One mechanism to molecularly explain the strong association of maternal anti-Ro60 Abs with cardiac disease in neonatal lupus (NL) is that these Abs initiate injury by binding to apoptotic cardiomyocytes in the fetal heart. Previous studies have demonstrated that β2-glycoprotein I (β2GPI) interacts with Ro60 on the surface of apoptotic Jurkat cells and prevents binding of anti-Ro60 IgG. Accordingly, the current study was initiated to test two complementary hypotheses, as follows: 1) competition between β2GPI and maternal anti-Ro60 Abs for binding apoptotic induced surface-translocated Ro60 occurs on human fetal cardiomyocytes; and 2) circulating levels of β2GPI influence injury in anti-Ro60–exposed fetuses. Initial flow cytometry experiments conducted on apoptotic human fetal cardiomyocytes demonstrated dose-dependent binding of β2GPI. In competitive inhibition experiments, β2GPI prevented opsonization of apoptotic cardiomyocytes by maternal anti-Ro60 IgG. ELISA was used to quantify β2GPI in umbilical cord blood from 97 neonates exposed to anti-Ro60 Abs, 53 with cardiac NL and 44 with no cardiac disease. β2GPI levels were significantly lower in neonates with cardiac NL. Plasmin-mediated cleavage of β2GPI prevented binding to Ro60 and promoted the formation of pathogenic anti-Ro60 IgG-apoptotic cardiomyocyte complexes. In aggregate these data suggest that intact β2GPI in the fetal circulation may be a novel cardioprotective factor in anti-Ro60–exposed pregnancies.

The identification of isolated congenital heart block in utero during the mid to late second trimester is almost universally associated with maternal Abs to a component of the SSA/Ro-SSB/La ribonucleoprotein complex, even in asymptomatic women. The cardiac disease of neonatal lupus (cardiac NL), although typically characterized by fibrosis of the atrioventricular node, can extend to the working myocardium and endocardium (1). Although the accessibility of maternal Ab to a normally sequestered intracellular Ag has been difficult to reconcile, apoptosis has been proposed as a cellular event that promotes the translocation of Ro and La proteins to the cell surface and binding by cognate Abs (2, 3). This notion led to the observation that healthy cardiomyocytes are capable of engulfing apoptotic cardiomyocytes and that binding of anti-Ro/La Abs to the apoptotic cardiomyocytes inhibits this physiologic process (4). Histological studies support the in vitro findings because hearts from fetuses dying with cardiac NL reveal exaggerated apoptosis, whereas apoptosis is rarely detected in healthy hearts from electively terminated age-matched fetuses (5).

The direct pathogenicity of maternal anti-Ro60 Abs has been questioned because cardiac NL occurs in only 2% of neonates born to mothers with the candidate Abs (1). Although Abs appear necessary, it is likely that fetal and environmental factors amplify the Ab effect to promote full expression of disease. A focus on β2-glycoprotein I (β2GPI) as a candidate fetal factor is supported by two recent observations, as follows: 1) Ro60 expressed on the surface of apoptotic Jurkat cells interacts with β2GPI; and 2) preincubation of the apoptotic cells with β2GPI significantly blocks the binding of anti-Ro60 Abs (6, 7). β2GPI is an abundant, positively charged protein composed of five short consensus repeats with a lysine patch adjacent to a hydrophobic C-terminal loop (residues 313–316) in domain V (8). Significantly lower levels of circulating β2GPI were reported in umbilical cord plasma compared with adult plasma (9), which may be relevant to the clinical observation that the maternal heart is not affected despite continuous exposure to the identical Abs. β2GPI has been implicated in the modulation of coagulation and fibrinolysis pathways (10) and is regulated by plasmin, which proteolytically cleaves β2GPI domain V (11, 12), the putative site for binding by Ro60 (6). Of further relevance to the pathogenesis of cardiac NL, the binding of anti-Ro60 IgG to apoptotic cardiomyocytes was recently shown to enhance the activity of urokinase plasminogen activator (uPA), which catalyzes the conversion of plasminogen to plasmin (13). This may in turn result in an amplification cycle whereby anti-Ro60 binding results in increased plasmin generation, cleavage of β2GPI, and further uncompeted binding by pathogenic Ab.

Accordingly, this study was initiated to evaluate the hypothesis that in utero levels of β2GPI influence pregnancy outcome in anti-Ro60–positive mothers. The relevance of the Ro60–β2GPI interaction to the pathogenesis of cardiac NL was approached by using the target cell, human fetal cardiomyocytes, and affinity-purified anti-Ro60 IgG from a mother of an affected child. The levels of β2GPI were measured by ELISA in umbilical cord blood from affected and unaffected neonates, each exposed to maternal anti-Ro60 Abs. The Ro60–β2GPI binding site was then mapped to determine whether cleavage of β2GPI by plasmin affects binding to Ro60.

