Interaction of the antiphospholipid syndrome autoantigen beta-2 glycoprotein I with DNA and neutrophil extracellular traps.

Beta-2 glycoprotein I (ß2GPI) is a phospholipid-binding plasma protein and prominent autoantigen in antiphospholipid syndrome (APS). Here, we tested the hypothesis that ß2GPI might bind to not only phospholipids, but also cell-free DNA and neutrophil extracellular traps (NETs). We report that ß2GPI interacts with cell-free DNA from different species, as well as NETs, in a dose-dependent manner, retarding their migration in an agarose-gel electrophoretic mobility shift assay. We confirm the direct binding interaction of ß2GPI with DNA and NETs by ELISA. We also demonstrate that ß2GPI colocalizes with NET strands by immunofluorescence microscopy. Finally, we provide evidence that ß2GPI-DNA complexes can be detected in the plasma of APS patients, where they positively correlate with an established biomarker of NET remnants. Taken together, our findings indicate that ß2GPI interacts with DNA and NETs, and suggest that this interaction may play a role as a perpetuator and/or instigator of autoimmunity in APS.

Necroptotic cell binding of ß2 -glycoprotein I provides a potential autoantigenic stimulus in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies against diverse self-antigens with damage to multiple organs. Immunization with the SLE autoantigen ß2 -glycoprotein I (ß2 GPI) and lipopolysaccharide (LPS), a known trigger of necroptosis, induces a murine model of SLE. We hypothesized that necroptotic cells, like apoptotic cells, provide a "scaffold" of cellular self-antigens, but, unlike apoptotic cells, necroptotic cells do so in a proinflammatory and immunogenic context. We demonstrate that ß2 GPI indeed binds to necroptotic cells and serves as a target for anti-ß2 GPI autoantibodies. We further demonstrate that necroptotic, but not apoptotic, cells promote antigenic presentation of ß2 GPI to CD4 T cells by dendritic cells. Finally, we show that ß2 GPI/LPS-immunized mice deficient in RIPK3 (receptor-interacting serine/threonine-protein kinase 3) or MLKL (mixed lineage kinase domain like), and consequently unable to undergo necroptosis, have reduced SLE autoantibody production and pathology. RIPK3-/- mice had low levels of SLE autoantibodies and no renal pathology, while MLKL-/- mice produced low levels of SLE autoantibodies initially, but later developed levels comparable with wild type (WT) mice and pathology intermediate to that of WT and RIPK3-/- mice. Serum cytokine levels induced by LPS tended to be lower in RIPK3-/- and MLKL-/- mice than in WT mice, suggesting that impaired proinflammatory cytokine production may impact the initiation of autoantibody production in both strains. Our data suggest that self-antigen (i.e. ß2 GPI) presented in the context of necroptosis and proinflammatory signals may be sufficient to overcome immune tolerance and induce SLE.

T cells from induced and spontaneous models of SLE recognize a common T cell epitope on ß2-glycoprotein I.

Systemic lupus erythematosus is a prototypic model for B-cell epitope spread in autoimmunity. Autoantibodies to numerous molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting phospholipid-binding proteins, particularly ß2-glycoprotein I. Notably, mice immunized with ß2-glycoprotein I and lipopolysaccharide develop a strong T cell response to ß2-glycoprotein I that is associated with autoantibody production and renal disease, similar to that seen in human SLE. Here we hypothesized that mice with murine systemic lupus erythematosus, whether induced or spontaneous, should have T cells that recognize ß2-glycoprotein I. We evaluated the response of splenic T cells from mice with induced (C57BL/6 and C3H/HeN) and spontaneous (MRL/lpr) systemic lupus erythematosus to peptides spanning the entire sequence of human ß2GPI. We found that mice with induced and spontaneous systemic lupus erythematosus recognize a common T cell epitope (peptide 31; LYRDTAVFECLPQHAMFG) in domain III of ß2-glycoprotein I. ß2GPI-reactive CD4+ T cells from the two models differed primarily in cytokine production: T cells from mice with induced SLE expressed IFN-γ, while T cells from MRL/lpr mice expressed both IL-17 and IFN-γ, indicating that IL-17-expressing T cells are not necessary for generating a ß2GPI-reactive T cell response. These data suggest that the generation of a ß2-glycoprotein I-reactive T cell response is shared by both induced and spontaneous models of systemic lupus erythematosus and that this T cell response may mediate epitope spread to autoantibodies in both models.

