Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

The dual role of innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

Antiphospholipid syndrome (APS), as a primary disease or a secondary syndrome in systemic lupus erythematosus (SLE), is characterized by the presence of antiphospholipid antibodies (aPL) and a clinical event. It is likely that both genetic and environmental factors lead to the development of aPL and progression to disease. However, the precise mechanisms are not known. We hypothesize that innate immune activation plays a dual role in APS and SLE, both in the production of aPL (i.e. "initiation" phase) and in the development of a clinical event (i.e. "effector" phase). We have shown that mice immunized with certain phospholipid-binding proteins (e.g. ß2-glycoprotein I (ß2GPI)), plus a concomitant trigger of innate immunity (e.g. a toll-like receptor 4 (TLR4) ligand), produce a strong ß2GPI-reactive T cell response, resulting in high levels of aPL as well as other SLE autoantibodies. We propose that ß2GPI, through its interaction with apoptotic cells, permits B cell epitope spread to multiple SLE autoantibodies. Innate immune activation is also implicated in a murine model of aPL-enhanced thrombus formation. This dual role of innate immune activation provides new insight into the mechanisms involved in the initiation of aPL and other SLE-related autoantibodies, as well as the development of aPL-mediated disease.

Phospholipid-binding proteins differ in their capacity to induce autoantibodies and murine systemic lupus erythematosus.

We have previously shown that immunization of nonautoimmune mice with the phospholipid-binding protein ß2-glycoprotein I (ß2GPI), in combination with lipopolysaccharide (LPS), induces a murine model of systemic lupus erythematosus (SLE), with sequential emergence of autoantibodies and glomerulonephritis. Here, we determine whether the paradigm for induction of murine SLE extends to other phospholipid-binding proteins. Mice were immunized with a phospholipid-binding protein (prothrombin (PT), protein S, or ß2GPI), or a nonphospholipid-binding protein (glu-plasminogen), in the presence of LPS. The breadth and degree of the autoantibody response, and the frequency of glomerulonephritis, varied among the three proteins, with ß2GPI being the most effective in inducing SLE-like disease. The phospholipid-binding proteins also differed in the pattern of serum cytokines they elicited. The most apparent difference between ß2GPI and the other phospholipid-binding proteins was in their ability to bind to LPS: ß2GPI bound to LPS, while PT and protein S did not. Our data suggest that binding to phospholipid(s) is a necessary, but not sufficient, condition for full induction of murine SLE. We propose that other properties, such as physiologic function, avidity for anionic phospholipids, and degree of interaction with other cell surface and/or circulating molecules (particularly LPS) may determine the range and severity of disease.

Anti-inflammatory cytokines, pro-fibrogenic chemokines and persistence of acute HCV infection.

Chemokines and cytokines play a vital role in directing and regulating immune responses to viral infections. Persistent hepatitis C virus (HCV) infection is characterized by the loss of anti-HCV cellular immune responses, while control of HCV infection is associated with maintenance of anti-HCV cellular immune responses. To determine whether plasma concentrations of 19 chemokines and cytokines controlling T-cell trafficking and function differed based on infection outcome, we compared them in at-risk subjects followed prospectively for HCV infection. Levels were compared over time in subjects who controlled HCV infection (Clearance) and subjects who developed persistent HCV infection (Persistence) at two time points during acute infection: (i) first viraemic sample (initial viraemia) and (ii) last viraemic sample in Clearance subjects and time-matched samples in Persistence subjects. At initial viraemia, increased pro-inflammatory tumour necrosis factor α (TNFα) plasma concentrations were observed in the Clearance group, while the plasma levels of anti-inflammatory interleukin (IL)-2, IL-10 and IL-13 were higher in the Persistence group. IL-13 was positively correlated with IL-2 and IL-10 at initial viraemia in the Persistence group. At the time of last viraemia, plasma levels of eotaxin, macrophage chemoattractant protein-4 (MCP-4), IL-5 and IL-10 were higher in the Persistence group and IL-10 and IL-5 levels were positively correlated. Collectively, these results suggest that the development of persistent infection is associated with an anti-inflammatory and pro-fibrogenic chemokine and cytokine profile that is evident at the onset of infection and maintained throughout acute infection.