Umbilical cord serum or plasma from neonates of anti-Ro60–positive mothers and serum from mothers were obtained, with informed consent. Pregnancies resulting in cardiac NL or no cardiac manifestations were identified from the Research Registry for Neonatal Lupus (14) and PR Interval and Dexamethasone Evaluation (15). Each database has Institutional Review Board approval for evaluation of deidentified information. Maternal serum and cord blood contained Abs reactive with Ro60 and/or Ro52 and La, as measured by a New York University Clinical Laboratory Improvement Amendments–approved laboratory. Total IgG and affinity-purified Abs against rRo60 (16) were prepared from maternal serum (4). Anti-β2GPI Abs were measured in maternal sera by QUANTA Lite β2GPI IgG ELISA (INOVA Diagnostics, San Diego, CA).

Healthy fetal hearts of gestational ages 16–24 wk were obtained after elective termination and receiving consent from the mothers through B. Poulos at the Human Fetal Tissue Repository of Albert Einstein College of Medicine in accordance with the guidelines of the Institute Review Board of New York University School of Medicine. Cells were isolated and cultured, as described (4). Apoptosis was induced with 1 μg/ml staurosporine for 4 h in serum-free DMEM or by treating cardiomyocytes with IFN-γ for 24 h and plating on poly–2-hydroxyethyl methacrylate in the presence of IFN-γ, TNF-α, and cycloheximide (4). Early and late apoptotic Jurkat cells were prepared, as described (17). Apoptosis was confirmed by flow cytometric analysis of phosphatidylserine exposure (annexin V binding).

Native purified human β2GPI (Haematologic Technologies, Essex Junction, VT) was added to apoptotic cardiomyocytes or Jurkat cells for 30 min at room temperature, washed, and detected by either polyclonal rabbit anti-β2GPI antiserum or anti-β2GPI domain I mAb (a gift of S. Krilis, University of New South Wales) (18) by flow cytometry (6). A proteolytically clipped form of β2GPI was generated with human plasmin (Sigma-Aldrich, St. Louis, MO) (19), and binding to apoptotic cells was determined (6). In competitive inhibition experiments, increasing concentrations of β2GPI (0.5–50 μg/ml) or IgG-depleted cord blood (diluted 1/10) were coincubated with 10 μg/ml affinity-purified anti-Ro60 IgG or healthy control IgG in the presence or absence of plasmin (0.2–3 μg/ml), and binding was assessed with an anti-human IgG-FITC by flow cytometry (17). To confirm the specificity of the β2GPI-mediated inhibition of anti-Ro60 IgG binding to apoptotic cardiomyocytes, recombinant human growth arrest-specific 6 (GAS6) or milk fat globule epidermal growth factor 8 (MFG-E8) was used in competitive inhibition experiments, as described above. Binding of GAS6 and MFG-E8 to apoptotic cardiomyocytes was detected with an anti–penta-His mAb (Qiagen) that recognizes the C-terminal 6-His tag in both proteins.

β2GPI levels in umbilical cord serum or plasma were determined by a capture ELISA adapted from McNally et al. (20). Briefly, rabbit polyclonal anti-β2GPI antiserum diluted 1/2000 in PBS was coated onto 96-well Nunc Maxisorp microtiter plates. All incubations were conducted at 37°C for 1 h. Wells were blocked with 2% BSA/PBS and then washed three times with PBS containing 0.05% Tween 20. A standard curve was constructed with known concentrations of purified human β2GPI. In some experiments, plasmin-cleaved β2GPI was added to ELISA plates. Umbilical cord serum or plasma was diluted 1/1000, 1/2000, and 1/4000 in 1% BSA/PBS. After extensive washing, anti-β2GPI mAb was applied and detected with alkaline phosphatase-conjugated anti-mouse IgG (Sigma-Aldrich).

To confirm ELISA findings, a panel of nine umbilical cord bloods, five from neonates affected by cardiac NL and four noncardiac NL neonates, was subjected to SDS-PAGE under nonreducing conditions and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat skim milk powder in PBS overnight at 4°C, washed three times in PBS/0.1% Tween 20, and probed with either rabbit anti-β2GPI antiserum (1/2000) or mAb (0.5 μg/ml) for 1 h at room temperature. Membranes were stained with IRDye 800CW anti-rabbit or anti-mouse IgG conjugates and analyzed on the Odyssey Infrared Imager (LI-COR Biosciences).

