We previously induced pathogenic antibodies against anionic phospholipids (PL) in experimental animals by immunization with lipid-free purified human beta2glycoprotein I (beta2GPI). We hypothesized that antiphospholipid antibodies (aPL) are induced by in vivo binding of foreign beta2GPI to self-PL, thus forming an immunogenic complex against which aPL antibodies are produced. If this hypothesis is true, other PL-binding proteins that are products of ubiquitous viral/bacterial agents may also induce aPL. To test this hypothesis, groups of NIH/Swiss mice were immunized with synthetic peptides of viral and bacterial origin that share structural similarity with the putative PL-binding region of beta2GPI. Compared with the control groups, animals immunized with the peptides produced significantly higher levels of aPL and anti-beta2GPI antibodies. These findings demonstrate that some PL-binding viral and bacterial proteins function like beta2GPI in inducing aPL and anti-beta2GPI production, and are consistent with a role for such viral and bacterial proteins in inducing aPL antibody production in humans.

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beta 2-Glycoprotein I (beta 2 GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. beta 2 GPI influence upon the reactive species production by Kupffer cells was evaluated in order to investigate whether beta 2 GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolymristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25 mol% phosphatidylserine (PS) or 50 mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). beta 2 GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat beta 2 GPI. Albumin (500 micrograms/ml) showed no effect upon chemiluminescence. beta 2 GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of beta 2 GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of beta 2 GPI. At a concentration of 125 micrograms/ml, beta 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of beta 2 GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of beta 2 GPI as a mediator of senescent cell removal.

The aim of this study was to investigate the interaction of antiphospholipid antibodies (aPL) from two different populations (patients with autoimmune or infectious disorders) with cardiolipin (CL) arranged in a defined bilayer. beta2-Glycoprotein I (beta2GPI), an apolipoprotein that plays a critical role in the aPL binding to phospholipids, was quantified by dot blot in purified IgG-aPL samples, further classified according to apparent avidity to CL. In solid-phase assays, beta2GPI increased, preferentially, the binding of low-avidity autoimmune aPL to CL but inhibited the binding of low-avidity syphilitic aPL. In the absence of beta2GPI, both autoimmune and infectious aPL induced the leakage of the entrapped fluorescent probe, carboxyfluorescein (CF), from small unilamellar vesicles containing CL. aPL-induced probe leakage was protein concentration-dependent and characterized by a lag-phase onset of 100-120 min. beta2GPI increased the leakage rate induced by low-avidity autoimmune aPL only and inhibited the leakage induced by all syphilitic aPL. The following conclusions were provided: (1) in the absence of beta2GPI, autoimmune and infectious aPL bind to CL in a bilayer, inducing liposome leakage; (2) the leakage mechanism induced by aPL is suggested to be intravesicular; (3) beta2GPI requirement for phospholipid binding in both solid and fluid phase is associated to aPL avidity; (4) CL alone or the CL-beta2GPI complex are the most likely epitopes for autoimmune aPL; (5) aPL from syphilis patients can only form the CL-aPL complex, supporting that beta2GPI is not (part of) the target epitope.

Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies that are detected in the serum of patients with a variety of conditions, including autoimmune (systemic lupus erythematosus), infectious (syphilis, AIDS) and lymphoproliferative disorders (paraproteinemia, myeloma, lymphocytic leukemias). Thrombosis, thrombocytopenia, recurrent fetal loss and other clinical complications are currently associated with a subgroup of aPL designating the antiphospholipid syndrome. In contrast, aPL from patients with infectious disorders are not associated with any clinical manifestation. These findings led to increased interest in the origin and pathogenesis of aPL. Here we present the clinical features of the antiphospholipid syndrome and review the origin of aPL, the characteristics of experimentally induced aPL and their historical background. Within this context, we discuss the most probable pathogenic mechanisms induced by these antibodies.