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Rheumatology Advance Access originally published online on June 3, 2008
Rheumatology 2008 47(8):1144-1150; doi:10.1093/rheumatology/ken120
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Natural antibodies against phosphorylcholine as potential protective factors in SLE

J. Su1,2,*, X. Hua1,2,*, H. Concha2, E. Svenungsson3, A. Cederholm1,2 and J. Frostegård1

1Rheumatology Unit, 2Center for Infectious Medicine, Department of Medicine, Karolinska University Hospital, Huddinge and 3Rheumatology Unit, Department of Medicine, Karolinska University Hospital, Solna, Stockholm, Sweden.

Correspondence to: J. Su, Department of Medicine, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. E-mail: jun.su{at}ki.se


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objective. We have recently reported that natural antibodies against phosphorylcholine (anti-PC) have atheroprotective properties. Here we compare anti-PC with other autoantibodies in SLE patients with and without cardiovascular disease (CVD).

Methods. Twenty-six women (52 ± 8.2 yrs) with SLE and a history of CVD (SLE cases) were compared with 26 age-matched women with SLE without CVD (SLE controls) and 26 age-matched population-based control women (controls). PC was conjugated with BSA (PC-BSA) or keyhole-limpet haemocyanin (PC-KLH). Anti-PC and antibodies against phosphatidylserine (anti-PS) and BSA (anti-BSA) were studied by ELISA. Anti-PC-IgG were extracted from intravenous immunoglobulin (IVIG). Activation of endothelial cells by platelet-activating factor (PAF) was studied with FACScan.

Results. IgG anti-PC-BSA and anti-PC-KLH were decreased among SLE-cases and SLE-controls as compared with controls (P < 0.005 and P < 0.05), respectively. SLE cases were more prevalent in the lowest 25th percentile of anti-PC-IgM (and IgG) as compared with controls (P < 0.05) but anti-PC-IgM levels did not differ significantly between groups. Among SLE controls, anti-PC-BSA were associated negatively with organ damage (SLICC) and disease activity (SLEDAI) (P < 0.05). Among SLE cases, anti-PC-BSA and anti-PC-KLH were associated negatively with SLICC (P = 0.021; P = 0.010) and anti-PC-BSA was negatively associated with SLEDAI (P < 0.039).

Anti-PS-IgG and anti-BSA-IgG were raised among SLE cases as compared with other groups (P < 0.05) and did not cross-react with anti-PC. Anti-PC-IgG could be extracted from IVIG and inhibited PAF-induced expression of adhesion molecules.

Conclusion. Low levels of anti-PC could be of importance in SLE. Anti-BSA and anti-PS and low levels of anti-PC could contribute to development of CVD in SLE.

KEY WORDS: Systemic lupus erythematosus, Atherosclerosis, Antibodies, Phosphorylcholine


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cardiovascular disease (CVD) is common among patients with SLE and associated with increased prevalence of atherosclerotic plaques, as determined by intima–media thickness (IMT) in the carotid artery [1]. This is an important clinical problem, but could also give information about how autoimmunity may influence atherosclerosis in the general population. CVD in SLE is related to both traditional risk factors including dyslipidaemia and non-traditional risk factors. These latter include increased levels of oxidized low-density lipoprotein (OxLDL); raised activity of inflammatory factors like the TNF and platelet-activating factor (PAF)-acetylhydrolase; inflammation as determined by CRP; homocysteine and aPLs [2–5]. We recently demonstrated a novel mechanism by which aPLs may cause CVD in SLE by decreasing binding of anti-thrombotic Annexin A5 to endothelium [6]. The aPL have also been implicated as a risk factor for CVD in the general population [7, 8].

Atherosclerosis is characterized by the presence of large numbers of macrophages and T cells and production of inflammatory cytokines in the atherosclerotic lesions [9]. One important antigen in atherosclerosis is OxLDL, which is immune-stimulatory, pro-inflammatory and is also taken up by macrophages in the artery wall. We and others demonstrated that many pro-inflammatory effects of OxLDL are caused by inflammatory phospholipids generated during LDL oxidation. These have PAF-like properties since a major ligand for the PAF receptor, phosphorylcholine (PC), is exposed on OxLDL [10, 11]. PC is an interesting molecule from an immunological point of view, since it is a target of an important group of natural antibodies against PC (anti-PC). In mice, anti-PC are known to protect against lethal infection with Streptococcus pneumoniae, but their role in humans is not very well characterized [12]. Interestingly, several studies indicate that immunization of experimental animals with OxLDL decreases atherosclerosis development [13, 14].