ß2-Glycoprotein I-Reactive T Cells in Autoimmune Disease.

Anti-phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are autoimmune diseases characterized by autoantibody production and autoantibody-related pathology. Anti-phospholipid antibodies (aPL) are found in all patients with APS and in 20-30% of individuals with SLE. aPL recognize a number of autoantigens, but the primary target in both APS and SLE is ß2-glycoprotein I (ß2GPI). The production of IgG aPL in APS and SLE, as well as the association of aPL with certain MHC class II molecules, has led to investigation of the role of ß2GPI-reactive T helper (Th). ß2GPI-reactive CD4 Th cells have been associated with the presence of aPL and/or APS in both primary APS and secondary APS associated with SLE, as well as in SLE patients and healthy controls lacking aPL. CD4 T cells reactive with ß2GPI have also been associated with atherosclerosis and found within atherosclerotic plaques. In most cases, the epitopes targeted by autoreactive ß2GPI-reactive CD4 T cells in APS and SLE appear to arise as a consequence of antigenic processing of ß2GPI that is structurally different from the soluble native form. This may arise from molecular interactions (e.g., with phospholipids), post-translational modification (e.g., oxidation or glycation), genetic alteration (e.g., ß2GPI variants), or molecular mimicry (e.g., microbiota). A number of T cell epitopes have been characterized, particularly in Domain V, the lipid-binding domain of ß2GPI. Possible sources of negatively charged lipid that bind ß2GPI include oxidized LDL, activated platelets, microbiota (e.g., gut commensals), and dying (e.g., apoptotic) cells. Apoptotic cells not only bind ß2GPI, but also express multiple other cellular autoantigens targeted in both APS and SLE. Dying cells that have bound ß2GPI thus provide a rich source of autoantigens that can be recognized by B cells across a wide range of autoantigen specificities. ß2GPI-reactive T cells could potentially provide T cell help to autoantigen-specific B cells that have taken up and processed apoptotic (or other dying) cells, and subsequently present ß2GPI on their surface in the context of major histocompatibility complex (MHC) class II molecules. Here, we review the literature on ß2GPI-reactive T cells, and highlight findings supporting the hypothesis that these T cells drive autoantibody production in both APS and SLE.

ß2-Glycoprotein I-specific T cells are associated with epitope spread to lupus-related autoantibodies.

Systemic lupus erythematosus (SLE) is a prototypic model for B cell epitope spread in autoimmunity. Autoantibodies to numerous and molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting the apoptotic cell-binding protein ß2-glycoprotein I (ß2GPI). Notably, mice immunized with ß2GPI and LPS display a remarkably similar pattern of autoantibody emergence to that seen in human SLE. Here, we used this model to investigate whether epitope spread to SLE-related autoantibodies is associated with a unique or limited ß2GPI-specific T cell response. We ask whether MHC class II haplotype and its associated T cell epitope restriction impact epitope spread to SLE-related autoantibodies. We found that ß2GPI/LPS-immunized mice produced similar SLE-related autoantibody profiles regardless of their ß2GPI T cell epitope specificity or MHC class II haplotype. Although ß2GPI T cell epitope specificity was clearly determined by MHC class II haplotype, a number of different ß2GPI T cell epitopes were associated with epitope spread to SLE-related autoantibodies. Notably, one ß2GPI T cell epitope (peptide 23, NTGFYLNGADSAKCT) was also recognized by T cells from an HLA-DRB1*0403(+) autoimmune patient. These data suggest that the generation of a ß2GPI-reactive T cell response is associated with epitope spread to SLE-related autoantibodies, independent of epitope specificity or MHC class II restriction. On the basis of these findings, we propose that factors enabling a ß2GPI-reactive T cell response may predispose individuals to the development of SLE-related autoantibodies independent of their MHC class II haplotype.

