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*
Department of Rheumatology, Hospital for Joint Diseases, New York University School of Medicine, New York, NY 10003;
Cardiology Division, Veterans Affairs Medical Center, Brooklyn, NY 11209; and
Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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), and 52ß (amino acids 169–245 deleted) as well as
with murine recombinant 52-kDa SSA/Ro. Control animals received
ß-galactosidase or a polypeptide encoded by pET-28 alone. Following
primary immunization and two boosters, high titer responses to the
respective Ags were established by ELISA, immunoblotting, and
immunoprecipitation. Sera from mice immunized with either human 52
or 52ß immunoprecipitated murine 52Ro. mRNA and protein expression of
52Ro was demonstrated in the newborn murine heart. A spectrum of
atrioventricular nodal conduction abnormalities was identified by
electrocardiogram. First-degree block was detected in 7% of 27
pups born to mothers immunized with 48La, 20% of 54 pups born to
60Ro-immunized mothers, 6% of 56 pups born to 52-immunized mothers,
7% of 86 pups born to 52ß-immunized mothers, and 9% of 22 pups born
to mothers immunized with murine 52Ro. Advanced conduction
abnormalities were only identified in offspring of 52- or
52ß-immunized mice. In the 52 group, one pup had complete block
and another had second-degree block (Wenckebach type); in the 52ß
group, five pups had complete block. Maternal Abs to the primary
immunogens were detected in the pups. No control had any conduction
abnormalities. This Ab-specific animal model provides strong evidence
for a pathogenic role of anti-SSA/Ro-SSB/La Abs, particularly 52Ro,
in the development of congenital heart block. The range and frequency
of conduction defects suggest that additional factors promote disease
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A molecular definition for all three target autoantigens has been
provided by the isolation of cDNA clones. 60-kDa SSA/Ro contains an
RNA-binding protein consensus motif (5, 6), which could account for its
direct interaction with small cytoplasmic hY RNAs (7). More recent
studies demonstrate that the "zinc finger" in human 60-kDa SSA/Ro
is not conserved across species (8). It has been suggested that 60-kDa
SSA/Ro may function as part of a novel quality control or discard
pathway for 5S ribosomal RNA production in Xenopus
oocytes (9). Anti-SSB/La Abs recognize a 48-kDa polypeptide that does
not share antigenic determinants with either 52-kDa or 60-kDa SSA/Ro
(10, 11). SSB/La facilitates the maturation of RNA polymerase III
transcripts, directly binds a spectrum of RNAs, and associates at least
transiently with 60-kDa SSA/Ro (12, 13). In addition to the
well-characterized 60-kDa SSA/Ro and 48-kDa SSB/La autoantigens,
another target of the autoimmune response in mothers whose children
have CHB is the 52-kDa SSA/Ro protein (14). The full-length protein,
52, 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 two coiled coils
with heptad periodicity (one being a leucine zipper with the potential
for intramolecular dimerization), and a C-terminal "ret finger
protein-like" domain (15, 16). An alternatively spliced
transcript, 52ß, has been identified recently (17) in which exon 4
encoding amino acids 169–245, inclusive of the leucine zipper and an
immunodominant epitope (18, 19), has been deleted. In vitro-translated
52ß is immunoprecipitated by antisera from mothers whose children
have CHB (17), which is consistent with reports of an additional
N-terminal epitope on 52 that is retained in 52ß (18, 19).
mRNA expression of 52ß is maximal in the human fetal heart between 14
and 16 wk of gestation (17, 20).
