies were found to reflect disease activity accurately in serial measurements.      

Deposition of antibodies to a soluble cytoplasmic antigen in the kidneys of patients with systemic lupus erythematosus

Two patients with systemic lupus erythematosus were studied whose sera contained precipitating anti-bodies to a soluble cytoplasmic antigen, termed Ro. A reduction in the amount of these antibodies in each case was accompanied by a deterioration in the clinical status with the development of nephritis leading to death. Acid elution of gamma globulin was performed from homogenates of the renal cortex, and in both instances antibodies to Ro were demonstrated in the eluates by double immunodiffusion. The titer of these antibodies was measured in both sera and eluates, and specific enrichment of anti-Ro in the eluates was demonstrated in both patients. This strongly suggests the direct participation of Ro-anti-Ro immune complexes in the progressive renal disease and may underlie the association seen here between the decreasing serum titers of antibodies to Ro and the clinical deterioration in these two cases.     

Anti-52 kDa Ro, anti-60 kDa Ro, and anti-La antibody profiles in neonatal lupus

OBJECTIVE: Studies suggest that anti-52 kDa Ro antibodies are more sensitive and specific than anti-60 kDa Ro antibodies for neonatal lupus. However, these studies mainly used immunoblot or ELISA using recombinant protein, which have poor sensitivity for anti-60 kDa Ro antibodies. In addition, the control patients were not disease matched. We reassessed the sensitivity and specificity of anti-52 kDa Ro, anti-60 kDa Ro, and anti-La, addressing these limitations. METHODS AND RESULTS: To assess sensitivity, 125 mothers of children with neonatal lupus (NLM) were recruited. All maternal sera were assessed using a commercial line immunoassay that uses natural 60 kDa Ro protein (Inno-Lia ANA Update, Innogenetics NV, Gent, Belgium). By this method, 96% of the sera had antibodies to 60 kDa Ro, 86% to 52 kDa Ro, and 78% to 48 kDa La. Immunoblot of 65 NLM showed significantly fewer positive results for anti-60 kDa Ro (p < 0.001) and anti-52 kDa Ro (p < 0.05). Sensitivity of the 3 antibodies was assessed in the symptomatic mothers of children with congenital heart block (CHB) (78 women) and disease matched controls with unaffected children (65 women) using Inno-Lia ANA Update. The sensitivity of each antibody was compared by multiple logistic regression to adjust for maternal disease. There was no significant difference between the groups for 60 kDa Ro or for anti-52 kDa Ro antibody. However, there was a significant difference for the anti-La antibody (p = 0.001), with an odds ratio of 3.59. This translates to an increase in risk from a published 2% for CHB in an anti-Ro-positive mother to 3.1% if the woman is also anti-La antibody-positive, and to a decrease in risk to 0.9% if anti-La-negative. CONCLUSION: Contrary to previous reports, 52 kDa Ro as detected by Inno-Lia ANA Update is not more specific for or frequent in CHB than 60 kDa Ro. However, the presence of anti-La antibodies significantly increases the risk for CHB.     

Clinical features of Sjögren’s syndrome associated with recurrent annular erythema

Our objective was to describe the clinical features of Sjögren’s syndrome (SS) with recurrent annular erythema which resembles subacute cutaneous lupus erythematosus (SCLE), and determine a possible association of anti-SS-A/Ro and/or SS-B/La antibody titers with the episodes of cutaneous manifestation. Recurrent annular erythema was observed in 4% (six patients; five females and one male) of our 143 patients diagnosed as primary SS. All the patients developed annular erythema on the facial area as their initial manifestation when they were between 21 and 31 years old. They had few subjective sicca symptoms, but both anti-SS-A/Ro and SS-B/La antibodies were positive and parotid sialography showed typical findings for SS (subclinical SS). Parotid gland swellings had developed in five of the patients during their follow-up periods, (3–12 years). In addition to the facial area, most patients repeated the cutaneous episodes on extremities and palmar, plantar or auricular areas. Skin biopsy was performed in three patients and the common findings were mononuclear cell infiltrations in the dermis with few epidermal changes. Transient leukopenia (four patients), low titers of anti-DNA antibodies (one patient) and chronic false-positive results of serological tests for syphilis (one patient) were observed. Two of our patients, therefore, temporarily fulfilled four items of the ARA classification criteria for systemic lupus erythematosus (SLE), if their facial erythema was considered as malar rash. Serum antibodies to 52 kDa SS-A/Ro peptides (80%) were more frequently detected in the five patients examined by enzyme-linked immunosorbent assays (ELISA), compared with those to 60 kDa SS-A/Ro peptides (40%). Furthermore, we could serially determine serum anti-SS-A/Ro and SS-B/La antibody titers in three patients by using ELISA for 3 or 4 years. Annular erythema usually developed when the antibody titers became relatively high and disappeared after the treatment with oral prednisolone which suppressed the antibody titers. We could observe five pregnancies in our three patients and all the patients developed annular erythema during their pregnant periods. Their five infants, however, were free from any complications such as neonatal lupus erythematosus and congenital heart block. We conclude that annular erythema is a rare manifestation of SS and develops in relatively young patients who are subclinical for sicca symptoms. The cutaneous episodes seemed to relate with anti-SS-A/Ro and/or SS-B/La antibody titers. Some of the erythema observed in SS patients may belong to SCLE, but they do not usually develop typical SLE. The possibility that SS may be more frequent in the SCLE patients remains to be determined.     

Anti-calreticulin segregates anti-Ro sera in systemic lupus erythematosus: anti-calreticulin is present in sera with anti-Ro alone but not in anti-Ro sera with anti-La or anti-ribonucleoprotein

OBJECTIVE: To examine a well characterized group of patients with systemic lupus erythematosus (SLE) for anti-calreticulin. METHODS: The sera of 77 patients with SLE were studied by immunodiffusion, solid phase immunoassay, and immunoblot for antibodies against ribonucleoprotein (RNP) autoantigens and calreticulin. RESULTS: Thirty-five had anti-calreticulin and 40 had anti-60 kDa Ro. There was no association of anti-60 kDa Ro and anti-calreticulin. However, among anti-60 kDa Ro positive sera that also contained either anti-La or anti-RNP, none of 18 had anti-calreticulin. All the remaining sera with anti-60 kDa Ro had anti-calreticulin and anti-52 kDa Ro. CONCLUSION: Anti-60 kDa Ro patients with SLE can be divided into those with anti-60 kDa Ro and either anti-La or anti-RNP or those with anti-60 kDa Ro, anti-52 kDa Ro, and anti-calreticulin.     

Neonatal lupus

Neonatal lupus erythematosus is characterized by congenital atrioventricular heart block and/or transient lupus skin lesions frequently similar to those seen in patients with subacute cutaneous lupus erythematosus. The mothers have anti-SSA (Ro) and/or anti-SSB (La) antibodies. The syndrome is important to recognize because newborns may develop complete heart block with congestive heart failure. However, in the majority of cases, neonatal lupus erythematosus is a relatively benign disorder and cutaneous lesions generally disappear completely by 6 months of age.