Journal
of Autoimmunity
(1991) $165176
Ro and La Antigens and Maternal Anti-La Idiotype on the Surface of Myocardial Fibres in Congenital Heart Block
Angela C. Horsfall, Patrick J.W. Venables, Pamela V. Taylor* and Ravinder N. Maini Kennedy Institute of Rheumatology, London W6 7DW, UK; *Department of Obstetrics and Gynaecology, Leeds University, Belmont Grove, Leeds UK (Received
30 April 1990 and accepted 23 August
1990)
Congenital complete heart block (CCHB) is a rare but potentially fatal disease of infants born to mothers with autoimmune disease where maternal autoantibodies to Ro (SS-A) are thought to cross the placenta and damage fetal cardiac tissue. We have adopted a novel approach to demonstrate the localization and specificity of maternal autoantibodies deposited in fetal heart. We raised an anti-idiotype against maternal antiLa antibodies, which reacted strongly with the surface immunoglobulin on the myocardial fibres from a CCHB heart but not a control fetal heart of the same age. Maternal immunoglobulin eluted from the CCHB heart reacted with La (SS-B) by ELISA. Using monoclonal and aflinity-purified antibodies to La and affinity-purified anti-R0 antibodies, both antigens were identified on the surface of the fibres of the affected heart. Surface coexpression of immunoglobulin, complement and Class II antigen, consistent with a local immune response, was also found. This is the first definitive demonstration of Ro and La antigens and specific maternal antiLa antibody and idiotype on the surface of myocardial fibres in CCHB. It suggests that induction of Ro and La antigens on the surface of myocardial fibres during fetal development may be critical in the localization of the specific autoantibodies and subsequent evolution of congenital complete heart block.
Introduction
Congenital heart block (CCHB) is a rare but potentially fatal disease which was initially recognized in infants of patients with systemic lupus erythematosus Correspondence and reprint requests to: Dr A. C. Horsfall, Clinical Immunology Division, The Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW, UK. This work was supported by the Arthritis and Rheumatism Council of Great Britain. 165 0896-841 l/91/010165+
12 $03.00/O
0 1991 Academic Press Limited
166 A. C. Horsfall et al. (SLE) [ 11. It is thought that damage to fetal cardiac tissues is caused by maternal autoantibodies [2] crossing the placenta, based on in vitro studies [3, 41 and on the finding of immunoglobulin and complement components within the myocardium and conduction systems [5]. Recent studies suggest that the maternal antibodies react with Ro and La. Over 90% of the mothers of affected offspring have circulating IgG anti-R0 (SS-A) antibodies and over 80% also have anti-La (SS-B) antibodies [6]. Titres of anti-Rio and anti-La antibodies in serum from CCHB babies are reported to be lower than in the mothers, suggesting that autoantibodies are removed from the circulation and deposited in the baby’s tissues [6]. Unaffected children have autoantibody titres comparable to maternal levels, and a study of HLA identical twins discordant for CCHB showed that the titre of anti-R0 in the affected twin was lower than in the nonaffected twin [7]. These data suggest that anti-R0 and anti-La are involved in CCHB, although there has been no conclusive demonstration to date that either autoantibody has a direct pathogenic role. This study of congenital heart block used a novel approach to demonstrate both the localization and specificity of maternal autoantibody with anti-idiotype reagents. We have also used monoclonal and af%nity-purified polyclonal antibodies to study the expression of the Ro and La antigens in fetal cardiac tissue. Materials and methods Cardiac tissue was obtained from a male infant who died within hours of a Caesarian delivery at a gestational age of 30 weeks. Fetal bradycardia was first detected at 28 weeks 5 days. A diagnosis of CCHB was confirmed by ECG at birth. No further organs were available for study. Control cardiac tissue was obtained at autopsy from a male fetus which died at 32 weeks’ gestation from placental haemorrhage. Tissue blocks and cryostat sections (6 urn) mounted on glass microscope slides were stored at - 70°C. The mother of the CCHB baby had a mild undifferentiated connective tissue disease with histological evidence of chronic immune sialadenitis, but failed to fulfil criteria for Sjogren’s syndrome. Her serum had IgG anti-La antibodies by immunodiffusion, ELISA [B] and immunoblotting of HeLa and MOLT4 T-cell extracts. Anti-60 kD Ro antibodies were detected by immunoblotting of HeLa cell extract and by ELISA [8] using bovine Ro (Cellon, Luxembourg). Anti-52 kD Ro antibodies were detected by immunoblotting of MOLT4 T-cell extract by Dr E. Chan (Scripps Clinic, La Jolla, CA). IgG anti-La antibodies were purified from post-partum maternal serum as previously described [9] and used to raise polyclonal anti-idiotypes in rabbits [lo]. Immunoglobulin levels were quantitated by inhibition ELISA [ 111. To characterize IgG anti-idiotypes, they were coated onto ELISA plates at 10 ug/ml and incubated with IgG prepared from serum samples from 25 patients fulfilling criteria for primary Sjiigren’s syndrome (6 with anti-R0 antibodies alone, 19 with anti-R0 and antiLa antibodies, all with rheumatoid factor), 6 patients with autoantibodies to other antigens (anti-dsDNA, Sm/nRNP and rheumatoid factors) and 21 normal healthy adults. All sera were depleted of rheumatoid factors by immunoabsorption with pooled normal human IgG. Dilutions of maternal IgG anti-La (immunizing idiotype) were assayed on each plate as an internal standard. Bound IgG was detected
Antigen and idiotype in congenital complete heart block
167
Table 1. Immunojhorescence Detect
Probe Anti-Id (maternal anti-La) Rabbit IgG’
anti-rabbit anti-rabbit
FITC’ FITC
Rabbit anti-C3c-FITC’ L2433 Fab’2 anti-Hu.Ig-TRITC4
anti-mouse-FITC’
Identify anti-La Id control complement HLA-DR Ig
Pooled hu.IgG-biotin Maternal anti-La-biotin Non-maternal anti-La-biotin Non-maternal anti-Ro-biotin
streptavidin-FITC5 or avidin-Texas Red5
control La antigen La antigen Ro antigen
gg:
anti-mouse-FITC
La antigen
anti-mouse-FITC
control
] Monoclonal anti-La (IgG,,)
KSm5 Monoclonal anti-Sm (IgG,,)
Suppliers: ‘Sigma; *Dako; ‘Kallestad; 4Amersham; ‘ATCC.
with goat anti-human y-chain-specific antibodies conjugated (Sigma, Poole, UK) previously absorbed with rabbit IgG.
to alkaline
phosphatase
Western blotting Gel electrophoresis (SDS-PAGE) of purified anti-La from the mother (C) and from an unrelated patient (E) with Sjdgren’s syndrome and normal human pooled IgG was carried out under reducing conditions and analysed by immunoblotting as previously described [9]. Blots were probed with rabbit anti-idiotype and developed with goat anti-rabbit y-chain-specific peroxidase conjugate (ICN, High Wycombe, Bucks, UK). ImmunojIuorescence Six micron cryostat sections were air dried, washed once in phosphate buffered saline (PBS), pH 7.2 and fixed in acetone and methanol (1 :l, v/v) on ice. Thirty microlitres of antibody (10 pg/ml) per section were allowed to incubate for 1 h at room temperature. Following washing for 20 min with two further changes of PBS, sections were incubated with fluorochrome-conjugated antibodies (Table 1) for 30 min at room temperature. After subsequent washing in PBS, the slides were mounted in 90% glycerol/PBS containing p-phenylenediamine (0.1 mg/ml) and viewed with a Leitz Laborlux fluorescent microscope with a x 50 water immersion lens. Maternal antibodies were eluted from three cardiac sections by applying to each section 100 ~1 of PBS containing 5% goat serum (PBS-GS), followed by 100 ~10.2 M glycine containing 0.5 M NaCl, pH 2.5 or 100 l.d3 M guanidine hydrochloride containing 5% goat serum, followed by 100 ~1PBS-GS for 10 min each prior to aspiration by pipette. Control sections were incubated with three consecutive applications of PBSGS. After dialysis against PBS for 48 h with three buffer changes, fractions were tested for specificity by ELISA and the sections examined by immunofluorescence.
