Departments of 1 Rheumatology and 2 Pediatrics, University Medical Centre, Vodnikova 62, 1000 Ljubljana, Slovenia 3 Department of Medicine II, Hokkaido University School of Medicine, Sapporo 060-8638, Japan 4 Department of Cell Chemistry, Institute of Cellular and Molecular Biology, Okayama University Medical School, Okayama 700-8558, Japan
Correspondence to: A. Ambroi
; E-mail: ales.ambrozic{at}mf.uni-lj.si
Transmitting editor: T. Saito
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Abstract |
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Keywords: allergy, antigen, antiphospholipid antibody, autoantibody, autoimmunity, epitope
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Introduction |
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The exact epitopic sites for antibodies against ß2GPI (anti-ß2GPI) have not yet been revealed, but it seems that domains I and IV are dominant in the binding of anti-ß2GPI from APS patients (1214). The physiological function of ß2GPI is still unclear. However, the interaction of ß2GPI and anti-ß2GPI apparently leads to a prothrombotic state, accelerated atherogenesis, and interferes with the clearance and processing of apoptotic cells.
Particularly during the first few months of life, intact macromolecules may pass through the epithelium of the gastrointestinal tract in amounts sufficient to effectively engage the T cell system (15). It has been shown that virtually all children initiate IgG1 production against nutritional antigens by 3 months of age, and this production typically peaks during infancy and wanes thereafter, whereas corresponding IgG4 responses continue to develop (16). This early pattern of transient Th2-dependent antibody production against non-pathogenic nutritional antigens fails to terminate in children with atopic diseases (17,18). Since ß2GPI has been remarkably conserved during the evolution of animal species, showing >80% homology between the amino acid sequences of human, bovine and rat counterparts (19), we have considered the possibility that in children with atopic diseases an exaggerated immune response to ß2GPI from milk or meat products could lead not only to the production of antibodies against bovine ß2GPI, but also toward human ß2GPI. The immunization of experimental animals with heterologous ß2GPI was shown to induce the production of antibodies specific for the administered antigen and a subset of antibodies that cross-reacted with homologous ß2GPI (20). To our knowledge, no links between anti-ß2GPI and allergic diseases have been reported yet.
We report here a high frequency of anti-ß2GPI in children with atopic dermatitis (AD), but not in those with other allergic diseases examined. Anti-ß2GPI in children with AD were found to recognize epitopes on domain V of ß2GPI, most probably in close vicinity of the phospholipid-binding site. This epitopic difference from anti-ß2GPI in APS and the inability of AD-associated anti-ß2GPI to bind ß2GPI in combination with phospholipids may provide clues to understanding their presumed non-thrombogenicity.
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Methods |
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mAb
Two monoclonal ß2GPI-dependent aPL were used as calibrators to establish standard curves for the anti-ß2GPI and anticardiolipin antibody (aCL) ELISA: HCAL, a chimeric IgG mAb consisting of human and
1 C regions and V regions from the mouse monoclonal aCL WBCAL-1 (21,22), and EY2C9, an IgM mAb derived from a patient with APS (23). Arbitrary units (AU) were set for the anti-ß2GPI and aCL ELISA by the concentrations of the mAb giving definite absorbance values. The activity of 1 ng/ml of HCAL was defined as 1 IgG AU (AUG) and the activity of 5 ng/ml of EY2C9 as 1 IgM AU (AUM). IgA AU (AUA) were set by the dilutions of a selected IgA anti-ß2GPI+ in-house standard from a patient with APS.
Three anti-ß2GPI mAb (Cof-19, Cof-22 and Cof-23) obtained from BALB/c mice immunized with human ß2GPI (12) were applied as positive controls.
Anti-ß2GPI ELISA
ß2GPI was purified from pooled human sera by affinity column chromatography (2) and coated at 10 µg/ml in PBS (pH 7.4, 50 µl/well) on Costar High Binding EIA/RIA plates (cat. no. 3590; Costar, Cambridge, MA) for 2 h at room temperature. After one wash with 300 µl of PBS containing 0.05% Tween 20 (PBS-T), 50 µl/well of the standards and test sera diluted 1:100 in PBS-T were applied for 30 min at room temperature. Following four washes, 50 µl/well of alkaline phosphatase-conjugated goat anti-human IgG, IgM or IgA (ACSC, Westbury, NY) diluted in PBS were added for 30 min at room temperature. After four washes, 100 µl/well of p-nitrophenylphosphate (Sigma, St Louis, MO) dissolved at 1 mg/ml in 1 M diethanolamine buffer (pH 9.8) was applied. OD at 405 nm was first measured after 15 min and then every 3 min by a microtiter plate reader (Rainbow Spectra Thermo; Tecan, Salzburg, Austria) until an optimal fitting to the predicted OD of the standards was obtained. All sera were tested in duplicates both in wells with and without ß2GPI (sample blank values). Specific absorbance was obtained by subtracting sample blank values from raw data.
