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Rheumatology 2001; 40: 1026-1032
© 2001 British Society for Rheumatology

Original Papers

IgG subclass distribution of antibodies against ß2-GP1 and cardiolipin in patients with systemic lupus erythematosus and primary antiphospholipid syndrome, and their clinical associations

M. Samarkos, K. A. Davies1, C. Gordon2, M. J. Walport1 and S. Loizou1

5th Department of Internal Medicine, Evangelismos Hospital, Athens, Greece,
1 Rheumatology Section, Division of Medicine, Hammersmith Hospital, Imperial College School of Medicine, London and
2 Department of Rheumatology, Division of Immunity and Infection, University of Birmingham, Birmingham, UK


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To determine the immunoglobulin G (IgG) subclass distribution of anticardiolipin (aCL) and anti-ß2-glycoprotein 1 (ß2-GP1) antibodies (aß2-GP1), and to examine possible associations between the different aß2-GP1 and aCL subclasses and the main clinical manifestations of the antiphospholipid syndrome (APS).

Methods. We studied 130 patients with systemic lupus erythematosus and 35 patients with primary APS. We used enzyme-linked immunosorbent assays to measure IgG aCL and aß2-GP1 and to determine the IgG subclass distribution of these two autoantibodies.

Results. When the number of patients positive for each subclass was examined, IgG3 and IgG2 aCL were more frequent (63.5 and 54.1% of patients were positive for the two subclasses, respectively), while for aß2-GP1 IgG2 was the most prevalent subclass (81.8% of patients were positive). IgG2 aCL was significantly associated with arterial thrombosis (P=0.023) and fetal loss (P=0.013), and IgG3 aCL was significantly associated with arterial thrombosis (P=0.0003) and fetal loss (P=0.045). IgG2 aß2-GP1 was associated with venous thrombosis (P=0.012) and IgG3 aß2-GP1 was associated with venous thrombosis (P=0.036) and fetal loss (P=0.024).

Conclusions. The IgG2 predominance of aß2-GP1 suggests that the antibody response against ß2-GP1 may be T-cell-independent. As IgG2 and IgG3 differ in their effector functions, their association with the same clinical manifestations (i.e. thrombosis and fetal loss) suggests that more than one mechanism may be involved in the pathogenesis of thrombosis and fetal loss in APS.

KEY WORDS: Systemic lupus erythematosus, Anticardiolipin/antiphospholipid antibodies, IgG subclasses.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Antiphospholipid antibodies (aPL) are a heterogeneous group of autoantibodies and are often found to be elevated in the sera of patients suffering from a wide range of clinical conditions, mainly autoimmune and infectious diseases [1–3]. In autoimmune diseases, aPL are associated with a spectrum of clinical manifestations, such as arterial and venous thrombosis, recurrent fetal loss and thrombocytopenia. The antiphospholipid syndrome (APS) is characterized by the combination of increased aPL titres with at least one of these manifestations.

Until relatively recently, aPL have been detected by a range of assays, each detecting a specific subgroup of aPL, e.g. anticardiolipin antibody (aCL)-specific enzyme-linked immunosorbent assays (ELISAs) and lupus anticoagulant assays [4–6], and it was thought that aPL were directed specifically against negatively charged phospholipids [1]. However, recent data support the view that, in autoimmune disease patients, the aPL detected by solid-phase assays do not bind to phospholipids alone but bind either to a complex of phospholipids and plasma proteins, such as ß2-glycoprotein 1 (ß2-GP1) and prothrombin, or bind directly to these proteins alone [6–8]. This has been shown to be the case in systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS) patients, in whom antibodies against ß2-GP1 (aß2-GP1) have been detected by the use of ß2-GP1 as the ligand in solid-phase ELISAs [9].

The immunoglobulin (Ig) G subclass distribution of autoantibodies may give insight into their mode of action, as IgG subclasses differ in their ability to activate complement and in their properties of binding to Fc{gamma} receptors [10, 11]. Furthermore, IgG subclass responses are greatly dependent on their target antigen: protein antigens generally elicit a T-cell-dependent IgG1 and IgG3 response, whereas carbohydrate antigens induce a T-cell-independent IgG2 response [12–14]. Hitherto, a relatively large number of studies have examined the prevalence of increased levels of antibodies against ß2-GP1 (aß2-GP1) in patients with SLE and/or the APS [15–18], but only one study has examined the IgG subclass distribution of aß2-GP1 [19]. In contrast, several studies on IgG aCL subclasses have been published, with conflicting results [20–23]. Interestingly, the most recent of these studies has reported an association between IgG2 aCL and the occurrence of thrombosis [23].