The dual role of innate immunity in the antiphospholipid syndrome.

The antiphospholipid syndrome (APS), as both a primary syndrome and a syndrome in association with systemic lupus erythematosus (SLE), can be a devastating disease. It is unclear what factors (genetic and/or environmental) lead to the generation of antiphospholipid antibodies (aPL). It is equally unclear why only certain individuals with aPL develop clinical events. We hypothesize that innate immune activation plays a critical role at two distinct stages of APS, namely, the initiation phase, in which aPL first appear, and the effector phase, in which aPL precipitate a thrombotic event. According to the model we propose, aPL alone are insufficient to cause thrombosis and a concomitant trigger of innate immunity, e.g. a toll-like receptor (TLR) ligand, must be present for thrombosis to occur. Here, we discuss our findings that mice immunized with beta(2)-glycoprotein I (beta(2)GPI) and lipopolysaccharide (LPS), a TLR ligand, produce high levels of aPL and other SLE-associated autoantibodies, and develop lupus-like glomerulonephritis. We also discuss our data showing that autoantibodies to heat shock protein 60 (HSP60), an 'endogenous TLR ligand', promote thrombus generation in a murine model of arterial injury. Thus, both pathogen-derived TLR ligands (e.g. LPS) and endogenous TLR ligands (e.g. HSP60) may contribute to the pathogenesis of APS. This putative dual role of innate immunity provides new insight into the generation of aPL as well as the enigma of why some individuals with aPL develop APS, while others do not.

Autoantibodies to heat shock protein 60 promote thrombus formation in a murine model of arterial thrombosis.

BACKGROUND AND OBJECTIVES: Anti-heat shock protein (HSP)60 autoantibodies are associated with atherosclerosis and are known to affect endothelial cells in vitro. However, their role in thrombus formation remains unclear. We hypothesized that anti-HSP60 autoantibodies could potentiate thrombosis, and evaluated the effect of anti-murine HSP60 antibodies in a ferric chloride (FeCl3)-induced murine model of carotid artery injury. METHODS: Anti-HSP60, or control, IgG was administered to BALB/c mice 48 h prior to inducing carotid artery injury, and blood flow was monitored using an ultrasound probe. RESULTS: Thrombus formation was more rapid and stable in anti-HSP60 IGG-treated mice than in controls (blood flow=1.7%+/-0.6% vs. 34%+/-12.6%, P=0.0157). Occlusion was complete in all anti-HSP60 IgG-treated mice (13/13), with no reperfusion being observed. In contrast, 64% (9/14) of control mice had complete occlusion, with reperfusion occurring in 6/9 mice. Thrombi were significantly larger in anti-HSP60 IgG-treated mice (P=0.0001), and contained four-fold more inflammatory cells (P=0.0281) than in controls. Non-injured contralateral arteries of anti-HSP60 IgG-treated mice were also affected, exhibiting abnormal endothelial cell morphology and significantly greater von Willebrand factor (VWF) and P-selectin expression than control mice (P=0.0024 and P=0.001, respectively). CONCLUSIONS: In summary, the presence of circulating anti-HSP60 autoantibodies resulted in increased P-selectin and VWF expression and altered cell morphology in endothelial cells lining uninjured carotid arteries, and promoted thrombosis and inflammatory cell recruitment in FeCl3-injured carotid arteries. These findings suggest that anti-HSP60 autoantibodies may constitute an important prothrombotic risk factor in cardiovascular disease in human vascular disease.

Efficient radiation production in long implosions of structured gas-puff Z pinch loads from large initial radius.

We have proposed and demonstrated successfully a new approach for generating high-yield K-shell radiation with large-diameter gas-puff Z pinches. The novel load design consists of an outer region plasma that carries the current and couples energy from the driver, an inner region plasma that stabilizes the implosion, and a high-density center jet plasma that radiates. It increased the Ar K-shell yield at 3.46 MA in 200 ns implosions from 12 cm initial diameter by a factor of 2, to 21 kJ, matching the yields obtained earlier on the same accelerator with 100 ns implosions. A new "pusher-stabilizer-radiator" physical model is advanced to explain this result.