The cut-off levels for anti-ß2GPI in children were determined based on the absorbance values of control healthy children. The distribution of absorbance values was significantly skewed on the linear scale and nearly symmetrical on the log scale, the cut-off points were defined as the mean + 2SD for the log-absorbance values. Expressed in previously defined AU, the cut-off points for IgG, IgM and IgA anti-ß2GPI in children were 4.2 AUG, 2.6 AUM and 4.0 AUA respectively (24).
Fluid-phase inhibition test with ß2GPI
Except for the preparation of test samples, the assay conditions were the same as described for the anti-ß2GPI ELISA. Tested sera or Cof mAb diluted to 50% maximal binding in 1% BSA (essentially fatty acid free, cat. no. A-6003; Sigma) in PBS-T were mixed with equal volumes of ß2GPI solutions in the same buffer. The final concentrations of ß2GPI were 100, 20, 4, 0.8, 0.16, 0.032 and 0 µg/ml. The mixtures were incubated for 45 min at room temperature, then 50 µl/well was applied to plate wells. OD was measured after 30 min of color development.
Anti-ß2GPI ELISA for IgG subclasses
Coating of ß2GPI, washing and application of serum samples were performed as in the anti-ß2GPI ELISA. Secondary mouse anti-human monospecific antibodies to IgG1 (diluted 1:250; clone HP 6069; Zymed, San Francisco, CA), IgG2 (diluted 1:500; clone MAB 1308; Chemicon, Temecula, CA), IgG3 (diluted 1:1000, clone HP-6050; Sigma) or IgG4 (diluted 1:1000, Clone HP-6025; Sigma) were applied in 50 µl/well aliquots for 1 h at room temperature. For color development, alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) and p-nitrophenylphosphate were used.
Anti-BSA ELISA and fluid-phase inhibition test with BSA
Costar High Binding microtiter plates were coated with 50 µl/well of BSA dissolved at 20 µg/ml in PBS for 2 h at room temperature. All further steps were as described for the anti-ß2GPI ELISA. OD was measured after 45 min of incubation at room temperature. The cut-off point was defined as for the anti-ß2GPI ELISA.
For the inhibition test, ß2GPI and BSA were coated on the same plate as described for the anti-ß2GPI and anti-BSA ELISA respectively. Inhibition mixtures were prepared in duplicates, one containing tested serum and BSA at concentrations up to 1 mg/ml in PBS-T and the other serum diluted in PBS-T only. Following incubation for 45 min at room temperature, the mixtures were applied to wells with ß2GPI, BSA or without an antigen. All further steps were as in the anti-ß2GPI ELISA.
ELISA and fluid-phase inhibition test with bovine ß2GPI
The purification of bovine ß2GPI, ELISA and inhibition test were performed as described for human ß2GPI, which was also used as a control protein. Inhibition mixtures containing either bovine or human ß2GPI in fluid phase were applied to parallel duplicate wells coated with either protein so that all four possible antigen combinations were tested on the same plate. The final concentrations of ß2GPI in mixtures were 200, 100, 20, 4, 0.8, 0.16 and 0 µg/ml.
Standard aCL ELISA
Costar Medium Binding EIA/RIA plates (cat. no. 3591) were coated with cardiolipin as in the standard aCL ELISA. After incubation with 120 µl/well of 10% FBS (Sigma) in PBS for 1 h at room temperature, the plates were washed once with 300 µl/well of PBS. Then 100 µl/well of standards and serum samples diluted 1:100 in PBS containing 10% FBS were applied for 2.5 h at room temperature. Plates were washed 4 times with PBS and 100 µl/well of alkaline phosphatase-conjugated rabbit anti-human IgG (ACSC) were added. Following 1-h incubation at room temperature, plates were washed 4 times and 100 µl/well of substrate were applied. OD was measured as described for the anti-ß2GPI ELISA.
