Restricted T-cell receptor beta-chain usage by T cells autoreactive to beta(2)-glycoprotein I in patients with antiphospholipid syndrome

We recently identified CD4(+) T cells that are autoreactive to beta(2)-glycoprotein I (beta(2)GPI) and that promote antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In this study, T-cell receptor (TCR) beta chains of beta(2)GPI-reactive T cells were examined in 8 beta(2)GPI-responders, including 5 patients with APS and 3 healthy subjects, using polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant beta(2)GPI. The TCR Vbeta segments that expanded oligoclonally after stimulation with beta(2)GPI varied among responders, but the Vbeta7 and Vbeta8 segments were commonly detected in 6 and 4 beta(2)GPI-responders, respectively. Analysis of the complementarity-determining region 3 sequence of beta(2)GPI-reactive T cells revealed limited diversity, and all Vbeta7(+) TCRs had an amino acid motif of TGxxN/Q or minor variations. The Vbeta8(+) TCRs had another motif, PxAxxD/E. Surprisingly, an identical Vbeta7(+) TCRbeta chain was used by beta(2)GPI-reactive T cells in 3 patients with APS. There was no apparent difference in the TCRbeta usage between APS patients and healthy responders. Some of the Vbeta7(+) TCRs with the TGxxN/Q motif detected by PCR-SSCP analysis were also used by beta(2)GPI-specific CD4(+) T-cell clones responsive to an immunodominant epitope containing the major phospholipid-binding site. Depletion of Vbeta7(+) or Vbeta8(+) T cells from the peripheral blood mononuclear cell cultures significantly inhibited in vitro anti-beta(2)GPI antibody production in response to beta(2)GPI. Our results indicate preferential usage of TCRbeta chains by beta(2)GPI-reactive T cells. These TCRbeta chains can be reasonable targets for TCR-based immunotherapy for patients with APS.     

T cells that are autoreactive to beta2-glycoprotein I in patients with antiphospholipid syndrome and healthy individuals

OBJECTIVE: To identify the T cells responsive to beta2-glycoprotein I (beta2GPI) that mediate antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). METHODS: In vitro proliferative responses and anti-beta2GPI antibody production induced by beta2GPI were examined in peripheral blood mononuclear cell (PBMC) cultures from 12 APS patients, 13 systemic lupus erythematosus patients without APS, and 12 healthy donors. RESULTS: Peripheral blood T cells from all subjects failed to respond to beta2GPI in its native form. In contrast, reduced beta2GPI was able to stimulate T cells not only from all 12 patients with anti-beta2GPI antibodies, but also from 10 of 25 individuals without anti-beta2GPI antibodies. The specificity of the responses to beta2GPI was confirmed by activation of the reduced beta2GPI-primed T cells by recombinant beta2GPI in secondary cultures. Characterization of the T cell response induced by beta2GPI revealed that the response was associated with the presence of the DR53-associated alleles, the responding T cells were CD4+ and restricted by HLA class II, and antigenic peptides were located in domains IV and/or V. Anti-beta2GPI antibody production was induced specifically in anti-beta2GPI antibody-positive patients, in PBMC cultures with reduced beta2GPI. Anti-beta2GPI antibodies produced in vitro recognized beta2GPI immobilized with cardiolipin or beta2GPI coated on "high-binding" polystyrene plates. CONCLUSION: These results strongly suggest that CD4+ and HLA class II-restricted T cells responsive to beta2GPI are involved in the production of antiphospholipid antibodies in APS patients.     