Antiphospholipid antibodies are associated with enhanced oxidative stress, decreased plasma nitric oxide and paraoxonase activity in an experimental mouse model.

OBJECTIVE: Oxidative stress contributes to atherosclerosis, and evidence of enhanced oxidative stress exists in antiphospholipid syndrome (APS). In a non-lupus murine model, we evaluated whether anticardiolipin (aCL) antibodies could affect the oxidant/antioxidant balance as an early biochemical step of APS. METHODS: Hybridomas producing human and murine aCL and anti-beta(2)-glycoprotein I (abeta2-GPI) monoclonal antibodies were injected into three groups of five female BALB/c severe combined immunodeficiency (SCID) mice. Corresponding hybridomas secreting non-antiphospholipid antibodies of the same isotype were employed as controls. Sera and organs were collected after 30 days. Paraoxonase (PON) activity, peroxynitrite, superoxide, nitric oxide (NO) and nitrotyrosine were measured in plasma. Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase (iNOS) was assessed by western blot and immunohistochemistry. RESULTS: PON activity and NO (sum of nitrate and nitrite) levels were reduced in the human aCL IgG group (P<0.002 and P<0.04, respectively), whilst peroxynitrite and superoxide and expression of total antioxidant capacity of plasma were increased (P<0.01). PON and NO were decreased in the murine abeta2-GPI IgG and IgM aCL groups (P<0.03 and P<0.05, respectively). Nitrotyrosine was elevated in the human aCL IgG group (P<0.03). Western blotting showed reduced iNOS expression in the hearts of the IgG aCL group, confirmed by immunostaining. PON inversely correlated with IgG aCL titres (P<0.001), superoxide (P<0.008) and peroxynitrite levels (P<0.0009). Peroxynitrite and total IgG aCL were independent predictors of PON (P<0.0009 and P<0.02, respectively). Superoxide was the only independent predictor of NO (P<0.008) and of nitrotyrosine (P<0.002). CONCLUSION: aCL antibodies are associated with the decreased PON activity and reduced NO that may occur in the preclinical phase of APS.

Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells.

The immunosuppressive effect of rapamycin is mediated by inhibition of interleukin-2-stimulated T cell proliferation. We report for the first time that rapamycin also inhibits growth factor-induced proliferation of cultured mouse proximal tubular (MPT; IC(50) ~1 ng/ml) cells and promotes apoptosis of these cells by impairing the survival effects of the same growth factors. On the basis of these in vitro data, we tested the hypothesis that rapamycin would impair recovery of renal function after ischemic acute renal failure induced in vivo by renal artery occlusion (RAO). Rats given daily injections of rapamycin or vehicle were subjected to RAO or sham surgery. Rapamycin had no effect on the glomerular filtration rate (GFR) of sham-operated animals. In rats subjected to RAO, GFR fell to comparable levels 1 day later in vehicle- and rapamycin-treated rats (0.25 +/- 0.08 and 0.12 +/- 0.05 ml. min(-1). 300 g(-1), respectively) (P = not significant). In vehicle-treated rats subjected to RAO, GFR increased to 0.61 +/- 0.08 ml. min(-1). 300 g(-1) on day 3 (P < 0.02 vs. day 1) and then rose further to 0.99 +/- 0.09 ml. min(-1). 300 g(-1) on day 4 (P < 0.02 vs. day 3). By contrast, GFR did not improve in rapamycin-treated rats subjected to RAO over the same time period. Rapamycin also increased apoptosis of tubular cells while markedly reducing their proliferative response after RAO. Furthermore, rapamycin inhibited activation of 70-kDa S6 protein kinase (p70(S6k)) in cultured MPT cells as well as in the renal tissue of rats subjected to RAO. We conclude that rapamycin severely impairs the recovery of renal function after ischemia-reperfusion injury. This effect appears to be due to the combined effects of increased tubular cell loss (via apoptosis) and profound inhibition of the regenerative response of tubular cells. These effects are likely mediated by inhibition of p70(S6k).

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of murine lupus.

