Role of the MyD88 transduction signaling pathway in endothelial activation by antiphospholipid antibodies

Objective To investigate the ability of human anti-beta 2-glycoprotein I (anti-beta 2 GPI) antibodies to recognize the cofactor adherent on endothelial cells (EC) and to modulate endothelial functions. Methods Six human affinity-purified polyclonal anti-beta 2 GPI IgG and 2 IgM monoclonal antibodies (MAb) were obtained from patients with the antiphospholipid syndrome. The antibodies were tested for their ability to 1) bind to endothelial monolayers through the adherent beta 2 GPI and 2) modulate endothelial adhesion molecule expression and interleukin-6 (IL-6) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) secretion. Results The affinity-purified IgG and the MAb with anti-beta 2 GPI activity, but not the respective controls, displayed EC binding, which declined on cells incubated in serum-free medium and was restored in a dose-dependent manner by exogenous human beta 2 GPI. After EC binding, both polyclonal and monoclonal antibodies up-regulated adhesion molecule expression. Anti-beta 2 GPI MAb also significantly increased IL-6 and 6-keto-PGF1 alpha secretion. Conclusion These findings support the hypothesis that anti-beta 2 GPI antibodies bind and activate EC through the adherent cofactor beta 2 GPI, likely leading to a procoagulant state.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780400322

Endothelial cells as target for antiphospholipid antibodies : Human polyclonal and monoclonal anti-β2-glycoprotein I antibodies react in vitro with endothelial cells through adherent β2-glycoprotein I and induce endothelial activation

Antiphospholipid antibodies (aPL) are associated with poor obstetric outcome, such as recurrent abortions, fetal death, growth retardation, and early preeclampsia (1). Passive transfer of whole immunoglobulin fractions from aPL-positive sera has been found to induce fetal loss and growth retardation in pregnant naive mice, suggesting a direct pathogenetic role (2–4).

Although it has been assumed that aPL are directed against anionic PL, current advances in the field suggest that antibodies to PL-binding plasma proteins, such as β2-glycoprotein I (β2GPI), can be detected in standard aPL assays (5). Antibodies specific for β2GPI have been identified and found to be associated with the clinical manifestations of the antiphospholipid syndrome (APS) (6–22). The in vivo immunohistologic demonstration of β2GPI on trophoblast surfaces (23,24) and the induction of fetal loss by anti-β2GPI antibodies in experimental animal models (25,26) suggested a role of anti-β2GPI antibodies in fetal loss. Moreover, even murine and human aPL monoclonal antibodies (mAb) specifically reacting with anionic PL in the absence of any plasma cofactor have been shown to produce fetal loss, growth retardation, placental deposition, and necrosis in experimental animal models (3,27,28).

Although experimental models have emphasized the role of thrombotic phenomena in placental tissue (4,27), studies in humans have shown that thrombotic events cannot account for all of the histopathologic findings in placentae from women with the APS (29,30). The possibility of direct villous and extravillous trophoblastic damage by aPL through the recognition of phosphatidylserine (PS) exposed during syncytium formation has been suggested (31). Reported direct effects of aPL on trophoblasts have included inhibition of the intercytotrophoblast fusion process (31), of human chorionic gonadotropin (hCG) or placental lactogen secretion (31,32), and/or of trophoblast invasiveness (31). Furthermore, whole IgG fractions from APS patient sera or xenogenic murine anti-PS mAb have been shown to displace annexin V from trophoblasts (and endothelial cell surfaces in the case of human IgG), thus creating conditions favorable to procoagulant state in vitro (31,33).

The purpose of the present study was to investigate the in vitro ability of IgG from sera containing high levels of aPL to bind human trophoblast cells and to affect hCG secretion and invasiveness. Furthermore, to identify whether specific effects were related to individual antibody subpopulations, human mAb reacting with β2GPI or with anionic PL in the absence of any plasma cofactor were investigated for their ability to reproduce the binding to trophoblast cell membranes and the modulation of hormone secretion as well as invasiveness. From our results, it appears that trophoblast cells might represent one target for circulating aPL reacting with β2GPI and/or with “pure” anionic PL (whose binding is independent of any plasma cofactor) and that such antibodies affect trophoblast differentiation–related activities.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/1529-0131%28200001%2943%3A1%3C140%3A%3AAID-ANR18%3E3.0.CO%3B2-P

ANTIPHOSPHOLIPID ANTIBODIES AFFECT TROPHOBLAST GONADOTROPIN SECRETION AND INVASIVENESS BY BINDING DIRECTLY AND THROUGH ADHERED β2-GLYCOPROTEIN I

Objective

To clarify the association of autoantibodies against prothrombin with the clinical manifestations of the antiphospholipid syndrome (APS) and with the presence of lupus anticoagulant (LAC).