Modified aCL ELISA
Sumilon S microtiter plates (Sumitomo Bakelite, Tokyo, Japan) were coated with 40 µl/well of 50 µg/ml cardiolipin (Sigma) in ethanol by evaporation at 4°C overnight. After blocking with 200 µl/well of 1% BSA in PBS (1% BSA/PBS) for 1 h at room temperature, the plates were washed 3times with 300 µl/well of PBS-T. Then 50 µl/well of standards and serum samples diluted 1:100 in 1% BSA/PBS containing 10 µg/ml of ß2GPI were applied for 2 h at room temperature. Plates were washed 3 times and 50 µl/well of alkaline phosphatase-conjugated mouse anti-human IgG (Southern Biotechnology, Birmingham, AL) were added for 1 h at room temperature. After washing, the substrate was added and OD was measured as described for the anti-ß2GPI ELISA.
The cut-off values in the aCL ELISA, determined by the same method as in the anti-ß2GPI ELISA, were 13.9 AUG and 5.6 AUG for the standard and modified method respectively; 13.9 AUG corresponded to 7.6 GPL international standard IgG aCL units (25).
ELISA and inhibition tests with domain-deleted mutants (DM) of human ß2GPI
Recombinant DM of ß2GPI, lacking one or more domains, as well as whole molecule ß2GPI (WM) were produced by the baculovirus/Sf 9 insect cell vector expression system (12,26). The ELISA with different DM (DM II-V, DM III-V, DM IV-V and DM V lacking domains at the N-terminal region or DM I-III and DM I-IV lacking domains at the C-terminal region) as antigens was performed as outlined for the anti-ß2GPI ELISA. Cof mAb and rabbit polyclonal anti-ß2GPI served as positive controls.
Inhibition of binding to solid-phase ß2GPI was tested with recombinant domain V of ß2GPI (DM V) in fluid phase at its final concentrations of 50, 20, 4, 0.8, 0.16 and 0 µg/ml.
ELISA with proteolytically cleaved ß2GPI
Cleaved ß2GPI was prepared by treatment with human plasmin as described (27). The ELISA was performed on Costar High Binding plates using intact serum purified ß2GPI on the same plate for comparison.
Statistical analysis
Statistical tests were performed using subroutines from the statistical analysis package by MS Excel 6.0 for Windows. Chi-square test, t-test assuming equal or unequal variances and linear regression analysis were used when appropriate. Differences were considered significant whenever P < 0.05.
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Results |
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Inhibition of anti-ß2GPI with ß2GPI in fluid phase
The binding of IgG anti-ß2GPI from the children with AD to ß2GPI on microtiter plates was inhibited in a dose-dependent manner by ß2GPI in solution. Fifty percent inhibition was achieved by ß2GPI concentrations between 15 and 30 µg/ml (0.30.6 µM). These concentrations were 100-fold higher than the IC50 of the Cof mAb (0.10.35 µg/ml). Anti-ß2GPI from the APS patients and HCAL mAb showed only 1535% inhibition at 100 µg/ml (2 µM) of ß2GPI. Representative inhibition profiles are presented in Fig. 2.
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Binding to BSA in and cross-reactivity of anti-BSA with ß2GPI
Because of the high background binding exhibited by many sera of the atopic children in preliminary experiments using BSA as the blocking agent, we presumed that some sera may also contain anti-BSA. In fact, increased levels of IgG anti-BSA were found in 24 of 71 sera (34%) from atopic children. The frequency of anti-BSA was significantly higher in the children with AD (19 of 45 = 42%) than in the other atopic children (five of 26 = 19%, P = 0.038). No correlation between the absorbance values for anti-ß2GPI and anti-BSA in the children with atopic diseases was observed. There was also no association between the presence of anti-ß2GPI and anti-BSA in the children with AD.
Anti-BSA in 13 selected atopic children were inhibited in a dose-dependent manner by fluid-phase BSA (IC50 = 0.2 20 µg/ml BSA). BSA had no influence on the detection of anti-ß2GPI, thus excluding cross-reactivity of anti-BSA with ß2GPI.
Binding of anti-ß2GPI from atopic children to bovine ß2GPI
All 19 sera from the AD children containing antibodies toward human ß2GPI bound efficiently to bovine ß2GPI, suggesting a cross-reactivity. Five of 16 randomly selected sera among atopic children negative against human ß2GPI expressed a substantial binding to bovine ß2GPI. HCAL and EY2C9 mAb bound both antigens similarly to anti-ß2GPI from the children with AD. In addition, anti-ß2GPI from the APS patients bound efficiently both antigens, although some sera expressed a higher binding to human than to bovine ß2GPI (Fig. 3A).