The present study is the first in which the IgG subclass distributions of aß2-GP1 and aCL have been determined simultaneously in the sera of SLE and PAPS patients. We also attempted to clarify whether there were any differences in subclass distribution in different patient subgroups (e.g. in patients with high vs low autoantibody levels). Additionally, we examined possible associations between the four aß2-GP1 and aCL subclasses, and any of the main clinical manifestations of the APS.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
We studied retrospectively 130 patients (120 female, 10 male; age 19–83 yr, median 43 yr) with SLE from the Department of Rheumatology, Division of Immunity and Infection, University of Birmingham, and 35 patients with PAPS (29 female, 6 male; age 20–71 yr, median 39) from the Rheumatology Section, Imperial College School of Medicine at Hammersmith Hospital. All SLE patients fulfilled the American College of Rheumatology criteria [24] and all PAPS patients had elevated levels of IgG and/or IgM anticardiolipin antibodies or a positive lupus anticoagulant test, and at least one of the following clinical manifestations of the antiphospholipid syndrome: arterial thrombosis (peripheral arterial thrombosis, thrombotic stroke), venous thrombosis (deep vein thrombosis or pulmonary embolism), at least two fetal losses, and thrombocytopenia (platelet count less than 120 000/ml). The clinical data were obtained from medical records. Serum samples were stored at -70°C. The control group consisted of 88 healthy blood donors. At the time when blood was drawn, all patients had given informed consent for the use of their blood sample for research.

Concentrations of IgG and IgM aCL and aß2-GP1 were measured in all patients and controls. We also determined the IgG subclass distribution of aCL and aß2-GP1 in all patients who tested positive for the IgG isotype for each of these two autoantibodies.

aCL ELISA
aCL were measured using the method of Loizou et al. with minor modifications [25]. Briefly, microtitre plates (MP01, Life Sciences International, Basingstoke, UK) were coated with 2 µg/well of cardiolipin (Sigma Biosciences, Poole, Dorset, UK) in ethanol and were left to dry at 4°C. Subsequently the plates were blocked overnight with 5% adult bovine serum (ABS) in phosphate-buffered saline (PBS). Serum samples diluted 1:100 in 5% ABS/PBS were added and incubated for 2 h at room temperature. Alkaline phosphatase (AP)-conjugated goat anti-human IgG {gamma}-chain-specific or goat anti-human IgM µ-chain-specific (Sigma) antibodies were added for the determination of the respective isotypes. After the addition of p-nitrophenyl phosphate, the optical density at 405 nm was measured with an automated ELISA reader (MultiScan MCC/340; Titertek Life Sciences International). The levels of both aCL isotypes were calculated in arbitrary ELISA units (AEU) from an eight-point standard curve. Serum samples with low and high antibody levels were included as internal controls in all cases.

ß2-GP1 purification
ß2-GP1 was purified from normal human plasma using a modification of the method of Polz et al. [26]. The initial precipitation steps were followed by affinity chromatography on a heparin–Sepharose column (HiTrap Heparin; Pharmacia LKB Biotechnology, Uppsala, Sweden). A second affinity chromatography step was performed on a protein G column (HiTrap Protein G; Pharmacia). The final product was delipidated by mixing with butanol. The antigenic properties and purity of the final product were confirmed by double radial immunodiffusion, by ELISA against a rabbit anti-human ß2-GP1 antiserum (Dako, Glostrup, Denmark), and by sodium dodecyl sulphate polyacrylamide gel electrophoresis, which gave a single band at approximately 50 kDa (data not shown).

aß2-GP1 ELISA
Microtitre plates (Immulon 2; Dynex Technologies, Billingshurst, UK) were coated overnight at 4°C with 0.25 µg/well of our ß2-GP1 preparation in 0.2 M borate-buffered saline (BBS) pH 8.4, while an appropriate number of wells were coated with 0.2 M BBS alone for estimation of non-specific binding. Plates were subsequently blocked with 0.5% BSA, 0.4% Tween-20/PBS for 2 h at room temperature. Serum samples (50 µl) diluted 1:100 in 0.5% BSA, 0.4% Tween-20 in PBS were added and incubated at room temperature for 1 h. Alkaline phosphatase-conjugated goat F(ab')2 fragments of anti-human {gamma}-chain-specific IgG diluted 1:3000 or µ-chain-specific IgM diluted 1:1000 (Sigma) were added for the determination of the different isotypes. All washing steps were performed using 0.075% Tween-20/PBS. The final step was the same as for the aCL ELISA. Non-specific binding from non-antigen-coated wells was subtracted from all samples, and the levels of both aß2GP1 isotypes were calculated in AEU from a standard curve of eight points. Internal controls were included on each plate.

