52-kDa Ro/SSA epitopes preferentially recognized by antibodies from mothers of children with neonatal lupus and congenital heart block

Neonatal lupus erythematosus is a rare disorder caused by the transplacental passage of maternal autoantibodies. The 52-kDa Ro/SSA antigen (Ro52) ribonucleoprotein represents an antigenic target strongly associated with the autoimmune response in mothers whose children have neonatal lupus and cardiac conduction disturbances, mainly congenital heart block. The objective of this study was to identify putative Ro52/60-kDa Ro/SSA antigen (Ro60) epitopes associated with neonatal lupus and congenital heart block. The reactivity of IgG antibodies present in the sera from mothers with systemic lupus erythematosus and Sj?gren's syndrome and in the sera from asymptomatic mothers (a longitudinal study of 192 samples from 66 subjects) was investigated by ELISA using Ro52, Ro60 and 48-kDa La/SSB antigen proteins, as well as 45 synthetic peptides, 13-24 residues long, of Ro52/Ro60 proteins. One to 19 samples collected before, during and after pregnancy were available for each mother. Forty-three disease controls selected randomly and normal sera were tested in parallel. Although no differences were found between Sj?gren's syndrome and asymptomatic mothers of group I, who had at least one infant with neonatal lupus, and of group II, who had healthy babies only, significant differences were observed between lupus mothers from both groups. In the former group of lupus mothers, a significantly higher frequency of antibodies to Ro52 peptides 107-122 and 277-292 was observed. Between 18 and 30 weeks of gestation, the period of risk, there was clearly an elevated level of antibodies reacting with Ro52 peptides 1-13, 277-292 and 365-382. Antibodies to Ro52 peptide 365-382 have been shown previously to cross-react with residues 165-185 of the heart 5-HT4 serotoninergic receptor, and might be pathologically important. The level of these Ro52 antibody subsets decreased at the end of pregnancy and after delivery. IgG antibodies to Ro52 peptides 1-13, 107-122, 277-292 and 365-382 may therefore represent important biomarkers to predict a complication in pregnant lupus women with Ro52 antibodies.     

52-kDa Ro/SSA epitopes preferentially recognized by antibodies from mothers of children with neonatal lupus and congenital heart block

Neonatal lupus erythematosus is a rare disorder caused by the transplacental passage of maternal autoantibodies. The 52-kDa Ro/SSA antigen (Ro52) ribonucleoprotein represents an antigenic target strongly associated with the autoimmune response in mothers whose children have neonatal lupus and cardiac conduction disturbances, mainly congenital heart block. The objective of this study was to identify putative Ro52/60-kDa Ro/SSA antigen (Ro60) epitopes associated with neonatal lupus and congenital heart block. The reactivity of IgG antibodies present in the sera from mothers with systemic lupus erythematosus and Sjögren's syndrome and in the sera from asymptomatic mothers (a longitudinal study of 192 samples from 66 subjects) was investigated by ELISA using Ro52, Ro60 and 48-kDa La/SSB antigen proteins, as well as 45 synthetic peptides, 13–24 residues long, of Ro52/Ro60 proteins. One to 19 samples collected before, during and after pregnancy were available for each mother. Forty-three disease controls selected randomly and normal sera were tested in parallel. Although no differences were found between Sjögren's syndrome and asymptomatic mothers of group I, who had at least one infant with neonatal lupus, and of group II, who had healthy babies only, significant differences were observed between lupus mothers from both groups. In the former group of lupus mothers, a significantly higher frequency of antibodies to Ro52 peptides 107–122 and 277–292 was observed. Between 18 and 30 weeks of gestation, the period of risk, there was clearly an elevated level of antibodies reacting with Ro52 peptides 1–13, 277–292 and 365–382. Antibodies to Ro52 peptide 365–382 have been shown previously to cross-react with residues 165–185 of the heart 5-HT₄ serotoninergic receptor, and might be pathologically important. The level of these Ro52 antibody subsets decreased at the end of pregnancy and after delivery. IgG antibodies to Ro52 peptides 1–13, 107–122, 277–292 and 365–382 may therefore represent important biomarkers to predict a complication in pregnant lupus women with Ro52 antibodies.     

