Immune responses to native ß2-glycoprotein I in patients with systemic lupus erythematosus and the antiphospholipid syndrome

M. L. Davies, S. P. Young, K. Welsh1, M. Bunce1, B. P. Wordsworth2, K. A. Davies3, D. R. Wagenknecht4, E. Taylor, C. Gordon, S. Jobson5, D. Briggs5 and S. J. Bowman

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


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To identify HLA class II associations with anti ß2-glycoprotein I (ß2GPI) antibodies in a cohort of Caucasian patients with systemic lupus erythematosus (SLE) and to determine whether these HLA genotypes act as restriction elements for lymphocyte proliferation to native human ß2GPI in vitro.

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.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The antiphospholipid syndrome (APS) is characterized by serum autoantibodies directed against anionic phospholipids in association with clinical features of recurrent foetal loss arterial and/or venous thrombosis, thrombocytopenia and livedo reticularis [1]. Typically, it occurs as a primary entity or in association with systemic lupus erythematosus (SLE). These antibodies are generally class-switched, somatically hypermutated antibodies suggesting a T-cell-dependent process [1].

Although it is possible that T cells can recognize phospholipid fragments presented directly by HLA molecules, a more likely hypothesis is a hapten–carrier 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*0604–9 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*0604–9 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.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and controls
Two hundred and twenty-two Caucasian SLE patients were recruited (11 males, 211 females; median age 46 yr, range 17–80 yr; median disease duration 12 yr, range 0–59 yr). An additional 51 Caucasian SLE patients known to the University of Birmingham SLE service had died, were lost to follow-up or were otherwise unavailable for this study. We also identified 37 patients with the primary APS [11], of whom 20 participated in this study (median age 37 yr, range 18–75 yr; median disease duration 2 yr, range 0–12 yr). The HLA allele frequency data among controls were derived from 225 Caucasian consecutive cadaver donors identified via the Oxford Tissue Typing Service (109 males, 116 females; median age 46 yr, range 6–71 yr). Sixteen healthy Caucasian controls (14 females, two males; median age 37 yr, range 27–58 yr) working in the rheumatology departments donated blood for study in the proliferation assays. This project was approved by the South Birmingham Local Research Ethics Committee.

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 Ficoll–Hypaque 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 {chi}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.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Antibody status
Using a combination of an anticardiolipin/ß2GPI screening ELISA and an ELISA specific for IgG anti-ß2GPI antibodies, the SLE patients were divided into three groups: group 1 (18 patients) had IgG anti-ß2GPI antibodies (median titre 53 U/ml, range 15.1 to >100 U/ml). Twelve of these patients (67%) had clinical features of the antiphospholipid syndrome [11]. All except one had anticardiolipin antibodies in the screening ELISA and 13 of 15 (87%) testable samples were positive for circulating LAC. Group 2 (53 patients) had anticardiolipin antibodies in the screening ELISA but were negative for IgG anti-ß2GPI antibodies. Included in this group were four patients who were negative for anticardiolipin antibodies but had clinical features of the APS but were taking warfarin, so that LAC could not be tested. Thirty-one of these patients (58%) had clinical features of the APS. Eight of 39 (20%) testable samples from patients in this group had circulating LAC (all except one in patients with clinical features of the APS). The remaining 151 patients (group 3) were either negative (133 patients) or had only equivocally positive levels (18 patients) of anticardiolipin antibodies. Only two patients in group 3 (1.3%) had clinical features compatible with the APS (thrombosis in one and infertility in the other). None of the 18 equivocal samples were positive for anti-ß2GPI antibodies and none of 16 testable samples had circulating LAC. Twenty of the patient samples without anticardiolipin antibodies (group 3), taken from patients attending clinics at the same time as the patients with anti-ß2GPI antibodies, were tested for anti-ß2GPI antibodies and LAC. All were negative.

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 1Go. 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.