Soluble overlapping fragments of mouse Ro60 encompassing aa 82–244, 82–146, 82–192, 149–244, and 193–236 expressed as maltose-binding protein (MBP) fusion constructs were prepared from pMAL cDNAs (a gift of K. Kaufman, Oklahoma Medical Research Foundation) (21). All recombinant proteins were biotinylated with an amine reactive biotinylation reagent (Pierce, Rockford, IL), according to the manufacturer’s instructions. Binding of biotinylated MBP-Ro60 aa 82–244, 82–146, 82–192, 149–244, or 193–236 to native or plasmin-cleaved β2GPI was assessed by ELISA (6).

A homology model of the three-dimensional structure of human Ro60 was constructed based on the crystal structure of Xenopus laevis Ro60 using the Swiss Model Program (22). Molecular visualization and hypothetical docking experiments of Ro60 and the crystal structure of human β2GPI were performed using Swiss Pdb Viewer (23) and manual docking of the structures based on experimental mapping data, surface charge, and stereochemical complementarity.

Dichotomous variables (sex, race, maternal Ab status, dexamethasone use, and delivery method) between cardiac NL and noncardiac NL neonates were compared by Fisher’s exact test. Continuous variables (birth weight, gestational age, titers of anti-Ro60 Abs, and levels of β2GPI) were compared in neonates with cardiac NL and noncardiac NL by the unpaired t test. Levels of β2GPI in umbilical cord blood from twins were compared by paired t test. Multivariate regression analysis was conducted, including cardiac NL, dexamethasone use, and delivery method as predictor variables in the model. Two-sided p values <0.05 were considered statistically significant.

To test the hypothesis that β2GPI may be a protective factor in cardiac NL by preventing the formation of pathogenic anti-Ro60 IgG-apoptotic cardiomyocyte complexes in the fetal heart, initial experiments assessed the binding of β2GPI to apoptotic human fetal cardiomyocytes. Apoptosis was induced either by staurosporine (intrinsic pathway) or a loss of anchorage in the presence of IFN-γ, TNF-α, and cycloheximide (extrinsic pathway). The latter treatment induced annexin V positivity consistent with apoptosis in 75% of the cell population, of which 38 ± 5% were considered early apoptotic (propidium iodide [PI] negative) and 37 ± 13% late apoptotic (PI positive) (Fig. 1A). Staurosporine treatment generated ∼66% annexin V–positive cells, of which 29 ± 18% and 37 ± 4% were considered early and late apoptotic, respectively. Purified native human β2GPI bound to both early and late apoptotic cardiomyocytes in a saturable and dose-dependent manner, with a 2-fold increase in binding to late apoptotic cells compared with the early apoptotic population (Fig. 1B). Affinity-purified anti-Ro60 IgG from a mother of an infant with cardiac NL bound to both early and late apoptotic cardiomyocytes, with enhanced binding to the late apoptotic population. Increasing the concentration of β2GPI revealed a dose-dependent inhibition of anti-Ro60 IgG binding to early and late apoptotic cardiomyocytes (Fig. 1C). These findings confirm that β2GPI competes with anti-Ro60 IgG for binding to the surface of apoptotic cells and extends the observation to human fetal cardiomyocytes. To address the specificity of the Ro60–β2GPI interaction on apoptotic cardiomyocytes, competitive inhibition experiments were repeated using GAS6 or MFG-E8, two molecules that also bind apoptotic cells (24, 25). Neither GAS6 nor MFG-E8 altered anti-Ro60 IgG binding to apoptotic cardiomyocytes (Fig. 1D). Binding of GAS6 and MFG-E8 to apoptotic cardiomyocytes was confirmed with an anti–penta-His mAb (data not shown).

FIGURE 1.

Purified native human β2GPI binds to apoptotic human fetal cardiomyocytes and inhibits binding of maternal anti-Ro60 IgG. A, Apoptotic cardiomyocyte populations were gated based on annexin V and PI staining. Annexin V–positive, PI-negative cells (quadrant, Q4) were termed early apoptotic, and annexin V/PI–positive cells (Q2) were late apoptotic. B, β2GPI bound both early and late populations in a dose-dependent and saturable manner. C, Increasing concentrations of β2GPI inhibited the binding of anti-Ro60 IgG to both early and late apoptotic cardiomyocyte populations. D, The inhibition of anti-Ro60 IgG binding to apoptotic cardiomyocytes is specific for β2GPI as GAS6 and MFG-E8 do not alter Ab binding.

FIGURE 1.