We recently demonstrated that anti-PC, especially of IgM subclass, is atheroprotective, and predict a favourable outcome in atherosclerosis development in hypertensives [15]. In line with this are recent studies where passive transfer of anti-PC inhibits atherosclerosis development [16]. Little is known about anti-PC in SLE. Here we demonstrate that anti-PC are decreased in SLE, while anti-PS and anti-BSA (BSA is a carrier protein for PC in our studies) are raised. Extracted anti-PC inhibited PAF-induced endothelial activation. The implications of these findings are discussed.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Subjects
The study group consisted of 26 women with SLE with a history of CVD (myocardial infarction, angina pectoris, thromboembolic stroke or intermittent claudication), 26 age-matched women with SLE but without clinical manifestations of CVD and 26 age-matched control women who were recruited randomly from the Swedish population registry. Details of the recruitment and clinical characteristics of the three groups have been reported previously [4]. Routine measurements of SLE-related parameters, including disease activity measurements (SLEDAI), organ damage index (SLICC) autoantibodies and lipids have been reported previously [4]. All patients fulfilled the 1982 revised criteria of the ARA for classification of SLE [4]. The study was approved by the local ethics committee of the Karolinska Hospital. All subjects gave written informed consent before entering the study.

Carotid ultrasound
The right and left carotid arteries were examined by use of a duplex scanner (Acuson Sequoia, Mountain View, CA, USA) and the IMT was determined as described [17]. A plaque was defined as a local intima–media thickening, with a thickness >1 mm.

Determination of autoantibodies against PC-BSA, PC-KLH, PS and BSA
IgG and IgM antibodies to PC-BSA or PC-KLH, PS, BSA was determined by ELISA. F96 microtitre polysorp plates (Roskilde, Denmark), were coated with PC-BSA or PC-KLH (Biosearch Technologies, Inc., CA, USA) 10 µg/ml, BSA (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) 2 µg/ml; Immulon 1B plates (Thermo Labsystems, Franklin, MA, USA) were coated with PS (Sigma) 160 µg/ml, (PC-BSA, PC-KLH, BSA in PBS buffer; PS in ethanol and allowed to dry), incubated overnight at 4°C. According to the manufacturer's description, each KLH molecule contained eight PC and each BSA molecule contained two PC. After five washings with PBS, the plates were blocked with 2% PBS–BSA for 2 h at room temperature. Serum samples were diluted 1 : 30 in 0.2% BSA–PBS and added at coated plates 50 µl/well and washed as described earlier. The plates were incubated with 100 µl/well of alkaline phosphatase-conjugated goat anti-human IgG (Sigma) diluted 1 : 9000 or alkaline phosphatase-conjugated goat anti-human IgM (Sigma) diluted 1 : 7000, The reaction was developed with alkaline phosphatase substrate (Sigma) and optical density (OD) was read at 405 nm with an ELISA Multiscan Plus spectrophotometer (Molecular Devices Emax, San Francisco, CA, USA). All samples were measured in duplicates and the coefficient of variation was <15%.

Extraction of anti-PC IgG from IVIG
Column coupling
PC-KLH (Biosearch Technologies Inc., CA, USA) was diluted in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) at 1 mg/ml, then coupled to a HiTrap NHS column (Amersham Biosciences, Piscataway, NJ , USA) according to the manufacturer instruction. The coupled column was stored in 4°C.

Purification of anti-PC IgG
Human pooled immunoglobulin (Baxter Medical AB Torshamnsgatan 35 Stockholm, Sweden) was diluted in binding buffer (20 mM Na2HPO4) at 50 mg/ml and filtered through 0.45 µm filter before passing through pre-coupled PC-KLH Sepharose gel column. Washing and further elution were performed according to the manufacturer's recommendation. Shortly, anti-PC IgG was eluted by 0.1 M glycine–HCl buffer, the pH value was neutralization to 7.0 by 1 M Tris–HCL (pH 9.0). The purified fractions were desalted using PD-10 columns (Amersham Pharmacia Biotech AB, Warrenale, PA, USA). The antibody was concentrated by centrifugation with Centriprep Centrifugal Filter (Millipore, USA, Billerica, MA, USA), then stored in 4°C after filtered with 0.22 µm filter.

