Elena Raschi

Antiphospholipid antibodies affect trophoblast gonadotropin secretion and invasiveness by binding directly and through adhered β2‐glycoprotein I

OBJECTIVE To investigate the in vitro ability of antiphospholipid antibodies (aPL) to bind human trophoblast cells and to affect gonadotropin secretion and invasiveness. METHODS Antiphospholipid antibody IgG from women with recurrent miscarriages, beta2-glycoprotein I (beta2GPI)-independent IgG aPL human monoclonal antibody (mAb) (519), and IgM anti-beta2GPI human mAb (TMIG2) were investigated for their binding to trophoblasts cultured for various amounts of time, their ability to affect invasiveness of Matrigel-coated filters, and their release of human chorionic gonadotropin (hCG). RESULTS Polyclonal IgG aPL, as well as mAb 519 and TMIG2, bound to trophoblasts, the highest binding being found when cells displayed the greatest amount of syncytium formation. TM1G2 binding was found to be betaGPI dependent. Both polyclonal and monoclonal aPL, but not the controls, significantly reduced hCG release and Matrigel invasiveness. CONCLUSION These findings suggest that aPL recognition of both anionic PL and adhered beta2GPI on trophoblast cell structures might represent a potential pathogenetic mechanism for defective placentation in women with the antiphospholipid syndrome.

Statins prevent endothelial cell activation induced by antiphospholipid (anti–β2-glycoprotein I) antibodies: Effect on the proadhesive and proinflammatory phenotype

OBJECTIVE To investigate the ability of statins, the inhibitors of the hydroxymethylglutaryl-coenzyme A reductase enzyme, to affect endothelial cell activation induced by anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in vitro. METHODS Human umbilical vein endothelial cell (HUVEC) activation was evaluated as U937 monocyte adhesion, E-selectin, and intercellular adhesion molecule I (ICAM-1) expression by cell enzyme-linked immunosorbent assay and as interleukin-6 (IL-6) messenger RNA (mRNA) expression by RNA protection assay. E-selectin-specific nuclear factor kappaB (NF-kappaB) DNA-binding activity was evaluated by the gel-shift assay. HUVECs were activated by polyclonal affinity-purified IgG, human monoclonal IgM anti-beta2GPI antibodies, human recombinant IL-1beta, tumor necrosis factor alpha, or lipopolysaccharide (LPS). RESULTS Fluvastatin reduced, in a concentration-dependent manner (1-10 microM), the adhesion of U937 to HUVECs and the expression of E-selectin and ICAM-1 induced by anti-beta2GPI antibodies as well as by cytokines or LPS. Another lipophilic statin, simvastatin, displayed similar effects but to a lesser extent than fluvastatin. The inhibition of E-selectin expression exerted by fluvastatin was related to the impairment of NF-kappaB binding to DNA. Moreover, the drug attenuated the expression of IL-6 mRNA in HUVEC exposed to anti-beta2GPI antibodies or cytokines. Incubation of HUVECs with mevalonate (100 microM), concomitantly with fluvastatin, greatly prevented the inhibitory effect of statin. CONCLUSION Endothelial activation mediated by anti-beta2GPI antibody can be inhibited by statins. Because of the suggested role of endothelial cell activation in the pathogenesis of antiphospholipid syndrome (APS), our data provide, for the first time, a rationale for using statins as an additional therapeutic tool in APS.

Toll‐like receptor and antiphospholipid mediated thrombosis: in vivo studies

Objective: A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS–/–) mice and the association between tlr4 gene polymorphisms and APS in patients. Methods: IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS–/– mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin. Results: IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-β2glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS−/− mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS−/− mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls. Conclusions: These findings in LPS−/− mice and the reduction in the “protective” polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Antiphospholipid Antibodies and the Antiphospholipid Syndrome: Pathogenic Mechanisms

Antiphospholipid antibodies (Abs) are associated with thrombosis and are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome. It is generally accepted that the major autoantigen for aPL Abs is beta (2) glycoprotein I, which mediates the binding of aPL Abs to target cells (i.e., endothelial cells, monocytes, platelets, trophoblasts, etc.) leading to thrombosis and fetal loss. This article addresses molecular events triggered by aPL Abs on endothelial cells, platelets, and monocytes and complement activation, as well as a review of the current knowledge with regard to the putative receptor(s) recognized by aPL Abs on target cells as well as novel mechanisms that involve fibrinolytic processes. A section is devoted to the description of thrombotic and inflammatory processes that lead to obstetric complications mediated by aPL Abs. Based on experimental evidence using in vitro and in vivo models, new targeted therapies for treatment and/or prevention of thrombosis and pregnancy loss in antiphospholipid syndrome are proposed.