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Binding of IgG anti-ß2GPI from atopic children in aCL ELISA
Among 19 anti-ß2GPI+ sera from the children with AD, positive results were obtained in only three and two sera by the standard and modified aCL ELISA, respectively. Among anti-ß2GPI atopic children, three sera expressed positive values in the standard aCL ELISA and one serum in the modified aCL ELISA. No statistically significant difference in aCL positivity was observed between anti-ß2GPI+ and anti-ß2GPI sera.
Binding to DM of ß2GPI
Anti-ß2GPI from the children with AD recognized all DM containing domain V and showed no binding to DM without this domain (Fig. 4). Further, all tested anti-ß2GPI+ sera from the children with AD bound to solid-phase recombinant domain V. There was also a positive correlation between the absorbance values for binding to complete ß2GPI and domain V (r = 0.85, P < 0.0001) (Fig. 5A). The binding of anti-ß2GPI from the children with AD to solid-phase ß2GPI was efficiently and in a dose-dependent manner inhibited by domain V in the fluid phase (IC50 = 6 to 9 µg/ml = 0.4 to 0.7 µM) (Fig. 5B). The molar concentrations of domain V and complete ß2GPI giving 50% inhibition were comparable.
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Binding to proteolytically cleaved ß2GPI
The cleavage of ß2GPI practically abolished the binding of anti-ß2GPI from all 17 tested positive children with AD (mean OD 0.386 ± 0.183, range 0.1390.798 for native purified ß2GPI and 0.014 ± 0.018, range 00.060 for cleaved ß2GPI). Cof mAb, used as controls, bound to both forms of ß2GPI efficiently.
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Discussion |
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In this study we present the finding of a high frequency (42%) of IgG anti-ß2GPI in 45 children with AD. Furthermore, not only the high frequency but also the levels of IgG anti-ß2GPI were similar to those usually found in APS. Anti-ß2GPI was significantly associated with AD, as a low level of IgG anti-ß2GPI was detected in only one of 26 patients with other atopic diseases and in none of the 22 children who experienced a systemic anaphylactic reaction after stings of Hymenoptera insects.
Since the existence of anti-ß2GPI in AD children has not been associated with clinical manifestations of APS, we presumed that those antibodies may differ from anti-ß2GPI in APS patients. A series of experiments was performed to characterize the binding properties of anti-ß2GPI in AD in comparison with those in APS.
The specificity of anti-ß2GPI in AD was supported by the efficient inhibition of binding to solid-phase ß2GPI with ß2GPI in the fluid phase. Interestingly, >80% inhibition was obtained with 100 µg/ml of ß2GPI (Fig. 2), which was approximately half the physiologic concentration in human serum (28). In contrast, anti-ß2GPI from the APS patients was very weakly inhibited by fluid-phase ß2GPI. This difference could be attributed to the presumed conformational specificity of anti-ß2GPI from APS patients, recognizing ß2GPI epitopes that are fully expressed only when ß2GPI is bound to an appropriate negatively charged surface, or to differing affinities for fluid-phase ß2GPI.
Another important distinction between anti-ß2GPI in AD and APS was noted using the standard and modified aCL ELISA. Namely, anti-ß2GPI from the children with AD showed negligible binding in either of these assays, indicating that their target epitopes were not available when ß2GPI (from FBS or purified human protein) was combined with cardiolipin. Therefore, anti-ß2GPI in AD could not be classified as aCL. Antibodies reacting in anti-ß2GPI, but not in aCL, ELISA were reported in some APS patients and healthy individuals, but were not further characterized (29,30).
The assays with deletion mutants of ß2GPI provided further evidence for a distinct fine specificity of anti-ß2GPI in AD. By contrast to antibodies from the APS patients, for which the major epitope proved to reside within domains IIV (1214), the sera from the children with AD showed a reactivity restricted to domain V of ß2GPI. Furthermore, a high inhibition of binding to ß2GPI was obtained by isolated domain V, confirming that this domain possessed main epitope(s) for anti-ß2GPI in AD.