Anti-PC-IgG antibody characterization
The binding specificity of the purified anti-PC-IgG was measured by competition ELISA. The purified anti-PC-IgG was pre-incubated with indicated concentrations of competitors, including PC-KLH, phosphatidylcholine (PtC), KLH. The supernatants were added to microtitre plates (Nunc, Roskilde, Denmark) coated with PC-KLH (10 µg/ml), the amount of antibody bound were detected with alkaline phosphatase-conjugated goat anti-human IgG, colour was developed by adding the alkaline phosphatase substrate. The percentage of inhibition was calculated as (OD without competitor – OD with competitor) x 100%/OD without competitor.

Adhesion molecule expression by endothelial cells
Pooled human umbilical vascular endothelial cells (HUVECs) at passage 2 were purchased from Cascade Biologics, Inc. (Portland, OR, USA). Cultures were maintained in EGMTM phenol red-free medium (Clonetics, San Diego, CA, USA), containing 2% of fetal bovine serum and supplements, at 37°C under humidified 5% CO2 conditions. All experiments were performed at passages 3–5. HUVECs were seeded at 6 x 104 cells/2 ml density on 6-well plates (Nunc Inc., Naperville, IL, USA).

After allowing 24 h for cells attachment, the cells were incubated with PAF 1 µg/ml either with anti-PC-IgG 10 µg/ml or with commercially available human immunoglobulin (IVIG) Gammagard®S/D (Baxter, Inc.) 10 µg/ml. After 24 h incubation, detached floating cells were washed away, cell were harvested into BD Falcon tubes (Becton Dickinson, San Jose, CA, USA). After centrifuging at 410g for 5 min, cells were resuspended in 300 µl FACS buffer (1% FBS–PBS), incubated with 10 µl PE-conjugated anti-CD54 (eBiosceince, San Diego, CA, USA) and 10 µl FITC-conjugated anti-Human CD106 (Becton, Dickinson) for 30 min on ice. The intercellular adhesion molecule (ICAM-1) CD54 and the vascular cell adhesion molecule (VCAM-1) CD106 were studied with FACSCalibur flow cytometer (Becton Dickson) equipped with CellQuest Software (Becton Dickinson). For each sample, 10 000 cells were analysed.

Binding of Annexin A5 to endothelial cells has been described in detail previously [6]. Briefly, HUVECs were incubated with individual serum from the three groups studied, and Annexin A5 conjugated with FITC added and then binding wasdetermined by FACScan.

Statistical analysis
The statistics were computed using StatView software (SAS Institute AB, Göteborg, Sweden). Antibody levels were dichotomized at the 75th and 90th percentile or determined as continuous variables as indicated. Correlation analysis was performed using simple regression for normally distributed variables, and Spearman's correlation analysis for non-normally distributed variables. Skewed continuous variables were logarithmically transformed to attain a normal distribution. Study groups were compared using ANOVA for continuous variables with Fischer's protected least significant difference test as post hoc test and Chi-squared for categorical variables where comparisons between two groups were made using Fischer's exact test. The significance level was put at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Clinical, metabolical and laboratory characteristics
To give background information for the present study, basic clinical and metabolical characteristics are presented in Table 1, which is a modified version of what has been previously reported [4]. To summarize previous findings: SLE cases had raised levels of very low density lipoprotein (VLDL) and lipoprotein(a) [Lp(a)], but decreased levels of high density lipoprotein (HDL), raised inflammatory parameters including CRP, TNF and PAF-acetylhydrolase; elevated lupus anti-coagulants and antibodies against OxLDL; increased levels of circulating OxLDL [4, 5, 18]. There were no significant differences in blood pressure, smoking and prevalence of diabetes mellitus between the three groups.


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  		TABLE 1. Basic characteristics of study group

 
As reported previously, SLE cases had taken a higher cumulative prednisolone dose (P = 0.04); lipid-lowering agents (statins in all cases), anti-hypertensives, low-dose aspirin, warfarin and AZA were more common among SLE cases [4].

Determination of antibody levels
Antibody levels are presented in Table 2. Anti-PC-IgG were decreased among SLE controls and SLE cases as compared with controls for PC-BSA (95.9 ± 30.4 vs 98.5 ± 35.8; P = 0.002 and P = 0.001) and PC-KLH (100.1 ± 19.1 vs 100.9 ± 19.5; P = 0.03 and P = 0.04). Anti-PC-IgG values in the lowest 25th percentile were more common among SLE cases as compared with controls (P = 0.01) with PC-BSA as antigen but did not reach significance with PC-KLH. Values in the lowest 10th percentile were more common among SLE cases as compared with controls both when PC-BSA and PC-KLH were used as antigens (P = 0.05).