Methods

We examined 265 patients who visited our autoimmune disease clinic. IgG and IgM antiprothrombin antibodies were tested by enzyme-linked immunosorbent assay (ELISA) as either antiphosphatidylserine–prothrombin complex (aPS/PT) antibodies or as antibodies against prothrombin coated on irradiated ELISA plates (as antigen) (aPT). IgG, IgM, and IgA anticardiolipin (aCL) antibodies and their β2-glycoprotein I (β2GPI) dependency were also evaluated by ELISA. LAC was tested by 3 different methods.



Results

The presence of aPS/PT, but not of aPT, significantly correlated with the clinical manifestations of APS (odds ratio [OR] 4.39, 95% confidence interval [95% CI] 2.06–9.38), and aPS/PT antibodies were as specific as β2GPI-dependent aCL for APS (93.1% for both). IgG aPS/PT strongly correlated with the presence of LAC as detected using the dilute Russell viper venom time test (OR 38.2, 95% CI 13.4–109.1).



Conclusion

Antiprothrombin antibodies are heterogeneous and their clinical relevance depends on the method of detection applied. Positive results on the aPS/PT test can serve as a marker of thrombotic events in patients with autoimmune diseases.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/1529-0131%28200009%2943%3A9%3C1982%3A%3AAID-ANR9%3E3.0.CO%3B2-2

Association of autoantibodies against the phosphatidylserine-prothrombin complex with manifestations of the antiphospholipid syndrome and with the presence of lupus anticoagulant.

The antiphospholipid syndrome (APS) is characterised by both arterial and venous thrombosis, recurrent pregnancy loss and thrombocytopaenia in association with antiphospholipid antibodies (aPL). To explore further the pathogenesis of thrombosis in APS, we evaluated the behaviour of tissue factor (TF) pathway in patients with APS. Plasma antigen levels of soluble TF and tissue factor pathway inhibitor (TFPI), a physiological regulator of TF dependent coagulation activation, were measured in 57 APS patients (36 primary and 21 secondary to systemic lupus erythematosus). Significantly elevated levels of both TF and TFPI were found in APS patients compared with 25 healthy controls (279 +/- 15 vs. 217 +/- 17 pg/ml, p = 0.01; 56.24 +/- 2.00 vs. 47.92 +/- 2.22 ng/ml, p = 0.01, respectively), suggesting in vivo upregulation of TF pathway in patients with APS. By flow-cytometry, monocytes from a healthy donor displayed higher TF antigen expression when incubated in the presence of APS plasmas than in control plasmas (24.23 +/- 3.11 vs. 12.78 +/- 1.57%, p = 0.002). Peripheral blood mononuclear cells (PBMC) also expressed more procoagulant activity (PCA) when incubated in the presence of APS plasmas than in control plasmas (1.80 +/- 0.12 vs. 1.35 +/- 0.054, p = 0.001) implying that TF up-regulation in APS was reproducible in vitro. Human monoclonal anticardiolipin antibodies induced PCA on PBMC and also TF mRNA on both PBMC and human umbilical vein endothelial cells shown by reverse-transcription polymerase chain reaction. These data strongly suggest that the TF pathway is implicated in the pathogenesis of aPL related thrombosis.

The role of the tissue factor pathway in the hypercoagulable state in patients with the antiphospholipid syndrome

Objective To elucidate the specificity of anticardiolipin antibodies (aCL) from patients with the antiphospholipid syndrome (APS) to various phospholipids (PLs), DNA, and beta 2-glycoprotein I (beta 2-GPI). Methods Five monoclonal aCL were established from peripheral blood lymphocytes of 3 patients with the APS. The reactivity of monoclonal aCL with various PLs, with DNA, and with beta 2-GPI was examined by enzyme-linked immunosorbent assay (ELISA). Results All of the monoclonal aCL bound to anionic PLs, only in the presence of beta 2-GPI. Neither monoclonal aCL nor beta 2-GPI bound to DNA. Monoclonal aCL bound to solid-phase beta 2-GPI on polystyrene ELISA plates that had carboxyl groups on their surface, but did not react with solid-phase beta 2-GPI on ordinary polystyrene plates. A mixture of beta 2-GPI and CL inhibited the binding of monoclonal aCL to beta 2-GPI, but CL or beta 2-GPI alone did not. Conclusion Monoclonal aCL may recognize a cryptic epitope, which appears as a result of beta 2-GPI binding to anionic PLs or to polystyrene with carboxyl groups.

Kenji Ichikawa

Papers

beta 2-Glycoprotein I reactivity of monoclonal anticardiolipin antibodies from patients with the antiphospholipid syndrome