View this table:
[in this window]
[in a new window]
 
TABLE 1.  Frequency of patients possessing at least one copy of the HLA-DR and DQ alleles specified among SLE patients (SLE all, n=222) with anti-ß2GPI antibodies (ß2GPI, n=18), with anticardiolipin antibodies only (ACA, n=53), without either antibody (SLE, n=151) and controls (n=225)

 
Patients with anti-ß2GPI antibodies (group 1) had increased frequencies of HLA-DRB1*0401/4/8 (P=0.038) and DR6(13) (P=0.003), the latter due to an increase in DRB1*1302 (P=0.004). The presence of any one of the HLA-DR types (DRB1*0401/4/8, DR11 or DRB1*1302), however, showed the strongest association with anti-ß2GPI antibodies (89%) compared with controls (48%) (P=0.001). Although DQB1*03 alleles are in linkage disequilibrium with some HLA-DR4, DR8, DR11 and DR13 alleles, there was no significant difference between group 1 and controls for any DQ type.

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 dose–response curve for proliferation in response to exogenous purified human ß2GPI (Fig. 1Go). Maximal proliferation occurred with the addition of 10 µg/ml (equivalent to a total concentration of 30 µg/ml).



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 1.  Proliferative response in vitro to different concentrations of ß2GPI for a control sample ({blacksquare}), a patient with SLE and the APS ({blacktriangledown}) and a patient with PAPS ({square}). Data are mean c.p.m.; vertical bars show the standard deviation of triplicate samples.

 
Using this concentration, we identified in vitro proliferation (SI>=3) to native human ß2GPI in four out of 14 patients (29%) with anti-ß2GPI antibodies (six with the PAPS, four with SLE and the APS and four with SLE and anti-ß2GPI antibodies without clinical features of the APS), four out of 15 (27%) SLE patients with APS but without anti-ß2GPI antibodies, seven of 22 (32%) SLE patients without the APS and one of 15 (7%) controls (SLE vs controls P=0.07).

Patients with anti-ß2GPI antibodies had a higher median SI for proliferative responses to ß2GPI (2.185, interquartile range 1.55–3.37) compared with controls (1.36, interquartile range 1.09–2.14) (P=0.04) (Fig. 2Go). 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.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 2.  Proliferative responses in vitro to PHA, PPD, rabies protein and purified native ß2GPI among patient groups (group 1, SLE patients with anti-ß2GPI antibodies; n=14; group 2, SLE patients with anticardiolipin antibodies only, n=15; group 3, SLE patients without antibodies against either ß2GPI or cardiolipin, n=22) and controls (n=15). Data are mean SI for each group; vertical bars show the standard error of the mean.

 
No significant association was seen between proliferative responses to ß2GPI and the HLA genotypes of SLE patients: six of 21 patients with HLA-DR4 (DRB1*0401/4/8), DR11 and/or DRB1*1302 had an SI of >=3 to ß2GPI compared with seven of 30 patients without these HLA types (not significant). There was no significant difference in the median SI between patients with (1.76, interquartile range 1.26–3.12) and without (1.42, interquartile range 1.17–2.70) these HLA types.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The failure to clear material generated through programmed cell death (apoptosis) is believed to play a critical role in the pathogenesis of SLE [16, 17]. During apoptosis, some phospholipids, such as phosphatidylserine, are transported from the inner to the outer leaflet of the cell membrane [18]. Plasma proteins, such as ß2GPI, can then bind to the externalized phospholipids, and they have been identified as important cofactors for the binding of antiphospholipid antibodies [2]. They are therefore very attractive candidates for the T-cell antigens under these conditions in a hapten–carrier model.

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 dose–response 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.


    Acknowledgments
 
We are grateful to the Arthritis Research Campaign and the Medical Research Council (MRC), UK, Lupus UK and the Royal Society for funding this work. SJB was in receipt of an MRC Clinician Scientist Fellowship. We are grateful to Dr Stephen Gough for HLA-typing some of the patients and to Dr Tim Plant for the anticardiolipin antibody testing. We would like to thank the patients who participated in this study and our medical colleagues who contributed to the collection of clinical data. We are also grateful to Jan Skan and Stephanie Heaton for sample collection and identification. We thank Dr Jean Amiral, Serbio, Paris, France and Statens Serum Institut, Copenhagen, Denmark for their kind gifts of materials (see Patients and methods section).