Clinical and experimental data support a pathogenic role of SSA/Ro and SSB/La Abs in the development of CHB. Maternal IgG anti-SSB/La Ids were identified on the surface of fetal myocardial fibers (21), and anti-SSA/Ro Abs have been eluted from an affected fetal heart (22). Complete AV block is induced in the rabbit heart (23) as well as the human fetal heart (24) after perfusion of the aorta with sera containing anti-SSA/Ro Abs and affinity-purified anti-52-kDa SSA/Ro Abs. Furthermore, these same Ab preparations inhibit whole-cell and single-channel L-type calcium channels (23, 24). Despite these compelling observations, an animal model has yet to be established. Accordingly, this study was initiated to develop an Ab-specific murine model to correlate the arrhythmogenic effects of maternal autoantibodies with the in vivo genesis of CHB. This was approached by immunizing BALB/c mice with each of the components of the SSA/Ro-SSB/La complex. Mice were mated after the establishment of primary immune responses, and electrocardiograms (EKGs) were performed on all pups within 1 day of birth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on previously reported cDNA sequences, primers containing
internal restriction sites to facilitate in-frame directional cloning
were designed to amplify cDNA fragments encoding full-length human
48-kDa SSB/La, full-length 52, 52ß (an alternative form of 52-kDa
SSA/Ro in which amino acids 169–245 are deleted (17)), 60-kDa SSA/Ro,
murine 52-kDa SSA/Ro, and ß-galactosidase (ß-gal). cDNA
fragments were subcloned into pET-28a (Novagen, Madison, WI) for
expression of the 6x His-tagged proteins and subsequent
Ni2+ resin purification. DNA sequencing confirmed the
identity of each subclone. Escherichia coli DE3 were
transformed with each recombinant plasmid, and cultures were induced
with isopropyl thiogalactose for expression of the recombinant
proteins. Stored cells were thawed and lysed in 5 ml of 6 M
GuHCl, 0.1 M sodium phosphate, and 0.01 M Tris/HCl (pH 8.0). Following
sonication and centrifugation, supernatants were collected and added to
8 ml of a 50% slurry of Ni-nitrilotriacetic acid resin (Qiagen,
Chatsworth, CA). Recombinant proteins were incubated for 45 min with
the Ni-nitrilotriacetic acid resin, and the slurry was loaded onto a
1.5-cm diameter column. Columns were washed with 8 M urea, 0.1 M sodium
phosphate, and 0.01 M Tris/HCl (pH 8.0) until the A280 was <0.01. This
washing was followed by an additional wash with 8 M urea, 0.1 M sodium
phosphate, and 0.01 M Tris/HCl (pH 6.3) until the A280 was <0.01. The
recombinant proteins were eluted with 20 ml of 8 M urea, 0.1 M sodium
phosphate, and 0.01 M Tris/HCl (pH 5.9) followed by 20 ml of 8 M urea,
0.1 M sodium phosphate, and 0.01 M Tris/HCl (pH 4.5) and 20 ml of 6 M
GuHCl plus 0.2 M acetic acid (each collected in 3-ml fractions).
Elution fractions were analyzed by SDS-PAGE, and samples were dialyzed
in 6 M urea and 0.01 M Tris (pH 7.0). Estimations of protein
concentration were made using the bicinchoninic acid protein
assay (Pierce, Rockford, IL).
Immunizations of BALB/c mice
We immunized 6 to 8-wk-old female BALB/c mice (purchased from
Charles River, Wilmington, MA) with either recombinant human 48-kDa
SSB/La, 52 (full-length) SSA/Ro, 52ß SSA/Ro, 60-kDa SSA/Ro, or
murine 52-kDa SSA/Ro. For the initial immunization, 50 µg of
recombinant protein in CFA was injected i.p.; 25 µg of the same
preparation in CFA was administered s.c. to the right and left scapular
region. Control animals were given the same injections with recombinant
ß-gal or a Ni2+ affinity-purified polypeptide encoded by
pET-28 alone in CFA. The first two boosters were given at 7–10-day
intervals using 25 µg of recombinant protein in IFA injected i.p. and
12.5 µg of recombinant protein injected s.c. to the right and left
scapular region. Subsequent booster injections were continued with a
similar regularity until primary responses were detected in all mice of
a given group. Thereafter, booster injections were given approximately
every 3 wk. Test bleeds were performed every 7–10 days after
immunization and boosters.
ELISA and immunoblotting
These assays were performed as described previously (18), with some modifications. Briefly, wells were coated overnight with 0.1–0.2 µg of recombinant protein in PBS, washed with PBS containing 0.05% Tween 20 (PBS-Tween), blocked with 3% BSA/PBS-Tween, washed with PBS-Tween, and incubated with serial dilutions of Ab in PBS-Tween (1/50, 1/100, 1/500, and 1/1000) for 1 h at 22°C. Goat anti-mouse IgG (whole molecule) conjugated to alkaline phosphatase at a dilution of 1/30,000 was used at the second stage for 1 h at 22°C; OD was read at 405 nm. Murine antisera were tested in parallel against control polypeptides derived from the pET-28 vector alone. Results were considered positive if they were >2 SD greater than the mean obtained with sera from pET-28 control-immunized mice.