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  		TABLE 2. Antibody activities of IgG subclass against PC-BSA, PC-KLH, PS, BSA

 
Levels, determined as continuous variables, of anti-PC-IgM did not differ significantly either when PC-BSA or PC-KLH was used as antigen. However, IgM anti-PC-BSA values in the lowest 25th percentile and in the lowest 10th percentile were more common among SLE cases as compared with controls (P = 0.018). This also applied when PC-KLH were used as antigens (P = 0.05).

Anti-BSA-IgG differed completely from anti-PC-BSA and there was no cross-reactivity between these antigens (Fig. 1b). There was a striking association between anti-BSA IgG levels and CVD in SLE (Table 2) in contrast to anti-PC-BSA (Table 2). ANOVA analysis indicates that anti-BSA-IgG was raised among SLE cases as compared with SLE controls (176.8 ± 63.9 vs 142.9 ± 55.1; P = 0.02) and among SLE controls as compared with controls (142.9 ± 55.1 vs 115.4 ± 31.2; P = 0.045).


Figure 1
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  		FIG. 1. Inhibition by PC, PS and PPD of binding to PC-BSA-coated plates. In order to investigate the cross-reactivity of anti-PC, competition assay was performed. Pooled sera were pre-incubated overnight with different concentrations of PC, PS or PPD at 4°C and centrifuged at 16 000 g for 30 min (4°C). The supernatants were then tested for antibody binding to PC-BSA-coated plate. Results are presented as (OD without competitor – OD with competitor) x 100%/OD without competitor as shown in Fig. 1a. In order to investigate the cross-reactivity of anti-BSA and anti-PC, a competition ELISA was performed based on anti-BSA. Results are presented same as shown in Fig. 1b.

 
For anti-BSA-IgG, in the highest 25th percentile, SLE cases were more prevalent as compared with controls and SLE controls (P < 0.001 and P = 0.001). In the highest 10th percentile, SLE cases were more prevalent than controls (P = 0.02).

Levels of anti-PS-IgG was raised among SLE cases as compared with SLE controls (74.9 ± 35.3 vs 56.5 ± 19.6 P = 0.015) and controls (74.9 ± 35.3 vs 54.6 ± 21.8 P = 0.007). Likewise, if dichotomized into the highest 25th percentile, SLE cases were more prevalent than SLE controls (0.009) and non-significantly more prevalent than controls (0.066).

Also at the highest 10th percentile of anti-PS IgG, SLE cases were more common than SLE controls (P = 0.01) and controls, but this latter difference did not reach statistical significance (P = 0.09).

Associations between antibody levels and other measurements
Among SLE controls, anti-PC-BSA were associated negatively with organ damage SLICC values (R = –0.35; P = 0.037). Anti-PC-BSA was negatively associated with SLEDAI (R = –0.36; P = 0.039) while the negative association between anti-PC-KLH and SLICC or SLEDAI did not reach statistical significance (data not shown).

Among SLE cases, anti-PC-BSA and anti-PC-KLH were associated negatively with organ damage SLICC values (R = –0.44; P = 0.021 and R = –0.49; P = 0.010). Anti-PC-BSA was negatively associated with SLEDAI (R = –0.36; P = 0.039) while the negative association between anti-PC-BSA or anti-PC-KLH and SLEDAI did not reach statistical significance (data not shown).

Anti-BSA-IgG was negatively associated with triglycerides (TGs) among SLE cases (R = –0.49; P = 0.014) and controls (R = –0.40; P = 0.044) respectively and HDL was negatively associated with anti-BSA-IgG among controls (R = –0.49; P = 0.012).

Among SLE cases, anti-PC-BSA IgM and anti-PC-KLH IgM were both positively associated with binding of Annexin A5 to endothelium (R = 0.45; P = 0.022) and (R = 0.43; P = 0.029). Anti-PC-BSA IgG or anti-PC-KLH IgG was not associated with Annexin A5 binding.

There were no striking associations between anti-PS-IgG and other parameters.

There was no significant association between present or cumulative doses of prednisolone, AZA, cyclophosphamide or other treatment and antibody determinations (data not shown)

Age or mean IMT were not significantly associated with the measured antibodies (data not shown).