Purified native human β2GPI binds to apoptotic human fetal cardiomyocytes and inhibits binding of maternal anti-Ro60 IgG. A, Apoptotic cardiomyocyte populations were gated based on annexin V and PI staining. Annexin V–positive, PI-negative cells (quadrant, Q4) were termed early apoptotic, and annexin V/PI–positive cells (Q2) were late apoptotic. B, β2GPI bound both early and late populations in a dose-dependent and saturable manner. C, Increasing concentrations of β2GPI inhibited the binding of anti-Ro60 IgG to both early and late apoptotic cardiomyocyte populations. D, The inhibition of anti-Ro60 IgG binding to apoptotic cardiomyocytes is specific for β2GPI as GAS6 and MFG-E8 do not alter Ab binding.

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Having established in vitro proof-of-concept for the protective β2GPI hypothesis, translation to pathogenesis was sought by measurement of circulating β2GPI levels in affected and unaffected neonates exposed to maternal anti-Ro60 Abs. Because it is not feasible to obtain fetal blood during the critical time of heart injury (18–24 wk gestation), umbilical cord blood was used as a proxy to assess the potential influence of circulating β2GPI levels in the pathogenesis of disease. Umbilical cord blood [serum or plasma was previously reported to have no differences in β2GPI levels (20, 26)] from 97 anti-Ro60–exposed infants was studied, 53 with cardiac NL and 44 with no cardiac manifestations (Table I). Of the 53 affected children, 49 had congenital heart block (47, third degree; 2, second degree), 3 had an isolated cardiomyopathy, and 1 had sustained sinus bradycardia. Of the noncardiac NL neonates (many of whom had affected siblings), 38 were completely healthy, 5 had cutaneous NL, and 1 had hepatic/hematological manifestations of NL. The demographic characteristics, mother’s Ab status and medication, method of delivery, and infant birth weight and gestational age are shown in Table I. The sex, race, maternal Ab profile, and titer of anti-Ro60 Ab were not significantly different between affected and unaffected neonates. As expected, mothers of children with cardiac NL were more likely to have taken dexamethasone and deliver by C-section (p = 0.0001). Cardiac NL children were more frequently born prematurely and of lower birth weight than the noncardiac NL controls (p = 0.007).

Table I.
Clinical and demographic characteristics of anti-Ro60–exposed neonates with cardiac NL compared with those without cardiac NL
Cardiac NLa (N = 53)Noncardiac NLb (N = 44)p Value
Sex of child   0.22 
 Male 24 (45%) 25 (57%)  
 Female 29 (55%) 17 (39%)  
 NA 0 (0%) 2 (4%)  
Race/Ethnicity   0.78 
 White 43 (81%) 36 (82%)  
 Black 3 (6%) 3 (7%)  
 Hispanic 1 (2%) 4 (9%)  
 Asian 4 (7%) 1 (2%)  
 Other/NA 2 (4%) 0 (0%)  
Ab status   0.68 
 Anti-Ro+/La+ 29 (55%) 27 (61%)  
 Anti-Ro+/La 24 (45%) 17 (39%)  
Medication    
 Dexamethasone 26 (49%) 3 (7%) 0.0001 
Delivery    
 C-section 37 (70%) 10 (23%) 0.0001 
 Vaginal 3 (6%) 16 (36%)  
 NA 13 (24%) 18 (41%)  
Birth weight (g) 2682 (±647)c 3124 (±572)d 0.007 
Gestational age (wk) 37 (±2)e 38 (±2)f 0.018 
Cardiac NLa (N = 53)Noncardiac NLb (N = 44)p Value
Sex of child   0.22 
 Male 24 (45%) 25 (57%)  
 Female 29 (55%) 17 (39%)  
 NA 0 (0%) 2 (4%)  
Race/Ethnicity   0.78 
 White 43 (81%) 36 (82%)  
 Black 3 (6%) 3 (7%)  
 Hispanic 1 (2%) 4 (9%)  
 Asian 4 (7%) 1 (2%)  
 Other/NA 2 (4%) 0 (0%)  
Ab status   0.68 
 Anti-Ro+/La+ 29 (55%) 27 (61%)  
 Anti-Ro+/La 24 (45%) 17 (39%)  
Medication    
 Dexamethasone 26 (49%) 3 (7%) 0.0001 
Delivery    
 C-section 37 (70%) 10 (23%) 0.0001 
 Vaginal 3 (6%) 16 (36%)  
 NA 13 (24%) 18 (41%)  
Birth weight (g) 2682 (±647)c 3124 (±572)d 0.007 
Gestational age (wk) 37 (±2)e 38 (±2)f 0.018 

Birth weight and gestational age are presented as mean ± SD. All other data are reported as N (%).

a

Third degree block (n = 47); second degree (n = 2); cardiomyopathy (n = 3); sinus bradycardia (n = 1).

b

Rash (n = 5); liver complications (n = 1).

c

n = 36.

d

n = 29.

e

n = 44.

f

n = 31.