SDS immunoblots were performed as described previously (25). Recombinant proteins (1–2 µg/lane), human fetal heart extracts, or murine newborn heart extracts were separated by SDS-PAGE in 15% gels using a high ratio of acrylamide to bis-acrylamide under reducing conditions as detailed previously (26). Separated proteins were electrotransferred to a nitrocellulose sheet for 3 h at a constant voltage of 60 V. The nitrocellulose strips were incubated for 1 h in PBS-Tween containing 3% nonfat milk followed by incubation with a 1/100 dilution of sera and were washed extensively in the PBS-Tween solution. Membranes were then incubated for 1 h in blocking buffer containing a 1/5000 dilution of horseradish peroxidase-linked anti-mouse IgG (New England Bio-Labs, Beverly, MA). Filters were washed again in PBS-Tween, and detection was accomplished using the Phototope-horseradish peroxidase Western blot detection kit (New England Bio-Labs) according to the manufacturer’s instructions. Membranes were wrapped in Saran Wrap and exposed to x-ray film.
In vitro transcription/translation and immunoprecipitation
The Bluescript plasmids p52FL (containing the full-length cDNA for the 52-kDa SSA/Ro protein) (15), pASH9 (containing the full-length cDNA for 60-kDa SSA/Ro) (5), pM9 (containing the full-length cDNA for 48-kDa SSB/La) (10), and p52 M (containing the full-length cDNA for murine 52-kDa SSA/Ro) were linearized and used as substrates for in vitro transcription using T3 RNA polymerase. The resultant RNA was translated in vitro using a rabbit reticulocyte lysate (Promega, Madison, WI) in the presence of [35S]methionine (Trans 35S-label, 70% methionine, and 15% cysteine; ICN Biochemicals, Costa Mesa, CA) as described previously (18).
For immunoprecipitation, 10 µl of murine antisera (1/1 dilution with PBS and 0.1% sodium azide) were mixed with 50 µl of 50% protein A-Sepharose, 100 µl of 10 mg/ml BSA, 200 µl of reaction buffer (150 mM NaCl, 4 mM EDTA, 50 mM Tris-HCl (pH 7.4), 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), and 1–10 µl of labeled in vitro translation product. The reaction mixtures were rotated at 4°C for 1–3 h in 1.5-ml Eppendorf tubes. The beads were washed four times with chilled reaction buffer, eluted and boiled with reducing SDS sample buffer, and run on 15% high-ratio monomer/cross-linker acrylamide gels overnight at 7.5 mAmps. The gels were then stained with Coomassie blue and destained to visualize m.w. markers and ensure uniformity of Ig heavy and light chains precipitated in each reaction, dried with heat and vacuum, and placed with film at -70°C for periods of time ranging from overnight to 3 days.
RT-PCR analysis for murine 52-kDa SSA/Ro mRNA transcripts
First-strand synthesis was accomplished using 5 µg of
1-day-old mouse heart total RNA containing 40 U RNasin (Promega,
Madison, WI) as described previously (17). PCR was performed using a
TC9600 Cycler (Perkin-Elmer, Foster City, CA). Briefly, the 50 µl
reaction in 1x Taq buffer contained 2 U Taq
polymerase (Life Technologies, Gaithersburg, MD), 2.5 mM
MgCl2, 200 µM of deoxynucleoside triphosphate,
10 ng of cDNA, and 20 pmol of the following primers: m52–3
(5'-ATAGAATTCAAGATGTCACCCTCTACAACC-3'), a sense primer spanning the
ATG start site for translation, and m52–4
(5'-TTTATGTCGACTTTATTGTCCGGGAAGTACCGTCAG-3'), an antisense primer.