    Notes
 
Correspondence to: S. J. Bowman, Rheumatology Department, Division of Immunity and Infection, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Cuadrado MJ, Hughes GR. Hughes (antiphospholipid) syndrome. Clinical features. Rheum Dis Clin North Am2001;27:507–24.[ISI][Medline]
  2. McNeil HP, Simpson RT, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta2-glycoprotein 1 (apolipoprotein H). Proc Natl Acad Sci USA1990;87:4120–4.[Abstract]
  3. Inanc M, Donohoe S, Ravirajan CT, Radway-Bright EL, Mackie I, Machin S, Isenberg DA. Anti-ß2-glycoprotein I, anti-prothrombin and anticardiolipin antibodies in a longitudinal study of patients with systemic lupus erythematosus and the antiphospholipid syndrome. Br J Rheumatol1998;37:1089–94.[ISI][Medline]
  4. Visvanathan S, McNeil HP. Cellular immunity to ß2-glycoprotein-1 in patients with the antiphospholipid syndrome. J Immunol1999;162:6919–25.[Abstract/Free Full Text]
  5. Hattori N, Kaburaki J, Mimori T, Ikeda Y, Kawakami Y, Kuwana M. Identification of autoreactive T cells to ß2-glycoprotein I (ß2GPI) that mediate antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). Arthritis Rheum1999;42(Suppl. 9):S367.
  6. Sebastiani GD, Galeazzi M, Morozzi G, Marcolongo R. The immunogenetics of the antiphospholipid syndrome, anticardiolipin antibodies, and lupus anticoagulant. Semin Arthritis Rheum1996;25:414–20.[ISI][Medline]
  7. Galeazzi M, Sebastiani GD, Tincani A et al. HLA class II alleles associations of anticardiolipin and anti-ß2GPI antibodies in a large series of European patients with systemic lupus erythematosus. Lupus2000;9:47–55.[ISI][Medline]
  8. Arnett FC, Olsen ML, Anderson KL, Reveille JD. Molecular analysis of major histocompatibility complex alleles associated with the lupus anticoagulant. J Clin Invest1991;87:1490–5.[ISI][Medline]
  9. Caliz AR, Atsumi T, Kondeatis E et al. HLA class II gene polymorphisms in antiphospholipid syndrome: haplotype analysis in 83 caucasoid patients. Rheumatology2001;40:31–6.[Abstract/Free Full Text]
  10. Arnett FC, Thiagarajan P, Ahn C, Reveille JD. Associations of anti-ß2-glycoprotein 1 autoantibodies with HLA class II alleles in three ethnic groups. Arthritis Rheum1999;42:268–74.[ISI][Medline]
  11. Wilson WA, Gharavi AE, Koike T et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum1999;42:1309–11.[ISI][Medline]
  12. Bunce M, O'Neill CM, Barnardo MC et al. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens1995;46:355–67.[ISI][Medline]
  13. Mullighan CG, Bunce M, Welsh KI. High resolution HLA-DQB1 typing using the polymerase chain reaction and sequence-specific primers. Tissue Antigens1997;50:688–92.[ISI][Medline]
  14. Polz E, Wurm H, Kostner GM. Studies on the composition of the protein part of triglyceride rich lipoproteins of human serum: isolation of polymorphic forms of beta 2-glycoprotein-1. Artery1981;9:305–15.[ISI][Medline]
  15. Pilling D, Akbar AN, Bacon PA, Salmon M. CD4+ CD45RA+ T cells from adults respond to recall antigens after CD28 ligation. Int Immunol1996;8:101–6.[Abstract]
  16. Botto M, Dell'Agnola C, Bygrave AE et al. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nature Genet1998;19:56–9.[ISI][Medline]
  17. Utz PJ, Hottelet M, Schur PH, Anderson P. Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus. J Exp Med1997;185:843–54.[Abstract/Free Full Text]
  18. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol1992;148:2207–16.[Abstract/Free Full Text]
  19. Wilson WA, Scopelitis E, Michalski JP et al. Familial anticardiolipin antibodies and C4 deficiency genotypes coexist with MHC DQB1 risk factors. J Rheumatol1995;22:227–35.[ISI][Medline]
  20. Ito H, Matsushita S, Tokano Y et al. Analysis of T cell responses to the ß2-glycoprotein I-derived peptide library in patients with anti-ß2-glycoprotein I antibody associated autoimmunity. Human Immunol2000;61:366–77.[ISI][Medline]
Submitted 21 June 2001; Accepted 12 October 2001





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Disclaimer
Request Permissions
Google Scholar
Articles by Davies, M. L.
Articles by Bowman, S. J.
PubMed
PubMed Citation
Articles by Davies, M. L.
Articles by Bowman, S. J.
Related Collections
Systemic Lupus Erythematosus and Autoimmunity