168 A. C. Horsfall et al. Table
2. Autoantibody
profiles in mother and CCHB anti-R0
Immunodiffusion’* ELI SA’# Western Blot’#
infant
anti-La
Mother
Baby
Mother
Baby
256 18 u/ml 52kD 60 kD
16 NA NA
64 37u/ml 47kD
4 NA NA
*Serum taken at time of delivery. #Post-partum serum. ‘Reciprocal tiue by immunodiffusion against human spleen extract as a source of Ro antigens and rabbit thymus extract as a source of La antigens. 2ELISA using purified bovine 60 kD Ro antigen (Cellon, Luxembourg) and rabbit thymus extract La. Results expressed as units/ml by reference to standard curves. 3Molecular weights of peptides identified by Western blotting using MOLT4 T cell and HeLa cell extracts. NA: not available.
Anti-La antibodies from the mother of the CCHB baby and anti-La and anti-R0 antibodies from a patient with Sjiigren’s syndrome and a history of uncomplicated pregnancy were purified by afhnity chromatography against purified 60 kD bovine Ro [ 121 and rabbit thymus extract La as previously described 191. All three antibody preparations were labelled with NHS-LC-Biotin (Pierce, Rockford, IL) [9]. Two mouse monoclonal anti-La antibodies (SW1 and SWS) and one mouse monoclonal anti-Sm antibody (KSm5) prepared in this laboratory were also used [ 13,141. Biotin labelled antibodies were visualized with streptavidin Fluorescein isothiocyanate (FITC) or streptavidin Texas Red. Non-conjugated antibodies were visualized with goat anti-mouse or rabbit y-chain specific antibodies conjugated to FITC as appropriate. Table 1 lists the probes, detection systems and epitopes recognized in immunofluorescent studies and the sources of reagents. Results
At the time of delivery, titres of antibodies to both the Ro and La antigens were lower in the baby than in maternal serum, suggesting deposition of both antibodies in the fetus (Table 2). Western blotting of post-delivery serum showed that anti-R0 antibodies reacted with both the 60 kD and 52 kD components of the Ro antigen. Characterization
of anti-idiotype
In direct ELISA the binding of human IgG to immobilized rabbit anti-idiotype showed that cross-reactive idiotypes were present on IgG in sera containing both antiRo and anti-La antibodies and in six sera containing anti-R0 antibodies alone. Idiotypes were not detected in sera lacking autoantibody activity or on IgG with specificity for other autoantigens such as dsDNA or Sm/nRNP. The immunizing idiotype (maternal IgG anti-La) showed the highest binding to anti-idiotype (Figure 1).
Antigen and idiotype in congenital complete heart block
169
80-
i p .z z 3
:
60-
0.. 2.
0
t
0
0,
anti-La+ anti-Ra+
anti- Laanti-Ro+
anti- Laanti - Ro-
Figure 1. Binding of human IgG to rabbit IgG anti-La anti-idiotype. ELISA plates were coated with rabbit IgG anti-idiotype or non-immune rabbit IgG (10 pg/ml) and incubated with human IgG (100 pg/ ml) purified from serum samples of patients with autoimmune disease or from healthy individuals (closed circles). Open circles represent IgG from a patient with rheumatoid arthritis (rheumatoid factor positive), a patient with systemic lupus erythematosus (anti-Sm positive) and a patient with overlap disease (antinRNP positive). All patients with antibodies to the Ro and La antigens had Sjogren’s syndrome. Results are expressed as the percentage binding of each IgG sample compared to the immunizing maternal IgG anti-La (*).
Western blotting revealed that the idiotypes were located on the heavy chain of the variable region of aflinity purified anti-La antibodies (Figure 2). Anti-idiotype did not significantly inhibit the binding of anti-La antibodies to the La antigen, suggesting that the idiotype was located outside the antigen binding site. Demonstration
of anti-La
antibodies and idiotype on myocardialfibres
Anti-idiotype showed linear surface binding to myocardial fibres in CCHB (Figure 3a, Table 3) which was inhibited by pre-incubation with purified maternal anti-La (Figure 3b). The staining pattern was identical using anti-human IgG (Table 3). No IgM or IgA could be demonstrated (data not shown). Anti-idiotype did not react with control fetal heart (Figure 3c, Table 3). Treatment of CCHB cardiac sections by guanidine hydrochloride but not by glycine, pH 2.5, released maternal anti-La antibodies which could be detected by ELISA (Figure 4). The failure of glycine to elute anti-La IgG suggests that these are high affinity antibodies. Levels of anti-R0 antibodies were low in all samples (E,, less than 0.100 by ELISA) with no significant increase in the sample eluted with guanidine hydrochloride (data not shown). Expression
of Ro and La antigens
on myocardialjibres
Immunofluorescence on HEp 2 cells confirmed that the two monoclonal anti-La antibodies reacted with the nucleus [13] as did the a@inity purified polyclonal
170
A. C. Horsfall et al.
67
-
43
-
30
-
20
-
I4 -
IgG
E
C
Figure 2. Immunoblot of human IgG probed with anti-idiotype. Anti-La antibodies were immunopurified from maternal serum (C) and from the serum of an unrelated patient with Sjogren’s syndrome (E). These and normal pooled human IgG lacking anti-La activity were electrophoresed under reducing conditions, blotted onto nitrocellulose and probed with rabbit anti-idiotype. Molecular weight markers are indicated on the left ( x 10m3kD).
antibodies (Figure 5a). Affinity purified human polyclonal anti-60 kD Ro antibody gave a filamentous cytoplasmic perinuclear staining. There was also some intranuclear and nucleolar fluorescence (Figure 5b). Both mouse monoclonal (SWl) and human polyclonal anti-La antibodies reacted with the surface of myocardial fibres in CCHB, suggesting expression of the La antigen on the membrane of these cells (Figure 3d, Table 3). The fluorescence became stronger, particularly with the purified human antibodies, after probing sections treated with guanidine hydrochloride. Weak cytoplasmic staining of control fetal heart was found with anti-La antibodies (Figure 3e, Table 3). In CCHB sections a second monoclonal anti-La antibody (SW5) did not react L. with the surface of myocardial fibres but showed weak cytoplasmic fluoresce&e (Figure 3f, Table 3). Monoclonal anti-Sm antibodies did not react with the surface of the myocardial fibres but did give nuclear staining of the lymphocytic infiltrating cells (data not shown). Human anti-60 kD Ro antibodies reacted with the cytoplasm of myocardial fibres in both CCHB and the control fetal heart. Strong surface fluorescence compatible with membrane expression of Ro was seen in the CCHB heart with cytoplasmic and possible weak surface fluorescence in the control heart (Figure 3g and 3h, Table 3).
