Subclinical atherosclerosis in systemic lupus erythematosus and antiphospholipid syndrome: focus on ß2GPI-specific T cell response.

OBJECTIVE: Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. ß2 glycoprotein I (ß2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, ß2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to ß2GPI and its relationship with atherosclerotic process. APPROACH AND RESULTS: Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte ß2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to ß2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte ß2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte ß2GPI and TF with IMT or FMD was detected. ß2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to ß2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm. CONCLUSIONS: A significant percentage of patients with SLE and PAPS show a ß2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

High expression of beta2-glycoprotein I is associated significantly with the earliest stages of hepatitis B virus infection.

Human beta2-glycoprotein I (beta2-GPI) binds to recombinant hepatitis B surface antigen (rHBsAg) and can bind specifically to annexin II, which is located on the cell membrane of human hepatoma SMMC-7721 cells. Viral envelope proteins are essential for mediating cellular entry. The aim of this study was to investigate the role of beta2-GPI in the early stages of hepatitis B virus (HBV) infection. Western blot and qRT-PCR analyses revealed that beta2-GPI expression was upregulated in HepG2.2.15 cells at both the mRNA and protein level and was almost non-existent in 293T and CHO cells. Furthermore, annexin II was expressed at lower levels in HepG2.2.15 cells compared to L02, HepG2, and SMMC-7721 cells. Additionally, ELISA analyses demonstrated that beta2-GPI enhanced the ability of HBsAg to bind to cell surfaces, and there was differential adhesion to L02, HepG2, HepG2.2.15, and 293T cells. Western blot and ELISA were then performed to assess the effects of HBV and the HBsAg domain on beta2-GPI expression in co-transfected 293T cells. This study revealed that HBV and the large HBV envelope protein increased beta2-GPI expression. Further investigation indicated that beta2-GPI colocalized with HBsAg in the cytosol of HepG2.2.15 cells, with sodium taurocholate co-transporting polypeptide (NTCP) on the cell membrane in NTCP-complemented HepG2 cells, and with annexin II in the cytosol of HepG2 and HepG2.2.15 cells. These data suggest that high expression of beta2-GPI enhances HBsAg binding to cell surfaces, thus contributing to virus particle transfer to the NTCP receptor and interaction with annexin II for viral membrane fusion.

TLR2 is one of the endothelial receptors for beta 2-glycoprotein I.

During the antiphospholipid syndrome, beta2-gpI interacts with phospholipids on endothelial cell (EC) surface to allow the binding of autoantibodies. However, induced-pathogenic intracellular signals suggest that beta2-gpI associates also with a receptor that is still not clearly identified. TLR2 and TLR4 have long been suspected, yet interactions between TLRs and beta2-gpI have never been unequivocally proven. The aim of the study was to identify the TLR directly involved in the binding of beta2-gpI on EC surface. beta2-gpI was not synthesized and secreted by ECs in vitro, but rather taken up from FCS. This uptake occurred through association with TLR2 and TLR4 which partitioned together in the lipid rafts of ECs. After coimmunoprecipitation, mass-spectrometry identification of peptides demonstrated that TLR2, but not TLR4, was implicated in the beta2-gpI retention. These results were further confirmed by plasmon resonance-based studies. Finally, siRNA were used to obtain TLR2-deficient ECs that lost their ability to bind biotinylated beta2-gpI and to trigger downstream phosphorylation of kinases and activation of NFkappaB. TLR4 may upregulate TLR2 expression, thereby contributing to beta2-gpI uptake. However, our data demonstrate that direct binding of beta2-gpI on EC surface occurs through direct interaction with TLR2. Furthermore, signaling for anti-beta2-gpI may be envisioned as a multiprotein complex concentrated in lipid rafts on the EC membrane.

Beta2-glycoprotein I and its clinical significance: from gene sequence to protein levels.

In order to elucidate beta2-GPI at the DNA level and characterize its polymorphisms, mRNA expression, protein levels and clinical significance at each of these steps, a molecular review of beta2-GPI literature was performed. The human beta2-GPI complete nucleotide sequence has been reported and it consists of 8 exons separated by large introns. The beta2-GPI gene is polymorphic with four alleles. The distribution of point mutations can be significantly different between various racial populations. DNA variation studies of the beta2-GPI gene identified a total of 151 single-nucleotide polymorphisms, 26 of which are within regions with potential clinical significance. Southern blot analysis indicated the presence of one gene product only. An atypical TATA box and a hepatic nuclear factor-1 element are both essential for beta2-GPI promoter activity. Transcription factor binding sites for STAT, CREB, C/EBPbeta, NF-1, AP-1, NFAT, HNF-3beta and HNF-1 have been identified in the promoter region of the beta2-GPI gene by computer analysis. The beta2-GPI transcriptional signal of 1.5 kb was detected in Northern blot analysis and its 326-amino-acid sequence was found to be one of the most proline-rich eukaryotic proteins. Amino acid substitutions have been shown to be associated with loss of phospholipid binding, development and recognition of antiphospholipid antibodies.

Antiphospholipid antibodies mediate autoimmunity against dying cells.

The Antiphospholipid Syndrome (APS) is characterized by thrombosis and pregnancy loss, clinical events mediated by pathogenic anti-phospholipid autoantibodies (aPL). ß2-glycoprotein I (ß2GPI) is the major autoantigens recognized by aPL. ß2GPI is a cationic protein that binds to negatively charged surfaces such as those of apoptotic cells. This feature may lead to two major events: i) immunization with ß2GPI fosters the Fc-receptor-mediated uptake by antigen presenting cells of apoptotic material decorated with ß2GPI and the activation of ß2GPI-specific T cells which in turn provide help to ß2GPI-specific B cells for the production of anti-ß2GPI; ii) apoptotic bodies decorated with ß2GPI can be opsonized by anti-ß2GPI and shifted towards a pro-inflammatory clearance by macrophages; epitope spread can occur with the generation of autoimmunity against nuclear autoantigens. In the presence of a predisposing genetic background and of a particular cytokine environment (type I interferons), the sequential emergence of autoantibodies can evolve into overt clinical disease. The spectrum of clinical phenotypes of the patients can be modulated by several factors affecting the pathogenicity of anti-ß2GPI (e.g. domain specificity). We conclude that dying cells may play a dual role in APS: (I) as immunogen for the induction of aPL (etiology) and (II) as targets of aPL for the chronification of inflammation and the development of autoimmune diseases (pathology).