Of the multiple murine models of autoimmunity, the three most closely resembling human systemic lupus erythematosus (SLE) are the MRL/lpr, New Zealand Black/White F(1), and male BXSB. Although these strains share many disease characteristics, no common cellular defect has previously been found in prediseased mice from all these strains. We show in this study that macrophages from prediseased mice of all three SLE-prone strains, as well as macrophages from mice whose genomes contribute to the development of SLE (MRL/+, New Zealand White, New Zealand Black, female BXSB, and LG/J), have an identical and profound defect in cytokine expression that is triggered by apoptotic cells. Strikingly, none of 13 nonautoimmune strains tested exhibited this defect. Given that apoptotic Ags have been increasingly recognized as the target of autoantibodies, a defect in cytokine expression that is triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity.

Apoptosis and the antiphospholipid syndrome.

The target of many antiphospholipid autoantibodies (APA) has been shown to be a complex between anionic phospholipid (PL) and the plasma protein beta 2-glycoprotein I (beta 2-GPI), but the identity of the natural target(s) and/or immunogen for APA in vivo remains undetermined. The anionic PL of cell membranes represent important potential targets and immunogenes for APA. Although anionic PL are normally absent from the extracellular surface of cell membranes, they redistribute from the inner to the outer leaflet during apoptosis. We and others have shown that beta 2-GPI binds selectively to the surface of apoptotic, but not viable, cells, and that the binding of beta 2-GPI to the surface of apoptotic cells generates an epitope recognized by APA from patients with both primary antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). In this review, we discuss recent findings, which suggest not only that apoptotic cell-bound beta 2-GPI is injected by non-intravenous routes. We also review briefly the potential role of oxidation in generating epitopes responsible for the recognition and induction of APA. Taken together, we believe that the available evidence supports a role for apoptotic cells as far as targets of APA and possible players in the induction of APA.

Phospholipid-bound beta 2-glycoprotein I induces the production of anti-phospholipid antibodies.

Apoptotic-cell-bound beta2-glycoprotein I (beta2GPI), but not apoptotic cells or beta2GPI alone, can induce the production of anti-phospholipid (anti-PL) antibodies (Ab) in normal mice. Although it is presumed that beta2GPI binds to anionic phospholipid (PL) exposed on the apoptotic cell membrane, the precise nature of this complex and its immunogenicity is unclear. To address these issues, we investigated the structure and immunogenicity of human beta2GPI in the presence of different PL that may be expressed on the surface of apoptotic cells. BALB/c mice were immunized intravenously (iv) with beta2GPI in the presence of cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC), or PS/PC (25%/75%) vesicles. Cardiolipin+beta2GPI induced the highest levels of anti-beta2GPI and anti-CL IgG Ab and lupus anticoagulant (LA) activity, while beta2GPI with PC or PS/PC vesicles produced no significant anti-PL Ab. PS+beta2GPI was somewhat immunogenic, but less so than PG+beta2GPI. beta2GPI was immunogenic in the presence of native (CL(N)), but not hydrogenated (CL(H)), CL. Circular dichroism analysis demonstrated that the structure of beta2GPI was altered specifically by interaction with CL(N), but not other anionic PL, including CL(H). Similarly, the structure of CL(N)was affected by interaction with beta2GPI, as detected by(31)P nuclear magnetic resonance. These findings demonstrate that beta2GPI complexed with CL(N)is structurally altered, highly immunogenic, and induces the production of IgG anti-PL Ab. Furthermore, the structural modification and the generation of immunogenic epitopes on beta2GPI upon interaction with CL(N)require the presence of unsaturated fatty acid chains, suggesting a role for oxidation in this process.

Mouse proximal tubular cell-cell adhesion inhibits apoptosis by a cadherin-dependent mechanism.