Antigen and idiotype in congenital complete heart block
171
Table 3. Summary of immunojluorescent staining patterns in fetal heart sections CCHB heart Myocardial fibres Idiotype (anti-Id) Immunoglobulin (aIg) Antigen (a) La
Lymphocytic infiltrate -
-
+N +N +N+C +N +N +c +N
+c -
+++s ++s
Monoclonal SW 1 Monoclonal SW5 Maternal anti-La Non-maternal anti-La G.HCl elution fan&La Non-maternal anti-R0 (b) Ro Monoclonal KSm5 (c) Sm
++s SC +s+c +s ++s ++s++c -
Control heart
NT +s++c NT
Complement (aC3c)
+s
-
-
Class II (HLA DR)
+s
+s+c
-
S: surface, N: nuclear, C: cytoplasmic,NT: not tested.
Expression of class II and complement In common with other published reports, both immunoglobulin and complement components (C3c) could be demonstrated on myocardial fibres of the affected cardiac tissue (Table 3). Additionally, strong expression of MHC Class II antigens was observed on the surface of myocardial fibres and lymphocytes in CCHB but absent in control fetal heart (Figure 3i, Table 3).
Discussion This study has definitively demonstrated the expression of both the Ro and La antigens and anti-La antibody on the surface of myocardial fibres in fetal cardiac tissue with congenital complete heart block. We propose that anti-La antibodies are deposited in fetal cardiac tissue by binding to the La antigen expressed on the surface of myocardial fibres. Localization of maternal anti-La was detected by a novel approach using polyclonal anti-idiotypic antibodies and confirmed by eluting anti-La antibodies from cardiac sections. Furthermore, dissociation of antibodies from cardiac sections revealed La epitopes to which mouse monoclonal and human polyclonal anti-La antibodies were able to bind. By demonstrating specificity our data confirm and extend previous observations on IgG deposition in fetal hearts [ 15, 16,5]. In contrast to previous reports, no IgA or IgM deposits were found in the CCHB heart sections in this study [ 16,5]. Initially anti-R0 antibodies were thought to be important in the pathogenesis of CCHB based on the observations that all mothers of affected children had circulating
172
A. C. Horsfall et al.
Figure 3. Immunofluorescent photographs: (a) CCHB stained with anti-Id; (b) CCHB stained with anti-Id pre-incubated with purified maternal anti-La; (c) control fetal heart stained with anti-Id; (d) CCHB stained with monoclonal anti-La (SW 1); (e) control fetal heart stained with monoclonal anti-La; (fj CCHB stained with monoclonal anti-LA (SW5); (g) CCHB stained with anti-Ro; (h) control fetal heart stained with anti-Ro; (i) CCHB stained with antibody to HLA Class II (DR).
anti-Rio antibodies [2] and that titres of these antibodies were lower in affected infants consistent with deposition [6,7]. More recently, both anti-R0 and anti-La antibodies have been implicated in pathogenesis. A specific association of maternal antibodies
Antigen and idiotype in congenital complete heart block
173
600
500
400
8 w"
300
\ \ \ \ \ \ \ \
200
100
0
--_ 1st wash
_-2nd
3rd
wash
wash
Figure 4. Binding to La antigen of immunoglobulin eluted from CCHB cardiac sections measured by ELISA. Ordinate represents extinction at 405 nm x 10-3. Open circles linked by broken lines represent PBS washes of control fetal cardiac tissue. Closed circles linked by broken lines represent PBS washes of CCHB fetal cardiac tissue and closed circles linked by solid lines CCHB fetal cardiac tissue treated with 3 M guanidine hydrochloride in the second wash.
Figure 5. HEp 2 cells were stained with La and Ro specific antibodies affinity purified from the serum of a patient with Sjogren’s syndrome, biotinylated and detected with streptavidin FITC: (A) nuclear location of the La antigen and (B) cytoplasmic location of the Ro antigen.
for the human 52 kD Ro antigen has been reported and is particularly prevalent among maternal CCHB serum samples in combination with antibodies to the 48 kD La antigen [ 171.
174
A. C. Horsfall et al.
Our data is consistent with a role for anti-Rio as well as anti-La antibodies in the pathogenesis of CCHB. Because anti-idiotype appeared to bind anti-Rio, it is possible that some of the antibody detected on the surface of fibres had anti-R0 activity. Failure to detect anti-R0 antibodies in eluates from cardiac sections reflects the source of antigen used for ELISA (bovine 60 kD rather than human 52 kD). Anti52 kD Ro antibodies may well be present in eluates, but lack of cloned and/or purified human 52 kD Ro and specific antibodies imposed limitations on our study. Surface expression of the La antigen was detected using both polyclonal anti-La antibodies and the monoclonal antibody SWl. SW5 did not bind to the surface of CCHB myocardial fibres implying that the epitopes for this antibody, known to be different from SW1 [18], are blocked by maternal anti-La antibodies of the same epitope specificity. In favour of this interpretation was the finding that elution of the maternal immunoglobulin with anti-La activity increased the subsequent staining with SW5 and the polyclonal anti-La antibodies. In this study we found, in control fetal heart at 32 weeks’ gestation, that the Ro antigen was abundantly expressed in the cytoplasm, unlike the La antigen which was difficult to detect although normally located in the nucleus of resting cells. Previously we have shown that La is cytoplasmic in activated cells [ 131 and therefore one might expect that in a rapidly dividing tissue such as growing fetal heart the La antigen may also be cytoplasmic. In support of this was the finding of weak cytoplasmic fluorescence with one of the monoclonal anti-La antibodies. In a recent report we described the sequential expression of the La antigen in the nucleus, followed by the cytoplasm, and ultimately on the cell surface in virally infected cells [ 193. It is possible that a similar mechanism could operate in congenital heart block although whether the Ro antigen is similarly induced by viral infection is not known. However, our study shows that in normal fetal cardiac tissue at 32 weeks gestation La antigen was not expressed on the surface of the myocardial fibres. Thus the surface expression of the Ro and/or La antigens would be the critical event in causing maternal autoantibody binding and may account for the observation that only 5% of autoantibody positive patients have affected children [20]. Studies are now in progress to examine surface expression of these antigens in normal fetal tissue at different gestational times. Various factors have been implicated in CCHB, including the presence of maternal antibodies to Ro and La [6], previous history of CCHB births [21], Class II [22] and III [23] antigens and cytomegalovirus infection [24]. All these mechanisms are apparently contradicted by the study of HLA identical twins where both had circulating anti-Rio antibodies but only one developed CCHB. Lower titres of anti-R0 were found in the affected twin, implying deposition of anti-R0 [7]. In our study, both anti-R0 and anti-La antibodies were lower in the child than in maternal serum suggesting deposition of both antibodies. This does not negate the proposed hypothesis of surface expression of the La and/or Ro antigens following viral infection. Intra-uterine and maternally transmitted viral infections affecting one twin and not the other have been previously documented [25,26]. Whatever the explanation, it is clear that simply possessing anti-Rio and anti-La antibodies cannot be the sole requirement for the development of congenital heart block, as only 5% of antibody-positive patients have affected children [20]. Our demonstration of the Ro and La antigens and specific autoantibody on the surface
Antigen and idiotype in congenital complete heart block
of diseased fibres suggests that migration of these ribonucleoprotein constitutes an important part of the pathogenesis.