Amplification was started by heating for 2 min to 94°C. A total of 30
cycles followed, each consisting of 1 min at 94°C, 2 min at 55°C,
and 1.5 min at 72°C. The temperature was then held for 10 min at
72°C and cooled down to 4°C. A total of 6 µl of the respective
product was mixed with 1.5 µl of sample buffer (50% glycerol and
0.25% bromophenol blue in H2O) and applied to a 1.5–1.8%
agarose gel in Tris borate EDTA (TBE) buffer for analysis. To
determine the m.w. of the PCR products, 250 ng of øX174 DNA
HaeIII digest and 250 ng of 
/HindIII fragments
(Life Technologies) were run in parallel. The gels were subsequently
stained with ethidium bromide, and photographs were taken with Polaroid
film 667 (Cambridge, MA).
Positive control plasmids encoding human 52 and 52ß cDNAs and the
identical primers were run for direct comparison to ensure that the
RT-PCR products were accurate representations.
Blood collection
Blood was obtained from the tails of adult mice and by cardiocentesis under anesthesia (inhaled ethyl ether) in pups within the first day of life.
Electrocardiographic recordings
On the day of delivery, standard EKGs were performed. The pups were placed in the supine position, and leads I, II, III, aVR, aVL, and aVF were recorded with limb leads attached using miniature electrodes and a portable EKG machine. Paper speed settings were adjusted to 25 and 50 mm/s; voltage amplification was 20 mV. Each lead was recorded for at least 15 s to precisely identify P waves and QRS complexes, and map PR intervals. No anesthesia was necessary. PR prolongation was defined as >92 ms, which corresponded to the mean ± 2 SD obtained in the pups born to ß-gal-immunized mothers.
Statistical analysis
The Student t test for unpaired data was used to compare heart rates between the different groups. Fisher’s exact test was used to compare the frequency of first-degree block between control and experimental groups. p values of <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish a murine model of autoimmune-associated CHB, female
BALB/c mice were immunized with the following human recombinant
proteins: full-length 52 SSA/Ro (n = 8 mice), 52ß
SSA/Ro (n = 5), 60-kDa SSA/Ro (n = 7),
48-kDa SSB/La (n = 10), or murine 52-kDa SSA/Ro
(n = 5). Mice were bled every 7–10 days, and sera from
each mouse were separately analyzed for reactivity by ELISA,
immunoblotting, and immunoprecipitation. For each recombinant
immunogen, high-titer IgG responses were generated, which may indicate
limited B cell tolerance to these proteins in normal mice. No
anti-52-kDa SSA/Ro, -60-kDa SSA/Ro, or -48-kDa SSB/La responses
were detected in mice immunized with pET-28 alone (n =
7) or with ß-gal recombinant protein constructed in pET-28
(n = 3).
As described previously (27), primary immune responses to each of the
human recombinant proteins were readily demonstrated by ELISA and
immunoblotting (data not shown). To unambiguously demonstrate
the specificity of each Ab and evaluate reactivity to conformationally
dependent epitopes that might not be detected by ELISA or
immunoblotting, murine antisera were tested by
immunoprecipitation of [35S]methionine-labeled in vitro
translation products. This approach provided an independent assay of
reactivity, because the translation products were derived from
different vectors (pBluescript) than those used for the preparation of
the immunizing recombinant proteins, thus eliminating reactivity with
pET-28 vector-derived polypeptides or E. coli proteins.
Representative immunoprecipitations are shown in Fig. 1
. Mice immunized with recombinant
48-kDa SSB/La recognized in vitro-translated 48-kDa SSB/La
(lane 5). Mice immunized with recombinant full-length
52 SSA/Ro or the 52ß isoform recognized both 52 and 52ß
(lanes 10, 11, 14, and
15). Mice immunized with 60-kDa SSA/Ro recognized 60-kDa
SSA/Ro (lane 20). Immunoprecipitations were also
performed using antisera obtained from the F1 generations
as a proxy to determine whether the maternal Abs reactive with each of
the recombinant proteins were effectively transferred across the murine
placentae. All sera (collected in the first day of life) from several
pups born to mothers in each immunized group confirmed the presence of
the appropriate Ab. As exemplified in Fig. 1 (lanes
22 and 23), the serum obtained from a pup born to a
mother immunized with 52ß contained Abs that were reactive with 52
and 52ß.