Cross-reactivity between antibodies
Competition studies indicate that anti-PC were competed out by pre-incubation with itself (>70%), while PS and tuberculin-purified protein derivative (PPD) had no competitive capacity (<20%). Data is shown in Fig. 1a. Anti-BSA were competed out by pre-incubation with itself (>70%), while PC had no competitive capacity (<20%), as shown in Fig. 1b.

We extracted anti-PC-IgG from IVIG. The specificity of such polyclonal anti-PC-IgG antibodies was determined by a competition ELISA. Figure 2 shows that the binding of purified anti-PC-IgG is inhibited significantly by pre-incubation with different concentrations of PC-KLH. However, there was no association with other antigens.


Figure 2
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  		FIG. 2. In order to characterize purified anti-PC-IgG, competition ELISA was performed. Anti-PC IgG binding to PC-KLH is competed by PC-KLH or PtC, KLH. PC-KLH (10 µg/ml) was plated on microtitre wells overnight at 4°C. Anti-PC IgG was mixed with equal volumes of various competitors overnight at 4°C. The supernatants were added to microtitre palate. The bound anti-PC IgG was detected and expressed as (OD without competitor – OD with competitor) x 100%/OD without competitor.

 
Furthermore, anti-PC-BSA and anti-PC-KLH cross-react (Fig. 3) but not completely.


Figure 3
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  		FIG. 3. In order to investigate the cross-reactivity of anti-PC-BSA and anti-PC-KLH, competition assay was performed. Pooled sera were pre-incubated overnight with different concentrations of PC-BSA or PC-KLH at 4°C and centrifuged at 16 000 g for 30 min (4°C). The supernatants were then tested for antibody binding to PC-BSA-coated plate. Results are presented as (OD without competitor – OD with competitor) x 100%/OD without competitor.

 
Anti-PC-IgG inhibits the adhesion molecule expression of endothelial cells
To study the inhibition of anti-PC-IgG on PAF-induced ICAM-1 and VCAM expression by endothelial cells, HUVECs were incubated for 24 h with PAF either with anti-PC-IgG or, as a control, IVIG. ICAM-1 expression was significantly inhibited by anti-PC-IgG, while IVIG did not show any reduction of ICAM-1 expression (Fig. 4a). Similarly, anti-PC-IgG reduced the VCAM-1 expression by endothelial cells treated with PAF (Fig. 4b).


Figure 4
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  		FIG. 4. Anti-PC-IgG (10 µg/ml) mediated inhibition of ICAM-1and VCAM-1 induction induced by endothelial cells treated with PAF (1 µg/ml). (a) ICAM-1 expression by endothelial cells (grey) treated with PAF (red) is inhibited by anti-PC-IgG (green) as compared with IVIG (blue). (b) VCAM-1 expression by endothelial cells (grey) treated with PAF (red) is inhibited by anti-PC-IgG (green) as compared with IVIG (blue).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Antibodies against PC have been known for more than 20 yrs, though their clinical importance in general is not well characterized and their role in SLE is not known [12]. We recently demonstrated that anti-PC of IgM subclass (and trendwise IgG) are protection factors for atherosclerosis in patients with established hypertension [15].

Here we report that anti-PC-BSA IgG were negatively associated with SLICC and SLAM in both SLE groups with similar associations albeit somewhat weaker for anti-PC-KLH IgG. Anti-PC recognizes apoptotic cells and also OxLDL, and it is possible that anti-PC could contribute to clearance of these. Since apoptic function is generally believed to be disturbed in SLE, one possibility that deserves further study is that low anti-PC could contribute to decreased clearance, and thus predispose to some aspects of SLE. It is noteworthy that while an array of antibodies in SLE are reported to be raised, natural, germ-line encoded anti-PC are low.

We also report that anti-PC are less prevalent both among women with SLE and a history of CVD, and SLE patients without such a history as compared with control women. These differences were most pronounced for anti-PC IgG where mean levels were lower which was not the case for anti-PC IgM. However, low levels of both anti-PC IgM and anti-PC IgG were more common among SLE cases as compared with controls. PC is a hapten that needs a carrier to become immunogenic, e.g. exposed for the immune system. Similar results were obtained when PC was linked to two different carriers, BSA and KLH.