175
antigens
Acknowledgements
We are grateful to the Arthritis and Rheumatism Council, Great Britain, for financial support, to Drs J. B. Harley and K. K. Gaither, Oklahoma Medical Research Foundation, Oklahoma City, for affinity purification of anti-Rio antibodies from the serum of one of our patients, to Dr E. Chan, Scripps Clinic, La Jolla, for immunoblots of 52 kD Ro antigen, to our Departmental colleagues, Drs D. G. Williams and P. R. Smith, for preparing monoclonal anti-La antibodies and to Drs A. King and G. Batcup for providing clinical specimens from the heart block family. References 1. Chameides, L., R. C. Truex, V. Vetter, W. J. Rashkind, F. M. Galioto Jr, and J. A. Noonan. 1977. Association of maternal lupus erythematosus with congenital complete heart block. N. Eng1.J. Med. 297: 1204-1207 2. Scott, J. S., P. J. Maddison, P. V. Taylor, E. Esscher, 0. Scott, and R. P. Skinner. 1983. Connective tissue disease, antibodies to ribonucleoprotein and congenital heart block. N. England. J. Med. 309: 209-2 12 3. Taylor, I?. V. and J. S. Scott. 1986. Pathogenic effects of maternal antibody in isolated complete heart block. Presented at 6th International Congress of Immunology, Toronto, abstract. p. 437 4. Guarnieri, T., S. DaTorre, S. Myers, D. Brezinski, T. Provost, and E. Alexander. 1987. Antibody mediated heart block in neonatal lupus: an experimental model of differential age susceptibility. CircuZation 76(Suppl. IV): abstract 0319 5. Taylor, P. V., J. S. Scott, L. M. Gerlio, E. Esscher, and 0. Scott. 1986. Maternal antibodies against fetal cardiac antigens in congenital complete heart block. N. Engl. J. Med. 315: 667-672 6. Taylor, P. V., K. F. Taylor, A. Norman, S. Griffiths, and J. S. Scott. 1988. Prevalence of maternal Ro (SS-A) and La (SS-B) autoantibodies in relation to congenital heart block. Br.J. Rheumatol. 27: 128-132 7. Harley, J. B., J. L. Kaine, 0. F. Fox, M. Reichlin, and H. Gruber. 1985. Ro (SS-A) antibody and antigen in a patient with congenital complete heart block. Arthritis Rheum. 28: 1321-1325 8. Venables,P. J. W.,P. J. Charles,R. R. C. Buchanan, T. Y&P. A. Mumford, L. Schrieber, G. R. W. Room, and R. N. Maini. 1983. Quantitation and detection of isotypes of anti-SSB antibodies by ELISA and Farr assays using affinity purified antigens. An approach to the investigation of Sj6gren’s syndrome and systemic lupus erythematosus. Arthritis Rheum. 26: 143-152 9. Horsfall, A. C., C. M. Brown, and R. N. Maini. 1987. Purification of human autoantibodies from cross-linked antigen immunosorbents. J. Zmmunol. Meth. 104: 43-49 10. Horsfall, A. C., P. J. W. Venables, P. A. Mumford, and R. N. Maini. 1986. Restricted idiotypes on antibodies to the La (SS-B) antigen. Clin. Exp. Zmmunol. 63: 393-401 11. Plater-Zyberk, C., M. F. Clarke, K. Lam, P. A. Mumford, G. R. W. Room, and R. N. Maini. 1983. In vitro immunoglobulin synthesis by lymphocytes from patients with rheumatoid arthritis. I Effect of monocyte depletion and demonstration of an increased proportion of lymphocytes forming rosettes with mouse erythrocytes. Clin. Exp. Zmmunol. 52: 505-511 12. Gaither, K. K., 0. F. Fox, H. Yamagata, H. J. Mamula, M. Reichlin, and J. B. Harley. 1987. Implications of anti-Ro/SjGgren’s syndrome A antigen autoantibody in normal sera for autoimmunity. J. Clin. Invest. 79: 841-846
176
A. C. Horsfall
et al.
13. Smith, P. R., D. G. Williams, P. J. W. Venables, and R. N. Maini. 1985. Monoclonal antibodies to the Sjogren’s syndromeassociated antigen SS-B (Laj.3. Immunol. Meth. 77: 63-76
14. Williams, D. G., M. R. Stocks, P. R. Smith, and R. N. Maini. 1986. Murine lupus monoclonal antibodies deiine five epitopes on two different Sm polypeptides. fmmunology 58: 495-500 15. Lee, L. A., S. Coulter, S. Erner, and H. Chu. 1987. Cardiac immunoglobulin deposition in congenital heart block associated with maternal anti-R0 antibodies. Am. 3. Med. 83: 793-796
16. Litsey, S. E., J. A. Noonan, W. N. O’Connor, C. M. Cottrill, and B. Mitchell. 1985. Maternal connective tissue disease and congenital heart block: demonstration of immunoglobulin in cardiac tissue. N. EngZ.3. Med. 312: 98-100 17. Buyon, J. P., E. Ben-Chetrit, S. Karp, R. A. S. Roubey, L. Pompeo, W. H. Reeves, E. M. Tan, and R. Winchester. 1989. Acquired congenital heart block. Pattern of maternal antibody response to biochemically defined antigens of the SSA/Ro-SSB/La system in neonatal lupus. 3. Clin. Invest. 84: 627-634 18. Smith, P. R. 1986. Characterisation of the Sjogren’s syndrome antigens Ro and La. PhD Thesis. University of London, UK 19. Baboonian, C., P. J. W. Venables, J. Booth, D. G. Williams, L. M. Roffe, and R. N. Maini. 1989. Virus infection induces redistribution and membrane localisation of the nuclear antigen La (SS-B): a possible mechanism for autoimmunity. CZin. Exp. Immunol. 78: 454-459
20. Ramsey-Goldman, R., D. Horn, J.-S. Deng, G. C. Ziegler, L. E. Kahl, V. D. Steen, R. E. LaPorte, and T. A. Medsger. Jr. 1986. Anti-SS-A antibodies and fetal outcome in maternal systemic lupus erythematosus. Arthritis Rheum. 29: 1269-1273 21. Buyon, J., R. Roubey, S. Swersky, L. Pompeo, A. Parke, L. Baxi, and R. Winchester. 1988. Complete congenital heart block: risk of occurrence and therapeutic approach to prevention. 3. Rheumatol. 15: 1104-l 108 22. Watson, R. M., A. T. Lane, N. K. Barnett, W. B. Bias, F. C. Arnett, and T. T. Provost. 1984. Neonatal lupus erythematosus: A clinical, serological and immunogenetic study with review of the literature. Medicine (Baltimore) 63: 362-678 23. Arnaiz-Villena, A., J. J. Vazquez-Rodriguez, J. L. Vicario, P. Lavilla, D. Pascual, F. Moreno, and J. Martinez-Laso. 1989. Congenital heart block immunogenetics. Evidence of an additional role of HLA Class III antigens and independence of Ro antibodies. Arthritis
Rheum.
