Genesio Balestrieri

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.

Apoptotic cell clearance in systemic lupus erythematosus: I. Opsonization by antiphospholipid antibodies

OBJECTIVE To verify whether antiphospholipid antibodies (aPL) recognize and opsonize apoptotic human cells. METHODS Apoptosis was induced via CD95 crosslinking or ultraviolet irradiation. IgG and anti-beta2-glycoprotein I (anti-beta2-GPI) antibodies were purified from patient sera by affinity chromatography. The aPL that bound to apoptotic cells were assessed by flow cytometry, and the subdomains recognized were identified by confocal microscopy. Human macrophages were derived from monocytes, and their ability to phagocytose 3H-labeled apoptotic bodies, whether opsonized or not opsonized by aPL, was assessed. Tumor necrosis factor alpha (TNF alpha) secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS The aPL, but not control Ig or Ig from aPL-negative patients, bound to apoptotic cells, but not to viable cells. Nuclear antigens were not recognized. Opsonization of apoptotic cells by aPL substantially enhanced recognition and binding by scavenger macrophages, with massive TNF alpha secretion. CONCLUSION Antiphospholipid antibodies facilitate apoptotic cell clearance by macrophages and trigger TNF alpha release, possibly enhancing the immunogenicity of the autoantigens they contain.

Anti-beta 2-glycoprotein I antibodies: a marker of antiphospholipid syndrome?

Anticardiolipin (aCL) and anti-beta2-glycoprotein I(antiβ2GPI) antibodies have been shown in animal models as not cross-reacting antibody populations. This observation prompted us to prove if anti-β2GPI exist in human sera by using a reliable method and then to investigate if these are independent from aCl antibodies. We have developed a new ELISA for the detection of anti-β2GPI antibodies employing the coating of the protein in carbonate buffer to irradiated microtitre plates and the filtration of serum samples, that makes irrelevant the binding to the uncoated wells. IgG F(ab)2 fragments from IgG positive sera were shown bind β2GPI, providing that the binding was a specific antibody binding, mediated by the antigen binding site of the antibody molecule; moreover the antibodies were not able to differentiate native and delipidated β2GPI coated plates, making a possible role of a phospholipid contaminant unlikely. On the other hand, the phosphorus content of native as well as delipitated β2GPI was undetectable. IgG, but not IgM, anti-β2GPI antibodies were classically inhibited by the addition of soluble β2GPI, while cardiolipin liposomes appear to modify the reaction in a completely different way, possibly by the described interaction between cardiolipin and β2GPI. The application of the new ELISA to the study of patients has shown that: (1) the presence of anti-β2GPI is statistically associated with the presence of aCL antibodies (P < 0.0001), (2) anti-β2GPI antibodies are related to the classical features of antiphospholipid syndrome (thrombosis: P < 0.001; fetal loss: P < 0.001) while, in this series of patients, aCl antibodies are not (thrombosis: P < 0.126; fetal loss: P < 0.061).

Apoptotic cell clearance in systemic lupus erythematosus: II. Role of β2-glycoprotein I

OBJECTIVE To analyze the contribution of beta2-glycoprotein I (beta2-GPI) to apoptotic cell recognition by antiphospholipid antibodies (aPL) and macrophages from patients with autoimmune disease. METHODS Phosphatidylserine expression by Jurkat cells undergoing apoptosis upon CD95 crosslinking or ultraviolet irradiation was verified by confocal microscopy of cells stained with fluorescein isothiocyanate-labeled annexin V. Beta2-GPI was purified by heparin/cationic-exchange chromatography and was biotinylated or used to purify beta2-GPI-specific antibodies by affinity chromatography. Binding to apoptotic cells was assessed by flow cytometry. The clearance of 3H-labeled, apoptotic cells by macrophages was assessed by beta counting. RESULTS The array of epitopes generated by beta2-GPI association with apoptotic cells specifically targets their recognition and is required for their opsonization by human aPL. Nevertheless, beta2-GPI is not required for apoptotic cell clearance by human macrophages in the absence of aPL. CONCLUSION The proinflammatory clearance of aPL-opsonized apoptotic cells, but not the nonphlogistic clearance of apoptotic cells by scavenger macrophages, depends on beta2-GPI.