β2GPI levels as assessed by ELISA were significantly lower in the neonates with cardiac NL (133.6 ± 7.6 μg/ml) compared with those without cardiac NL (193.5 ± 10.6 μg/ml) (p < 0.0001) (Fig. 2A). Dexamethasone use and delivery status were also significantly associated with β2GPI (p = 0.05, p = 0.01, respectively). Gender, race, maternal Ab status, birth weight, and gestational age were not significantly correlated with β2GPI. In a multivariable regression analysis that included cardiac NL status, dexamethasone use, and delivery method as predictor variables in the model, β2GPI levels remained lower in cardiac NL compared with noncardiac NL (p = 0.06). However, the effects of dexamethasone use and delivery method were substantially diminished and no longer significantly associated with β2GPI levels after adjusting for cardiac NL status (p = 0.53 and p = 0.40, respectively). These results are most likely explained by the fact that dexamethasone is generally only given to treat cardiac NL once identified, not for prophylaxis, and C-section is more common for delivery of a baby with cardiac NL than an otherwise healthy baby. Four twin pairs discordant for cardiac NL were available for study (2 monozygotic and 2 dizygotic) and proved to be highly informative. In each pair, the twin affected by cardiac NL had lower levels of β2GPI than the unaffected twin (p = 0.04) (Fig. 2A). In the noncardiac NL group, there were no significant differences between β2GPI levels in the children affected by rash or those without any manifestation of NL (data not shown). Titers of anti-Ro60 Abs were equivalent in the sera obtained at the time of delivery from mothers of children with cardiac NL (23,932 ± 4,647, n = 47) compared with noncardiac NL (27,138 ± 6,623 n = 40) (p = 0.69) and in the umbilical cord bloods from neonates with cardiac NL (11,814 ± 2,021, n = 47) versus noncardiac NL neonates (14,286 ± 2,829, n = 39) (p = 0.47).

FIGURE 2.

β2GPI levels are lower in anti-Ro60–exposed neonates with cardiac NL compared with noncardiac NL. A, β2GPI concentration, measured by ELISA, in umbilical cord blood from children affected by cardiac NL (n = 53) and noncardiac NL controls (n = 44). Solid horizontal lines represent mean β2GPI concentration. Dashed lines connect β2GPI levels in umbilical cord blood from twins discordant for cardiac NL. B, Immunoblot analysis of a panel of nine umbilical cord blood samples (four noncardiac NL and five cardiac NL) probed with anti-β2GPI Ab were quantitatively consistent with those measured by ELISA.

FIGURE 2.

β2GPI levels are lower in anti-Ro60–exposed neonates with cardiac NL compared with noncardiac NL. A, β2GPI concentration, measured by ELISA, in umbilical cord blood from children affected by cardiac NL (n = 53) and noncardiac NL controls (n = 44). Solid horizontal lines represent mean β2GPI concentration. Dashed lines connect β2GPI levels in umbilical cord blood from twins discordant for cardiac NL. B, Immunoblot analysis of a panel of nine umbilical cord blood samples (four noncardiac NL and five cardiac NL) probed with anti-β2GPI Ab were quantitatively consistent with those measured by ELISA.

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To substantiate the ELISA findings regarding β2GPI concentration and to investigate functional relevance, a subset of umbilical cord blood from four unaffected controls and five cardiac NL children was selected for further analysis. Immunoblot results were consistent with those of the ELISA in that the density of bands was lower in the five umbilical cord bloods from the neonates with cardiac NL compared with the four bloods from the healthy neonates (Fig. 2B). Cord blood from an unaffected control (β2GPI = 367 μg/ml) and a neonate with cardiac NL (β2GPI = 154 μg/ml) was selected for competitive inhibition experiments to support the protective effects of higher circulating levels of β2GPI. Prior to the experiment, IgG was removed from each cord blood by protein A to eliminate the effect of endogenous maternal anti-Ro60 Abs. As assessed by flow cytometery, exogenously added affinity-purified anti-Ro60 IgG bound at higher levels to apoptotic cardiomyocytes in the presence of cardiac NL cord blood compared with an equal quantity of cord blood from the healthy neonate (data not shown).

It is plausible that concomitant levels of maternal anti-β2GPI Abs may impede the protective function of β2GPI and account for the lower levels of β2GPI measured in umbilical cord blood from neonates with cardiac NL. To test this consideration, 40 maternal sera obtained at the time of birth (20 cardiac NL, 17 noncardiac NL, and 3 discordant twins) were available for evaluation of Abs to β2GPI. Of 14 tested in which the associated cord blood level of β2GPI was low (<150 μg/ml), only 1 had anti-β2GPI Abs. Overall, only 2 of the 40 were positive for anti-β2GPI Abs (1 cardiac NL and 1 noncardiac NL). These data suggest that maternal anti-β2GPI Abs are not associated with a loss of the protective function of β2GPI or the low levels of β2GPI observed in the cord bloods.