32: 1421-1426
24. Lewis, P. E., R. C. Cefalo, and A. L. Zaritsky. 1980. Fetal heart block caused by cytomegalovirus. Am.3. Obstet. Gynecol. 136: 967-968 25. Cooper, L. Z. 1985. The history and medical consequences of rubella. Rev. Infect. Dis. 7(Suppl.): 2-15 26. Park, C. L., H. Streicher, and R. Rothberg. 1987. Transmission of human immunodeficiency virus from parents to only one dizygotic twin.3. CZin. Microbial. 25: 1119-l 121
of Autoimmunity
(1991) $165176
Ro and La Antigens and Maternal Anti-La Idiotype on the Surface of Myocardial Fibres in Congenital Heart Block
Angela C. Horsfall, Patrick J.W. Venables, Pamela V. Taylor* and Ravinder N. Maini Kennedy Institute of Rheumatology, London W6 7DW, UK; *Department of Obstetrics and Gynaecology, Leeds University, Belmont Grove, Leeds UK (Received
30 April 1990 and accepted 23 August
1990)
Congenital complete heart block (CCHB) is a rare but potentially fatal disease of infants born to mothers with autoimmune disease where maternal autoantibodies to Ro (SS-A) are thought to cross the placenta and damage fetal cardiac tissue. We have adopted a novel approach to demonstrate the localization and specificity of maternal autoantibodies deposited in fetal heart. We raised an anti-idiotype against maternal antiLa antibodies, which reacted strongly with the surface immunoglobulin on the myocardial fibres from a CCHB heart but not a control fetal heart of the same age. Maternal immunoglobulin eluted from the CCHB heart reacted with La (SS-B) by ELISA. Using monoclonal and aflinity-purified antibodies to La and affinity-purified anti-R0 antibodies, both antigens were identified on the surface of the fibres of the affected heart. Surface coexpression of immunoglobulin, complement and Class II antigen, consistent with a local immune response, was also found. This is the first definitive demonstration of Ro and La antigens and specific maternal antiLa antibody and idiotype on the surface of myocardial fibres in CCHB. It suggests that induction of Ro and La antigens on the surface of myocardial fibres during fetal development may be critical in the localization of the specific autoantibodies and subsequent evolution of congenital complete heart block.
Introduction
Congenital heart block (CCHB) is a rare but potentially fatal disease which was initially recognized in infants of patients with systemic lupus erythematosus Correspondence and reprint requests to: Dr A. C. Horsfall, Clinical Immunology Division, The Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW, UK. This work was supported by the Arthritis and Rheumatism Council of Great Britain. 165 0896-841 l/91/010165+
12 $03.00/O
0 1991 Academic Press Limited
166 A. C. Horsfall et al. (SLE) [ 11. It is thought that damage to fetal cardiac tissues is caused by maternal autoantibodies [2] crossing the placenta, based on in vitro studies [3, 41 and on the finding of immunoglobulin and complement components within the myocardium and conduction systems [5]. Recent studies suggest that the maternal antibodies react with Ro and La. Over 90% of the mothers of affected offspring have circulating IgG anti-R0 (SS-A) antibodies and over 80% also have anti-La (SS-B) antibodies [6]. Titres of anti-Rio and anti-La antibodies in serum from CCHB babies are reported to be lower than in the mothers, suggesting that autoantibodies are removed from the circulation and deposited in the baby’s tissues [6]. Unaffected children have autoantibody titres comparable to maternal levels, and a study of HLA identical twins discordant for CCHB showed that the titre of anti-R0 in the affected twin was lower than in the nonaffected twin [7]. These data suggest that anti-R0 and anti-La are involved in CCHB, although there has been no conclusive demonstration to date that either autoantibody has a direct pathogenic role. This study of congenital heart block used a novel approach to demonstrate both the localization and specificity of maternal autoantibody with anti-idiotype reagents. We have also used monoclonal and af%nity-purified polyclonal antibodies to study the expression of the Ro and La antigens in fetal cardiac tissue. Materials and methods Cardiac tissue was obtained from a male infant who died within hours of a Caesarian delivery at a gestational age of 30 weeks. Fetal bradycardia was first detected at 28 weeks 5 days. A diagnosis of CCHB was confirmed by ECG at birth. No further organs were available for study. Control cardiac tissue was obtained at autopsy from a male fetus which died at 32 weeks’ gestation from placental haemorrhage. Tissue blocks and cryostat sections (6 urn) mounted on glass microscope slides were stored at - 70°C. The mother of the CCHB baby had a mild undifferentiated connective tissue disease with histological evidence of chronic immune sialadenitis, but failed to fulfil criteria for Sjogren’s syndrome. Her serum had IgG anti-La antibodies by immunodiffusion, ELISA [B] and immunoblotting of HeLa and MOLT4 T-cell extracts. Anti-60 kD Ro antibodies were detected by immunoblotting of HeLa cell extract and by ELISA [8] using bovine Ro (Cellon, Luxembourg). Anti-52 kD Ro antibodies were detected by immunoblotting of MOLT4 T-cell extract by Dr E. Chan (Scripps Clinic, La Jolla, CA). IgG anti-La antibodies were purified from post-partum maternal serum as previously described [9] and used to raise polyclonal anti-idiotypes in rabbits [lo]. Immunoglobulin levels were quantitated by inhibition ELISA [ 111. To characterize IgG anti-idiotypes, they were coated onto ELISA plates at 10 ug/ml and incubated with IgG prepared from serum samples from 25 patients fulfilling criteria for primary Sjiigren’s syndrome (6 with anti-R0 antibodies alone, 19 with anti-R0 and antiLa antibodies, all with rheumatoid factor), 6 patients with autoantibodies to other antigens (anti-dsDNA, Sm/nRNP and rheumatoid factors) and 21 normal healthy adults. All sera were depleted of rheumatoid factors by immunoabsorption with pooled normal human IgG. Dilutions of maternal IgG anti-La (immunizing idiotype) were assayed on each plate as an internal standard. Bound IgG was detected
Antigen and idiotype in congenital complete heart block
167
Table 1. Immunojhorescence Detect
Probe Anti-Id (maternal anti-La) Rabbit IgG’
anti-rabbit anti-rabbit
FITC’ FITC
Rabbit anti-C3c-FITC’ L2433 Fab’2 anti-Hu.Ig-TRITC4
anti-mouse-FITC’
Identify anti-La Id control complement HLA-DR Ig
Pooled hu.IgG-biotin Maternal anti-La-biotin Non-maternal anti-La-biotin Non-maternal anti-Ro-biotin
streptavidin-FITC5 or avidin-Texas Red5
control La antigen La antigen Ro antigen
gg:
anti-mouse-FITC
La antigen
anti-mouse-FITC
control
] Monoclonal anti-La (IgG,,)
KSm5 Monoclonal anti-Sm (IgG,,)
Suppliers: ‘Sigma; *Dako; ‘Kallestad; 4Amersham; ‘ATCC.
with goat anti-human y-chain-specific antibodies conjugated (Sigma, Poole, UK) previously absorbed with rabbit IgG.
to alkaline
phosphatase
Western blotting Gel electrophoresis (SDS-PAGE) of purified anti-La from the mother (C) and from an unrelated patient (E) with Sjdgren’s syndrome and normal human pooled IgG was carried out under reducing conditions and analysed by immunoblotting as previously described [9]. Blots were probed with rabbit anti-idiotype and developed with goat anti-rabbit y-chain-specific peroxidase conjugate (ICN, High Wycombe, Bucks, UK). ImmunojIuorescence Six micron cryostat sections were air dried, washed once in phosphate buffered saline (PBS), pH 7.2 and fixed in acetone and methanol (1 :l, v/v) on ice. Thirty microlitres of antibody (10 pg/ml) per section were allowed to incubate for 1 h at room temperature. Following washing for 20 min with two further changes of PBS, sections were incubated with fluorochrome-conjugated antibodies (Table 1) for 30 min at room temperature. After subsequent washing in PBS, the slides were mounted in 90% glycerol/PBS containing p-phenylenediamine (0.1 mg/ml) and viewed with a Leitz Laborlux fluorescent microscope with a x 50 water immersion lens. Maternal antibodies were eluted from three cardiac sections by applying to each section 100 ~1 of PBS containing 5% goat serum (PBS-GS), followed by 100 ~10.2 M glycine containing 0.5 M NaCl, pH 2.5 or 100 l.d3 M guanidine hydrochloride containing 5% goat serum, followed by 100 ~1PBS-GS for 10 min each prior to aspiration by pipette. Control sections were incubated with three consecutive applications of PBSGS. After dialysis against PBS for 48 h with three buffer changes, fractions were tested for specificity by ELISA and the sections examined by immunofluorescence.