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SSA/Ro is only 81% (29). Because the primary Ab responses generated to
human 52 SSA/Ro may represent alloreactivity rather than true
autoreactivity, murine sera were also tested against murine 52-kDa
SSA/Ro. Mice immunized with either human 52 or 52ß
immunoprecipitated in vitro-translated mouse 52-kDa SSA/Ro (Fig. 2, lanes 2 and 4).
Mice immunized with murine 52-kDa SSA/Ro recognized both human and
murine 52-kDa SSA/Ro (data not shown).
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To confirm that the murine Ab responses were directed against the
Ags present in the target tissue, mRNA and protein expression of 52-kDa
SSA/Ro was evaluated in murine hearts isolated from 1-day-old pups.
Using primers based on murine sequences, mRNA transcripts for murine
52-kDa SSA/Ro were readily amplified from mouse heart cDNA (Fig. 3A, lane 1). Mice
immunized with human 52-kDa SSA/Ro recognized the respective Ags in
MOLT-4 and mouse heart lysates (Fig. 3B, lanes 3
and 4).
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Offspring were obtained by breeding female mice with syngeneic
males. The data on the number of fertile females and pups born in each
group are summarized in Table I. The mean
heart rate (in beats per minute (bpm)) was not significantly different
between the experimental groups (48La, 312 bpm ± 91 SD; 52Ro,
363 bpm ± 131 SD; 52ßRo, 329 bpm ± 101 SD; 60Ro,
285 bpm ± 120 SD; and murine 52Ro, 286 bpm ± 88 SD)
and the ß-gal group (321 bpm ± 124 SD). Pups born to mothers
immunized with vector control had a higher mean heart rate when
compared with the other groups (390 bpm ± 96 SD;
p < 0.01). PR intervals could be reliably measured in
all offspring of mothers immunized with 48-kDa SSB/La, murine 52-kDa
SSA/Ro, and vector control as well as in 11 of 21, 47 of 56, 74 of 86,
and 50 of 54 pups born to mice immunized with ß-gal, human 52,
52ß, or 60-kDa SSA/Ro, respectively. The mean PR interval was similar
in the experimental groups (48La, 59 ± 19 ms; 52Ro, 55 ±
24 ms; 52ßRo, 59 ± 22 ms; 60Ro, 69 ± 27
ms; and murine 52Ro, 60 ± 21 ms) compared with
ß-gal group (58 ± 17 ms), whereas pups in the vector
control group had a lower mean PR interval (45 ± 12 ms;
p < 0.01). First-degree block was defined as a PR
interval of >92 ms (2 SD above the mean PR in offspring of
ß-gal-immunized mothers). As seen in Fig. 4, no pups in either control group had a
PR interval that was >90 ms. In contrast, first-degree block was found
in 2 pups born to 48-kDa SSB/La-immunized mothers, 3 born to
52-immunized mothers, 5 born to 52ß-immunized mothers, 10 born
to 60-kDa SSA/Ro-immunized mothers, and 2 born to murine 52-kDa
SSA/Ro-immunized mothers. In total, 22 (10%) of 220 pups in the
experimental groups (in which PR intervals could be measured)
demonstrated first-degree block compared with none of 43 pups whose
mothers were immunized with vector alone (two-tailed p
value = 0.03, Fisher’s exact test).
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-immunized mothers had advanced
heart block (one complete and another with second-degree block
(Wenckebach type)). Each of these pups was born to a different
52-immunized mother. Complete block was also detected in 5 of 86
pups born to 52ß-immunized mice. Three of these pups were from the
same litter. Representative EKGs are shown in Figs. 5 and 6. No
second- or third-degree block was found in the offspring of mothers
immunized with 48-kDa SSB/La, 60-kDa SSA/Ro, or murine 52-kDa SSA/Ro.
