Neonatal lupus erythematosus (LEN) is an autoimmune disease caused by the transplacental transfer of autoantibodies to the fetus, resulting in characteristic clinical manifestations, mainly cardiac and cutaneous. The incidence rate of LEN is about 1 in 12,500 to 20,000 live births, being a little higher in females and premature babies(1). However, the true prevalence is not established due to a significant proportion of undetected cases.
The pathogenesis of neonatal lupus is not fully elucidated; however, the onset of the disease is related to the passive transplacental transfer of maternal antinuclear autoantibodies. This group of antibodies is mainly directed against several extractable nuclear antigens (ENAs): Sm, RNP, SS-A/Ro, SS-B/La, Scl-70, Jo-1, but those that have key roles in the pathogenesis of neonatal lupus are anti-Ro/SS-A, anti-La/SS-B and anti-RNP antibodies(2). Autoimmune congenital heart block (CHB) occurs in 2% of anti-Ro/SS-A-exposed pregnancies, and the recurrence rate is nine times higher in subsequent pregnancies(3).
In this article, we provide a glimpse of the transport mechanism of maternal IgG class antibodies through the human placental and of the effects of transplacental transfer of maternal antinuclear autoantibodies in neonatal lupus.
Transplacental transport of antinuclear autoantibodies and their pathogenic role
The onset of neonatal lupus is related to antibodies transferred from the mother and belonging to the class of immunoglobulins G, in which four subclasses are differentiated: IgG1, IgG2, IgG3 and IgG4. IgG antibodies are transported by transcytosis with the involvement of neonatal receptors for the Fc fragments of IgG antibodies, present on placental trophoblast cells. The neonatal Fc receptor (FcRn) is a heterodimeric glycoprotein with a structure similar to that of major histocompatibility complex (MHC) but, unlike MHC class I, FcRn is unable to bind peptides (antigens); however, it interacts with the Fc IgG and albumin. The FcRn-IgG interaction and the FcRn-albumin interaction take place in an acidic environment (optimal at a pH of 5-6.5) and not in a physiological environment. The initial hypothesis reported on the IgG transport was focused on the placenta, and later additional data on receptors were collected(4,5).
The mechanism of transport of maternal IgG class antibodies through the human placental syncytiotrophoblast begins with the absorption of IgG into the cell by liquid-phase endocytosis. Then, in the acidic endosomal environment, IgG molecules are bound to FcRn and are thus protected against degradation by lysosomal enzymes. FcRn-IgG complexes are transferred inside the chorionic villous stroma, while IgG molecules not bound to FcRn are directed to the degradation pathway. FcRn releases IgG into the chorionic villous stroma (pH 7.4) and, after IgG dissociation, the FcRn receptor returns to the surface of the apical syncytiotrophoblast. As a result of this mechanism, fetal serum antibodies can reach slightly higher concentrations than maternal serum, as it is the case with the IgG1 subclass, while for the IgG2 subclass the fetal serum antibody concentration may be lower, and the levels of IgG3 and IgG4 in maternal and fetal serum are equal(6).
Anti-SSA autoantibodies are anti-Sjögren’s-syndrome-related antigen A autoantibodies, also known as anti-Ro, similar names including anti-SSA/Ro, anti-Ro/SSA, anti-SS-A/Ro, and anti-Ro/SS-A. Anti-Ro antibodies transferred via placenta are involved in the pathogenic mechanisms of the CHB. These autoantibodies can link cross-reactive epitopes represented by calcium regulatory molecules, such as L and T type ion channels, in the fetal heart(7). Altered calcium homeostasis and cardiac cell apoptosis are pathogenic consequences.
Anti-Ro60 and anti-La autoantibodies can bind similar antigens on the surface of apoptotic cells, this triggering an inflammatory process. This stage is probably associated with the prolongation of AV conduction(8). Depending on the presence of fetal susceptibility genes, local inflammation may be resolved with the normalization of fetal AV conduction or may be amplified and persist, leading to processes of fibrosis and calcification, with the development of permanent fetal AV block. HLA-DRB1*04 and HLA-Cw*05 alleles are associated with a higher risk of CHB, while DRB1*13 and Cw*06 appear to have a protective effect(9).
In addition to the inflammatory process generated by transplacental anti-Ro autoantibodies, apoptotic cardiomyocytes, complement C4 fraction deposition, calcification and fibrosis processes(10,11) and macrophage activation with the release of proinflammatory and profibrotic cytokines such as TNFa and TGFb are also involved in the pathogenic mechanisms(12,13).
Siglec-1-positive macrophages are detected in fetal heart lesions with CHB(14), the expression of the sialic acid binding immunoglobulin-like lectin Siglec-1 being increased by type 1 interferon which upregulates Ro52 and stimulates apoptosis(15).
Another possible cardiological complication related to maternal anti-SS-A antibodies is endocardial fibroelastosis, its severe consequence being represented by dilated cardiomyopathy(2).