Anti-endothelial cell IgG fractions from systemic lupus erythematosus patients bind to human endothelial cells and induce a pro-adhesive and a pro-infiammatory phenotype in vitro

Affinity purified immunoglobulin G (IgG) fractions from systemic lupus erythematosus (SLE) patients positive for anti-endothelial cell antibodies (AECA) bind human umbilical vein endothelial cell (HUVEC) monolayers. In vitro incubation of serial protein concentrations of SLE AECA IgG induces a dose-dependent endothelial activation: i) increase of functional adhesion of the monocytic cell line U937; ii) upregulation of E-Selectin, ICAM-1, VCAM-1 expression evaluated by a cell solid-phase enzyme linked immunoassay; and iii) increased secretion of interleukin (IL)-6 in the culture supernatants. Control experiments carried out with HUVEC monolayers incubated with IgG fractions from normal healthy controls or from AECA negative SLE sera do not affect at all endothelial adhesion molecule expression or pro-inflammatory cytokine secretion. The AECA IgG effects are not related to both anti-phospholipid or anti-DNA activities. Taken together the findings suggest that these autoantibodies might be important in recruiting and in activating mononuclear leukocytes responsible for vessel wall infiltration and raise the possibility that AECA might display a pathogenic role in SLE vessel damage.

Endothelial cells as a target for antiphospholipid antibodies: role of anti-beta 2 glycoprotein I antibodies.

PROBLEM: To investigate the role of the plasma cofactor for antiphospholipid antibodies in antibody binding to endothelial cells.

Toll-like receptors: another player in the pathogenesis of the anti-phospholipid syndrome.

The anti-phospholipid syndrome (APS) is a systemic autoimmune disease characterized by an adaptive immune response against self phospholipid (PL)--binding proteins. Although APS is considered as an autoantibody-mediated disease, there is now evidence that anti-phospholipid antibodies (aPL) are necessary but not sufficient to trigger some of the clinical manifestations of the syndrome. For example, mediators of the innate immunity are recognized to be additional second hits able to induce the thrombotic events in the presence of aPL. Finally, environmental agents – in particular infectious ones – were reported to act as triggers for the production of autoantibodies cross-reacting with PL-binding proteins as well as inflammatory stimuli that potentiate the aPL thrombogenic effect. Altogether, these findings suggest a role for the innate immunity in APS pathogenesis. Toll-like receptors (TLR) are receptors that induce prompt inflammatory responses and mediate functional activation in immune effector cells. There is evidence that aPL, and in particular anti-β2 glycoprotein I (β2GPI) antibodies, may activate endothelial cells and monocytes through TLR-4-dependent signalling. Whether or not TLR may behave as surface receptors for β2GPI is still matter of research. Drugs or molecules able to interfere with TLR involvement may represent new therapeutic approaches for APS.

ANTIPHOSPHOLIPID ANTIBODIES AND THE ENDOTHELIUM

The interaction between aPL (particularly anti-beta 2GPI antibodies) and endothelium does represent a potential pathogenetic mechanism for the thrombotic manifestations of the syndrome. The autoantibody-mediated EC activation probably plays a role in sustaining the appearance of a proadhesive, proinflammatory, and procoagulant phenotype. The heterogeneity of the APS clinical manifestations is likely linked to the varied effects that aPL can induce on ECs and to the different functions that ECs display depending on the anatomic localization.

Endothelium as a target for antiphospholipid antibodies.

Several, and not necessarily alternative, pathogenic mechanisms have been suggested to sustain the thrombophilic diathesis of the anti-phospholipid syndrome. Among them, interference of anti-phospholipid antibodies with cell acting in the coagulation cascade likely plays a major role. Anti-phospholipid antibodies have been shown to react with endothelial cells mainly by reacting with beta 2 glycoprotein I expressed on the cell membrane surface. Beta 2 glycoprotein I can adhere to endothelial cell surface through the Annexin II receptor and through negatively charged structures (heparin-like molecules) that are bound by the phospholipid-binding site of the molecule. The autoantibody binding involves a yet unknown receptor that activates a signalling pathway able to translocate NFkappaB from the cytoplasm to the nucleus and to activate genes for adhesion molecule, pro-inflammatory cytokine and Tissue Factor up-regulation. The ultimate effect is the induction of a pro-inflammatory and a pro-coagulant endothelial phenotype that has been reproduced both in vitro and in vivo experimental models. Additional effects of anti-phospholipid-mediated endothelial cell activation are the interference with the protein C/S system, with the Annexin V binding, the up-regulation of endothelin I synthesis and the induction of apoptosis. Altogether these effects cooperate in switching endothelium from an anti-coagulant to a pro-coagulant surface.

β2-Glycoprotein I as a 'cofactor' for anti-phospholipid reactivity with endothelial cells

β2-glycoprotein I (β2GPI) is a cofactor for anti-phospholipid (aPL) binding to cardiolipin (CL)-coated plates. β2GPI is also able to bind to endothelial cell (EC) membranes as supported by in-vivo as well as by in-vitro studies. The PL-binding site in the fifth domain of the molecule is involved in the adhesion to endothelium. Actually, specific mutations in this molecular portion abolish endothelium binding and a synthetic peptide spanning the sequence Glu274 –Cys288 of the CL-binding site displays comparable adhesion to EC monolayers. Heparan sulphate appears to be one of the anionic EC membrane structures with which cationic β2GPI interacts, as supported by studies with heparitinase-treated EC. β2GPI binding to EC might be related to its activity as endothelial growth factor or as a lipid-carrying glycoprotein. Adhesion of β2GPI to endothelial membranes offers suitable epitopes for circulating aPL that, once bound, can induce cell activation