Rheumatology Department, Division of Immunity and Infection, University of Birmingham Medical School, Birmingham,
1 Oxford Tissue Typing Unit, Churchill Hospital, Oxford,
2 Wellcome Trust Genetics Centre, Churchill Hospital, Oxford,
3 Imperial College School of Medicine, London, UK,
4 St Francis Hospital, Indianapolis, USA and
5 Histocompatibility and Immunogenetics Laboratory, National Blood Service, Birmingham, UK
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Abstract |
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Methods. Anti-ß2GPI antibodies were detected in patient sera using enzyme-linked immunosorbent assays (ELISAs). HLA class II alleles (DRB1, DQB1) were determined by polymerase chain reaction-based DNA genotyping. In vitro peripheral blood mononuclear cell (PBMC) responses to native human ß2GPI were measured in a 7-day proliferation assay.
Results. We identified three groups of Caucasian SLE patients using these ELISAs. In group 1, 16 out of 18 SLE patients (89%) with anti-ß2GPI antibodies were positive for HLA-DRB1*0401/4/8, DR11 or DRB1*1302 (P=0.001 vs controls) compared with 23 out of 53 patients (43%) in group 2 with anti-cardiolipin antibodies only, 57 out of 151 patients (38%) in group 3 (SLE patients without anticardiolipin antibodies) and 109 out of 225 controls (48%). Fourteen patients with anti-ß2GPI antibodies had greater median stimulation indices to ß2GPI in vitro compared with the 15 controls studied (P=0.04).
Conclusion. The HLA class II and PBMC proliferation data suggest that ß2GPI may be both a T- and B-cell autoantigen in SLE.
KEY WORDS: SLE, ß2GPI, APS, HLA, T cell.
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Introduction |
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Although it is possible that T cells can recognize phospholipid fragments presented directly by HLA molecules, a more likely hypothesis is a haptencarrier model in which phospholipid-binding plasma proteins, such as ß2-glycoprotein I (ß2GPI) [25] and prothrombin [3], are the source of the antigenic peptides leading to the generation of antibodies to both the phospholipid and its binding proteins.
HLA association data supports a T-cell-mediated hypothesis. HLA-DR4, DR53 and DQB1*0301 (linked to DR4) are increased in frequency in northern European APS patients and HLA-DR4, DR7 and DR53 in patients from southern Europe [6, 7]. Increased frequencies of the HLA-DQB1*0301 and HLA-DQB1*06 alleles have also been reported in patients whose antiphospholipid antibodies possess in vitro lupus anticoagulant (LAC) activity [8]. In the UK, an association between anti-ß2GPI antibodies and HLA-DRB1*1302 DQB1*06049 and DR7 has been reported in patients with the primary APS [9] and in a study from the USA of patients from three ethnic groupings, 16 of 41 (39%) of anti-ß2GPI antibody-positive white patients were HLA-DR4-positive and 13 of 41 (32%) DQB1*0302-positive [10]. No increase in the frequency of DR7 was seen in this latter study [10].
Taken together, these studies suggest relatively modest but consistent HLA associations with DR4 DQB1*03 (either *0301 or *0302 in different studies) and DRB1*1302 DQB1*06049 haplotypes and associations with DR7 in some studies but not others.
We set out to identify whether these HLA associations could be confirmed in a cohort of Caucasian patients with SLE and the APS in the UK and whether these same HLA class II alleles act as restriction elements for cellular immune responses in vitro to ß2GPI, thereby supporting the hypothesis that ß2GPI is a T-cell antigen in SLE patients with anti-ß2GPI antibodies.
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Patients and methods |
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Autoantibody testing
All serum samples used in this study were stored at -20°C before use. All patients were screened for anticardiolipin/anti-ß2GPI antibodies using a commercial enzyme-linked immunoassay (ELISA) (Binding Site, Birmingham, UK) that includes foetal calf serum as a source of ß2GPI and is referenced against the Louisville International Reference Sera. In this assay, a titre of <10 IgG anticardiolipin (phospholipid) binding activity/IgM anticardiolipin (phospholipid) binding activity (GPL/MPL international units) is considered negative, 10 <20 GPL/MPL as a weak positive and
20 as a positive result. A dilute Russell viper's venom test for LAC was performed in all patients with anticardiolipin antibodies except for 14 patients who were on warfarin and five other patients who could not be tested for logistical reasons.
Samples positive for anticardiolipin antibodies in the screening ELISA (or who had circulating LAC or clinical features suggestive of the APS) were tested with an anti-ß2GPI antibody ELISA (Shield Diagnostics, Dundee, UK) for the presence of elevated titres of immunoglobulin G (IgG) anti-ß2GPI antibodies (>15 U/ml). A titre of >100 U/ml is treated as equal to 100 U/ml for descriptive purposes in this study. This ELISA has been validated by the manufacturer using a reference antibody (HCAL; courtesy of Professor T. Koike, Sapporo, Japan).