Interaction of ß2-glycoprotein I with lipopolysaccharide leads to Toll-like receptor 4 (TLR4)-dependent activation of macrophages.

ß(2)-Glycoprotein I (ß(2)GPI) is an abundant plasma protein that binds to the surface of cells and particles expressing negatively charged lipids, but its physiological role remains unknown. Antibodies to ß(2)GPI are found in patients with anti-phospholipid syndrome, a systemic autoimmune disease associated with vascular thrombosis and pregnancy morbidity. Although it has been suggested that anti-ß(2)GPI antibodies activate endothelial cells and monocytes by signaling through TLR4, it is unclear how anti-ß(2)GPI antibodies and/or ß(2)GPI interact with TLR4. A number of mammalian proteins (termed "endogenous Toll-like receptor (TLR) ligands") have been reported to bind to TLR4, but, in most cases, subsequent studies have shown that LPS interaction with these proteins is responsible for TLR activation. We hypothesized that, like other endogenous TLR ligands, ß(2)GPI interacts specifically with LPS and that this interaction is responsible for apparent TLR4 activation by ß(2)GPI. Here, we show that both LPS and TLR4 are required for ß(2)GPI to bind to and activate macrophages. Untreated ß(2)GPI stimulated TNF-α production in TLR4-sufficient (but not TLR4-deficient) macrophages. In contrast, neither polymyxin B-treated nor delipidated ß(2)GPI stimulated TNF-α production. Furthermore, ß(2)GPI bound to LPS in a specific and dose-dependent manner. Finally, untreated ß(2)GPI bound to the surface of TLR4-sufficient (but not TLR4-deficient) macrophages. Polymyxin B treatment of ß(2)GPI abolished macrophage binding. Our findings suggest a potential new biological activity for ß(2)GPI as a protein that interacts specifically with LPS and point to the need to evaluate newly discovered endogenous TLR ligands for potential interactions with LPS.

Association of autoantibodies to heat-shock protein 60 with arterial vascular events in patients with antiphospholipid antibodies.

OBJECTIVE: Anti-heat shock protein 60 autoantibodies (anti-Hsp60) are associated with cardiovascular disease and are known to affect endothelial cells in vitro, and we have recently shown that anti-Hsp60 promote thrombosis in a murine model of arterial injury. Based on those findings, we undertook the present study to investigate the hypothesis that the presence of anti-Hsp60, alone or in combination with other thrombogenic risk factors, is associated with an elevated risk of vascular events. METHODS: The study population was derived from 3 ongoing cohort studies: 2 independent systemic lupus erythematosus (SLE) registries and 1 cohort comprising SLE patients and non-SLE patients. Data from a total of 402 participants were captured; 199 of these participants had had confirmed vascular events (arterial vascular events in 102, venous vascular events in 76, and both arterial and venous vascular events in 21). Anti-Hsp60 were detected by enzyme-linked immunoassay, and association with vascular events was assessed by regression analysis. RESULTS: Multiple regression analysis revealed that arterial vascular events were associated with male sex, age, and hypertension. Analyses of the vascular events according to their origin showed an association of anti-Hsp60 with arterial vascular events (odds ratio 2.26 [95% confidence interval 1.13-4.52]), but not with venous vascular events. Anti-Hsp60 increased the risk of arterial vascular events (odds ratio 5.54 [95% confidence interval 1.89-16.25]) in antiphospholipid antibody (aPL)-positive, but not aPL-negative, individuals. CONCLUSION: We demonstrate that anti-Hsp60 are associated with an increased risk of arterial vascular events, but not venous vascular events, in aPL-positive individuals. These data suggest that anti-Hsp60 may serve as a useful biomarker to distinguish risk of arterial and venous vascular events in patients with aPL.

T cells demonstrate a Th1-biased response to native beta2-glycoprotein I in a murine model of anti-phospholipid antibody induction.