Molecular mechanisms of congenital heart block

Autoantibody-associated congenital heart block (CHB) is a passively acquired autoimmune condition associated with maternal anti-Ro/SSA antibodies and primarily affecting electric signal conduction at the atrioventricular node in the fetal heart. CHB occurs in 1–2% of anti-Ro/SSA antibody-positive pregancies and has a recurrence rate of 12–20% in a subsequent pregnancy. Despite the long-recognized association between maternal anti-Ro/SSA autoantibodies and CHB, the molecular mechanisms underlying CHB pathogenesis are not fully understood, but several targets for the maternal autoantibodies in the fetal heart have been suggested. Recent studies also indicate that fetal susceptibility genes determine whether an autoantibody-exposed fetus will develop CHB or not, and begin to identify such genes. In this article, we review the different lines of investigation undertaken to elucidate the molecular pathways involved in CHB development and reflect on the hypotheses put forward to explain CHB pathogenesis as well as on the questions left unanswered and that should guide future studies.     

Down-regulation of L-type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits.

Congenital heart block is considered a model of passively acquired autoimmune disease in which the mother generates anti-SSA/Ro and/or anti-SSB/La antibodies that cross the placenta and presumably injure the heart of developing fetus. CHB is accompanied by ECG abnormalities including AV block, sinus bradycardia, and ventricular dysfunction. Our previous data indicate that these abnormalities are caused by maternal autoantibody-mediated disturbance of L-type Ca channels. To investigate the consequence of chronic exposure of L-type Ca channels in newborn pups to maternal autoantibodies during pregnancy, we immunized female rabbits with human 52 kDa-SSA/Ro (Ro52) recombinant protein. ECG revealed that pups from the immunized group had varying degrees of conduction defects. In addition, I(CaL) density and protein were reduced in hearts of pups from the immunized group. Sera and purified IgG from immunized rabbits inhibited I(Ba) recorded from oocytes with expressed alpha(1C) and beta(2a) subunits of L-type Ca channel. Pups born to Ro52 immunized mothers exhibited down-regulation of L-type calcium channels in heart. The data provide new insight into the pathogenesis of congenital heart block.     

SS-A/Ro52, an autoantigen involved in CD28-mediated IL-2 production

An autoantibody against SS-A/Ro52 (Ro52) is most frequently found in the sera of patients with Sj?gren's syndrome, systemic lupus erythematosus, and congenital heart block from anti-Ro52 Ab-positive mother. However, the physiological function of the autoantigen SS-A/Ro52 has not yet been elucidated. In this study, we describe the role of Ro52 protein in T cell activation. Overexpression of SS-A/Ro52 in Jurkat T cell resulted in enhanced IL-2 production following CD28 stimulation. Furthermore, transfection of anti-Ro52-specific small RNA duplexes partially blocked the expression of native and overexpressed Ro52 in Jurkat T cell, resulting in decreased IL-2 production via CD28 pathway in these cells. Finally, intracellular localization of Ro52 dramatically changed following CD28 stimulation. Our data reveal a novel function of Ro52 in CD28-mediated pathway, which eventually contributes to cytokine production and expression of the T cell biological programs.     

Congenital heart block: evidence for a pathogenic role of maternal autoantibodies