Anti-SS-A antibodies – also named anti-Ro after the patient’s name (Rose) in which they were initially detected – are directed against four different antigens, each consisting of a complex of low-molecular-weight RNA (micro-RNA) and proteins, with a molecular weight of 45, 52, 54 and 60 kDa, respectively. Only antibodies to SS-A/Ro52 and SS-A/Ro60 molecules are used in current medical practice, although older studies do not distinguish between Ro52 and Ro60 antibodies, defining them together as anti-SS-A/Ro(2,16).
Ro60 is described as an RNA-binding protein(17), while the Ro52 protein is a ubiquitin E3 ligase, which targets cytosolic virus-antibody complexes(18).
Anti-Ro52 antibodies are directed against the Ro52 antigen belonging to the tripartite motif protein TRIM21 receptor family and E3 ubiquitin ligase family. It has a role in the ubiquitination of proteins, initiating proinflammatory activities and cellular apoptosis(19-22).
After it is stimulated by interferon and binds to TLR (Toll receptors), the TRIM21 receptor also interacts with the transcription factors for the interferon regulatory factor. Substrates reported for Ro52 replication include IRF3, IRF5, IRF7 and IRF8, and these transcription factors regulate the level of type 1 interferon and cytokine production(19).
In addition, Ro52/TRIM21 may regulate T cell activation or proliferation, and overexpression of this receptor may increase the IL-2 synthesis(23).
There is a relationship between anti-Ro52 antibodies and late-onset systemic lupus erythematosus, with photosensitivity and hematological disorders(24).
The most significant clinical correlation of anti-Ro52 antibodies is with CHB. When analyzing the serological profile of mothers of infants with CHB, it was found that 95% of them have an increased titer of anti-Ro52 antibodies, and the frequency of Ro60 and La antibodies is 63% and 58%, respectively(25).
Anti-Ro52 antibodies are also found in other conditions, including interstitial lung disease(26), primary biliary cirrhosis and autoimmune hepatitis(27).
A sensitive and specific method for these autoantibodies detection is the enzyme-linked immunosorbent assay (ELISA), but the indirect immunofluorescence (IIF) method can also be used. Line-blot immunoassay (LIA) is also suitable for routine evaluation of autoantibodies to extractable nuclear antigens(16,28-31).
Antibodies to the anti-Ro52 p200 epitope (anti-amino acid antibodies in positions 200-239 of the Ro52 antigen) are most likely capable of causing fetal CHB and have been shown to be a high-risk factor for cardiac damage. ELISA may be used to detect antibodies binding the p200 peptide. In combination with fetal Doppler echocardiography, the determination of Ro52-p200 antibody levels may prove a valuable clinical tool to identify pregnancies where the risk for CHB is high, and allow the treatment before the condition has progressed into a complete AV block(32).
Anti-Ro60 antibodies are directed against Ro60, a protein with a molecular weight of 60 kD, consisting of two domains, one similar in structure to the von Willebrand factor and participating in cell adhesion, and the other being an alpha helix structure responsible for binding nucleotide acids(33).
The Ro60 antigen is a protein in the hY-RNA complex, with a role in RNA degradation(2,33).
Initially, molecular mimicry between nuclear antigen 1 of Epstein-Barr virus (EBNA1) and Ro60 protein was thought to be involved in the production of anti-Ro60 antibodies, but significant amino acid homologies were not found(2,34).
Subsequently, molecular mimicry with the Coxsackie virus protein was discussed in patients with Sjögren’s syndrome(35).
A new hypothesis revealed that the proteins of some commensal bacteria might contain epitopes that mimic regions of the human Ro60. A von Willebrand factor A (vWFA) peptide fraction is associated with the microorganism Capnocytophaga ochracea in the oral cavity and it is a potent activator of Ro60-reactive T cells. Another stimulating factor is the bacterium Escherichia coli which expresses vWFA(36).
These results indicate that commensal peptides can activate Ro60-reactive T cells(37).
Some species of human commensal bacteria that colonize the skin, mouth and intestines, namely Corynebacterium, Propionibacterium and Bacteroides, encode Ro60 orthologs with sequence similarities to human Ro60(38).
The salivary microbiome may be associated with the development of autoreactivity. The comparison of the von Willebrand factor domains present in human Ro60 with Lautropia mirabilis proteins also supports the molecular mimicry hypothesis(39).
Anti-SS-B antibodies, also called anti-La (anti-Lane antibodies) or, in combination, anti-La/SS-B, are targeted against the 48 kDa molecular weight phosphoprotein associated with RNA polymerase III(40).
They are present in patients with Sjögren’s syndrome and in patients with systemic lupus erythematosus (10-20%). In Sjögren’s syndrome, antibodies against La/SS-B are almost always present together with antibodies against Ro/SS-A(41).
The presence of anti-La antibodies in the absence of anti-Ro antibodies is very unusual, and cases of CHB associated with anti-La antibody positivity alone represent less than 1% of the incidence of autoimmune congenital heart blocks in the scientific literature(42).