HLA-DR and DQ genotyping
DNA typing for HLA-DRB1 and DQB1 alleles was performed on peripheral blood stored in EDTA at -20°C as described previously [12, 13].
Antigens and mitogens
Antigens and mitogens used in the proliferation assays included rabies protein (gift of Statens Serum Institut, Copenhagen, Denmark; final concentration 5 µg/ml), tetanus toxoid (TT) (Statens Serum Institut, Copenhagen, Denmark; final concentration 10 µg/ml) purified protein derivative (PPD) (Statens Serum Institut, 10 µg/ml), human ß2GPI (gift of Dr J Amiral, Serbio, Paris, France, purified using standard methods [14]), used at a final concentration of 10 µg/ml unless otherwise indicated, and phytohaemagglutinin (PHA) (Murex Diagnostics, Dartford, UK; final concentration 1 µg/ml).
PBMC proliferation assay
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral venous blood using FicollHypaque density centrifugation. Cells (2x105 per well) were cultured in flat-bottomed 96-well plates (200 µl total volume per well) for 7 days at 37°C in a 5% CO2 humidified atmosphere with antigen or mitogen in RPMI medium supplemented with 10% normal human serum (NHS) and 1% GPS (glutamine, penicillin and streptomycin). Monoclonal anti-CD28 antibody (Becton Dickinson, Oxford, UK) was added at a final concentration of 0.05 µg/ml as described previously [15] in order to overcome any costimulatory defects in patients with SLE. Cultures were performed in triplicate for TT, PPD and PHA and a minimum of five wells for ß2GPI and rabies protein. Tritiated thymidine (0.4 µCi) was added to each well for the final 18 h of culture. After harvesting, the number of counts per minute (c.p.m.) was measured using a Wallace Betaplate ß-scintillation counter. Results are expressed as either c.p.m. or a stimulation index (SI=c.p.m. with cells plus antigen/c.p.m. with cells alone). Data from four patients and one control for whom in vitro proliferative responses to recall antigens (TT or PPD) or mitogens (PHA) could not be demonstrated were excluded.
Statistical analysis
Non-parametric tests were used to compare differences in continuous variables and the 2 test was used to compare the frequencies of HLA alleles between groups. A P value of <0.05 was regarded as significant. As we were testing previous hypotheses, no corrections were made for the number of HLA alleles studied. Odds ratios and 95% confidence intervals are quoted where appropriate.
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Results |
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Thirteen of the 20 patients with the primary APS (PAPS) who were studied had anti-ß2GPI antibodies (median titre 52, range 18 to >100 U/ml). Eleven of these 13 had circulating LAC compared with two of five testable samples among the seven PAPS patients without anti-ß2GPI antibodies.
HLA class II associations with anti-ß2GPI antibodies
The HLA genotyping results for the patient and control groups are given in Table 1. SLE patients as a group had an increased frequency of HLA-DR3(17) (P<0.001) and to a lesser degree DR2(15) (P=0.018) compared with controls. Conversely, the frequencies of HLA-DR1 (P<0.001), HLA-DR7 (P=0.004) and DRB1*1301 (P=0.008) were reduced. The HLA-DQ data paralleled these findings, with an increased frequency of HLA-DQ2 (P<0.001) and a reduced frequency of DQ5 (P<0.001). The overall frequency of DQ6 (P=0.016) was reduced compared with controls.
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Although we only examined 20 PAPS patients, eight out of 13 (61%) with anti-ß2GPI antibodies possessed one of these alleles compared with two out of seven without (29%) and 48% of controls. These numbers are too small for rigorous statistical analysis.
Patients with anticardiolipin antibodies but without IgG anti-ß2GPI antibodies did not differ in respect to the frequency of any HLA-DR or DQ type compared with the remainder of the SLE patient group.
PBMC responses to ß2GPI in vitro
ß2GPI is a normal constituent of human serum that is present at a concentration of 200 µg/ml. Because normal human serum was used in the proliferative assay at a final concentration of 10%, this equates to a final concentration of 20 µg/ml of ß2GPI. We calculated a doseresponse curve for proliferation in response to exogenous purified human ß2GPI (Fig. 1). Maximal proliferation occurred with the addition of 10 µg/ml (equivalent to a total concentration of 30 µg/ml).
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Patients with anti-ß2GPI antibodies had a higher median SI for proliferative responses to ß2GPI (2.185, interquartile range 1.553.37) compared with controls (1.36, interquartile range 1.092.14) (P=0.04) (Fig. 2). A similar result was obtained by combining group 1 with group 2 (P=0.03 compared with controls). In contrast, the responses to PPD (P=0.03) and rabies protein (P=0.05) were greater in controls. SLE patients as a group (n=51) had reduced proliferative responses to PPD (P=0.002), rabies protein (P=0.05) and PHA (P=0.04) compared with controls.
