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Neonatal Lupus Erythematosus
Neonatal lupus erythematosus (NLE) is a disease of the developing fetus and neonate defined by characteristic clinical features in the presence of specific maternal autoantibodies. The first reported case of congenital heart block (CHB) associated with maternal autoimmune disease (i.e., Mikulicz syndrome or likely Sjögren syndrome [SS]) was published in 1901. However, it was not until the 1950s that it was generally recognized that autoantibodies in the mother were associated with NLE and it was another 20 to 30 years until anti-Ro and anti-La antibodies were reported to be associated with CHB. The rash of cutaneous NLE (cNLE) was first reported in 1954 in a child born to a mother with an autoimmune disease. It was not until 1981, however, that the association of cNLE and maternal anti-Ro antibodies was described.
NLE is considered a model of passively acquired autoimmunity. The transplacental passage of maternal-specific autoantibodies is necessary but not sufficient to cause the disease. The autoantibodies associated with NLE are directed against a group of small cytoplasmic and nuclear ribonucleoproteins (RNPs): Ro/SSA and La/SSB. Rarely, cNLE has been associated with isolated anti-U1RNP antibodies. The most common clinical manifestations of NLE are cardiac, dermatological, hematological, and hepatic. The term NLE is misleading as the affected child does not have systemic lupus erythematosus (SLE), and the mother is frequently healthy, without any symptoms of an autoimmune disease, or may have an autoimmune disease other than SLE.
Etiology and Pathogenesis
Autoantigens
RoRNP
Ro60/Trove2
The autoantigens associated with NLE are the Ro and La proteins, which are present in all cells. The first Ro protein identified was a 60-kD polypeptide (Ro60) and is encoded by the TROVE2 gene. Isolation and cloning of Ro60 identified a zinc finger and an RNA-binding protein consensus motif. ,
Crystallographic studies demonstrated a ring-shaped protein with two overlapping RNA binding sites that may subserve different functions depending on the cellular location of Ro60. One important function of Ro60 is to protect cells from damage from ultraviolet irradiation. While in the nucleus, Ro60 likely plays a role in RNA quality control. Ro binds to a class of noncoding RNAs (YRNAs) on the outer surface of the ring on the lower stem. The binding to YRNAs allows for the translocation of Ro60 from the nucleus to the cytoplasm. This translocation occurs via several proteins of which a zipcode-binding protein (ZBP) is likely the most important. , During apoptosis, YRNAs are required for translocation of Ro60 to the cell surface that may be pivotal to the formation of immune complexes on apoptotic cells and a Toll-like receptor (TLR)-dependent proinflammatory cascade. Murine studies have suggested that Ro60 may protect against the development of autoantibodies by sequestering defective RNPs. Ro60 may alter interferon-regulated genes via interaction with Alu RNAs.
La48
The second RoRNP protein recognized was the 48-kD La protein. Although it is at least transiently associated with Ro60, La does not share antigenic determinants with either Ro60 or Ro52. , La consists of at least two structural domains, each of which contains a distinct antigenic binding site. La is mainly found in the nucleus and its nuclear localization is dependent on the C-terminus but not the N-terminus (RNP-consensus motif). Similar to Ro60 it can appear on the cell surface during cell stress or apoptosis and therefore be a direct target for autoantibodies. La facilitates maturation and termination of RNAs.
Ro52/Trim21
A third recognized target of the autoimmune response in NLE is a 52-kD polypeptide, Ro52. Although two isoforms of Ro52 (52α and 52β) have been recognized, in postnatal life the 52α is the predominant form (now called Trim21 ). The 52β is derived from the splicing of exon 4 encoding aa168-245 that includes the leucine zipper. This results in a smaller protein with a predicted molecular weight of 45,000 and lacking a leucine zipper. The 52β transcript is the predominant transcript in earlier second trimester gestation when maternal antibodies begin to gain access to the fetal circulation, whereas by 18 weeks’ gestation 52α becomes the predominant or sole transcript.
The full-length protein, Ro52α/Trim21 has three distinct domains: an N-terminal region rich in cysteine/histidine motifs containing two distinct zinc fingers known as RING finger and B-box, a central region containing a leucine zipper with potential for intramolecular dimerization, and a C-terminal ret finger protein (rfp)-like domain. Ro52 is an E3 ubiquitin ligase that catalyzes the ubiquitination of several proteins, including Ro52 itself. Other important functions of Trim21/Ro52 include regulation of proinflammatory cytokine production ; the production of type 1 interferon and cytokine via its ubiquitination of interferon regulatory factors including IRF3, IRF5, IRF7, and IRF8 (reviewed ); regulation of the innate immune response to intracellular double-stranded DNA ; and modulation of follicular B-cell homeostasis and immunoglobulin production.
Calcium Channels
Calcium (Ca ++ ) channels are important in maintaining cardiac rhythm, and antibodies against components of these ion channels have been hypothesized to be important in CHB, the most important manifestation of NLE. , Specifically, L-type channels α 1c and α 1D subunits, and T-type Ca ++ channels α 1G subunit, have been shown to be present in both adult and fetal conducting tissue. This issue is addressed later in the chapter.