Adhesion of epithelial cells to matrix is known to inhibit apoptosis. However, the role of cell-cell adhesion in mediating cell survival remains uncertain. Primary cultures of mouse proximal tubular (MPT) cells were used to examine the role of cell-cell adhesion in promoting survival. When MPT cells were deprived of both cell-matrix and cell-cell adhesion, they died by apoptosis. However, when incubated in agarose-coated culture dishes (to prevent cell-matrix adhesion) and at high cell density (to allow cell-cell interactions), MPT cells adhered to one another and remained viable. Expression of E-cadherin among suspended, aggregating cells increased with time. A His-Ala-Val (HAV)-containing peptide that inhibits homophilic E-cadherin binding prevented cell-cell aggregation and promoted apoptosis of MPT cells in suspension. By contrast, inhibition of potential beta(1)-integrin-mediated interactions between cells in suspension did not prevent either aggregation or survival of suspended cells. Aggregation of cells in suspension activated phosphatidylinositol 3-kinase (PI3K), an event that was markedly reduced by the presence of the HAV peptide. LY-294002, an inhibitor of PI3K, also inhibited survival of suspended cells. In summary, we provide novel evidence that MPT cells, when deprived of normal cell-matrix interactions, can adhere to one another in a cadherin-dependent fashion and remain viable. Survival of aggregated cells depends on activation of PI3K.

Albumin is a major serum survival factor for renal tubular cells and macrophages through scavenging of ROS.

We have previously shown that lysophosphatidic acid (LPA), an abundant serum lipid that binds with high affinity to albumin, is a potent survival factor for mouse proximal tubular cells and peritoneal macrophages. We show here that BSA also has potent survival activity independent of bound lipids. Delipidated BSA (dBSA) protected cells from apoptosis induced by FCS withdrawal at concentrations as low as 1% of that in FCS. dBSA did not activate phosphatidylinositol 3-kinase, implying that its survival activity occurs via a mechanism distinct from that for most cytokines. On the basis of the following evidence, we propose that dBSA inhibits apoptosis by scavenging reactive oxygen species (ROS): 1) FCS withdrawal leads to ROS accumulation that is inhibitable by dBSA; 2) during protection from apoptosis, sulfhydryl and hydroxyl groups of dBSA are oxidized; and 3) chemical blockage of free sulfhydryl groups or preoxidation of dBSA with H(2)O(2) removes its survival activity. Moreover, dBSA confers almost complete protection from cell death in a well-established model of oxidative injury (xanthine/xanthine oxidase). These results implicate albumin as a major serum survival factor. Inhibition of apoptosis by albumin occurs through at least two distinct mechanisms: carriage of LPA and scavenging of ROS.

Reversible bilateral hydronephrosis without obstruction in hepatitis B-associated polyarteritis nodosa.

The manifestations of polyarteritis nodosa (PAN) are varied, but urological abnormalities other than ureteric stenosis and orchitis have not been described. We report a case of hepatitis B-associated PAN with bilateral hydronephrosis without obstruction. Retrograde urography conclusively demonstrated the absence of obstruction. Vasculitis-related myopathy, or neuropathy of the ureter, is the most likely cause of this finding. The patient was treated with high-dose steroids, cyclophosphamide, and plasmapheresis with resolution of hydronephrosis. Although the patient required dialysis at initiation of therapy, she went on to recover sufficient renal function to discontinue dialysis. We review the literature on the treatment of hepatitis B-associated PAN and discuss the pitfalls in diagnosis of this condition.

Apoptotic cells as immunogen and antigen in the antiphospholipid syndrome.

Apoptotic cell antigens have been increasingly recognized as the targets of autoantibodies across a broad spectrum of autoimmune diseases, including systemic lupus erythematosus (SLE) and the antiphospholipid (aPL) syndrome. In this review, we will focus on one set of apoptotic antigens, namely, those targeted in the aPL syndrome. Here we discuss the biology of aPL autoantibodies and recent work from our and other laboratories demonstrating that apoptotic cells express unique antigen(s) that serve(s) as both immunogen and antigen for aPL autoantibodies. Specific features or events occurring at the surface of apoptotic cells, which may influence immunogenicity and/or antigenicity, will also be discussed. Finally, we will speculate on the broader implications of these findings for the development of systemic autoimmunity as seen in SLE.

The role of apoptosis in autoimmunity: immunogen, antigen, and accelerant.