Domain V of ß2GPI was shown to contain a potent phospholipid-binding site including the C-terminal loop (9). The proteolytic cleavage of the C-terminal loop between Lys317 and Thr318 by plasmin abolished the binding of anti-ß2GPI from the children with AD. The integrity of the phospholipid-binding site was also critical for the binding of anti-ß2GPI from APS patients in some (8,10,27,31), but not all studies (32). The lack of binding to cleaved ß2GPI was attributed to either a conformational change of ß2GPI (27) or direct disruption of the epitopic site in domain V (8,10,31). However, anti-ß2GPI from APS patients bound to ß2GPI combined with phospholipids (8,10,27,31), which was not the case for anti-ß2GPI in AD. The failure of anti-ß2GPI from children with AD to bind cleaved ß2GPI or ß2GPI associated with cardiolipin suggested that their epitopes were located at or at least in close vicinity to the phospholipid-binding site in domain V. We may also assume that a sterical hindrance caused by the binding of such antibodies to ß2GPI interferes with the attachment of ß2GPI to phospholipids, as already noted for Cof-18 mAb, which also recognizes domain V of ß2GPI (12). Since the adhesion of ß2GPI to negatively charged phospholipids and subsequent binding of anti-ß2GPI appears to be the pathogenic sequence of events in APS, the inability of AD-associated anti-ß2GPI to bind ß2GPI in complex with phospholipids may be an explanation for their presumed non-thrombogenicity.
The mechanisms responsible for the occurrence of IgG anti-ß2GPI in AD are not clear. Several interdependent factors are probably involved. In virtually all sera from children with AD having antibodies against human ß2GPI we found also a similar reactivity with bovine ß2GPI. However, a specific binding to bovine ß2GPI was also observed in some sera from children with atopic diseases without antibodies against human ß2GPI. The inhibition tests with human and bovine ß2GPI implied that children with AD harbored two subsets of anti-ß2GPI: one recognizing specifically bovine ß2GPI, found also in other atopic children, and the other cross-reactive with human and bovine ß2GPI, resembling anti-ß2GPI from APS patients in our study and previous reports (33). An important factor responsible for the occurrence of antibodies against bovine ß2GPI might be repeated exposure to nutritional ß2GPI early in life, when the intestinal mucosa is more permissive for large molecules (15). It is conceivable that in infancy the ingested bovine or some other type of ß2GPI (from dietary products) could act as a peroral immunization agent inducing a transient production of species-specific anti-ß2GPI. In children with AD this humoral immune response may spread also to human ß2GPI. An additional explanation for the development of antibodies against human ß2GPI in children with AD might also be related to a deficiency of a specific regulatory T cell function in AD (34).
It has already been shown that exposure to cow milk during the first few months of life results in the initiation of immune responses towards ß-lactoglobulin, BSA and -casein. The specific IgG antibody production typically peaked during early infancy, with particularly high levels in children with AD, and declined thereafter (35,36). In our study as well, the AD children having IgG anti-ß2GPI were of a significantly younger age than those without anti-ß2GPI. Antibodies against BSA were also detected in our atopic children, particularly in AD. Although their frequency was similar to that of anti-ß2GPI, the potential cross-reactivity was excluded.
Anti-ß2GPI in children with AD were restricted to the IgG1 subclass. The IgG subclass distribution of antibodies reactive with dietary proteins, notably ß-lactoglobulin, has already been investigated. In two studies, significantly higher levels of specific IgG1 and IgG4 were found in infants with elevated IgE. This parallelism in IgG1, IgG4 and IgE responses was ascribed to the influence of IL-4 (37,38). It is tempting to speculate that the elevated levels of IgG1 anti-ß2GPI in children with AD were also a result of a genetically governed Th2 skewing of the response to new nutritional antigens in atopy prone infants.
At present we have no evidence that anti-ß2GPI in AD can influence allergic manifestations or induce APS. We are also not aware of any relation between AD and APS, considering patients personal and family history.
In conclusion, we found a high frequency of IgG anti-ß2GPI in children with AD and no clinical signs of APS. These anti-ß2GPI could not be classified as classic aPL (aCL), as they did not bind to ß2GPI associated with cardiolipin. The proteolytic cleavage of the C-terminal region of ß2GPI in close vicinity to the phospholipid-binding site abolished the binding of anti-ß2GPI from children with AD, implying that the epitope resided near the phospholipid-binding region in domain V. Further analyses and prospective studies are needed for definite conclusions on the clinical relevance of anti-ß2GPI in AD. The investigation of interactions between dietary ß2GPI and the human immune system may provide insights into the regulation of autoimmune anti-ß2GPI response.
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Acknowledgements |
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Abbreviations |
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aCLanticardiolipin antibody
ADatopic dermatitis
anti-ß2GPIantibodies against human ß2GPI
aPLantiphospholipid antibodies
APSantiphospholipid syndrome
AUarbitrary units
AUAIgA AU
AUGIgG AU
AUMIgM AU
DMrecombinant domain-deleted mutant of human ß2GPI
WMrecombinant whole molecule ß2GPI
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References |
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