There was no difference in the titer (as measured by ELISA) of the
immune responses to the respective Ags in mothers of affected pups
compared with mothers of unaffected pups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A spectrum of AV nodal conduction abnormalities was identified on
the EKG tracings obtained from the F1 generation of mice
immunized with components of the SSA/Ro-SSB/La complex but was not
identified in control groups. First-degree AV block was observed in 7%
of pups born to mothers immunized with 48-kDa SSB/La, in 6–7% of pups
born to 52- and 52ß-immunized mothers, in 20% of pups born to
60-kDa SSA/Ro-immunized mothers, and in 9% of pups born to murine
52-kDa SSA/Ro-immunized mothers. Second-degree block was observed in
2% of offspring from 52-immunized mothers. Third-degree block was
observed in 2–6% of the pups from 52- and 52ß-immunized
mothers.
A comparison of the frequency of second- and third-degree AV block observed in this inbred murine model with the disease in humans is difficult. Genetics notwithstanding, the frequency depends upon the denominator (i.e., the number of mothers at risk and/or the number of children born to these mothers). In one retrospective study of CHB, the rate was 7 of 259 live births in mothers with SLE (30); in those mothers with anti-SSA/Ro Abs, the rate was 6 of 79 (30). In another prospective study, none of 26 gravid lupus patients with anti-SSA/Ro and/or -SSB/La Abs gave birth to a child with CHB (31). Most recently, Simmons-O’Brien et al. (32) detected one case of CHB in 113 pregnancies of 43 anti-SSA/Ro Ab-positive women between 20 and 40 yr of age. Further confounding the estimation of risk is the fact that many mothers are asymptomatic; therefore, the identification of anti-SSA/Ro-SSB/La Abs is first made at the time heart block is detected in utero. Lee estimates the risk of CHB in a mother with the candidate Abs to be 1% (2) as calculated from the given rate of CHB in 1 per 15,000–20,000 live births (33) and the 0.5% frequency of anti-SSA/Ro Abs in asymptomatic pregnant women (34). Although data on the frequency of anti-52-kDa (vs. 60-kDa) SSA/Ro Abs in healthy pregnant women are unknown, the advanced conduction defects in the murine offspring approximated those reported for most series in humans. Expression of disease did not correlate with titer of immune response, which is also consistent with human disease (35). The heterogeneity of the fetal response remains unexplained, but it is hoped that the availability of this murine model will facilitate such studies.
In humans, the characteristic cardiac conduction abnormality associated with maternal anti-SSA/Ro-SSB/La Abs is complete AV block, rather than the first-degree block noted in the mice. This may be accounted for by differences in the pathogenetic properties of human compared with murine Abs or by differences in the response and repair of the fetal heart between the two species. Alternatively, it may simply be that first-degree block is underestimated in humans because it is not clinically detected as a bradyarrhythmia. In support of the latter possibility, routine EKGs are not performed on otherwise healthy infants of asymptomatic mothers and often not on infants whose mothers are known to have a rheumatic disorder, even if the candidate autoantibodies are established. However, there have been rare reports of autoimmune-associated first-degree AV block (4, 36). One illustrative case identified in the Research Registry for Neonatal Lupus supports the applicability of the mouse model to the disease in humans. A mother with an undifferentiated autoimmune syndrome and documented anti-SSA/Ro Abs gave birth to a child whose EKG demonstrated first-degree block, as noted by a prolonged PR interval for age, at 11 h of life. By 18 mo, the block had progressed to second-degree, and the child required a pacemaker (36).
Two previous publications have addressed the development of a murine
model of CHB; one used a completely different strategy. Kalush et al.
reported that offspring of BALB/c mice immunized with the monoclonal
anti-DNA Id 16/6 had conduction abnormalities (37). Of 31 pups born
to mothers with experimental SLE, 8 had first-degree heart block, 2 had
second-degree heart block, 2 had complete block, 10 had bradycardia,
and 8 demonstrated widening of the QRS complex. None of these disorders
could be detected in the 20 offspring of healthy control mice. One of
the difficulties in interpreting these findings is that the immunized
mothers synthesized a variety of autoantibodies, including those
reactive with 16/6 Id, ss/dsDNA, Sm, RNP, cardiolipin,
SSA/Ro, and SSB/La. In an earlier pilot study by our group in which
female BALB/c mice were immunized with human 52-kDa SSA/Ro, 2 of 20
pups had CHB (24). The results reported herein confirm these findings
and provide extensive characterization of the Ab reactivities as well
as novel data regarding immunization with human 52ß SSA/Ro, 48-kDa
SSB/La, and 60-kDa SSA/Ro and with murine 52 SSA/Ro.