Excessive exposure to anionic surfaces maintains autoantibody response to beta(2)-glycoprotein I in patients with antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an autoimmune prothrombotic disorder associated with autoantibodies to phospholipid (PL)-binding proteins, such as beta(2)-glycoprotein I (beta(2)GPI). We have recently reported that binding of beta(2)GPI to anionic PL facilitates processing and presentation of the cryptic beta(2)GPI epitope that activates pathogenic autoreactive T cells. To clarify mechanisms that induce sustained presentation of the dominant antigenic beta(2)GPI determinant in patients with APS, T-cell proliferation induced by beta(2)GPI-treated phosphatidylserine liposome (beta(2)GPI/PS) was evaluated in bulk peripheral blood mononuclear cell cultures. T cells from patients with APS responded to beta(2)GPI/PS in the presence of immunoglobulin G (IgG) anti-beta(2)GPI antibodies derived from APS plasma, and this response was completely inhibited either by the depletion of monocytes or by the addition of anti-FcgammaRI antibody. These findings indicate that efficient presentation of the cryptic determinants can be achieved by monocytes undergoing FcgammaRI-mediated uptake of beta(2)GPI-bound anionic surfaces in the presence of IgG anti-beta(2)GPI antibodies. Finally, beta(2)GPI-bound oxidized LDL or activated platelets also induced the specific T-cell response. Continuous exposure to these anionic surfaces may play a critical role in maintaining the pathogenic anti-beta(2)GPI antibody response in patients with APS.     

Anti-beta2-glycoprotein I antibodies in pediatric systemic lupus erythematosus and antiphospholipid syndrome

OBJECTIVE: To determine whether serum beta2-glycoprotein I antibody (anti-beta2GPI) detection improves identification of pediatric subjects at risk for antiphospholipid syndrome (APS). METHODS: Serum antiphospholipid antibodies (aPL) were identified by anticardiolipin enzyme-linked immunosorbent assay (ELISA), lupus anticoagulant assays, and syphilis screening in children with primary APS, systemic lupus erythematosus (SLE), or SLE plus APS. Anti-beta2GPI level and isotype were determined by beta2GPI ELISA and correlated with clinical manifestations and other aPL assays. RESULTS: One hundred-ten subjects under 22 years of age and of mixed ethnicity were evaluated. Fifty-seven had SLE (including 14 with APS), 25 had primary APS, 16 had SLE-like APS, 6 were healthy children with aPL detected incidentally, 4 had other rheumatic diseases and 2 had other conditions. Anti-beta2GPI were detected in 48% of SLE subjects and did not improve aPL detection over standard tests. Anti-beta2GPI were associated with stroke (P = 0.014), but not with other APS manifestations, and were rarely detected in primary APS. Among subjects with APS manifesting as chronic thrombocytopenia, anti-beta(2)GPI distinguished subjects with SLE from those with primary APS. CONCLUSIONS: With the exception of stroke, anti-beta2GPI detection does not improve identification of pediatric APS over that of traditional aPL assays. Anti-beta2GPI are rare in pediatric primary APS, but may predict evolution of chronic thrombocytopenia to SLE.     

Correlation between beta2-glycoprotein I valine/leucine247 polymorphism and anti-beta2-glycoprotein I antibodies in patients with primary antiphospholipid syndrome.

OBJECTIVES: Beta2-Glycoprotein I (beta2GPI) exon 7 polymorphism leads to a valine leucine amino acid exchange at position 247 in domain 5 of beta2GPI, between the phospholipid binding site and the cryptic site of the epitopes for anti-beta2GPI antibodies. Therefore, position 247 polymorphism may affect the conformational change of beta2GPI and the exposure of the epitopes for anticardiolipin antibodies (aCL) (= anti-beta2GPI antibodies). In this study we analysed the genetic polymorphism of beta2GPI in a British cohort of well-defined antiphospholipid syndrome (APS) patients. METHODS: This study comprised 88 Caucasoid patients with APS [57 with primary APS and 31 with APS secondary to systemic lupus erythematosus (SLE)]. Polymorphism assignment was determined by polymerase chain reaction followed by allele-specific restriction enzyme digestion (PCR-RFLP). The presence of anti-beta2GPI antibodies was detected by ELISA utilizing irradiated ELISA plates. RESULTS AND CONCLUSIONS: Anti-beta2GPI antibodies were present in 28 of 57 primary APS patients (49%) and in 19 of 31 secondary APS patients (61%). The allele containing valine247 was significantly more frequent in primary APS patients with anti-beta2GPI antibodies than in controls (OR = 2.51, 95%, CI 1.03-6.13, P = 0.0396) or in primary APS patients without anti-beta2GPI antibodies (OR = 2.92, 95% CI 1.16-7.39, P = 0.0204). This tendency was not found in the secondary APS group. In conclusion, the beta2GPI polymorphism, valine/leucine247, is correlated with anti-beta2GPI antibody production in patients with primary APS, and valine247 may be important in the formation of beta2GPI antigenicity.     