During pregnancy in autoimmune conditions, maternal autoantibodies are transported across the placenta and may affect the developing fetus. Congenital heart block (CHB) is known to associate with the presence of anti-Ro/SSA and anti-La/SSB antibodies in the mother and is characterized by a block in signal conduction at the atrioventricular (AV) node. The mortality rate of affected infants is 15% to 30%, and most live-born children require lifelong pacemaker implantation. Despite a well-recognized association with maternal anti-Ro/La antibodies, CHB develops in only 1% to 2% of anti-Ro-positive pregnancies, indicating that other factors are important for establishment of the block. The molecular mechanisms leading to complete AV block are still unclear, and the existing hypotheses fail to explain all aspects of CHB in one comprehensive model. In this review, we discuss the different specificities of maternal autoantibodies that have been implicated in CHB as well as the molecular mechanisms that have been suggested to operate, focusing on the evidence supporting a direct pathogenic role of maternal antibodies. Autoantibodies targeting the 52-kDa component of the Ro antigen remain the antibodies most closely associated with CHB. In vitro experiments and animal models of CHB also point to a major role for anti-Ro52 antibodies in CHB pathogenesis and suggest that these antibodies may directly affect calcium regulation in the fetal heart, leading to disturbances in signal conduction or electrogenesis or both. In addition, maternal antibody deposits are found in the heart of fetuses dying of CHB and are thought to contribute to an inflammatory reaction that eventually induces fibrosis and calcification of the AV node, leading to a complete block. Considering that CHB has a recurrence rate of 12% to 20% despite persisting maternal autoantibodies, it has long been clear that maternal autoantibodies are not sufficient for the establishment of a complete CHB, and efforts have been made to identify additional risk factors for this disorder. Therefore, recent studies looking at the influence of genetic and environmental factors will also be discussed.Autoantibody-associated congenital heart block (CHB) is a passively acquired autoimmune condition in which maternal autoantibodies are thought to initiate conduction disturbances in the developing fetal heart. Hallmarks of autoantibody-associated CHB are the presence of immune complex deposits, inflammation, calcification, and fibrosis in the fetal heart and a block in signal conduction at the atrioventricular (AV) node in an otherwise structurally normal heart. Clinical signs most commonly develop during weeks 18 to 24 of pregnancy. Although autoantibody-associated CHB may initially be detected as a first- or second-degree AV block, most of the affected pregnancies will present with fetal bradycardia in third-degree (complete) AV block, and ventricular rates typically are between 50 and 70 beats per minute. A complete AV block is a potentially lethal condition associated with significant morbidity, and the majority of affected children require permanent pacemaker implantation [1-3].Whereas complete AV block is the major manifestation of autoantibody-associated CHB, other cardiac abnormalities are increasingly being recognized. Transient first-degree AV block has been shown to occur in up to 30% of fetuses of mothers with anti-SSA/Ro 52-kDa antibodies [4]. The presence of sinus bradycardia [5-7] and prolongation of the QTc interval [8,9] have also been reported; however, these findings were not replicated in another recent study [10]. Endocardial fibroelastosis and cardiomyopathy have been reported in both the presence and absence of conduction abnormalities and are associated with a poor prognosis [11-14].Since the initial observation that sera of mothers of children with CHB contain anti-SSA/Ro antibodies, the association between maternal autoantibodies and CHB has been extensively studied. Most of the current knowledge comes from the comparative analysis of sera of women with affected or healthy infants, and additional information has been generated through the use of animal models. Nevertheless, the pathogenic molecular mechanisms of autoantibody-associated CHB remain unclear. Because the risk for CHB in an anti-SSA/Ro-positive pregnancy is only 1% to 2% [5,15], the need for a better marker not only for pregnancies at risk but also for the identification of other risk factors influencing the development of CHB is still important. This review will give a broad perspective of the maternal antibodies that have been associated with CHB and then will focus on the antibody specificities that have been more specifically implicated in the pathogenesis of the disease through in vitro and in vivo studies. The current hypotheses for autoantibody-associated CHB development will be discussed with an emphasis on the potential molecular targets for maternal antibodies in the fetal heart before mentioning other risk factors that have recently come to light.     

β2-glycoprotein I and protection from anti-SSA/Ro60-associated cardiac manifestations of neonatal lupus