Autoantibodies
The following paragraphs review the history of the search for pathogenic autoantibodies ( Table 25.1 ). Unfortunately, there are important limitations in interpreting many reports that include small numbers of patients and failure to use only serum samples obtained during the pregnancy when comparing autoantibodies from mothers of affected and unaffected children. The problem with this latter approach is that the autoantibody repertoire may change over time.
TABLE 25.1
Autoantibodies in Neonatal Lupus Erythematosus
| Most Important to Test for the following: |
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| Antibodies: Good Specificity but Low Sensitivity |
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| Antibodies: Not Validated After Initial Studies |
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∗ Affinity-purified Ro antigen is almost exclusively Ro60 with minimal if any Ro52 antigen.
Anti-Ro60 Antibodies
The first antibodies to be recognized to be associated with the development of NLE were directed against Ro60 and La48. , , However, it soon became apparent that not all children born to mothers with anti-Ro60 antibodies developed NLE, and that although it is a sensitive marker for the development of NLE, the presence of anti-Ro60 antibodies had poor specificity to identify at-risk pregnancies. Furthermore, the presence of these antibodies alone could not differentiate fetuses/infants at risk for different manifestations of NLE. , , In order to increase the specificity of anti-Ro antibodies as markers for NLE, investigators use different immunological methods in an attempt to determine the autoantibody repertoire.
Initial studies used the relatively insensitive method of an immunodiffusion assay that was replaced by an enzyme-linked immunosorbent assay (ELISA), and more recently by chemiluminescent assay (CIA), using either affinity-purified or recombinant proteins. Prior to the production of recombinant Ro proteins, an RNA immunoprecipitation assay was used to distinguish anti-Ro60 from anti-Ro52 antibodies. However, it is not commercially available and is only mentioned because it was used in research that helped determine the importance of the anti-Ro52 antibody response in the development of CHB. It is important to note which assay is used when analyzing results, as the type of protein used may give varying results. For technical reasons, all ELISAs or CIAs for Ro52 use recombinant proteins, whereas assays for anti-La48 or anti-Ro60 antibodies may use either affinity-purified or recombinant proteins. Assays for anti-Ro antibodies using affinity-purified proteins cannot identify anti-Ro52 antibodies.
The easiest and least expensive assay to screen pregnant women for the risk of delivering a child with NLE is the anti-Ro ELISA or CIA. These assays have excellent sensitivity at greater than 99% to identify all fetuses with CHB but poor specificity. Therefore other autoantibodies have been examined to increase the specificity without decreasing the sensitivity. A prospective study of mothers at risk to deliver a child with cardiac NLE showed that the titer of anti-Ro antibodies was important in determining the risk of developing cardiac NLE. Specifically, none of the mothers with low titer anti-Ro60 antibodies delivered a child with cardiac NLE, whereas the pregnancies of 5% of mothers with moderate to high titer anti-Ro60 levels were complicated by fetal cardiac NLE. Based on these data it was suggested that mothers with low levels of anti-Ro antibodies were at a very low risk of delivering a child with cardiac NLE and therefore may require less intensive screening. This has been confirmed in independent cohorts from other centers.
Anti-Ro52 Antibodies
Antibodies to Ro52 are found in 70% to 100% of mothers of children with CHB, and although less sensitive than anti-Ro60 antibodies, they are more specific for the development of CHB. , , Epitope mapping of the anti-Ro52 antibody response revealed an immunodominant region, spanning aa169-291, a region containing the leucine zipper (aa220-232), an epitope recognized by the sera of a majority of mothers of children with CHB. Examination of the fine specificity of this response showed that the dominant antibody response in mothers of children with CHB was directed against a polypeptide consisting of aa200-239 (p200), whereas a reduced risk of CHB was found when the dominant response was directed against aa176-196 (p176) and aa197-232 (p197).
These intriguing data led to multiple replication studies to address the sensitivities and specificities of these antibodies in CHB. Overall, anti-p200 antibodies were more specific but less sensitive than anti-Ro60 or anti-Ro52 antibodies for the risk of delivering a child with CHB compared with delivering an unaffected child. , , Translational studies showed that anti-p200 antibodies and anti-Ro52 antibodies both bound the surface of nonpermeabilized apoptotic but not healthy human fetal cardiocytes, suggesting that they may be pathogenic in CHB. An in vivo rodent model and an in vitro culture system showed that anti-p200 antibodies can bind neonatal rodent cardiocytes and alter Ca ++ homeostasis. ,
Anti-La48 Antibodies
One of the earliest studies suggested that the presence of both anti-Ro60 and anti-La48 maternal antibodies was sensitive and more specific than the presence of anti-Ro60 antibodies alone for identifying children at risk of CHB. A subsequent study showed that antibodies directed against a small La polypeptide named DD were found only in the sera of mothers of children with NLE and not in sera from mothers of unaffected children. Although this finding was specific for NLE, it had only 30% sensitivity, as many mothers without anti-DD antibodies delivered children with NLE, and anti-DD antibodies were present in only 30% of children with NLE. Subsequent larger studies showed that anti-La48 antibodies were present in up to 50% of sera of mothers of children with CHB but were less sensitive than either anti-Ro60 or anti-Ro52 antibodies for predicting the delivery of a child with CHB. , Therefore testing for the presence of anti-La48 antibodies alone is not a good screening tool to determine at-risk pregnancies. Whether the presence of isolated anti-La antibodies (without anti-Ro antibodies) can result in cardiac NLE is controversial. A few cases have been reported, but it remains unclear whether the assays used were sensitive enough to detect anti-Ro antibodies and/or whether maternal antibodies were measured many years after delivery of a child with CHB (creating the possibility of false negative results).