aCL and aß2-GP1 IgG subclasses
In preliminary experiments we established the equipotency of the four anti-human IgG subclass-specific monoclonal antibodies [10]. Four microtitre plates were each coated with a different purified human myeloma IgG subclass (IgG1{kappa} to IgG4{kappa}; all from Binding Site, Birmingham, UK) at concentrations of 0.025–0.8 µg/ml. After blocking, chequerboard dilutions (1:250–1:100 000) of each of the respective murine anti-human subclass-specific monoclonal antibodies were added to each plate (anti-IgG1 IUIS/WHO clone 8c/6–39, anti-IgG2 clone HP6014, anti-IgG3 clone HP6050 and anti-IgG4 clone HP 6025; all from Sigma) followed by an AP-conjugated rabbit anti-mouse IgG. The optimum dilution for each subclass-specific antibody was the dilution which gave a similar optical density for the same concentration for each of the different myeloma subclasses (Fig. 1Go). These were 1:500 for anti-IgG1, 1:6000 for anti-IgG2, 1:500 for anti-IgG3 and 1:50 000 for anti-IgG4.



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  		FIG. 1. Binding curves of IgG subclass-specific monoclonal antibodies, each at its optimum dilution, to respective myeloma proteins coated on the microtitre plate.

 
The microtitre plates used, antigen coating and blocking steps for the IgG subclass ELISAs were the same as those used for the total IgG aCL and aß2-GP1 ELISAs, except that all serum samples to be tested were diluted 1:50 and added to each of four plates used for the determination of each of the four subclasses. This was followed by addition of the appropriate concentration (as determined above) for each of the subclass-specific antibodies, to one of each of the four plates. After 2 h of incubation at room temperature, AP-conjugated rabbit anti-mouse IgG (Sigma) was added, followed by addition of the chromogenic substrate. The colour reaction on all four plates was stopped simultaneously. Two wells on each plate were coated with 0.5 µg/well human IgG subclass calibrator (Binding Site), which served as an interplate control. After subtraction of non-specific binding, the net absorbances for the four IgG subclasses were summed (total absorbance), and for each subclass we calculated the absorbance as the percentage of the total absorbance of the antigen-specific IgG.

To measure rheumatoid factor (RF), we used a semiquantitative latex agglutination kit (Rapitex RF; Behringwerke, Marburg, Germany).

Normal ranges
Patient sera were considered positive if the IgG and IgM aCL and aß2-GP1 levels were more than four standard deviations (SD) above the mean level of 88 healthy blood donors. Patient sera were considered positive for one subclass if the percentage levels of this subclass exceeded the upper limit of the accepted normal range (Fig. 2Go) for each of the respective subclasses in normal total serum [10, 11].



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  		FIG. 2. Subclass distribution of IgG aCL (a) and aß2-GP1 (b) in the total study population. Total IgG is the sum of the absorbance of all the subclasses. For each subclass, the ratio of its absorbance to the total absorbance is shown as a percentage. Columns represent mean and bars represent 95% CI.

 

Statistical analysis
The results were analysed with the statistical package Prism v2.0 (GraphPad Software, San Diego, CA, USA). We used the Mann–Whitney test to compare means and Spearman's rank sum test for correlations between numerical variables. To compare proportions and detect associations between nominal variables, we used Pearson's two-tailed {chi}2 test or Fisher's exact test as appropriate. A result was considered statistically significant when P<0.05.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
IgG and IgM aCL and aß2-GP1
Our normal range for IgG aCL was less than 14 AEU and for IgM aCL less than 10 AEU. The numbers of patients positive for each isotype in the SLE and PAPS groups are shown in Table 1Go.


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  		TABLE 1. Frequencies of IgG and/or IgM aCL- and aß2-GP1-positive patients

 
Our normal range for IgG aß2-GP1 was less than 12.5 AEU and for IgM aß2-GP1 less than 9.5 AEU. The numbers of positive patients for each isotype in the two patient groups (SLE and PAPS) are shown in Table 1Go. When patients positive for only IgG or IgM aCL were examined, it was found that levels of IgG aß2-GP1 were higher in PAPS than in SLE patients (54.1 vs 9.6 AEU; Mann–Whitney test, P<0.0001); in the same subgroup of patients (positive for only IgG or IgM aCL), more PAPS than SLE patients were positive for IgG aß2-GP1 (15/34 vs 10/57; {chi}2=7.54, P=0.006).