One mechanism to molecularly explain the strong association of maternal anti-Ro60 Abs with cardiac disease in neonatal lupus (NL) is that these Abs initiate injury by binding to apoptotic cardiomyocytes in the fetal heart. Previous studies have demonstrated that β(2)-glycoprotein I (β(2)GPI) interacts with Ro60 on the surface of apoptotic Jurkat cells and prevents binding of anti-Ro60 IgG. Accordingly, the current study was initiated to test two complementary hypotheses, as follows: 1) competition between β(2)GPI and maternal anti-Ro60 Abs for binding apoptotic induced surface-translocated Ro60 occurs on human fetal cardiomyocytes; and 2) circulating levels of β(2)GPI influence injury in anti-Ro60-exposed fetuses. Initial flow cytometry experiments conducted on apoptotic human fetal cardiomyocytes demonstrated dose-dependent binding of β(2)GPI. In competitive inhibition experiments, β(2)GPI prevented opsonization of apoptotic cardiomyocytes by maternal anti-Ro60 IgG. ELISA was used to quantify β(2)GPI in umbilical cord blood from 97 neonates exposed to anti-Ro60 Abs, 53 with cardiac NL and 44 with no cardiac disease. β(2)GPI levels were significantly lower in neonates with cardiac NL. Plasmin-mediated cleavage of β(2)GPI prevented binding to Ro60 and promoted the formation of pathogenic anti-Ro60 IgG-apoptotic cardiomyocyte complexes. In aggregate these data suggest that intact β(2)GPI in the fetal circulation may be a novel cardioprotective factor in anti-Ro60-exposed pregnancies.     

Immunization with short peptides from the 60-kDa Ro antigen recapitulates the serological and pathological findings as well as the salivary gland dysfunction of Sjogren's syndrome

Sj?gren's syndrome is a poorly understood autoimmune inflammatory illness that affects the salivary and lacrimal glands as well as other organ systems. We undertook the present study to determine whether mice immunized with short peptides from the 60-kDa Ro (or SSA) Ag, which is a common target of the autoimmunity of Sj?gren's syndrome, develop an illness similar to Sj?gren's syndrome. BALB/c mice were immunized with one of two short peptides from 60-kDa Ro that are know to induce epitope spreading. The animals were analyzed for the presence of anti-Ro and anti-La (or SSB) in the sera by immunoblot and ELISA. Salivary glands were collected and examined by histology after H&E staining. Salivary lymphocytes were purified and studied for cell surface makers by fluorescence-activated cell sorting. Timed stimulated salivary flow was measured. As reported previously, BALB/c mice immunized with 60-kDa Ro peptides developed an immune response directed against the entire Ro/La ribonucleoprotein particle that was similar to that found in humans with lupus or Sj?gren's syndrome. Functional studies showed a statistical decrease in salivary flow in immunized mice compared with controls. Furthermore, there were lymphocytic infiltrates in the salivary glands of immunized animals that were not present in controls. The infiltrates consisted of both CD4- and CD8+ T lymphocytes as well as B lymphocytes. BALB/c mice immunized with 60-kDa Ro peptides develop anti-Ro, salivary gland lymphocyte infiltrates, and salivary dysfunction that is highly reminiscent of human Sj?gren's syndrome.     

Analysis of the molecular composition of Ro ribonucleoprotein complexes. Identification of novel Y RNA-binding proteins.

Human Ro ribonucleoproteins (RNPs) are composed of one of the four small Y RNAs and at least two proteins, Ro60 and La; association of additional proteins including the Ro52 protein and calreticulin has been suggested, but clear-cut evidence is still lacking. Partial purification of Ro RNPs from HeLa S100 extracts allowed characterization of several subpopulations of Ro RNPs with estimated molecular masses of between 150 and 550 kDa. The majority of these complexes contained Ro60 and La, whereas only a small proportion of Ro52 appeared to be associated with Ro RNPs. To identify novel Y RNA-associated proteins in vitro, binding of cytoplasmic proteins to biotinylated Y RNAs was investigated. In these reconstitution experiments, several proteins with estimated molecular masses of 80, 68, 65, 62, 60 and 53 kDa, the latter two being immunologically distinct from Ro60 and Ro52, respectively, appeared to bind specifically to Y RNAs. Furthermore, autoantibodies to these proteins were found in sera from patients with systemic lupus erythematosus. The proteins bound preferentially to Y1 and Y3 RNA but, with the exception of the 53-kDa protein, only weakly to Y4 RNA and not at all to Y5 RNA. Coprecipitation of the 80, 68, 65, and 53-kDa proteins by antibodies to Ro60 and La was observed, suggesting that at least a proportion of the novel proteins may reside on the same particles as La and/or Ro60. Finally, the binding sites for these proteins on Y1 RNA were clearly distinct from the Ro60-binding site involving a portion of the large central loop 2, which was found to be indispensable for binding of the 80, 68, 65 and 53-kDa proteins, as well as the stem 3-loop 3 and stem 2-loop 1 regions. Interestingly, truncation of the La-binding site resulted in decreased binding of the novel proteins (but not of Ro60), indicating La to be required for efficient association. Taken together, these results suggest the existence of further subpopulations of Ro RNPs or Y RNPs, consistent with the heterogeneous characteristics observed for these particles in the biochemical fractionation experiments.     