Binding of beta 2-glycoprotein I to anionic phospholipids facilitates processing and presentation of a cryptic epitope that activates pathogenic autoreactive T cells

Antiphospholipid syndrome (APS) is an autoimmune prothrombotic disorder in association with autoantibodies to phospholipid (PL)-binding plasma proteins, such as beta(2)-glycoprotein I (beta(2)GPI). We have recently found that CD4(+) T cells autoreactive to beta(2)GPI in patients with APS preferentially recognize a cryptic peptide encompassing amino acid residues 276-290 (p276-290), which contains the major PL-binding site, in the context of DR53. However, it is not clear how previously cryptic p276-290 becomes visible to the immune system and elicits a pathogenics autoimmune response to beta(2)GPI. Here we show that presentation of a disease-relevant cryptic T-cell determinant in beta(2)GPI is induced as a direct consequence of antigen processing from beta(2)GPI bound to anionic PL. Dendritic cells or macrophages pulsed with PL-bound beta(2)GPI induced a response of p276-290-specific CD4(+) T-cell lines generated from the patients in an HLA-DR-restricted and antigen-processing-dependent manner but those with beta(2)GPI or PL alone did not. In addition, the p276-290-reactive T-cell response was primed by stimulating peripheral blood T cells from DR53-carrying healthy individuals with dendritic cells bearing PL-bound beta(2)GPI in vitro. Our finding is the first demonstration of an in vitro mechanism eliciting pathogenic autoreactive T-cell responses to beta(2)GPI and should be useful in clarifying the pathogenesis of APS.     

Suppressed intrinsic fibrinolytic activity by monoclonal anti-beta-2 glycoprotein I autoantibodies: possible mechanism for thrombosis in patients with antiphospholipid syndrome

beta2-glycoprotein I (beta2GPI) bears the epitope(s) for autoimmune anticardiolipin antibodies (aCL) frequently present in patients with antiphospholipid syndrome (APS). beta2GPI is involved in coagulation and fibrinolytic systems, including inhibition of contact activation. Coagulation factor XII is an initiator of intrinsic coagulation and also of intrinsic fibrinolysis. We investigated the effect of aCL (= anti-beta2GPI antibodies), regarding intrinsic fibrinolysis using autoimmune monoclonal anti-beta2GPI antibodies derived from a patient with APS or from an NZW/BXSB-F1 mouse. We developed a chromogenic assay system to determine intrinsic fibrinolytic activity. The reaction was activated by kaolin in the euglobulin fraction. Exogenous beta2GPI slightly suppressed intrinsic fibrinolytic activity of the euglobulin fraction from normal plasma. Human monoclonal anti-beta2GPI antibody (EY2C9) and mouse monoclonal anti-beta2GPI antibody (WBCAL-1) in the presence of beta2GPI decreased the activity. In this system, the suppression remained significant in the presence of an excess of exogenous activated factor XII. Euglobulin fractions from APS patients' plasma paralleled low activities of intrinsic fibrinolysis compared with those from healthy subjects. Our results suggest that beta2GPI and anti-beta2GPI antibodies suppress intrinsic fibrinolytic activities. This suppression was not only due to inhibition of factor XII activation but was also related to function of activated factor XII (XIIa). These phenomena partly explain the mechanisms of thrombosis in APS.     

Anti-beta2-glycoprotein I antibodies in complex with beta2-glycoprotein I can activate platelets in a dysregulated manner via glycoprotein Ib-IX-V