Molecular studies were conducted to assess the β2GPI–Ro60 interaction and factors that may affect this interaction and ultimately generate cardiac vulnerability. β2GPI was previously shown to bind within Ro60 aa 82–244, an immunodominant region of the molecule (22, 27). To more precisely map the β2GPI binding site on Ro60, soluble rMBP fusion proteins expressing fragments of Ro60 within aa 82–244 were used in direct binding experiments. β2GPI bound MBP-Ro60 aa 82–192 and aa 82–146, but not MBP-Ro60 aa 149–244 or aa 193–236 or MBP control (Fig. 3A). These results indicate that β2GPI binding occurs predominantly within Ro60 aa 82–146, an α helical region of the protein that forms a distinct cleft containing clusters of hydrophobic residues interspaced by acidic amino acids (Fig. 3A). To map the β2GPI binding site on Ro60 expressed on the surface of apoptotic cells, flow cytometry competitive inhibition experiments were performed with β2GPI and the Ro60 subfragments. MBP-Ro60 aa 82–192 and MBP-Ro60 aa 82–146 reduced the binding of β2GPI to late apoptotic Jurkat cells by 35.3 ± 2.7% and 18.8 ± 0.5%, respectively, whereas MBP-Ro60 aa 149–244 and aa 193–236 had a negligible effect on β2GPI binding (6.1 ± 0.3% and 6.5 ± 3.4% inhibition, respectively). These data support Ro60 aa 82–146 as being the predominant β2GPI binding region. The 2-fold greater inhibition observed with MBP-Ro60 aa 82–192 compared with the truncated MBP-Ro60 aa 82–146 suggests that β2GPI may favor the conformation presented by the larger Ro60 fragment.

FIGURE 3.

The Ro60 binding site on β2GPI is within the plasmin cleavage site, Lys317-Thr318. A, The β2GPI binding site on Ro60 was measured by ELISA using rMBP expressing various regions of Ro60. Ro60 fragments encompassing aa 82–244, aa 82–192, and aa 82–146 bound to purified native human β2GPI compared with MBP-Ro60 aa 149–244, aa 193–236, and MBP control. Values are the mean ± SD of triplicate determinations (n = 3). B, Molecular surface models of Ro60 and β2GPI with the β2GPI fifth domain oriented toward a cleft in Ro60 formed by the mapped aa 82–146 region. Surface colors are calculated using a coulombic algorithm for electrostatic potential in which blue is positive charge, white is neutral, and red is negatively charged. C, The binding of β2GPI to apoptotic cardiomyocytes is abrogated by plasmin cleavage. D, Plasmin reverses the β2GPI-mediated inhibition of anti-Ro60 binding to apoptotic cells. Representative flow cytometry dot plots depicting the binding of anti-Ro60 IgG (green) to late apoptotic cells (n = 5).

FIGURE 3.

The Ro60 binding site on β2GPI is within the plasmin cleavage site, Lys317-Thr318. A, The β2GPI binding site on Ro60 was measured by ELISA using rMBP expressing various regions of Ro60. Ro60 fragments encompassing aa 82–244, aa 82–192, and aa 82–146 bound to purified native human β2GPI compared with MBP-Ro60 aa 149–244, aa 193–236, and MBP control. Values are the mean ± SD of triplicate determinations (n = 3). B, Molecular surface models of Ro60 and β2GPI with the β2GPI fifth domain oriented toward a cleft in Ro60 formed by the mapped aa 82–146 region. Surface colors are calculated using a coulombic algorithm for electrostatic potential in which blue is positive charge, white is neutral, and red is negatively charged. C, The binding of β2GPI to apoptotic cardiomyocytes is abrogated by plasmin cleavage. D, Plasmin reverses the β2GPI-mediated inhibition of anti-Ro60 binding to apoptotic cells. Representative flow cytometry dot plots depicting the binding of anti-Ro60 IgG (green) to late apoptotic cells (n = 5).