Correlation between aCL and aß2-GP1
In PAPS patients, IgG and IgM aCL levels were positively correlated with IgG and IgM aß2-GP1 levels respectively (Spearman's rank correlation test, P<0.0001 for IgG and P=0.03 for IgM), whilst in SLE patients a significant correlation was only seen between IgM aCL and IgM aß2-GP1 levels (P<0.0001). When IgG aß2-GP1 and IgG aCL levels were compared in SLE patients who were positive for only IgG aß2-GP1, a strong correlation was also seen (P=0.0042). Only nine of the 40 (22.5%) IgG or IgM aß2-GP1-positive patients were negative for both IgG and IgM aCL.

IgG aCL subclasses
The percentage levels for each of the IgG aCL subclasses in all 74 IgG aCL-positive patients [mean value and 95% confidence intervals (CI)] were as follows: IgG1, 40.1% (33.6–46.7%); IgG2, 32.8% (26.6–39%); IgG3, 23.7% (18.6–28.8%); and IgG4, 3.3% (1.3–5.2%), as shown inFig. 2a. Although IgG1 was the predominant subclass in terms of mean percentage levels, when we considered positivity for each subclass, 47 out of 74 (63.5%) patients were positive for IgG3 and 40 out of 74 (54.1%) patients were positive for IgG2 (Table 2Go). We categorized all our IgG aCL-positive patients as ‘weakly positive’ when the level was lower than 40 AEU and ‘moderately to highly positive’ when the level was higher than 40 AEU. Moderately and highly positive IgG aCL patients had significantly higher mean IgG1 levels (45.8 vs 30.9%, P=0.03) and lower IgG3 levels (18.8 vs 30.4%, P=0.006) than weakly positive IgG aCL patients.


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  		TABLE 2. Numbers of patients positive for each IgG subclass and autoantibody

 

IgG aß2-GP1 subclasses
The IgG subclass distribution of in the IgG aß2-GP1 positive patient population was skewed towards IgG2. Mean values and 95% CI were as follows: IgG1, 24.4% (15.9–32.8%); IgG2, 70.2% (61.7–79.1%); IgG3, 5.0% (1.5–8.4%); and IgG4, 0.4% (0.1–0.7%) (Fig. 2b). When the number of patients positive for each subclass was examined, IgG2 was the predominant subclass, as 27 out of 33 patients (81.8%) were positive (Table 2Go). IgG aß2-GP1 positive patients were classed as ‘weakly positive’ when the level was lower than 30 AEU and ‘moderately to highly positive’ when the level was above 30 AEU. There was no difference in the subclass distribution between patients who were weakly and moderately to highly positive for IgG aß2-GP1.

Comparison of IgG aCL and aß2-GP1 subclasses
The rankings of IgG aCL and aß2-GP1 subclass concentrations in our total population of patients were found to be different. For the percentage of total IgG aCL level, the ranking for each of the four subclasses was IgG1>IgG2>IgG3>IgG4, whereas for aß2-GP1 it was IgG2>IgG1>IgG3>IgG4. When the number of patients positive for each subclass was examined, IgG2 and IgG3 aCL were more frequently elevated, whereas IgG2 was the most prevalent subclass for aß2-GP1. The relative concentrations of IgG subclasses for aCL were different in patients who were also positive for IgG aß2-GP1 in comparison with IgG aß2-GP1-negative patients. Patients who were positive for IgG aß2-GP1 had significantly higher levels of IgG2 aCL than patients who were negative for IgG aß2-GP1 (47.4 vs 26.7%, P=0.003). We also found that patients positive for both IgG aß2-GP1 and aCL generally had higher mean IgG2 aß2-GP1 (76.4 vs 56.5%) levels and lower mean IgG1 levels (18.1 vs 37.9%) than patients who were positive for IgG aß2-GP1 alone, although these differences did not reach statistical significance (Table 3Go).