OBJECTIVE: Results of previous studies suggest that anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in complex with beta2GPI activate platelets in a dysregulated manner, potentially contributing to the prothrombotic tendency associated with the antiphospholipid syndrome (APS). We undertook this study to investigate the possible contribution of the GPIb-IX-V receptor to platelet activation mediated by the anti-beta2GPI antibody-beta2GPI complex. METHODS: In vitro methods were used in the present study. The interaction between beta2GPI and the GPIbalpha subunit of the GPIb-IX-V receptor was delineated using direct binding and competitive inhibition assays. The interaction between the anti-beta2GPI antibody-beta2GPI complex and platelets was studied using a novel method in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by two methods; one involved measuring thromboxane B2 production and the other involved assessment of the activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta intracellular signaling pathway. The contribution of the GPIbalpha receptor to platelet activation induced by the anti-beta2GPI antibody-beta2GPI complex was assessed by observing the influence of 2 anti-GPIbalpha antibodies (AK2 and SZ2) directed against distinct epitopes. RESULTS: This study showed that beta(2)GPI could bind to the GPIbalpha receptor. The anti-beta2GPI antibody-beta2GPI complex was able to activate platelets, and this effect was inhibited by anti-GPIbalpha antibody directed against epitope Leu-36-Gln-59, but not by anti-GPIbalpha antibody directed against residues Tyr-276-Glu-282. CONCLUSION: Our findings show that inappropriate platelet activation by the anti-beta2GPI antibody-beta2GPI complex via the GPIbalpha receptor may contribute to the prothrombotic tendency associated with APS.     

Valine/Leucine247 polymorphism of beta2-glycoprotein I in patients with antiphospholipid syndrome: lack of association with anti-beta2-glycoprotein I antibodies

In antiphospholipid syndrome (APS) the presence of anti-beta2-glycoprotein I (beta2GPI) antibodies is strongly associated with thromboembolic complications. It has been suggested that the common beta2GPI Valine/Leucine247 (Val/Leu247) polymorphism could be found more commonly in APS and might influence the generation of anti-beta2GPI antibodies. Therefore we studied beta2GPI Val/Leu247 single-nucleotide polymorphism (SNP) by PCR in 338 patients with various autoimmune diseases (46 with secondary and 84 with primary APS) and 147 sex and age-matched healthy controls. In all patients lupus anticoagulant, anticardiolipin and anti-beta2GPI antibodies (both IgG and IgM) were also determined. All patients and controls were Caucasians. Frequencies of the SNP genotypes in patients did not depart from genetic equilibrum and did not differ from those found in controls. There was also no association between the presence of beta2GPI Val/Leu247genotypes and the presence or absence of lupus anticoagulant, anticardiolipin antibodies, anti-beta2GPI antibodies or clinical APS symptoms in all patients studied. In conclusion, among the exclusively Caucasian, Polish population of autoimmune patients beta2GPI Val/Leu247SNP has the same distribution as in healthy subjects and does not influence the production of anti-beta2GPI antibodies.     

Update on antiphospholipid antibodies

The association of antibodies with an apparent specificity for anionic phospholipids with thrombosis, fetal loss, thrombocytopenia, and certain other clinical manifestations is now well-recognized as the antiphospholipid syndrome (APS). Recent advances in our understanding of the antibodies and antigens involved include discovery of the crystal structure of beta2-glycoprotein I, (beta2GPI), genetic studies of beta2GPI polymorphisms, and the development of anti-beta2GPI and antiprothrombin immunoassays as clinical laboratory tests. The identification of antigen-specific T cells in APS patients has stimulated interest in the role of the cellular immune response in the syndrome. Clinical research in APS will also benefit from the development of preliminary classification criteria.     

Mechanisms of disease: antiphospholipid antibodies-from clinical association to pathologic mechanism

The discovery that antiphospholipid antibodies recognize plasma proteins that bind to phospholipids rather than recognizing phospholipids themselves has been a major advance in research into antiphospholipid syndrome (APS). It is now established that beta2-glycoprotein I (beta2 GPI) is the most important antigen for antiphospholipid antibodies. However, the possible pathologic mechanism is still much debated. This is mainly because not all patients with anti-beta2 GPI antibodies show clinical symptoms that are related to APS. Several reports indicate that anti-beta2 GPI antibodies with lupus anticoagulant (LA) activity are clinically of much importance. Most patients with LA caused by anti-beta2 GPI antibodies suffer from thrombosis as a result of recognition of the first domain of beta2 GPI by these antibodies. In the search for a pathologic mechanism that might explain the high occurrence of thrombosis in patients with anti-domain I antibodies (LA-causing anti-beta2 GPI antibodies), it was found that these antibodies show increased resistance to the anticoagulant activity of annexin A5. We have shown that the same population of antibodies also displays increased resistance to activated protein C. Owing to the diversity of clinical symptoms related to APS, it is likely that other pathologic mechanisms also contribute to the occurrence of APS-related symptoms.     