Clinical significance of antibodies to Ro52/TRIM21 in systemic sclerosis

Introduction

Autoantibodies to Ro52 recently identified as TRIM21 are among the most common autoantibodies in systemic autoimmune rheumatic diseases, but their clinical association remains poorly understood. We undertook this study to determine the clinical and serologic associations of anti-Ro52/TRIM21 antibodies in patients with systemic sclerosis (SSc).

Methods

Detailed clinical data and sera from 963 patients with SSc enrolled in a multicenter cohort study were collected and entered into a central database. Antibodies to Ro52/TRIM21 and other autoantibodies were detected with an addressable laser-bead immunoassay and different enzyme-linked immunosorbent assay (ELISA) systems. Associations between anti-Ro52/TRIM21 antibodies and clinical and other serologic manifestations of SSc were investigated.

Results

Anti-Ro52/TRIM21 antibodies were present in 20% of SSc patients and overlapped with other main SSc-related antibodies, including anti-centromere (by immunofluorescence and centromere protein (CENP)-A and CENP-B ELISA), anti-topoisomerase I, anti-RNA polymerase III, and anti-Pm/Scl antibodies. Anti-Ro52/TRIM21 antibodies were strongly associated with interstitial lung disease (odds ratio (OR), 1.53; 95% confidence interval (CI), 1.11 to 2.12; P = 0.0091) and overlap syndrome (OR, 2.06; 95% CI, 1.01 to 4.19; P = 0.0059).

Conclusions

Anti-Ro52/TRIM21 antibodies were the second most common autoantibodies in this SSc cohort. In SSc, anti-Ro52/TRIM21 antibodies may be a marker of interstitial lung disease and overlap syndrome.     

A novel role of endothelin-1 in linking Toll-like receptor 7-mediated inflammation to fibrosis in congenital heart block

Autoimmune associated congenital heart block (CHB) may result from pathogenic cross-talk between inflammatory and profibrosing pathways. Incubation of macrophages with immune complexes (IC) composed of Ro60, a target of the pathologic maternal autoantibodies necessary for CHB, hY3 ssRNA, and affinity-purified anti-Ro60 antibody induces the Toll-like receptor 7 (TLR7)-dependent generation of supernatants that provoke a fibrosing phenotype in human fetal cardiac fibroblasts. We show herein that these cells are a major source of TGFβ and that endothelin-1 (ET-1) is one of the key components responsible for the profibrosing effects generated by stimulated macrophages. Supernatants from macrophages incubated with IC induced the fibroblast secretion of TGFβ, which was inhibited by treating the macrophages with an antagonist of TLR7. Under the same conditions, the induced fibroblast secretion of TGFβ was decreased by inhibitors of the ET-1 receptors ETa or ETb or by an anti-ET-1 antibody but not by an isotype control. Exogenous ET-1 induced a profibrosing phenotype, whereas fibroblasts transfected with either ETa or ETb siRNA were unresponsive to the profibrosing effects of the IC-generated macrophage supernatants. Immunohistochemistry of the hearts from two fetuses dying with CHB revealed the presence of ET-1-producing mononuclear cells in the septal region in areas of calcification and fibrosis. In conclusion, these data support a novel role of ET-1 in linking TLR7 inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB.     