Anti-phospholipid syndrome (APS) is an autoimmune disorder characterized by the presence of autoantibody (AAb) to phospholipid (PL)-binding proteins, such as beta2-glycoprotein I (beta2GPI), and clinical manifestations including thrombosis and/or recurrent pregnancy loss. beta2GPI-reactive T cells are clearly implicated in the generation of these AAb, but the mechanism responsible for their activation remains unclear. We hypothesized that immunization of mice with human beta2GPI, in the context of a potent innate immune activator lipopolysaccharide (LPS), would generate not only high titers of anti-PL AAb, but also a strong beta2GPI-specific T cell response. Healthy, nonautoimmune C57BL/6 mice were immunized repeatedly with human beta2GPI in the presence of LPS. High titers of anti-PL to beta2GPI appeared after the second immunization, with T cell reactivity to beta2GPI detectable only after the fourth immunization. Splenic T cells from these mice proliferated in response to native beta2GPI, alone or bound to anionic PL. These T cells produced IL-2 and IFN-gamma, but not IL-4 or IL-10, indicating a Th1 bias of the beta2GPI-specific response. These findings suggest that T cells responsive to beta2GPI may become activated in APS patients by exposure to their cognate Ag in the context of innate immune activation and a pro-inflammatory environment.

Immunization with an apoptotic cell-binding protein recapitulates the nephritis and sequential autoantibody emergence of systemic lupus erythematosus.

The initial events predisposing to loss of tolerance in patients with systemic lupus erythematosus (SLE) are largely unknown, as are the events that precipitate the transition from preclinical to overt disease. We hypothesized that induction of murine SLE would require tipping the balance between tolerance and immunity in two ways: 1) an immunogen that could take advantage of apoptotic cells as a scaffold for epitope spread, and 2) an immune activator that would generate a strong and persistent T cell response to the inciting immunogen. We show that immunization of C57BL/6 and BALB/c mice with human beta(2)-glycoprotein I, an apoptotic cell-binding protein, in the presence of LPS induces a long-lived, potent response to beta(2)-glycoprotein I that results in epitope spread to multiple SLE autoantigens. SLE-specific autoantibodies emerged in a sequential manner that recapitulated the order seen in human SLE. Moreover, immunized mice developed overt glomerulonephritis closely resembling human lupus nephritis.

Antiphospholipid antibodies and thrombosis: association with acquired activated protein C resistance in venous thrombosis and with hyperhomocysteinemia in arterial thrombosis.

Although antiphospholipid antibodies (aPL) are associated with thrombosis, it is not known who with aPL is at higher risk for thrombosis. It was the aim of this cross-sectional study to investigate how thrombophilic factors contribute to venous or arterial thrombosis in aPL-positive individuals. In outpatient test centres at two tertiary care hospitals, two hundred and eight (208) persons requiring aPL testing were matched by age, gender and centre to 208 persons requiring a complete blood count. Persons were classified as aPL-positive (having anticardiolipin, lupus anticoagulant and/or anti-beta(2)-glycoprotein I antibodies) or aPL-negative. Several thrombophilic factors were studied using logistic regression modelling. Results showed that the aPL-positive group had three-fold more events (37%) than the aPL-negative group (12%). In unadjusted analyses, clinically important associations were observed between factor V Leiden and venous thrombosis, hyperhomocysteinemia and arterial thrombosis, and activated protein C resistance (APCR) and venous thrombosis (OR, 95% CI = 4.00, 1.35-11.91; 4.79, 2.03-11.33; and 2.03, 1.03-3.97, respectively). After adjusting for recruitment group, persons with both APCR and aPL had a three-fold greater risk (OR, 95% CI = 3.31, 1.30-8.41) for venous thrombosis than those with neither APCR nor aPL. Similarly, after adjusting for hypertension, family history of cardiovascular disease, gender and recruitment group, persons with both hyperhomocysteinemia and aPL had a five-fold increased risk (OR, 95% CI = 4.90, 1.37-17.37) for arterial thrombosis compared to those with neither risk factor. In conclusion, APCR phenotype and hyperhomocysteinemia are associated with a higher risk of venous and arterial thrombosis, respectively, in the presence of aPL.