Statins prevent endothelial cell activation induced by antiphospholipid (anti-beta2-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.     

TNFalpha DNA vaccination prevents clinical manifestations of experimental antiphospholipid syndrome

Naked DNA encoding TNFalpha was introduced to BALB/c mice with experimental antiphospholipid syndrome (APS) induced by beta2GPI. Administration of naked DNA encoding TNFalpha resulted in the generation of immunological memory to its gene product, associated with elevated circulating anti-TNFalpha antibodies. Enriched IgG fraction of the mouse anti-TNFalpha was biologically active since it prevented endothelial cell activation by TNFalpha e.g., inhibition of monocyte adhesion to activated endothelial cells (HUVEC). Mice immunized with beta2GPI, vaccinated with TNFalpha DNA at an early stage of disease development, showed decreased titres of circulating anti-beta2GPI antibodies as compared to the group of mice vaccinated with a control naked DNA. The reduction of antiphospholipid antibody production was followed by amelioration of the foetal loss, increased platelet count to normal values as well as normalization of the prolonged aPTT. APS mice which were introduced to the TNFalpha DNA vector at a later stage of the disease development, showed less improvement in their clinical manifestations. The current study suggests a way in which a DNA vaccine can be employed for induction of a protective immunity in experimental APS.     

Some antiphospholipid antibodies recognize conformational epitopes shared by beta2-glycoprotein I and the homologous catalytic domains of several serine proteases

OBJECTIVE: To test the hypothesis that some antiphospholipid antibodies (aPL) in patients with the antiphospholipid syndrome (APS) recognize a conformational epitope shared by beta2-glycoprotein I (beta2GPI; the major autoantigen for the antiphospholipid antibodies) and the homologous catalytic domains of several serine proteases (such as thrombin, activated protein C [APC], and plasmin) involved in hemostasis. METHODS: We generated 4 new IgG monoclonal aPL (2 screened against beta2GPI, 1 against thrombin, and 1 against protein C) from 2 APS patients. The monoclonal antibodies (mAb) were analyzed for binding to beta2GPI, thrombin, APC, and plasmin, as well as for anticardiolipin antibody (aCL) activity. To demonstrate a shared epitope between beta2GPI and a serine protease, 1 mAb was studied by cross-inhibition analysis. RESULTS: Both of the IgG anti-beta2GPI mAb bound to thrombin, APC, and plasmin. On the other hand, the 1 anti-thrombin mAb and the 1 anti-protein C mAb also bound to beta2GPI. Moreover, the binding of 1 cross-reactive mAb to beta2GPI was inhibited by alpha-thrombin (which contains only the catalytic domain of thrombin). All 4 mAb displayed aCL activity. CONCLUSION: Taken together with the findings that some aCL bind to several serine proteases that participate in hemostasis and share homologous catalytic domains, these data demonstrate that some aCL in APS patients recognize one or more conformational epitopes shared by beta2GPI and the catalytic domains of disease-relevant serine proteases.     

The molecular basis of antiphospholipid syndrome

We herein review evidence that the phospholipid-binding protein beta2 glycoprotein-1 (beta2GPI) is a causative autoantigen in APS. Recent work suggests that the molecular regions in beta2GPI that facilitate autoimmunization are those that promote binding to negatively charged phospholipids by means of strong positive (anionic) charge and hydrophobicity. Although many common infections can cause antiphospholipid antibodies to be produced in humans, such postinfectious aPL are rarely associated with thromboses or pregnancy morbidity, the central features of antiphospholipid syndrome (APS). We propose that the causes of APS include those infectious agents that mimic the above molecular domains in beta2GPI. In people who are susceptible to APS, tolerance to self-beta2GPI and phospholipids is likely to be broken by foreign bacterial or viral proteins that contain such beta2GPI-like epitopes.     