The PC head group is one of the neoantigens exposed on OxLDL that can elicit an immune response. PC is also exposed on bacteria as S. pneumoniae, gingival and other pathogens including nematodes and on apoptotic cells. An innate immune response may have developed to help clearance of such compounds and anti-PC have been linked to pneumonia but also to oral and other pathogens, and interestingly, anti-PC is also present in animals raised under sterile conditions [19–22].

SLE is a chronic inflammatory disease characterized by a perturbed immune response against self with production of high levels of an array of autoantibodies believed to contribute to the clinical symptoms in SLE. In sharp contrast, and surprisingly, antibodies against PC instead are low in the SLE patients in our study. Interestingly, an autoantibody that we have used to detect OxLDL in the circulation recognizes PC on OxLDL, and OxLDL measured by this method was raised in SLE cases [4] and also associated with SLE in general [23]. Based on these findings, we suggested that lipid peroxidation could be one important factor not only in SLE-related CVD but also in SLE in general. Our earlier findings indicate that OxLDL can promote T-cell activation [24], monocyte differentiation [25] and activation of endothelial cell adhesiveness [26]. Decreased levels of anti-PC could thus predispose to a defective clearance of obnoxious particles as OxLDL.

Our finding may indicate that low anti-PC levels could predispose to CVD in SLE in general. However, since anti-PC were also low among SLE controls, clearly other factors are needed to explain why CVD in SLE is developed more prematurely in some patients. Such factors include both traditional risk factors like hypertension, dyslipidaemia and non-traditional risk factors such as aPL, increased LDL oxidation and systemic inflammation with raised CRP and increased activity in the TNF system and other inflammatory enzymes like (PAF)-acetylhydrolase [1].

We also demonstrate that specific anti-PC IgG could be extracted from pooled human IG (IVIG) and that these antibodies inhibit PAF-induced endothelial activation, as determined by expression of VCAM and ICAM-1.

SLE is characterized by vascular inflammation in many organs. Expression and up-regulation of adhesion molecules is basic to migration of inflammatory cells into the tissues. ICAM-1 and VCAM-1 appear to be the predominant adhesion molecules at inflammatory sites. PAF is an important pro-inflammatory phospholipid, which shares many of its effects with OxLDL, having PC as a major epitope that binds to the PAF receptor. PAF is also produced by activated immune competent cells including endothelial cells [10, 11]. PAF and PAF-like lipids most likely play an important role in the inflammation typical of atherosclerosis and inhibition of PAF by interfering with the PAF receptor leads to decreased atherosclerosis in mice models [27, 28]. In the present study, we therefore used PAF to stimulate HUVECs expressing ICAM-1 and VCAM-1, and demonstrate that anti-PC-IgG can suppress PAF-induced up-regulation of these adhesion molecules. This study indicates that anti-PC-IgG has anti-inflammatory effects by means of inhibiting of adhesion molecule expression by endothelial cells and could be one mechanism explaining the anti-atherosclerotic effects of anti-PC.

It is thus possible that anti-PC could protect against atherosclerosis both at an early and later stage through inhibition of PAF (or PAF-like lipids). Since vascular inflammation is a common feature in SLE and PAF is increased in active SLE [29], low anti-PC levels could, in principle, predispose to vascular inflammation in SLE.

Further support for an atheroprotective role of anti-PC comes from a recent report where passive immunization with anti-PC was demonstrated to inhibit atherosclerosis in a mouse model [16].

Here we report that anti-PC were negatively associated with both SLICC and SLAM, in both SLE groups when analysed separately. This was somewhat more pronounced for anti-PC-BSA. Low anti-PC levels could thus have a more general importance in SLE. Whether this is a general feature of anti-PC in SLE could be studied in a larger and randomized study. If anti-PC production is decreased in SLE, this could represent an important immunological aberration, which could even have a genetic background. Low anti-PC levels could also be caused by consumption of the antibodies. SLE patients have a defective clearance of apoptotic cells and cells undergoing apoptosis sequentially express a range of oxidation-specific neo-self PC determinants, which bind to anti-PC [21]. Anti-PC have been reported to be self-binding, participate in immunological networks and to be able to present antigen to T cells. It is therefore possible that a perturbation in natural antibodies as anti-PC could play a role in the immune dysregulation present in SLE and thus in the pathogenesis in SLE, a possibility that is now studied in our laboratory in larger patient cohorts.