168 A. C. Horsfall et al. Table
2. Autoantibody
profiles in mother and CCHB anti-R0
Immunodiffusion’* ELI SA’# Western Blot’#
infant
anti-La
Mother
Baby
Mother
Baby
256 18 u/ml 52kD 60 kD
16 NA NA
64 37u/ml 47kD
4 NA NA
*Serum taken at time of delivery. #Post-partum serum. ‘Reciprocal tiue by immunodiffusion against human spleen extract as a source of Ro antigens and rabbit thymus extract as a source of La antigens. 2ELISA using purified bovine 60 kD Ro antigen (Cellon, Luxembourg) and rabbit thymus extract La. Results expressed as units/ml by reference to standard curves. 3Molecular weights of peptides identified by Western blotting using MOLT4 T cell and HeLa cell extracts. NA: not available.
Anti-La antibodies from the mother of the CCHB baby and anti-La and anti-R0 antibodies from a patient with Sjiigren’s syndrome and a history of uncomplicated pregnancy were purified by afhnity chromatography against purified 60 kD bovine Ro [ 121 and rabbit thymus extract La as previously described 191. All three antibody preparations were labelled with NHS-LC-Biotin (Pierce, Rockford, IL) [9]. Two mouse monoclonal anti-La antibodies (SW1 and SWS) and one mouse monoclonal anti-Sm antibody (KSm5) prepared in this laboratory were also used [ 13,141. Biotin labelled antibodies were visualized with streptavidin Fluorescein isothiocyanate (FITC) or streptavidin Texas Red. Non-conjugated antibodies were visualized with goat anti-mouse or rabbit y-chain specific antibodies conjugated to FITC as appropriate. Table 1 lists the probes, detection systems and epitopes recognized in immunofluorescent studies and the sources of reagents. Results
At the time of delivery, titres of antibodies to both the Ro and La antigens were lower in the baby than in maternal serum, suggesting deposition of both antibodies in the fetus (Table 2). Western blotting of post-delivery serum showed that anti-R0 antibodies reacted with both the 60 kD and 52 kD components of the Ro antigen. Characterization
of anti-idiotype
In direct ELISA the binding of human IgG to immobilized rabbit anti-idiotype showed that cross-reactive idiotypes were present on IgG in sera containing both antiRo and anti-La antibodies and in six sera containing anti-R0 antibodies alone. Idiotypes were not detected in sera lacking autoantibody activity or on IgG with specificity for other autoantigens such as dsDNA or Sm/nRNP. The immunizing idiotype (maternal IgG anti-La) showed the highest binding to anti-idiotype (Figure 1).
Antigen and idiotype in congenital complete heart block
169
80-
i p .z z 3
:
60-
0.. 2.
0
t
0
0,
anti-La+ anti-Ra+
anti- Laanti-Ro+
anti- Laanti - Ro-
Figure 1. Binding of human IgG to rabbit IgG anti-La anti-idiotype. ELISA plates were coated with rabbit IgG anti-idiotype or non-immune rabbit IgG (10 pg/ml) and incubated with human IgG (100 pg/ ml) purified from serum samples of patients with autoimmune disease or from healthy individuals (closed circles). Open circles represent IgG from a patient with rheumatoid arthritis (rheumatoid factor positive), a patient with systemic lupus erythematosus (anti-Sm positive) and a patient with overlap disease (antinRNP positive). All patients with antibodies to the Ro and La antigens had Sjogren’s syndrome. Results are expressed as the percentage binding of each IgG sample compared to the immunizing maternal IgG anti-La (*).
Western blotting revealed that the idiotypes were located on the heavy chain of the variable region of aflinity purified anti-La antibodies (Figure 2). Anti-idiotype did not significantly inhibit the binding of anti-La antibodies to the La antigen, suggesting that the idiotype was located outside the antigen binding site. Demonstration
of anti-La
antibodies and idiotype on myocardialfibres
Anti-idiotype showed linear surface binding to myocardial fibres in CCHB (Figure 3a, Table 3) which was inhibited by pre-incubation with purified maternal anti-La (Figure 3b). The staining pattern was identical using anti-human IgG (Table 3). No IgM or IgA could be demonstrated (data not shown). Anti-idiotype did not react with control fetal heart (Figure 3c, Table 3). Treatment of CCHB cardiac sections by guanidine hydrochloride but not by glycine, pH 2.5, released maternal anti-La antibodies which could be detected by ELISA (Figure 4). The failure of glycine to elute anti-La IgG suggests that these are high affinity antibodies. Levels of anti-R0 antibodies were low in all samples (E,, less than 0.100 by ELISA) with no significant increase in the sample eluted with guanidine hydrochloride (data not shown). Expression
of Ro and La antigens
on myocardialjibres
Immunofluorescence on HEp 2 cells confirmed that the two monoclonal anti-La antibodies reacted with the nucleus [13] as did the a@inity purified polyclonal
170
A. C. Horsfall et al.
67
-
43
-
30
-
20
-
I4 -
IgG
E
C
Figure 2. Immunoblot of human IgG probed with anti-idiotype. Anti-La antibodies were immunopurified from maternal serum (C) and from the serum of an unrelated patient with Sjogren’s syndrome (E). These and normal pooled human IgG lacking anti-La activity were electrophoresed under reducing conditions, blotted onto nitrocellulose and probed with rabbit anti-idiotype. Molecular weight markers are indicated on the left ( x 10m3kD).
antibodies (Figure 5a). Affinity purified human polyclonal anti-60 kD Ro antibody gave a filamentous cytoplasmic perinuclear staining. There was also some intranuclear and nucleolar fluorescence (Figure 5b). Both mouse monoclonal (SWl) and human polyclonal anti-La antibodies reacted with the surface of myocardial fibres in CCHB, suggesting expression of the La antigen on the membrane of these cells (Figure 3d, Table 3). The fluorescence became stronger, particularly with the purified human antibodies, after probing sections treated with guanidine hydrochloride. Weak cytoplasmic staining of control fetal heart was found with anti-La antibodies (Figure 3e, Table 3). In CCHB sections a second monoclonal anti-La antibody (SW5) did not react L. with the surface of myocardial fibres but showed weak cytoplasmic fluoresce&e (Figure 3f, Table 3). Monoclonal anti-Sm antibodies did not react with the surface of the myocardial fibres but did give nuclear staining of the lymphocytic infiltrating cells (data not shown). Human anti-60 kD Ro antibodies reacted with the cytoplasm of myocardial fibres in both CCHB and the control fetal heart. Strong surface fluorescence compatible with membrane expression of Ro was seen in the CCHB heart with cytoplasmic and possible weak surface fluorescence in the control heart (Figure 3g and 3h, Table 3).