Anti-phospholipid antibodies (aPL) and apoptosis: prothrombin-dependent aPL as a paradigm for phospholipid-dependent interactions with apoptotic cells.

The natural targets of anti-phospholipid antibodies (aPL) and the stimuli that induce them remain unknown. Apoptotic cells have been proposed as both potential targets and immunogens for anti-phospholipid antibodies. Demonstration of selective recognition by anti-phospholipid antibodies provides support for apoptotic cells as antigenic targets. Here, we summarize data showing that prothrombin (PT) binds to apoptotic, but not viable, cells, and that apoptotic-cell bound prothrombin provides a target for human polyclonal and murine monoclonal lupus anticoagulant (LA) antibodies. We discuss findings for two monoclonal lupus anticoagulant antibodies that have high (antibody 29J3-62) or low (antibody 29I4-24) affinity, respectively, for soluble prothrombin. Despite their very different affinities for soluble prothrombin, both monoclonal antibodies reacted similarly with prothrombin bound to phospholipid or apoptotic cells. Furthermore, both antibodies enhanced the binding of prothrombin to apoptotic cells. We propose that the recognition of apoptotic cells by these prothrombin-dependent monoclonal antibodies provides a paradigm for other anti-phospholipid autoantibodies. 29I4-24 is prototypical of phospholipid-dependent anti-phospholipid antibodies, while 29J3-62 represents a prototype for phospholipid-independent anti-phospholipid antibodies. Proteins such as prothrombin and beta2-glycoprotein I (beta2GPI) bind to apoptotic cells, thereby enhancing the recognition of apoptotic cells by anti-phospholipid antibodies. Furthermore, anti-phospholipid antibodies potentiate the interaction of these proteins with apoptotic cells. While it is unclear whether apoptotic cells are the inducing stimuli in patients with anti-phospholipid antibodies or even whether anti-phospholipid antibodies interact with apoptotic cells in vivo, it is nonetheless clear that anti-phospholipid antibodies have the potential to affect both the procoagulant activity and the uptake and clearance of apoptotic cells.

Prothrombin binds to the surface of apoptotic, but not viable, cells and serves as a target of lupus anticoagulant autoantibodies.

Anti-phospholipid Ab (aPL) are a heterogeneous group of autoantibodies directed against various combinations of phospholipids (PL) and PL-binding proteins. Lupus anticoagulant (LA) Ab, a subset of aPL, exhibit anticoagulant properties in vitro, but are procoagulant in vivo. Most LA Ab are specific for either beta(2)-glycoprotein I (beta(2)GPI) or prothrombin (PT), two PL-binding proteins. We have previously shown that beta(2)GPI and beta(2)GPI-dependent aPL bind specifically to apoptotic, but not viable, thymocytes. In this study, we demonstrate that PT, like beta(2)GPI, binds selectively to the surface of apoptotic, but not viable, Jurkat cells. Furthermore, PT supports the binding of systemic lupus erythematosus-derived polyclonal and murine monoclonal LA Ab to apoptotic cells. Two LA mAb, which differed dramatically in their relative affinities for PT, were studied. Although one mAb (29J3-62) had a high affinity for PT alone, the other (29I4-24) showed minimal reactivity with PT alone and required PL for elevated binding. Monovalent fragments of 29I4-24 reacted with PL-bound PT with high affinity, suggesting that this mAb recognizes a PL-dependent epitope. Despite these differences, PT-dependent binding of both mAb to apoptotic cells was 30-fold greater than that to viable cells. Moreover, binding of PT to apoptotic cells was, itself, increased in the presence of bivalent, but not monovalent, forms of either mAb. In summary, our data demonstrate the following: 1) specific binding of PT to apoptotic cells, an effect enhanced by PT-dependent LA Ab; 2) heterogeneity of PT-dependent LA Ab; and 3) potential pathogenicity of Ab of either low or high affinity for PT.