Cytoplasmic relocation of Daxx induced by Ro52 and FLASH

The RING-finger protein Ro52/TRIM21 is known to be an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. We recently showed that Ro52 is an E3 ubiquitin ligase and localizes to cytoplasmic bodies that are highly motile along the microtubule network. To expand our knowledge of Ro52, we searched partners co-operating with Ro52. We performed a yeast two-hybrid screening of a human brain cDNA library with Ro52 as bait. This screening identified several genes encoding Ro52-interacting proteins, including the apoptosis-related proteins, Daxx and FLASH. Further yeast two-hybrid assays revealed that Daxx binds to the B30.2 domain of Ro52 and that FLASH binds to coiled-coil domains of Ro52 through its death-effector domain-recruiting domain. These results suggest that Ro52, Daxx, and FLASH form heteromeric protein complexes. Indeed, this was supported by results of immunoprecipitation experiments in which we found that Daxx is co-immunoprecipitated with Ro52 in the presence of overexpressed FLASH. Importantly, our fluorescence microscopy revealed that, although Daxx is predominantly located in the nucleus, overexpression of both Ro52 and FLASH leads to relocation of Daxx into the cytoplasm. Thus, Ro52 seems to co-operate with FLASH to induce cytoplasmic localization of Daxx in cells.     

Ro60 peptides induce antibodies to similar epitopes shared among lupus-related autoantigens

The coexistence of autoantibodies to ribonucleoproteins (RNP) in sera of patients with systemic lupus erythematosus has been attributed to intermolecular determinant spreading among physically associated proteins. Recently, we showed that murine Ab responses to rRo60 or Ro60 peptides were diversified unexpectedly to small nuclear RNP. In this investigation, the mechanisms for this autoantibody diversification were examined. Intramolecular determinant spreading was demonstrated in mice immunized with human or mouse Ro60316-335. Immune sera depleted of anti-peptide Ab immunoprecipitated Ro60-associated mY1 and mY3 RNA and remained reactive to a determinant on Ro60128-285. Absorption with the immunogen depleted the immune sera completely of anti-Golgi complex Ab (inducible only with human Ro60316-335) and anti-La Ab, and reduced substantially Ab to SmD and 70-kDa U1RNP. Mouse rRo60 completely inhibited the immune sera reactivity to La, SmD, and 70-kDa U1RNP. However, La, SmD, and 70-kDa U1RNP preferentially inhibited the antiserum reactivities to these Ags, respectively. Affinity-purified anti-La Ab were reactive with Ro60, La, SmD, and 70-kDa U1RNP. These results provide evidence that a population of the induced autoantibodies recognized determinants shared by these autoantigens. Lack of sequence homology between Ro60316-335 and La, SmD, or 70-kDa U1RNP suggests that these determinants are conformational. Interestingly, similar cross-reactive autoantibodies were found in NZB/NZW F1 sera. Thus, a single molecular mimic may generate Ab to multiple RNP Ags. Furthermore, cross-reactive determinants shared between antigenic systems that are not associated physically (Ro/La RNP and small nuclear RNP) may be important in the generation of autoantibody diversity in systemic lupus erythematosus.     

The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death

Patients affected by Sj?gren's syndrome and systemic lupus erythematosus (SLE) carry autoantibodies to an intracellular protein denoted Ro52. Although the serologic presence of Ro52 autoantibodies is used clinically for diagnostic purposes, the function of the protein or why it is targeted as an autoantigen in several rheumatic conditions has not been elucidated. In this study, we show that the expression of Ro52 is significantly increased in PBMC of patients with Sj?gren's syndrome and SLE, and demonstrate that Ro52 is a RING-dependent E3 ligase involved in ubiquitination. Overexpression of Ro52, but not of Ro52 lacking the RING domain, in a mouse B cell line lead to decreased growth in steady state and increased cell death after activation via the CD40 pathway. The role of Ro52 in activation-mediated cell death was further confirmed as a reduction in Ro52 expression restored cell viability. These findings suggest that the increased expression of the Ro52 autoantigen in patients may be directly involved in the reduced cellular proliferation and increased apoptotic cell death observed in Sj?gren's syndrome and SLE, and may thus contribute to the autoantigenic load and induction of autoimmune B and T cell responses observed in rheumatic patients.     