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  		TABLE 3. IgG subclass distribution in relation to aCL and aß2-GP1 profile

 

Clinical associations
Complete clinical records were available for 157 patients (122 with SLE and 35 with PAPS). Of these patients, 24 (15.3%) had a history of arterial thrombosis, 22 (14.0%) of venous thrombosis, 19 (13.8%) of recurrent fetal loss and eight (5.1%) of thrombocytopenia. IgG2 aCL was significantly associated with arterial thrombosis ({chi}2=5.15, P=0.023) and fetal loss (Fisher's exact test, P=0.013) and IgG3 aCL was significantly associated with arterial thrombosis ({chi}2=12.9, P=0.0003) and fetal loss ({chi}2=4.0, P=0.045). IgG2 aß2-GP1 was associated with venous thrombosis (Fisher's exact test, P=0.012), and IgG3 aß2-GP1 was associated with venous thrombosis (Fisher's exact test, P=0.036) and fetal loss (Fisher's exact test, P=0.024).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
In the present study we measured IgG and IgM aCL and aß2-GP1 levels retrospectively, in the same serum sample, in a population of 130 SLE and 35 PAPS patients. We have also determined simultaneously the IgG subclass distribution of aCL and aß2-GP1 in patients with elevated titres of IgG aCL or aß2-GP1. Our findings (Table 1Go) are broadly in agreement with previous reports [15, 17], although higher prevalences for IgG aß2-GP1 in SLE have been reported previously [9, 18, 27]. These reported variations in IgG aß2-GP1 levels could be due to differences in the sensitivity of the assays used, differences in the definition of the cut-off point for positivity, and differences in the selection and ethnic composition of the patient populations studied. In our aß2-GP1 ELISA, non-specific binding was subtracted and the cut-off point for all assays was set at four standard deviations above the mean, which increased the specificity of our assays.

We found that raised IgG aß2-GP1 levels were more frequent in patients with PAPS than in SLE patients; the same was true when we examined patients who were positive for only IgG or IgM aCL. Levels of IgG and IgM aCL correlated positively with levels of aß2-GP1 in PAPS patients (P<0.0001 for IgG, P=0.03 for IgM) but not in SLE patients. The lack of correlation in the SLE group may be attributed to the fact that a significant proportion of patients in this group had very low levels of IgG aß2-GP1. Conversely, a strong correlation was found between IgG aß2-GP1 and aCL levels (P=0.0042) when we examined SLE patients who were positive for only IgG aß2-GP1.

The determination of the IgG subclass distribution of antibodies is technically challenging because of inherent variations in the detection sensitivity of the different subclass-specific monoclonal antibodies used and the lack of appropriate standards [10]. This problem can be alleviated by establishing the equipotency of the subclass-specific monoclonal antibodies used [10] or by using hapten-specific chimaeric IgGs as controls [28]. In most published IgG subclass studies, levels of each subclass were expressed as a percentage of the total antigen-specific IgG activity [10], or the subclass-specific activity was reported as positive or negative when compared with a normal serum pool [23]. The second method of reporting subclass results has the disadvantage of not being able to provide an accurate picture of the exact relationships between the different subclasses, as each subclass is assayed independently. We therefore chose the first of these approaches and, as preparation of hapten-specific chimaeric IgG controls was beyond the scope of this study, we chose to establish the equipotency of the monoclonal antibodies used (see Patients and methods). We used the total IgG subclass distribution of normal subjects as our reference range as this did not differ from that previously reported for SLE patients [23, 29]. Interference from RF is a potential problem as we used whole immunoglobulins and not Fab fragments in our assays. There were no RF-positive patients in the PAPS group in the present study. However, especially for aCL ELISA assays, earlier studies have suggested that the presence of RF might interfere in IgM aCL assays in sera that are positive for IgG aCL [30, 31]. As RFs are frequently IgM antibodies, there should be no interference in assays using anti-IgG as the conjugated antibody. We are therefore confident that our subclass assays were free of any interference by the presence of RF in our patients’ sera.

Various combinations of IgG aCL subclass distributions have been reported in SLE and PAPS [20–23]. Most previous studies have found increased levels of IgG1 and IgG3 [20–22], although a recent study reported IgG2 as the predominant major IgG subclass for aCL [23]. We found that the percentage mean levels were higher for IgG1 aCL, but IgG2 and IgG3 were more prevalent in terms of subclass positivity (Fig. 2aGo, Table 2Go). The predominance of IgG1 was more obvious in patients with high levels of IgG aCL. Our most interesting finding was that patients who were positive for IgG aß2-GP1 had significantly higher levels of IgG2 aCL than patients who were negative for IgG aß2-GP1 (Table 3Go). This suggests that aCL measured by conventional ELISAs are heterogeneous autoantibodies, and that this heterogeneity may be related to their reactivity with ß2-GP1 in solid-phase assays.

