CD39 and CD73 activity are protective in a mouse model of antiphospholipid antibody-induced miscarriages
Introduction
Antiphospholipid syndrome (APS) is an autoimmune disorder in which antiphospholipid antibodies (aPL-ab) are implicated in the development of venous and/or arterial thrombosis and pregnancy morbidity. It affects 0.5–1% of the population worldwide [1]. APS may coexist with other autoimmune diseases including systemic lupus erythematosus (SLE), or may present as the sole clinical syndrome known as primary APS. aPL-ab, which include lupus anticoagulant, moderate to high titres of anticardiolipin (aCL) antibodies and anti-β2-glycoprotein I (β2GP1) antibodies, interfere with components of the coagulation cascade [2].
Serum aPL-ab are present in about 1–5% of healthy women, and in 15% of women with recurrent first trimester losses. Obstetric APS is defined by ≥ 3 unexplained spontaneous abortions before 10th week of gestation, ≥1 unexplained deaths of morphologically normal foetuses at or after the 10th week of gestation, or one or more premature births before the 34th week as a result of preeclampsia and/or placental insufficiency [3].
Inflammation, complement activation and leukocyte activation contribute to both the aetiology and pathophysiology of thrombotic and obstetric APS [2]. Specifically, aPL-ab enhance platelet aggregation and activation by blocking the interaction of β2GP1 with von Willebrand factor (vWF) and enhancing the expression of platelet membrane glycoprotein GPIIb/IIIa. Additionally, by interacting with β2GP1 expressed on the endothelial cell membrane, these antibodies create a pro-inflammatory and pro-coagulant endothelium by upregulating cellular adhesion molecules and promoting the secretion of pro-inflammatory cytokines such as interleukin (IL)-6 and tumour necrosis factor (TNF)-α. aPL-ab cause increased endothelial and monocytic expression of tissue factor (TF), the primary initiator of the clotting cascade [4]. IgG purified from APS patient sera increases TF activity on normal monocytes in vitro [5]. Complement activation by aPL-ab also promotes thrombosis by increasing TF expression through the binding of C5a and membrane attack complex to specific receptors on endothelial cells. Anti-C5 monoclonal antibodies prevented thrombophilia in aPL-ab treated mice, suggesting that C5a–C5a receptor interactions are critical mediators of aPL-ab-induced pregnancy morbidity [6]. Additionally, Rand et al. recently showed that APS patient plasma constituents induce complement activation [7]. Despite several mechanisms characterized for aPL-ab mediated pathology, not all carriers of aPL-ab develop clinical manifestations of APS [3]; indeed aPL-ab are not sufficient to initiate thrombosis in vivo, and for clotting to occur a “second hit” or a secondary pro-coagulant factor is required [8].
Human studies have demonstrated the presence of inflammation and complement in the placenta from APS-related foetal loss [9] and complement activation in plasma [10], [11]. Using passive transfer of APS sera from patients into pregnant mice, Girardi et al. demonstrated that inhibition of trophoblastic complement activation by heparin is an important mechanism that underpins the success of heparin therapy in the prevention of APS-miscarriages [12], [13]. APS sera also induce leukocyte-TF expression initiating superoxide generation in the leukocytes, which contributes to decidual inflammation and foetal resorption [14]. Thus there is considerable merit in the study of strategies that inhibit monocyte and endothelial cell activation, TF expression and complement activation for the management of APS-related sequelae of placental failure, as well as thrombosis at other sites.
Recent studies show that lipid peroxidation is a predictor for endothelial dysfunction in APS [15] and primary APS patients have increased plasma levels of F2-isoprostanes [16]. aPL-ab cross-react with oxidized low-density lipoprotein (LDL) causing oxidative stress within the placental vascular endothelium, which is another causative or contributing factor in placental insufficiency, pre-eclampsia, and foetal growth restriction. Lipid peroxidation of LDL results in formation of oxidized LDL (oxLDL) comprising a variety of highly reactive breakdown products of polyunsaturated fatty acid including malondialdehyde and 4-hydroxynonenal (4-HNE). oxLDL interacts with β2GPI to form oxLDL/β2GPI complexes. These complexes promote endothelial dysfunction and lead to atherothrombotic disease as well as serve as neo-epitopes for aPL-ab [17].