Antigen and idiotype in congenital complete heart block
171
Table 3. Summary of immunojluorescent staining patterns in fetal heart sections CCHB heart Myocardial fibres Idiotype (anti-Id) Immunoglobulin (aIg) Antigen (a) La
Lymphocytic infiltrate -
-
+N +N +N+C +N +N +c +N
+c -
+++s ++s
Monoclonal SW 1 Monoclonal SW5 Maternal anti-La Non-maternal anti-La G.HCl elution fan&La Non-maternal anti-R0 (b) Ro Monoclonal KSm5 (c) Sm
++s SC +s+c +s ++s ++s++c -
Control heart
NT +s++c NT
Complement (aC3c)
+s
-
-
Class II (HLA DR)
+s
+s+c
-
S: surface, N: nuclear, C: cytoplasmic,NT: not tested.
Expression of class II and complement In common with other published reports, both immunoglobulin and complement components (C3c) could be demonstrated on myocardial fibres of the affected cardiac tissue (Table 3). Additionally, strong expression of MHC Class II antigens was observed on the surface of myocardial fibres and lymphocytes in CCHB but absent in control fetal heart (Figure 3i, Table 3).
Discussion This study has definitively demonstrated the expression of both the Ro and La antigens and anti-La antibody on the surface of myocardial fibres in fetal cardiac tissue with congenital complete heart block. We propose that anti-La antibodies are deposited in fetal cardiac tissue by binding to the La antigen expressed on the surface of myocardial fibres. Localization of maternal anti-La was detected by a novel approach using polyclonal anti-idiotypic antibodies and confirmed by eluting anti-La antibodies from cardiac sections. Furthermore, dissociation of antibodies from cardiac sections revealed La epitopes to which mouse monoclonal and human polyclonal anti-La antibodies were able to bind. By demonstrating specificity our data confirm and extend previous observations on IgG deposition in fetal hearts [ 15, 16,5]. In contrast to previous reports, no IgA or IgM deposits were found in the CCHB heart sections in this study [ 16,5]. Initially anti-R0 antibodies were thought to be important in the pathogenesis of CCHB based on the observations that all mothers of affected children had circulating
172
A. C. Horsfall et al.
Figure 3. Immunofluorescent photographs: (a) CCHB stained with anti-Id; (b) CCHB stained with anti-Id pre-incubated with purified maternal anti-La; (c) control fetal heart stained with anti-Id; (d) CCHB stained with monoclonal anti-La (SW 1); (e) control fetal heart stained with monoclonal anti-La; (fj CCHB stained with monoclonal anti-LA (SW5); (g) CCHB stained with anti-Ro; (h) control fetal heart stained with anti-Ro; (i) CCHB stained with antibody to HLA Class II (DR).
anti-Rio antibodies [2] and that titres of these antibodies were lower in affected infants consistent with deposition [6,7]. More recently, both anti-R0 and anti-La antibodies have been implicated in pathogenesis. A specific association of maternal antibodies
Antigen and idiotype in congenital complete heart block
173
600
500
400
8 w"
300
\ \ \ \ \ \ \ \
200
100
0
--_ 1st wash
_-2nd
3rd
wash
wash
Figure 4. Binding to La antigen of immunoglobulin eluted from CCHB cardiac sections measured by ELISA. Ordinate represents extinction at 405 nm x 10-3. Open circles linked by broken lines represent PBS washes of control fetal cardiac tissue. Closed circles linked by broken lines represent PBS washes of CCHB fetal cardiac tissue and closed circles linked by solid lines CCHB fetal cardiac tissue treated with 3 M guanidine hydrochloride in the second wash.
Figure 5. HEp 2 cells were stained with La and Ro specific antibodies affinity purified from the serum of a patient with Sjogren’s syndrome, biotinylated and detected with streptavidin FITC: (A) nuclear location of the La antigen and (B) cytoplasmic location of the Ro antigen.
for the human 52 kD Ro antigen has been reported and is particularly prevalent among maternal CCHB serum samples in combination with antibodies to the 48 kD La antigen [ 171.
174
A. C. Horsfall et al.
Our data is consistent with a role for anti-Rio as well as anti-La antibodies in the pathogenesis of CCHB. Because anti-idiotype appeared to bind anti-Rio, it is possible that some of the antibody detected on the surface of fibres had anti-R0 activity. Failure to detect anti-R0 antibodies in eluates from cardiac sections reflects the source of antigen used for ELISA (bovine 60 kD rather than human 52 kD). Anti52 kD Ro antibodies may well be present in eluates, but lack of cloned and/or purified human 52 kD Ro and specific antibodies imposed limitations on our study. Surface expression of the La antigen was detected using both polyclonal anti-La antibodies and the monoclonal antibody SWl. SW5 did not bind to the surface of CCHB myocardial fibres implying that the epitopes for this antibody, known to be different from SW1 [18], are blocked by maternal anti-La antibodies of the same epitope specificity. In favour of this interpretation was the finding that elution of the maternal immunoglobulin with anti-La activity increased the subsequent staining with SW5 and the polyclonal anti-La antibodies. In this study we found, in control fetal heart at 32 weeks’ gestation, that the Ro antigen was abundantly expressed in the cytoplasm, unlike the La antigen which was difficult to detect although normally located in the nucleus of resting cells. Previously we have shown that La is cytoplasmic in activated cells [ 131 and therefore one might expect that in a rapidly dividing tissue such as growing fetal heart the La antigen may also be cytoplasmic. In support of this was the finding of weak cytoplasmic fluorescence with one of the monoclonal anti-La antibodies. In a recent report we described the sequential expression of the La antigen in the nucleus, followed by the cytoplasm, and ultimately on the cell surface in virally infected cells [ 193. It is possible that a similar mechanism could operate in congenital heart block although whether the Ro antigen is similarly induced by viral infection is not known. However, our study shows that in normal fetal cardiac tissue at 32 weeks gestation La antigen was not expressed on the surface of the myocardial fibres. Thus the surface expression of the Ro and/or La antigens would be the critical event in causing maternal autoantibody binding and may account for the observation that only 5% of autoantibody positive patients have affected children [20]. Studies are now in progress to examine surface expression of these antigens in normal fetal tissue at different gestational times. Various factors have been implicated in CCHB, including the presence of maternal antibodies to Ro and La [6], previous history of CCHB births [21], Class II [22] and III [23] antigens and cytomegalovirus infection [24]. All these mechanisms are apparently contradicted by the study of HLA identical twins where both had circulating anti-Rio antibodies but only one developed CCHB. Lower titres of anti-R0 were found in the affected twin, implying deposition of anti-R0 [7]. In our study, both anti-R0 and anti-La antibodies were lower in the child than in maternal serum suggesting deposition of both antibodies. This does not negate the proposed hypothesis of surface expression of the La and/or Ro antigens following viral infection. Intra-uterine and maternally transmitted viral infections affecting one twin and not the other have been previously documented [25,26]. Whatever the explanation, it is clear that simply possessing anti-Rio and anti-La antibodies cannot be the sole requirement for the development of congenital heart block, as only 5% of antibody-positive patients have affected children [20]. Our demonstration of the Ro and La antigens and specific autoantibody on the surface
Antigen and idiotype in congenital complete heart block
of diseased fibres suggests that migration of these ribonucleoprotein constitutes an important part of the pathogenesis.