While pups from each of the experimentally immunized groups developed
some degree of AV block, only those born to mothers immunized with
human 52-kDa SSA/Ro, particularly the ß isoform, demonstrated
complete AV dissociation. Although the number of pups is too small to
draw definitive conclusions, several explanations may contribute to
this apparent selectivity. First, it has been demonstrated previously
that affinity-purified anti-52-kDa SSA/Ro Abs induce AV block in a
working heart model and inhibit whole-cell and single-channel slow,
inward calcium currents (24). Second, 52-kDa SSA/Ro is the most
prevalent Ag recognized on immunoblots by sera from mothers whose
children have CHB, although in vitro-translated 60-kDa SSA/Ro is
immunoprecipitated by all of these sera (25, 38, 39). Third, the mRNA
expression of 52ß relative to 52 in the human heart is greatest
between 14 and 16 wk of fetal life (20), albeit not yet established in
the mouse. A particular difficulty in attributing pathogenicity to
anti-52-kDa SSA/Ro Abs is that the majority of mice demonstrate
spreading of the immune response over time (most readily observed from
48-kDa SSB/La to 52-kDa SSA/Ro) (27). The absence of serious conduction
abnormalities in the 48-kDa SSB/La and murine 52-kDa SSA/Ro groups of
mice is unexplained but may relate to the limited number of pups.
However, this absence may also reflect differences in Ab titer,
affinity, or epitope specificity generated in response to a primary
exogenous immunogen compared with a "secondary" self Ag. Further
work with increased numbers of immunized mothers and early matings
before detectable spreading should more precisely define Ab specificity
and pathogenicity.
In conclusion, this murine model provides strong evidence for a pathogenic role of Abs reactive with SSA/Ro and SSB/La in the development of CHB. The spectrum of conduction abnormalities was varied and included a greater incidence of first-degree AV block than that reported for humans. The rate of second- and third-degree block approximates the 1–5% risk for a mother with anti-Ro/La Abs to have a child with CHB and suggests that additional factors are required to promote disease expression. Histologic evaluation of the affected hearts and more precise identification of the fine specificity of the arrhythmogenic Abs should provide invaluable clues to the pathophysiology of autoimmune-associated CHB.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jill P. Buyon, Department of Rheumatology, Room 1608, Hospital for Joint Diseases, 301 East 17th Street, New York, NY 10003.
3 Abbreviations used in this paper: CHB, congenital heart block; SLE, systemic lupus erythematosus; AV, atrioventricular; EKG, electrocardiogram; ß-gal, ß-galactosidase; bpm, beats per minute.
Received for publication May 19, 1998. Accepted for publication July 24, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A D Askanase, D M Friedman, J Copel, M R Dische, A Dubin, T J Starc, M C Katholi, and J P Buyon Spectrum and progression of conduction abnormalities in infants born to mothers with anti-SSA/Ro-SSB/La antibodies Lupus, March 1, 2002; 11(3): 145 - 151. [Abstract] [PDF]
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 J. P. Buyon, D. Nugent, E. Mellins, and C. Sandborg Maternal Immunologic Diseases and Neonatal Disorders NeoReviews, January 1, 2002; 3(1): e3 - 10. [Full Text] [PDF]
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 G.-Q. XIAO, Y. QU, K. HU, and M. BOUTJDIR Down-regulation of L-type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits FASEB J, July 1, 2001; 15(9): 1539 - 1545. [Abstract] [Full Text] [PDF]
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M.-E. Miranda-Carus, A. D. Askanase, R. M. Clancy, F. Di Donato, T.-M. Chou, M. R. Libera, E. K. L. Chan, and J. P. Buyon Anti-SSA/Ro and Anti-SSB/La Autoantibodies Bind the Surface of Apoptotic Fetal Cardiocytes and Promote Secretion of TNF-{alpha} by Macrophages J. Immunol., November 1, 2000; 165(9): 5345 - 5351. [Abstract] [Full Text] [PDF]
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