In general, PC is an interesting compound from an immunological point of view, being an immunodominant determinant of pneumococcal teichoic acids. Anti-PC can protect mice against lethal infection with bacteria like S. pneumoniae [30]. Antibodies to PC are present in mice developed under sterile conditions and are germ-line encoded and therefore designated as natural antibodies [31]. PC has several properties that could in principle both promote and protect against disease depending on pathogen and type of inflammatory reaction [12].

SLE is a prototypical autoimmune disease, with protean manifestations, and typical of SLE there is a raised production of autoantibodies of different specificities. It is therefore surprising that anti-PC are not raised but decreased in both SLE groups. A caveat is that our SLE patients are not an unselected cohort but instead a nested case–control study, designed specifically to determine risk factors and mechanisms for CVD in SLE. Whether decreased anti-PC level is a general feature of SLE should therefore be addressed in larger unselected studies.

Anti-PC-BSA IgM and anti-PC-KLH IgM were both positively associated with binding of Annexin V to endothelium, which represents an opposite reaction as compared with our recent finding that anti-phospholipid antibodies against cardiolipin (aCL) are associated with decreased Annexin V binding, which is associated with CVD in SLE [6]. This finding may indicate that anti-PC IgM could have a protective effect also on atherothrombosis, e.g. Late-stage atherosclerosis and plaque rupture and not only on atherosclerosis.

It should also be noted that the cause of the low antibody levels against these antigens in individuals with increased atherosclerosis could be complex and different possibilities are not mutually exclusive. These include consumption into the atherosclerotic lesions, which are known to contain OxLDL epitopes, or formation of immune complexes containing PC or OxLDL. Still, independent of mechanism, low anti-PC could play a pathogenic role.

Antibodies against phosphatidylserine (anti-PS) with a serine group instead of PC at the sn-3 position in the phospholipid, could not compete out anti-PC. Anti-PS were raised among SLE cases and differed significantly from SLE controls and controls. Anti-PS thus appears to be an important antibody in predisposing to CVD in SLE, most likely through its pro-thrombotic effects. The exact role of interactions between PS and Annexin A5, which binds to PS is not clear.

Since BSA was the carrier protein for PC, we also tested anti-BSA per se, and one surprising finding is that anti-BSA-IgG are raised among SLE cases. These antibodies appear to be specific since they could be pre-absorbed by pre-incubation with BSA. Anti-BSA have been discussed as a possible marker and underlying aetiological agent in insulin-dependent diabetes mellitus [32]. Earlier reports suggest that not only antibodies to bovine proteins including milk and ovalbumin, but also immunoglobulin could play a role in autoimmune conditions including SLE [33, 34]. A recent study suggests that anti-BSA appears in patients with a compromised epithelial permeability, and reflect a defect in immunological tolerance and a predisposition to autoimmunity [35]. We here report that anti-BSA-IgG was significantly raised among SLE cases as compared with SLE controls, which were in their turn significantly higher than among controls. This finding is compatible with an aberrant immune reactivity against BSA in SLE.

In summary, our findings indicate that atheroprotective anti-PC could be extracted from IVIG and inhibited PAF-induced endothelial activation as determined by expression of VCAM and ICAM-1. Both SLE cases and SLE controls had, surprisingly, low anti-PC levels and we hypothesize that this could contribute both to CVD in SLE and to SLE per se.

In contrast, IgG anti-PS and anti-BSA were raised among SLE cases and associated with CVD and could thus be important risk factors for CVD in SLE patients.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We are grateful to Kerstin Jensen-Urstad for ultrasound determinations and Jill Gustafsson and Eva Jemseby for their help with management of patient cohorts and blood sampling.

Funding: This work was supported by the King Gustaf V 80th Birthday Fund, the Swedish Rheumatism Association, The Swedish Science Fund, The Torsten and Ragnar Soderberg Foundation, Center of Gender-related Medicine at Karolinska Institutet, the Swedish Society of Medicine, the Swedish Heart-Lung Foundation and CIDaT. This work was supported by grants from the 6th Framework Program of the European Union, Priority 1: Life Sciences, Genomics and Biotechnology for Health (grant LSHM-CT-2006-037227 CVDIMMUNE).

Disclosure statement: J.F. is a co-inventor on pending patents on the role of anti-PC in CVD. All other authors have declared no conflicts of interest.


   Notes
 
*J. Su and X. Hua equally contributed to this work. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 27 October 2007; revised version accepted 21 February 2008.
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