175
antigens
Acknowledgements
We are grateful to the Arthritis and Rheumatism Council, Great Britain, for financial support, to Drs J. B. Harley and K. K. Gaither, Oklahoma Medical Research Foundation, Oklahoma City, for affinity purification of anti-Rio antibodies from the serum of one of our patients, to Dr E. Chan, Scripps Clinic, La Jolla, for immunoblots of 52 kD Ro antigen, to our Departmental colleagues, Drs D. G. Williams and P. R. Smith, for preparing monoclonal anti-La antibodies and to Drs A. King and G. Batcup for providing clinical specimens from the heart block family. References 1. Chameides, L., R. C. Truex, V. Vetter, W. J. Rashkind, F. M. Galioto Jr, and J. A. Noonan. 1977. Association of maternal lupus erythematosus with congenital complete heart block. N. Eng1.J. Med. 297: 1204-1207 2. Scott, J. S., P. J. Maddison, P. V. Taylor, E. Esscher, 0. Scott, and R. P. Skinner. 1983. Connective tissue disease, antibodies to ribonucleoprotein and congenital heart block. N. England. J. Med. 309: 209-2 12 3. Taylor, I?. V. and J. S. Scott. 1986. Pathogenic effects of maternal antibody in isolated complete heart block. Presented at 6th International Congress of Immunology, Toronto, abstract. p. 437 4. Guarnieri, T., S. DaTorre, S. Myers, D. Brezinski, T. Provost, and E. Alexander. 1987. Antibody mediated heart block in neonatal lupus: an experimental model of differential age susceptibility. CircuZation 76(Suppl. IV): abstract 0319 5. Taylor, P. V., J. S. Scott, L. M. Gerlio, E. Esscher, and 0. Scott. 1986. Maternal antibodies against fetal cardiac antigens in congenital complete heart block. N. Engl. J. Med. 315: 667-672 6. Taylor, P. V., K. F. Taylor, A. Norman, S. Griffiths, and J. S. Scott. 1988. Prevalence of maternal Ro (SS-A) and La (SS-B) autoantibodies in relation to congenital heart block. Br.J. Rheumatol. 27: 128-132 7. Harley, J. B., J. L. Kaine, 0. F. Fox, M. Reichlin, and H. Gruber. 1985. Ro (SS-A) antibody and antigen in a patient with congenital complete heart block. Arthritis Rheum. 28: 1321-1325 8. Venables,P. J. W.,P. J. Charles,R. R. C. Buchanan, T. Y&P. A. Mumford, L. Schrieber, G. R. W. Room, and R. N. Maini. 1983. Quantitation and detection of isotypes of anti-SSB antibodies by ELISA and Farr assays using affinity purified antigens. An approach to the investigation of Sj6gren’s syndrome and systemic lupus erythematosus. Arthritis Rheum. 26: 143-152 9. Horsfall, A. C., C. M. Brown, and R. N. Maini. 1987. Purification of human autoantibodies from cross-linked antigen immunosorbents. J. Zmmunol. Meth. 104: 43-49 10. Horsfall, A. C., P. J. W. Venables, P. A. Mumford, and R. N. Maini. 1986. Restricted idiotypes on antibodies to the La (SS-B) antigen. Clin. Exp. Zmmunol. 63: 393-401 11. Plater-Zyberk, C., M. F. Clarke, K. Lam, P. A. Mumford, G. R. W. Room, and R. N. Maini. 1983. In vitro immunoglobulin synthesis by lymphocytes from patients with rheumatoid arthritis. I Effect of monocyte depletion and demonstration of an increased proportion of lymphocytes forming rosettes with mouse erythrocytes. Clin. Exp. Zmmunol. 52: 505-511 12. Gaither, K. K., 0. F. Fox, H. Yamagata, H. J. Mamula, M. Reichlin, and J. B. Harley. 1987. Implications of anti-Ro/SjGgren’s syndrome A antigen autoantibody in normal sera for autoimmunity. J. Clin. Invest. 79: 841-846
176
A. C. Horsfall
et al.
13. Smith, P. R., D. G. Williams, P. J. W. Venables, and R. N. Maini. 1985. Monoclonal antibodies to the Sjogren’s syndromeassociated antigen SS-B (Laj.3. Immunol. Meth. 77: 63-76
14. Williams, D. G., M. R. Stocks, P. R. Smith, and R. N. Maini. 1986. Murine lupus monoclonal antibodies deiine five epitopes on two different Sm polypeptides. fmmunology 58: 495-500 15. Lee, L. A., S. Coulter, S. Erner, and H. Chu. 1987. Cardiac immunoglobulin deposition in congenital heart block associated with maternal anti-R0 antibodies. Am. 3. Med. 83: 793-796
16. Litsey, S. E., J. A. Noonan, W. N. O’Connor, C. M. Cottrill, and B. Mitchell. 1985. Maternal connective tissue disease and congenital heart block: demonstration of immunoglobulin in cardiac tissue. N. EngZ.3. Med. 312: 98-100 17. Buyon, J. P., E. Ben-Chetrit, S. Karp, R. A. S. Roubey, L. Pompeo, W. H. Reeves, E. M. Tan, and R. Winchester. 1989. Acquired congenital heart block. Pattern of maternal antibody response to biochemically defined antigens of the SSA/Ro-SSB/La system in neonatal lupus. 3. Clin. Invest. 84: 627-634 18. Smith, P. R. 1986. Characterisation of the Sjogren’s syndrome antigens Ro and La. PhD Thesis. University of London, UK 19. Baboonian, C., P. J. W. Venables, J. Booth, D. G. Williams, L. M. Roffe, and R. N. Maini. 1989. Virus infection induces redistribution and membrane localisation of the nuclear antigen La (SS-B): a possible mechanism for autoimmunity. CZin. Exp. Immunol. 78: 454-459
20. Ramsey-Goldman, R., D. Horn, J.-S. Deng, G. C. Ziegler, L. E. Kahl, V. D. Steen, R. E. LaPorte, and T. A. Medsger. Jr. 1986. Anti-SS-A antibodies and fetal outcome in maternal systemic lupus erythematosus. Arthritis Rheum. 29: 1269-1273 21. Buyon, J., R. Roubey, S. Swersky, L. Pompeo, A. Parke, L. Baxi, and R. Winchester. 1988. Complete congenital heart block: risk of occurrence and therapeutic approach to prevention. 3. Rheumatol. 15: 1104-l 108 22. Watson, R. M., A. T. Lane, N. K. Barnett, W. B. Bias, F. C. Arnett, and T. T. Provost. 1984. Neonatal lupus erythematosus: A clinical, serological and immunogenetic study with review of the literature. Medicine (Baltimore) 63: 362-678 23. Arnaiz-Villena, A., J. J. Vazquez-Rodriguez, J. L. Vicario, P. Lavilla, D. Pascual, F. Moreno, and J. Martinez-Laso. 1989. Congenital heart block immunogenetics. Evidence of an additional role of HLA Class III antigens and independence of Ro antibodies. Arthritis
Rheum.
32: 1421-1426
24. Lewis, P. E., R. C. Cefalo, and A. L. Zaritsky. 1980. Fetal heart block caused by cytomegalovirus. Am.3. Obstet. Gynecol. 136: 967-968 25. Cooper, L. Z. 1985. The history and medical consequences of rubella. Rev. Infect. Dis. 7(Suppl.): 2-15 26. Park, C. L., H. Streicher, and R. Rothberg. 1987. Transmission of human immunodeficiency virus from parents to only one dizygotic twin.3. CZin. Microbial. 25: 1119-l 121