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In a previous study, Ro60 was shown to bind β2GPI via its fifth domain (aa 244–326) (6), a structurally distinct part of the molecule defined by a lysine-rich patch and hydrophobic loop. To predict potential Ro60 binding sites within domain V of β2GPI, hypothetical docking experiments using a previously reported homology model of human Ro60 (22) and human β2GPI (8) were performed using Swiss PdbViewer. Kyte-Doolittle plots reflected a high degree of charge complementarity in the primary sequences of Ro60 aa 82–146 and β2GPI domain V. More specifically, manual docking taking into account both conformation complementarity and electrostatics indicated a potential surface interaction between the relatively acidic cleft formed by the mapped Ro60 aa 82–146 and a basic region adjacent to the β2GPI domain V hydrophobic loop (Fig. 3B). Interestingly, the Ro60 binding site on β2GPI was within the plasmin cleavage site, Lys317-Thr318. This suggests that proteolytic cleavage of β2GPI by plasmin may abrogate binding to Ro60 and promote the formation of pathogenic anti-Ro60 apoptotic cardiomyocyte complexes. Precedent for this possibility is that proteolytic cleavage of β2GPI by plasmin prevents binding to phospholipids (11).

To determine whether plasmin cleavage of β2GPI results in a loss of specificity for Ro60 on apoptotic cardiomyocytes, a plasmin-cleaved form of β2GPI was prepared (19) and binding to Ro60 was assessed. rMBP-Ro60 bound to immobilized native β2GPI by ELISA (OD 1.0 ± 0.02 compared with MBP control 0.1 ± 0.01), but did not bind to the plasmin cleaved β2GPI (OD 0.1 ± 0.02). Flow cytometry experiments confirmed that plasmin cleavage of β2GPI resulted in reduced binding to Ro60 on the apoptotic cardiomyocytes (Fig. 3C). To determine the effect of plasmin-cleaved β2GPI on the opsonization of apoptotic cells by anti-Ro60 Ab, maternal anti-Ro60 IgG was coincubated with either native or cleaved β2GPI and added to late apoptotic cardiomyocytes. To assess whether plasmin reverses the protective effect of β2GPI, anti-Ro60 IgG and native β2GPI in the presence or absence of plasmin were incubated together with apoptotic cells. As expected, the opsonization of apoptotic cardiomyocytes by anti-Ro60 IgG (mean fluorescence intensity [MFI] 3445 ± 102) was significantly inhibited by β2GPI (MFI 436 ± 90) (p = 0.006). The addition of plasmin to this system reversed the protective effect of β2GPI and promoted anti-Ro60 IgG opsonization at levels similar to those achieved in the absence of β2GPI (MFI 3378 ± 66) (Fig. 3D).

Plasmin cleavage of β2GPI could account for the reduced binding of umbilical cord β2GPI in the quantitative ELISA (Fig. 2A). However, plasmin-cleaved β2GPI could not be directly measured by ELISA (due to unavailability of monospecific reagent). Therefore, plasmin-cleaved β2GPI was compared with native purified β2GPI on ELISA. Native β2GPI gave a stronger signal on ELISA compared with cleaved β2GPI (range of reduction in binding 19–51%, p = 0.0001). The reduction in binding was presumably due to the loss of an epitope that was recognized by the coating anti-β2GPI polyclonal Ab because immobilized plasmin-cleaved β2GPI showed similar reactivity with an anti-β2GPI domain I mAb as native β2GPI (OD 1.57 ± 0.14 versus 1.54 ± 0.14, n = 3). This discrepancy in binding between native and cleaved β2GPI led to the speculation that there might be increased cleavage of β2GPI in fetuses with cardiac NL. To determine whether lower levels of β2GPI in umbilical cord blood from cardiac NL cases were due to a reduced concentration of β2GPI or increased cleavage of β2GPI by plasmin, immunoblots were repeated using the same panel of nine umbilical cord bloods (used in Fig. 2A), but probed with the anti-β2GPI domain I mAb (which detects both intact and cleaved β2GPI equally). Membranes probed with this mAb still revealed lower levels of β2GPI in the umbilical cord bloods from the five neonates with cardiac NL compared with the four noncardiac NL neonates equivalent to the results obtained when the membranes were probed with the polyclonal Ab (Fig. 2A). This finding suggests that the lower levels of β2GPI observed by ELISA and immunoblot are due to a lower concentration of circulating β2GPI protein rather than increased levels of plasmin-cleaved β2GPI in neonates with cardiac NL.

In the current study, we show that β2GPI competes with maternal anti-Ro60 IgG for binding to the surface of human fetal cardiomyocytes rendered apoptotic. A reasonable extension of these data to the pathogenesis of cardiac NL is the prediction that fetal levels of circulating β2GPI represent a protective factor that averts tissue injury by displacing Ab binding. The displacement by β2GPI may explain, in part, the absence of anti-Ro60 IgG binding to apoptotic cells in vivo in a murine transplacental model (28), or passive transfer xenograft model (29).