Arterial disease in lupus and secondary antiphospholipid syndrome: association with anti-beta2-glycoprotein I antibodies but not with antibodies against oxidized low-density lipoprotein

The prevalence and clinical significance of antibodies against beta2- glycoprotein I (anti-beta2GPI) and antibodies against oxidized low- density lipoprotein (anti-ox-LDL) were evaluated as potential indicators of arterial disease in patients with systemic lupus erythematosus (SLE) and SLE with secondary antiphospholipid syndrome (APS). IgG anti-beta2GPI and IgG anti-ox-LDL were measured by enzyme- linked immunosorbent assay (ELISA) in serum samples from 118 patients with SLE, including 40 with secondary APS. IgG anti-beta2GPI were positive in 17% (20/118) of SLE patients. The presence and titres of IgG anti-beta2GPI were strongly associated with a history of arterial thrombosis. Haemolytic anaemia was also significantly associated with the presence of IgG anti-beta2GPI. The prevalence of IgG anti-ox-LDL was 53% (63/118), but there was no association with arterial thrombosis. No correlation between the values of anti-ox-LDL and those of anti-beta2GPI was found. These results suggest that IgG anti- beta2GPI could be a marker for arterial thrombosis in SLE patients, while IgG anti-ox-LDL were not associated with arterial disease in this group of lupus patients.      

Heterogeneity of antibodies to beta2-glycoprotein 1 from patients with systemic lupus erythematosus

We studied antibodies to beta2-glycoprotein 1 (anti-beta2GP1) from 72 patients with systemic lupus erythematosus (SLE) with or without antiphospholipid syndrome (APS) or with or without anticardiolipin antibodies (aCL). Fifteen patients had APS and positive antiphospholipid antibodies [clinical APS(+)/aPL(+)], 12 patients had APS, negative serum IgG and IgM aCL, antiphosphatidylethanolamine, anti-phosphatidylserine and no lupus anticoagulant [clinical APS(+)/ aPL(-)]. A third group included 16 patients without APS but high aCL levels [clinical APS(-)/ aPL(+)]. In a fourth group we studied 29 patients without clinical manifestations of APS or aCL [clinical APS(-)/aPL(-)]. One hundred anticardiolipin and VDRL-negative normal sera were studied as controls. IgG antibodies to cardiolipin proper in a bovine beta2GP-free system, to human beta2GP1 immobilized on cardiolipin or to human beta2GP1 alone were detected in all sera by ELISA using irradiated and nonirradiated plates from two manufacturers. Sera from APS(+)/aPL(+) patients showed IgG binding to CL, CL + beta2GP1 and beta2GP1 in irradiated and nonirradiated plates. APS(+)/ aPL(-) sera had more significant IgG binding to beta2GP1 than normal controls when studied in both irradiated or nonirradiated plates (P = 0.001). This binding was inhibited by solid-phase cardiolipin in a dose-dependent manner. Sera from the APS(-)/aPL(+) subgroup had comparable IgG activity in both the CL and CL + beta2GP1 assays, while no anti-beta2GP1 activity was detected in these sera. Sera from the clinical APS(-)/aPL(-) patients were negative in the three ELISA systems. Antibodies to human beta2GP1 from SLE patients recognize various epitopes. Those from APS(+)/ aPL(+) patients appear to react with an epitope boosted by cardiolipin in addition to another one present in the native protein. In contrast, anti-beta2GP1 from patients with APS(+)/aPL(-) are blocked by cardiolipin, suggesting that their epitope is the phospholipid-binding site.     

Anti-beta 2-glycoprotein I antibodies: a useful marker for the antiphospholipid syndrome

We studied anti-beta 2-glycoprotein I antibodies (a beta 2GPI) in autoimmune disease patients to evaluate their relationship to clinical findings. Seventy-nine systemic lupus erythematosus (SLE) patients [44 with antiphospholipid antibodies (aPL)], 21 with primary antiphospholipid syndrome (APS), eight asymptomatic individuals with aPL and 60 controls were studied. Sixteen SLE patients (14 with aPL and two without aPL) and six with primary APS had a beta 2GPI. A significant relationship was found between a beta 2GPI and aPL (P < 0.01). In SLE, a significant correlation was found between previous thrombosis or thrombocytopenia and a beta 2GPI or a beta 2GPI + aPL, but not between fetal losses and a beta 2GPI. These data suggest that a beta 2GPI may be useful in the study of APS.      