Extracellular purines (ATP, ADP, and adenosine) are important signalling molecules that mediate diverse cellular functions after binding to purine receptors on the cell surface. In particular, they have a profound impact on thrombotic and inflammatory processes. ATP and ADP are released by activated leukocytes, endothelial cells and platelets. The prominent effect of ATP on leukocytes is to stimulate chemotaxis, initiate inflammatory cytokine release and generate reactive oxygen species, all events serving to accelerate inflammatory injury. The most important role of ADP is to bind to the P2Y1 and P2Y12 receptors and mediate a positive feedback amplification of platelet aggregation initiated by several agonists [18].
Extracellular ATP/ADP levels are tightly regulated by CD39 and CD73. CD39 (NTPDase1) is a cell-membrane anchored enzyme expressed on the extracellular surface of endothelial cells and particularly in human vascular and placental trophoblastic tissues, wherein it modulates ATP-dependent trophoblastic functions [19]. It is also the primary nucleotide-inactivating enzyme on neutrophils, monocytes and some subsets of T- and B- lymphocytes [18]. CD39 hydrolyses ATP and ADP to AMP, which is then hydrolysed to adenosine by the ecto-5′-nucleotidase CD73 [20], [21]. Extracellular adenosine interacts with 4 types of adenosine receptors, A1, A2A, A2B, and A3, which are widely expressed G protein-coupled signal transducers. CD39 and CD73 are viewed as ‘immunological switches’ that shift ATP-driven pro-inflammatory immune cell activity toward an anti-inflammatory and antithrombotic state mediated by adenosine [22].
All four adenosine receptors are expressed by endothelial cells and syncytiotrophoblast cells of the placenta, and expression is augmented during pre-eclampsia [23]. Adenosine specifically protects from APS-mediated injury by suppressing the production of pro-inflammatory cytokines IL-12 and TNFα [24]. Adenosine has an anticoagulant function on monocytes and endothelial cells by suppressing TF expression via its interaction with receptors A2A and A3. Indeed, Dilazep, an adenosine uptake inhibitor, increases extracellular adenosine and attenuates aPL-ab induced TF expression on monocytes in vitro [5]. Here we utilised CD39-transgenic (Tg) and CD39-null mice as well as CD73-null mice in a model of APS-foetal loss to assess whether reduction in decidual CD39 or CD73 activity could serve as a potential “second hit” trigger for this disease.
Section snippets
Patient information and production of aPL-ab and normal human IgG
Informed consent was obtained before collecting blood from patients with APS and normal human subjects. 100 ml of blood was collected from each patient and allowed to clot at room temperature, following which serum was extracted by centrifugation at 1500g for 15 min at room temperature. Polyclonal IgG was purified from pooled sera of two healthy individuals and used as control. aPL-ab was obtained from three (2 female and 1 male) patients with APS characterized by thromboses (arterial or
Results
In this study we investigated the role of CD39 and CD73 in a mouse model of APS-induced miscarriage. We confirmed that CD39 and CD73 are expressed in decidua of placentae from WT BALB/c mice (Fig. 1a i and ii). Examination of the uteri of WT C57BL/6 mice on day 15 of pregnancy showed formation of 8–12 foetuses per mouse; saline administration did not result in any detectable miscarriages within this timeframe (not shown). Non-immune IgG administration to WT or CD39−/− C57BL/6 mice did not cause
Discussion
The current treatment protocol for APS comprising of heparin plus aspirin is effective in preventing only 54% of miscarriages [28]. Moreover, high intensity anticoagulation is associated with significant side effects of bleeding. Thus there is an unmet need to identify new pathways that modify this disease for better prognostication and identification of new therapeutic targets.
We demonstrated that both CD39 and CD73 are expressed in decidua of BALB/c mice (Fig. 1a). The CD39-CD73 axis promotes
Acknowledgements
This work was supported by NHMRC Project Grant 566705 (KD, PC and HN). We acknowledge assistance from the staff at Bioresources Centre, St. Vincent's Hospital for animal husbandry and Jelena Kezic of the Monash Histology platform for assistance with Histology. The authors acknowledge Monash Micro Imaging, Monash University, for the provision of instrumentation, training and technical support.
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