Anti-U1RNP Antibodies
Cutaneous involvement in neonatal lupus has been reported in a minority of infants exposed to only anti-U1RNP antibodies. A few of these infants also presented with thrombocytopenia, anemia, elevated liver enzymes, and/or hepatomegaly, further supporting the association of isolated anti-U1RNP antibodies with non-cNLE. Transient prolongation of the atrioventricular (AV) interval in utero has been reported in one fetus exposed to anti-U1RNP antibodies (but not to anti-Ro or anti-La antibodies) but no PR prolongation or heart block was found after birth. A child born to a mother with only anti-U1RNP antibodies died with complete CHB, and an autopsy showed loss and calcification of myocytes in the area of the bundle of His without involvement of the AV nodal tissue. This is the first case of CHB with histological demonstration of calcification of the conducting system and the presence of low titer anti-U1RNP antibodies without anti-Ro antibodies.
Anti-L-Type and Anti-T-Type Calcium Channel Antibodies
Sera containing anti-Ro/La antibodies were shown to react with, and alter the function of, subunits of the L-type and T-type Ca ++ channels. The major target of the anti-T-type Ca ++ channel antibodies was an epitope present on an extracellular loop (p305) of the channel (vide infra). However, a subsequent study failed to show that the presence of maternal antibodies directed against the p305 epitope of T-type Ca ++ channel was a good marker for the risk of CHB.
Other Autoantibodies
The search for new cardiac antigen targets led investigators to examine whether anti-Ro52 antibodies cross-react with the serotoninergic 5-hydroxytryptamine receptor (anti-5-HT 4 R), which is present in human fetal atrium and may be important in atrial arrhythmias and fetal cardiac development. , However, only a minority of sera from mothers of children with CHB had anti-5-HT 4 R antibodies, suggesting that these antibodies are not necessary for the development of the CHB in the majority of patients. ,
Calreticulin, a Ca ++ -binding protein that is important in cardiac development, was initially thought to be part of a Ro protein. Elevated anticalreticulin antibody levels have been reported in mothers of children with NLE compared with levels in healthy subjects, but not compared with that of healthy pregnant women.
Other antigens present on the fetal heart or placenta have been suggested to be important in the development of, or protection from, CHB. A subset of anti-La antibodies was found to cross-react with laminin (present on the placenta), which may alter the autoantibody repertoire in the fetal circulation and therefore affect binding to the target fetal tissue. Anti-laminin autoantibodies bind to fetal but not adult heart and cardiac tissue, and ERV-3 and laminin are maximally expressed between 11 and 17 weeks’ gestation. , However, the presence or absence of these antibodies has not been predictive of the development of CHB.
Two other autoantibodies examined in NLE and CHB were anti-p57 recombinant protein and anti–α-fodrin antibodies. Anti-p57 antibodies were present in one-third of sera of mothers of children with NLE, but were almost always associated with anti-Ro antibodies and did not add to sensitivity and specificity testing. Anti–α-fodrin antibodies, present in sera of patients with SS, were demonstrated to be present in the sera of mothers of children with NLE; however, measurement of these antibodies has not been pursued as they do not add to the sensitivity or specificity of autoantibody testing over routine anti-Ro and anti-La antibody testing.
Maternal Factors
As NLE occurs in only a small proportion of infants born to mothers with anti-Ro antibodies, it is felt that other maternal factors may be important in the development of this disease. Studies of the impact of maternal age on the risk of CHB have yielded conflicting results: a Swedish study reported that older maternal age was associated with the delivery of children with CHB, but a study from Japan showed the opposite. Interestingly, the mean maternal ages of mothers in both groups in the Japanese study were older than those in the Swedish studies. One could suspect that women with an established autoimmune disease may have a pathogenic antibody repertoire and/or higher levels of autoantibodies and would therefore be more at risk of having a child with NLE. However, this was not the case, as 65% to 80% of mothers of cardiac NLE were asymptomatic/or had minimal symptoms of an autoimmune disease. Interestingly, anti-Ro antibody–positive women whose infants had only cardiac disease were more likely (75%) to be asymptomatic than those whose infants had only cutaneous manifestations (42%). As cNLE may be unnoticed/misdiagnosed in babies of asymptomatic mothers who do not know that they are anti-Ro antibody positive, the smaller proportion of asymptomatic women in the group of mothers whose children have cNLE could represent a sampling bias. When an established autoimmune disease is present in the mother, SS, SLE, and undifferentiated connective tissue disease are most commonly seen. Further research is needed to understand the impact of maternal health on the risk and type of NLE manifestations.
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