The hypothesis of β2GPI as a protective factor in cardiac NL is supported by our finding that levels of circulating β2GPI are lower in neonates with cardiac NL compared with unaffected neonates. Several factors could account for the lower levels of β2GPI observed in the neonates with cardiac NL. Previous studies of cardiac tissue from fetuses dying with cardiac NL demonstrated increased apoptotic cells in the septal region (5). Therefore, decreased levels of β2GPI might be secondary to absorption from the circulation by binding to the apoptotic cells. However, this possibility is unlikely for several reasons, as follows: 1) apoptosis occurs earlier in development and not necessarily at term; 2) the binding of β2GPI should be displaced by anti-Ro60 Abs (if equivalent as in the case of twins) in both affected and unaffected fetuses. Alternatively, lower levels of β2GPI may be a primary factor in Ab-mediated injury due to a genetic predisposition and not a secondary effect. It is known that plasma levels of β2GPI vary among individuals (ranging from undetectable to 350 μg/ml in adults), which may be under genetic control (30). For example, substitution of C with A at the β2GPI transcriptional initiation site is a causative mutation that affects gene expression at the transcriptional level and ultimately β2GPI plasma concentrations (31). Although it has been reported that African Americans have lower levels than Caucasians due to genetic polymorphisms, the lowest levels detected were not present in the few African American patients (data not shown). The disparate levels in the discordant monozygotic twins suggest that genetics do not fully account for reduced β2GPI. Finally, maternal anti-β2GPI Abs were not associated with a loss of the protective function of β2GPI or the low levels of β2GPI observed in the umbilical cord bloods.

Molecular studies revealed the β2GPI binding site on Ro60 to be within aa 82–146. In a recent report, we showed that this region of Ro60 also contains an apotope that is recognized by 45% of patients with systemic lupus erythematosus and isolated anti-Ro60 responses (22). However, β2GPI inhibited the opsonization of apoptotic cells by IgG from patients with anti-Ro60 autoantibodies, not just those with reactivity to the aa 82–146 apotope (data not shown). This suggests that β2GPI inhibits the binding of anti-Ro60 IgG by steric hindrance, whereupon binding to Ro60 aa 82–146, β2GPI undergoes a conformational change and masks other Ro60 apotopes. Interestingly, Ro60 binds to β2GPI within the plasmin cleavage site, residues 317–318, of domain V. Based on these findings, it is likely that a neonate with less circulating β2GPI would be more vulnerable to the effects of plasmin cleavage than a neonate with higher levels of β2GPI. These findings are in accord with those of Briasouli et al. (32), who demonstrated that binding of anti-Ro60 Abs to apoptotic cardiomyocytes results in an increased and altered distribution of uPAR as well as enhanced activation of the uPA. In binding experiments using FACS analysis, these authors confirmed that preincubation of apoptotic cardiomyocytes with β2GPI markedly decreased the binding of IgG from a mother whose child had cardiac NL to the apoptotic cardiomyocytes. Consistent with the notion that the binding of anti-Ro60 contributes to a pathway leading to an enhanced activation of the uPA protease, β2GPI added prior to the cardiac NL-IgG attenuated plasminogen activation.

Whereas the clinical association between maternal anti-Ro60 autoantibodies and cardiac NL is strong (33), the rarity and unique fetal vulnerability suggest that other variables are required for disease expression. Both the levels of circulating β2GPI and extent of plasmin cleavage may be highly permissive with regard to the balance of anti-Ro60 Ab binding. Either intrinsically lower levels based on genetic predisposition or cleavage generated by plasminogen activation would confer loss of protection in fetuses exposed to maternal anti-Ro60 Abs. Because Ro60 is the first of the Ro/La autoantigens to be exposed on the surface of cells undergoing apoptosis (17), it is plausible that preventing the formation of pathogenic anti-Ro60 apoptotic cell complexes with β2GPI may facilitate clearance and protect the fetal heart from Ab-mediated tissue injury.

We thank Prof. Steven Krilis and Dr. Bill Giannakopoulos for discussion and expertise regarding cleaved β2GPI.

This work was supported by Australian National Health and Medical Research Council Postgraduate Training Fellowship Grant 595989 (to J.H.R.), Australian National Health and Medical Research Council Grant 595907 (to T.P.G.), and National Institutes of Health Grants RO1 AR42455-16 and N01-AR-4-2271 (to J.P.B. and R.M.C.).

Abbreviations used in this article:

GAS6

human growth arrest-specific 6

β2GPI

β2-glycoprotein I

MBP

maltose-binding protein

MFG-E8

milk fat globule epidermal growth factor 8

MFI

mean fluorescence intensity

NL

neonatal lupus

PI

propidium iodide

uPA

urokinase plasminogen activator.

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The authors have no financial conflicts of interest.