Anti-RNP antibodies are directed against the RNP antigen, which belongs to a group of small nuclear ribonucleoproteins (snRNPs) that contain high uridine RNA (U-RNA) and various 70 kDa (U1) molecular weight base proteins, 33 kDa (protein A) and 22 kDa (protein C). Antibodies against nuclear ribonucleoprotein (nRNP) may be detected by indirect immunofluorescence (antigen-tissue substrates and HEp 2 cells). For a more accurate identification, ELISA and Western Blot methods are used.
Anti-U1-snRNP antibodies are detected in high titers in 95% to 100% of patients with mixed connective tissue disease. These autoantibodies may also be present in patients with systemic lupus erythematosus, rheumatoid arthritis or Sjögren’s syndrome, but in such patients they do not correlate with the disease activity(41).
Mothers with anti-Ro, anti-La antibodies or in some cases with anti-RNP antibodies may develop rheumatic diseases (systemic lupus erythematosus, Sjögren’s syndrome, rheumatoid arthritis), or have minimal symptoms (photosensitivity, Raynaud’s syndrome, myalgia, artalgia), or may be asymptomatic, being investigated after the onset of lupus in the newborn. The probability of developing lupus erythematosus within 10 years after the birth of a child with neonatal lupus was calculated at 18.6%, and the probability of developing Sjogren’s syndrome in such a patient is 27.9%(43).
Because most women with antibodies have children who do not develop neonatal lupus, it is believed that there are a number of factors, most likely genetic or environmental, necessary for the onset of neonatal lupus. A correlation was observed between the specificity and the level of antibodies from the mother crossing the placental barrier and the risk of developing fetal CHB(1,44).
Regarding the clinical manifestations, it is important to underline that some are reversible, such as skin, hematological, hepatic, neurological impairment(45-48), but some are irreversible, such as the heart lesions(49,50).
Cutaneous manifestations are photosensitivity and transient rash, erythematous-squamous or erythematous-annular lesions, which may occur from birth or in the first weeks of life. Periorbital erythema – called “raccoon’s eye” or “owl’s eye” – is more common(51).
From a histological point of view, the lesions are similar to those of subacute lupus, with hyperkeratosis, atrophy, basal degeneration and intracellular edema. Skin lesions persist during the elimination of circulating maternal antibodies and usually resolve spontaneously, without leaving scars, and therefore do not require drug treatment(52,53).
The spectrum of cardiac abnormalities includes conduction disorders (transient arrhythmias, grade I, II or III atrioventricular block) and cardiomyopathies (dilated cardiomyopathy, endocardial fibroelastosis)(54,55).
The specific heart disorder in neonatal lupus is the CHB, considered the most severe clinical manifestation due to irreversibility and increased mortality and morbidity. A congenital atrioventricular block occurs between 18 and 24 weeks of gestation in the absence of other structural cardiac abnormalities, and may present as grade I, II and especially III AV block(37,56,57).
Autopsies data reveal areas of fibrosis and calcifications at the atrioventricular node, which is an additional argument for the irreversibility of heart damage in severe cases(59,60).
The negative prognostic factors in the evolution of CHB include: low gestational age (<20 weeks) at the onset of symptoms, generalized fetal edema, cardiomyopathy, fibroelastosis, decreased heart rate (<50 bpm) and impaired left ventricular function(61,62).
In utero exposure to maternal Ro/La autoantibodies may lead to fetal CHB, recent studies indicating interferon (IFN) activation in fibroblasts of the fetal heart. In both maternal and neonatal peripheral blood mononuclear cells, IFN-a production is induced by anti-Ro/La positive plasma.
Increased expression of IFN-regulated genes and elevated plasma IFN-a levels are revealed not only in anti-Ro/La positive women, but also in their newborns. The IFN scores of neonates (calculated using 13 IFN-regulated genes) born to mothers receiving immunomodulatory treatment are similar to those of controls, despite maternal persistent IFN activation(63).
Maternal autoantibodies biomarkers can identify women at a higher risk for delivering a child with CHB. The combination of anti-Ro52 and anti-Ro60 antibodies, especially in high levels, could recognize women at risk for fetal autoimmune CHB. Whether anti-p200 antibody should be tested as biomarker of CHB, over standard commercial ELISA, is still debated. The PR interval measurement by weekly fetal echocardiogram from 16 to at least 24 weeks of gestation is strongly recommended for CHB prenatal diagnosis. Some limitations are due to the difficult identification of first-degree atrioventricular block and to the possible occurrence of a complete block from a normal rhythm in only few days. Maternal pharmacological prophylaxis with hydroxychloroquine from the tenth week of gestation has an important role in preventing CHB in pregnant women with high risk for recurrent CHB(3).
The good knowledge of pathogenic mechanisms, the identification of patients at risk for neonatal lupus, the adequate screening for pregnant women with risk factors, together with an interdisciplinary collaboration, involving obstetricians, pediatricians, pediatric cardiologist and neonatologist, are important for the optimal care of these patients. n
Conflict of interests: The authors declare no conflict of interests.