The immunologic basis of systemic autoimmune diseases such as systemic lupus erythematosus (SLE) is complex and multifaceted. Recent advances in the field of apoptosis have suggested new paradigms for the development of autoimmunity. This review examines the role that apoptosis plays in maintaining immunologic tolerance to self-antigens, and how abnormalities in the regulation of apoptosis can lead to a breakdown in self-tolerance. This article also examines the increasing recognition of apoptotic cell antigens as the targets of autoantibodies and discusses the possibility that the autoimmune response characteristic of SLE is specifically directed against apoptotic cells. In addition, we will describe some of the features that distinguish nonpathogenic anti-DNA autoantibodies from those which deposit in the kidney and lead to lupus nephritis. Finally, we will attempt to synthesize the vast body of data connecting apoptosis and SLE into a single hypothesis in which we suggest that apoptotic cells are a primary source of immunogen, and that abnormalities in the handling of apoptotic cells can lead to a breakdown in self-tolerance.

Role of superoxide in apoptosis induced by growth factor withdrawal.

We have examined the role of reactive oxygen species (ROS) in apoptosis induced by growth factor deprivation in primary cultures of mouse proximal tubular (MPT) cells. When confluent monolayers of MPT cells are deprived of all growth factors, the cells die by apoptosis over a 10- and 14-day period. Both epidermal growth factor (EGF) and high-dose insulin directly inhibit apoptosis of MPT cells deprived of growth factors. Growth factor deprivation results in an increase in the cellular levels of superoxide anion while apoptosis of MPT cells induced by growth factor withdrawal is inhibited by a number of antioxidants and scavengers of ROS. Growth factor deprivation also results in activation of caspase activity, which is inhibited by EGF and high-dose insulin as well as by the ROS scavengers and antioxidants that inhibit apoptosis. The cell-permeant caspase inhibitor, z-Val-Ala-Asp-CH2F (zVAD-fmk), prevents the increase in caspase activity and markedly inhibits apoptosis induced by growth factor deprivation. However, zVAD-fmk had no effect on the increased levels of superoxide associated with growth factor deprivation. Thus we provide novel evidence that ROS play an important role in mediating apoptosis associated with growth factor deprivation. ROS appear to act upstream of caspases in the apoptotic pathway. We hypothesize that oxidant stress, induced by growth factor withdrawal, represents a signaling mechanism for the default pathway of apoptosis.

Induction of anti-phospholipid autoantibodies by beta2-glycoprotein I bound to apoptotic thymocytes.

The target of many anti-phospholipid autoantibodies (aPL) has been shown to be a complex between anionic phospholipid and the plasma protein beta2-glycoprotein I (beta2GPI) or the protein beta2GPI alone. As aPL binding studies have been performed almost exclusively in vitrothe identity of the natural target and/or immunogen for aPL in vivo remains undetermined. The anionic phospholipids of cell membranes represent an important potential target and immunogen for aPL. Although anionic phospholipids are normally absent from the extracellular surface of cell membranes, they redistribute from the inner to the outer leaflet during apoptosis. We have previously shown that beta2GPI binds selectively to the surface of apoptotic, but not viable, cells, and that binding of beta2GPI to the surface of apoptotic cells generates an epitope recognized by aPL from patients with primary aPL syndrome and systemic lupus erythematosus. We show here that immunization of non-autoimmune mice with beta2GPI combined with, or bound to, apoptotic cells induces aPL and lupus anticoagulant activity. Generation of aPL required heterologous beta2GPI, and occurred upon immunization with apoptotic cells and beta2GPI by three different routes of administration. Importantly, for intravenous immuniz-ations, generation of aPL occurred only when apoptotic cells and beta2GPI were injected together, but not when either was injected alone, suggesting that cell-bound beta2GPI is the true immunogen for production of aPL. Unlike other models of induced aPL, adjuvant was not an absolute requirement. Induced aPL reacted with murine, as well as bovine, beta2GPI, suggesting that heterologous beta2GPI bound to apoptotic cells can break tolerance and induce auto-antibodies reactive with autologous beta2GPI. Combined with our previous data, these results show that apoptotic cells can serve as both immunogens and natural targets for aPL.