Significance of valine/leucine247 polymorphism of beta2-glycoprotein I in antiphospholipid syndrome: increased reactivity of anti-beta2-glycoprotein I autoantibodies to the valine247 beta2-glycoprotein I variant

OBJECTIVE: To clarify the consequences of the valine/leucine polymorphism at position 247 of the beta(2)-glycoprotein I (beta(2)GPI) gene in patients with antiphospholipid syndrome (APS), by investigating the correlation between genotypes and the presence of anti-beta(2)GPI antibody. The reactivity of anti-beta(2)GPI antibodies was characterized using recombinant Val(247) and Leu(247) beta(2)GPI. METHODS: Sixty-five Japanese patients with APS and/or systemic lupus erythematosus who were positive for antiphospholipid antibodies and 61 controls were analyzed for the presence of the Val/Leu(247) polymorphism of beta(2)GPI. Polymorphism assignment was determined by polymerase chain reaction followed by restriction enzyme digestion. Recombinant Val(247) and Leu(247) beta(2)GPI were established to compare the reactivity of anti-beta(2)GPI antibodies to beta(2)GPI between these variants. The variants were prepared on polyoxygenated plates or cardiolipin-coated plates, and the reactivity of a series of anti-beta(2)GPI antibodies (immunized anti-human beta(2)GPI monoclonal antibodies [Cof-19-21] and autoimmune anti-beta(2)GPI monoclonal antibodies [EY1C8, EY2C9, and TM1G2]) and IgGs purified from patient sera was investigated. RESULTS: A positive correlation between the Val(247) allele and the presence of anti-beta(2)GPI antibodies was observed in the patient group. Human monoclonal/polyclonal anti-beta(2)GPI autoantibodies showed higher binding to recombinant Val(247) beta(2)GPI than to Leu(247) beta(2)GPI, although no difference in the reactivity of the immunized anti-beta(2)GPI between these variants was observed. Conformational optimization showed that the replacement of Leu(247) by Val(247) led to a significant alteration in the tertiary structure of domain V and/or the domain IV-V interaction. CONCLUSION: The Val(247) beta(2)GPI allele was associated with both a high frequency of anti-beta(2)GPI antibodies and stronger reactivity with anti-beta(2)GPI antibodies compared with the Leu(247) beta(2)GPI allele, suggesting that the Val(247) beta(2)GPI allele may be one of the genetic risk factors for development of APS.     

Lymphocyte subpopulations and intima media thickness in primary antiphospholipd syndrome

The aim of this study was to evaluate a possible association between lymphocyte subsets and intima media thickness (IMT) of carotid arteries in primary antiphospholipid syndrome (PAPS). We used a cross-sectional study on PAPS patients (n = 18) and healthy controls (n = 16). IgG anti-cardiolipin antibody (aCL), IgG anti-beta2glycoprotein-I (anti-beta2GPI), IgG anti-beta2glycoprotein-I complexed to oxidized low-density lipoprotein (oxLDL) and to a specific oxidized moiety of LDL (oxLig1), and beta2GPI-oxLDL were measured by ELISA. Lymphocyte immunophenotyping was performed using pairs of monoclonal antibodies directly labelled with fluorescein isothiocyanate, or phycoerythrin or phycoerythrin-Texas-red-X. Intima media thickness (IMT) of carotid arteries was determined by high-resolution sonography. Total peripheral blood lymphocytes did not differ between PAPS and controls. Memory CD4+/CD45RO + T cells were lower in PAPS than controls (P = 0.0007) as well as CD16+56+ natural killer cells (P = 0.02). In PAPS memory T CD45RO + cells positively correlated with IgG anti-beta2GPI-oxLigl (P = 0.002) and to IMT of carotid arteries (common carotid P = 0.02, bifurcation P = 0.007). Naive CD4+/CD45RA+ T cells inversely correlated with beta2GPI-oxLDL (P = 0.009). The relation between IgG anti-beta2GPI-oxLig1 and IMT of carotid arteries with memory CD45RO + T lymphocytes suggests a role for the latter in PAPS related atherogenesis.