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Discussion |
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This model implies that peptides derived from processed ß2GPI are presented to helper T lymphocytes in the peptide-binding groove of HLA class II molecules. The identification of HLA class II associations with the presence of these autoantibodies supports this model. In this study we have confirmed the associations between anti-ß2GPI antibodies and the presence of HLA-DR4 (DRB1*0401/4/8) and DRB1*1302 reported previously in different studies [9, 10]. We were unable to identify a bias to either DQB1*0301 [8] or DQB1*0302 [7, 10]. In agreement with Arnett et al. [10], we found no association with DR7 [7, 9], and were unable to support the hypothesis that the specific amino acid sequence 71 TRAELDT 77, shared by HLA-DQB1*03 and DQB1*06 (including DQB1*0602 linked to DRB1*1501) plays a critical role [19]. We have, however, raised the novel possibility of a modest association with DR11 (which is usually linked to DQB1*0301 in Caucasian haplotypes). Because of the tight linkage between DR and DQ specificities for the HLA alleles of interest, it is not possible, however, to determine if any of the putative restriction elements are at the DR or DQ locus using this approach.
We next set out to address the hypotheses that T-cell proliferative responses to ß2GPI are restricted by the same MHC (major histocompatibility complex) molecules and are associated with the presence of anti-ß2GPI antibodies in individual patients. One methodological issue is that normal human serum contains 200 µg/ml ß2GPI. Conventional in vitro assays of T-cell proliferation generally use growth medium containing a final concentration of 10% foetal bovine serum (FBS) or NHS, which exposes the T cells to 20 µg/ml of ß2GPI before additional antigen is added. In the first paper to study T-cell responses to ß2GPI, Visvanathan and McNeil [4] used a serum-free assay in order to address this problem. They demonstrated that eight of 18 patients with the APS (but none of the controls) responded to purified ß2GPI or normal serum added to the serum-free medium, with an optimum final concentration of purified ß2GPI of 25 µg/ml. Hattori et al. [5] adopted a different approach by comparing proliferation in cultures with and without 10% FBS or NHS. As they found no differences in proliferation in these circumstances, they concluded, in contrast to Visvanathan and McNeil, that there is no response to native ß2GPI. They identified proliferative responses to 10 µg/ml trypsin-digested or reduced ß2GPI in medium containing 10% FBS in all 12 APS patients with anti-ß2GPI antibodies studied, but also four of 13 SLE patients without anti-ß2GPI antibodies and six of 12 healthy donors.
In order to address this issue, we calculated a doseresponse curve of proliferation in response to purified native human ß2GPI (gift of Dr J. Amiral, Serbio, Paris, France or IDRL, Birmingham, UK) in a 7-day proliferation assay using PBMCs from a healthy control and two APS patients with medium containing 10% NHS. Maximal proliferation occurred with the addition of 10 µg/ml ß2GPI. This would be equivalent to a total concentration in the assay of 30 µg/ml native ß2GPI. The results are in line with the data of Visvanathan and McNeil [4].
Using this approach, we identified proliferative responses (defined as S >3) to native ß2GPI in 15 out of 51 SLE patients studied (29%) compared with one of 15 controls (7%) (P=0.07). This finding suggests that proliferative responses are found in controls and also raises the possibility that they may be more frequent in SLE patients in general. In support of this observation, modest but significant differences in the median SIs between patients with anti-ß2GPI antibodies (group 1) and controls (P=0.04) and between patients with anticardiolipin antibodies (groups 1 and 2 combined) were demonstrated, supporting the hypothesis that a T-cell response directed against ß2GPI could be involved in the generation of anti-ß2GPI/anticardiolipin antibodies. The magnitudes of the responses, however, are small and are comparable to those of a naive T-cell antigen (rabies protein) rather than a recall antigen (TT or PPD).
Given the modest HLA associations in this and previous studies, it is likely that any HLA association with the T-cell response to native ß2GPI will be relatively weak. In a Japanese population, an alternative approach using a synthetic peptide library has been used to identify specific peptide epitopes of ß2GPI [20]. As in this study, no clear restriction of the proliferative responses by a single or limited number of HLA restriction elements was identified.
In summary, our data confirm the association between anti-ß2GPI antibodies and the HLA-DR4 (DRB1*0401/4/8) and DRB1*1302 haplotypes in Caucasians and demonstrates that patients with anti-ß2GPI antibodies have greater proliferative responses to native ß2GPI in vitro. These data support the hypothesis that native human ß2GPI may be both a T- and a B-cell autoantigen in SLE/PAPS.
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Acknowledgments |
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Notes |
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References |
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