The only other study that has explored the IgG subclass distribution of aß2-GP1 reported the predominance of IgG2 [19]. In the present study also, IgG2 was found to be the most predominant subclass, although we also observed a higher proportion of IgG1 aß2-GP1 (24.4%) than Arvieux and colleagues [19], who had previously found that IgG1 accounted for only 6% of the total IgG aß2-GP1. The IgG aß2-GP1 subclass distribution was different in patients positive for both IgG aß2-GP1 and aCL compared with patients who were positive for IgG aß2-GP1 but negative for IgG aCL (Table 3Go).

Protein antigens usually elicit a T-cell-dependent antibody response which is restricted to the IgG1 and IgG3 subclasses of IgG [13, 14], whereas carbohydrate antigens elicit a T-cell-independent antibody response that is commonly restricted to the IgG2 subclass. In the present study we observed the relatively unusual situation of a protein antigen (ß2-GP1) eliciting a T-cell-independent response. A possible explanation is that the carbohydrate chains of ß2-GP1 are involved in its recognition by B-cells. Previous observations that monoclonal antibodies bind equally well to native and N-glyconase-digested ß2-GP1 in vitro contradict this hypothesis [32]. Additionally, Arvieux et al. [19] have presented evidence against the involvement of carbohydrates in the binding sites of ß2-GP1 for aß2-GP1. There is considerable evidence that aß2-GP1 from patients with the APS are of low affinity and that they can bind to ß2-GP1 molecules when these are clustered on a phospholipid membrane [33]. Additionally, aß2-GP1 affinity increases when ß2-GP1 is dimerized [34]. Consequently, one could assume that when ß2-GP1 is bound to phospholipid surfaces (e.g. on the surface of apoptotic cells) a repetitive epitope may be formed, which can elicit a T-cell-independent antibody response.

The IgG subclass response to an antigen is also influenced by other factors, such as the cytokine environment [35], the chronicity of antigenic stimulation [36] and the genetic background of the subject [37]. There are only a few longitudinal studies of the IgG subclass distribution of autoantibodies, and most of these have suggested that the isotype pattern remains constant [38]. Consequently, a study of the IgG subclass distribution of aCL and aß2-GP1 over time in relation to disease duration and severity (which was outside the scope of the present study) would be very interesting.

Although IgG2 aCL has been reported to be associated with arterial and/or venous thrombosis [23], there are no studies which have specifically examined the clinical associations of IgG aß2-GP1 subclasses. In the present study, we have shown associations for IgG2 and IgG3 aCL and aß2-GP1 subclasses with thrombosis and fetal loss. Associations of IgG2 and IgG3 aCL with fetal loss have not been reported previously, and the associations we found in the present study between IgG2 and IgG3 aß2-GP1 and (some or all of) the clinical manifestations of the APS are reported here for the first time. It is notable that, while IgG2 and IgG3 aCL were associated with arterial thrombosis, IgG2 and IgG3 aß2-GP1 were found to be more specifically associated with venous thrombosis. Arvieux et al. [19] suggested that, although the predominance of the IgG2 subclass of aß2-GP1 was probably of clinical significance, the involvement of Fc{gamma} receptors in the pathogenicity of these antibodies was unlikely. Furthermore, as IgG2 and IgG3 are known to differ in their effector functions, the associations found here for both of them with the same clinical manifestations suggest that more than one mechanism is involved in the pathogenesis of thrombosis and fetal loss in APS.

The results of the present study support the view that aCL and aß2-GP1 belong to two related but distinct groups of heterogeneous autoantibodies. The IgG subclass distribution of aCL appears to be dependent on the presence or absence of elevated IgG aß2-GP1 in patients’ sera. The IgG subclass distribution of aß2-GP1 suggests that the antibody response against ß2-GP1 may be T-cell-independent. Significant associations were found between individual IgG subclasses and the main clinical manifestations of the APS. Further research is needed, focusing on the mechanisms of induction and potential pathogenicity of aß2-GP1 and aCL, on the importance of aß2-GP1 in patients with aCL occurring in non-autoimmune diseases, and—in the context of SLE and PAPS patients—on changes in the IgG subclass distribution of the antibodies with time and the duration and severity of disease.


   Notes
 
Correspondence to: M. Samarkos, 5th Department of Internal Medicine, Evangelismos Hospital, 45–47 Ipsilantou Street, Athens 10676, Greece. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 4 July 2000; Accepted 27 March 2001


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