Prevalence and pattern of cognitive impairment in systemic lupus erythematosus patients with and without overt neuropsychiatric manifestations

The prevalence and pattern of cognitive impairment in systemic lupus erythematosus (SLE) patients with (NPSLE) and without (nSLE) overt neuropsychiatric manifestations were investigated. Fifty-two nSLE patients, 23 NPSLE patients and 27 healthy controls were evaluated with a battery of standardized neuropsychological and psychological tests. Disease duration, disease activity index, and current corticosteroid therapy were collected. Cognitive impairment was identified in 14 (26.9%) and in 12 (52.2%) of subjects with nSLE and NPSLE, respectively. Both SLE groups showed a significant impairment compared with controls on tasks assessing verbal and non-verbal long-term memory, and visuoconstructional abilities. In addition, NPSLE patients reported worse performances than both nSLE patients and controls on task evaluating short-term visuospatial memory. NPSLE subjects were significantly more anxious and depressed compared to both nSLE subjects and controls. By multivariate analysis, only depression levels, among clinical variables, significantly predicted cognitive performance. This study shows that cognitive impairment occurs frequently in both nSLE and NPSLE subjects. The higher frequency in NPSLE may be related to coexisting depressive disturbances.

Anti-endothelial cell antibodies in patients with Wegener's granulomatosis and micropolyarteritis.

Anti‐endothelial cell antibodies (AECA) have been detected by cell surface radioimmunoassay innine out of 15 patients with micropolyarteritis (MPA) and in two out of five patients with Wegenersgranulomatosis. AECA mostly belonged to the IgG isotype and were present in the active phase ofthe diseases. These antibodies were not detectable in 10 sera from patients with essential mixedcryoglobulinaemia. suggesting that they were not a mere epiphenomenon consequent to theinflammatory vascular injury. The binding activity was not related to ABH antigens or to HLA class Iantigens displayed by resting human endothelial cells in culture and was not influenced by removingimmune complexes. Absorption of the anti‐neutrophil cytoplasmic antibodies (ANCA). present inMPA and Wegeners granulomatosis sera, did not affect the endothelial binding. AECA‐positive seradid not display lytic activity against endothelial cells, neither alone nor after addition of freshcomplement or normal human peripheral blood mononuelear cells. Although AECA arc notcytolytic for endothelial cell monolayers in vitro, the reactivity against intact endothelial cells suggeststheir possible involvement in in vivo pathological processes affecting vascular structures in smallvessel primary vasculitides.

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.

Dendritic cell presentation of antigens from apoptotic cells in a proinflammatory context: Role of opsonizing anti-β2-glycoprotein I antibodies

OBJECTIVE To verify whether opsonization of apoptotic cells skews the outcome of apoptotic cell antigen presentation by dendritic cells (DCs). METHODS RMA cells, which were engineered with a mutant ovalbumin (OVA) protein and were devoid of the leader secretory sequence (OVA-RMA), underwent ultraviolet irradiation to induce apoptosis. Binding of anti-beta2-glycoprotein I antibodies (anti-beta2GPI) and phagocytosis of apoptotic cells were assessed by flow cytometry and confocal microscopy. Presentation of processing antigens and major histocompatibility complex (MHC) class II-restricted or MHC class I-restricted antigens was assessed using OVA-specific T cell hybridomas. RESULTS Anti-beta2GPI facilitated presentation of epitopes from internalized apoptotic cells to MHC class II-restricted, but not to class I-restricted, T lymphocytes. DCs challenged with supernatants of apoptotic cells did not activate OVA-specific T cells, making it unlikely that anti-beta2GPI complexed with antigen released from dying cells plays a role in antigen presentation. DCs challenged with low numbers of anti-beta2GPI-opsonized apoptotic cells secreted interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-10 in an autocrine/paracrine manner. CONCLUSION Opsonization influences the outcome of the disposal of low numbers of apoptotic cells by DCs. This implies that soluble factors bound to apoptotic cells modulate their immunogenicity.

Human anticardiolipin monoclonal autoantibodies cause placental necrosis and fetal loss in BALB/c mice

OBJECTIVE To analyze the structure, specificity, and in vivo pathogenetic potential of 2 human anticardiolipin (aCL) monoclonal antibodies (MAb). METHODS Human aCL IgG MAb were generated from hybridized Epstein-Barr virus-induced B cell lines from a healthy subject (MAb 519) and from a patient with primary antiphospholipid syndrome (MAb 516). Studies of antigen-binding specificity and analysis of Ig V-gene mutations were carried out. The MAb were independently injected into mated female BALB/c mice, and their effect on pregnancy outcome was compared with that of MAb 57, a highly mutated and antigen-selected human IgG1lambda rabies virus antibody. RESULTS Both MAb 519 and MAb 516 utilized minimally mutated V(H)DJ(H) and VkappaJkappa gene segments and bound cardiolipin and other anionic phospholipids in the absence of beta2-glycoprotein I (beta2-GPI). The mice injected with aCL MAb displayed a significantly higher rate of fetal resorption and a significant reduction in fetal and placental weight as compared with those injected with MAb 57. These findings were accompanied by a finding of placental human IgG deposition and necrosis in the aCL MAb-treated animals. CONCLUSION The results of this study indicate that human aCL IgG that are beta2-GPI independent can induce pathology.