Anti-alpha-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-alpha-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sj?gren's syndrome (SjS). We looked (in Nijmegen) for anti-alpha-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS 6, rheumatoid arthritis [RA] 12, systemic lupus erythematosus [SLE] 6, and scleroderma 6) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-alpha-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-alpha-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-alpha-fodrin antibodies, respectively. The ELISAs for alpha-fodrin showed six (Nijmegen) and four (Hanover) anti-alpha-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-alpha-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-alpha-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-alpha-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-alpha-fodrin autoantibodies does not add much to the diagnosis of Sj?gren's syndrome.     

Transdifferentiation of cardiac fibroblasts,a fetal factor in anti-SSA/Ro-SSB/La antibody-mediated congenital heart block

The signature lesion of autoantibody-associated congenital heart block (CHB) is fibrosis of the conducting tissue. To date, participation of myofibroblasts in the cascade to injury has been unexplored. The importance of myofibroblast/macrophage cross-talk is demonstrated by the novel finding of these cell types in the heart of a neonate dying of CHB. This clue to pathogenesis prompted consideration of the mechanism by which maternal anti-SSA/Ro-SSB/La Abs initiate an inflammatory response and promote fibrosis. Isolated cardiocytes from 16-24 wk abortuses were rendered apoptotic by exposure to poly (2-) hydroxyethylmethacrylate; flow cytometry confirmed surface expression of Ro/La. Apoptotic cardiocytes were incubated with affinity-purified Abs to 52 and 60 kDa Ro from CHB mothers (opsonized) or IgG fractions from healthy donors (nonopsonized). Macrophages cultured with opsonized apoptotic cardiocytes expressed proinflammatory markers, supported by a three-fold increase in active alpha(V)beta(3) integrin. Fetal cardiac fibroblasts exposed to supernatants obtained from macrophages incubated with opsonized apoptotic cardiocytes (but not nonopsonized) dramatically increased expression of the myofibroblast marker alpha-smooth muscle actin (SMAc). The "opsonized" supernatant reversed an inhibitory effect of the "nonopsonized" supernatant on proliferation of fibroblasts (120 vs 69%, p < 0.05). Parallel experiments examined the effects of two cytokines and their neutralizing Abs on fibroblasts. TGFbeta1 increased SMAc staining but decreased proliferation. TNF-alpha did not affect either readout. Addition of anti-TGFbeta1 Abs to the "opsonized" supernatant blocked SMAc expression but increased proliferation, while anti-TNF-alpha blocking Abs had no effects. These data suggest that transdifferentiation of cardiac fibroblasts to a scarring phenotype is a pathologic process initiated by maternal Abs.     

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.     

Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of TNF-alpha by macrophages

Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16-24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-alpha from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.     

Dynamic movements of Ro52 cytoplasmic bodies along microtubules

The RING-finger protein Ro52/TRIM21 is known as an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. Recently, Ro52 has been shown to localize to distinct structures called cytoplasmic bodies and function as an E3 ubiquitin ligase. However, the Ro52 cytoplasmic bodies have not been well characterized. In this study, we investigated the Ro52 cytoplasmic bodies using fluorescence microscopy. This analysis revealed that the Ro52 cytoplasmic bodies are diffusely located in the cytoplasm and exist independently of TRIM5α cytoplasmic bodies. Our results further showed that the Ro52 cytoplasmic bodies are not stained with MitoTracker dye and are not colocalized with the proteasome subunit Rpt5, the caveolae component caveolin-1, the endosome markers (EEA1, Rab5, and Rab7), and the lysosome marker LAMP2. These results indicate that the Ro52 cytoplasmic bodies are not mitochondria, proteasome-enriched structures, caveolae, endosomes, or lysosomes. Importantly, the Ro52 cytoplasmic bodies are highly motile and are located along the microtubule network. These results suggest that the Ro52 cytoplasmic bodies are unidentified structures that are transported along the microtubule network.