Complement activation has emerged as a common event in pregnancy loss, but the downstream mediators and effectors of placental and fetal damage induced by aPL remains unknown. Knowing that TF expression is a characteristic feature associated with aPL, we sought to investigate whether TF contributes to aPL-induced fetal loss in mice.
To test this hypothesis we used a mouse model of aPL-induced pregnancy loss.19,20
In this model, passive transfer of human and murine aPL induces fetal loss and growth restriction, thereby showing a direct pathogenic role for aPL.19,20
Mice that received aPL-IgG showed strong TF staining throughout the decidua and on embryonic debris. In contrast, mice treated with control IgG (NH-IgG) displayed weak TF staining, which was restricted to the ectoplacental cone region and intact embryo. Surprisingly, there was no increase in fibrin staining associated with increased TF staining in deciduas from aPL-treated mice.45
In addition, no thrombi were observed in deciduas from aPL-treated mice. The absence of fibrin deposition and thrombi suggests that TF-dependent activation of coagulation does not mediate aPL-induced pregnancy loss.
To assess the importance of TF in aPL-induced fetal injury, we inhibited TF with a monoclonal anti-mTF antibody 1H1.46
TF blockade prevented fetal death in aPL-treated mice (). The pregnancy outcomes in mice treated with both aPL-IgG and 1H1 mAb were comparable to that observed in mice receiving NHIgG alone. In deciduas from aPL-treated mice there was increased C3b deposition and neutrophil infiltration as we previously described.19,20
Blockade of TF with anti-TF mAb 1H1 diminished aPL-induced C3 deposition and neutrophil infiltration. The reduction in inflammation and fetal injury by anti-mTF antibody 1H1 shows that TF is a crucial effector molecule in aPL-induced pregnancy loss.
Figure 2 Blockade of TF with a monoclonal antibody in wild type mice or a genetic reduction of TF prevented aPL-induced pregnancy loss. (A) Mice that received aPL-IgG had a high frequency of fetal resorption than those that received normal human IgG (P < (more ...)
Similar levels of aPL were detected in plasma and deciduas from aPL+1H1 and aPL+rat IgG-treated mice showing that anti-TF 1H1 antibodies do not interfere with binding of aPL. As an alternative strategy to show that TF is required for aPL-induced fetal loss, we studied pregnancy outcomes in mice expressing low levels of TF.47
Low TF mice express very low levels of human TF (hTF) from a transgene in the absence of murine TF(mTF-/-
Low TF females were mated with low TF males (mTF-/-
). We found that low TF mice treated with aPL-IgG were protected from fetal loss compared with control mice (). Serum t1/2
and peak levels of aPL were comparable in low TF mice and control mice. The reduction of TF expression completely rescued embryos and diminished C3 deposition and neutrophil infiltration in deciduas from aPL-treated mice to the level of mice treated with aPL-IgG and anti-TF mAb. In addition, low TF females mated with wild type males were protected from aPL-induced pregnancy loss, showing that maternal TF is crucial for pathology in this model.
Some studies suggest that aPL induce a procoagulant response in endothelial cells and monocytes through interaction with toll-like receptor 4 (TLR4).48,49
Given these studies and the capacity of TLR4 signaling to induce TF expression on monocytes and endothelial cells, we examined the role of TLR4 in aPL-induced pregnancy loss and increased decidual TF expression. TLR4-/-
mice were not protected from aPL-induced pregnancy loss, and maternal-embryo units showed TF staining of deciduas and embryo destruction comparable to aPL-treated wild type mice.
We then studied whether aPL binding to other surface receptors may be sufficient to cause miscarriage. To examine the possibility that direct binding of aPL induce TF expression on trophoblasts, we used two different mouse monoclonal aPL that recognise phospholipids on trophoblast cells50
but differ in their Fc domain and thus differ in their complement activation capacity. FB1 is an IgG2b that can activate complement via the classical pathway, and FD1 is an IgG1 that can not activate complement. Pregnant mice treated with FB1 had a four-fold increase in fetal resorption frequency, which could be prevented by blocking complement activation with the C3 convertase inhibitor Crry-IgG. In contrast, FD1, which shows similar binding to deciduas, did not induce miscarriages. Immunohistochemical analysis of decidual tissue showed an increase in TF staining only in mice treated with FB1, which was markedly decreased by inhibiting complement. Minimal TF staining was present in mice treated with FD1 indicating that aPL binding to trophoblasts is not sufficient to induce TF expression, and that complement activation is required for aPL-induced TF increase in deciduas.
Knowing that TF expression is dependent on complement activation and neutrophils, we were able to show that TF expression in neutrophils is a result of C5a-C5aR interaction.
Experiments performed in TFfloxed/floxed
/LysMCre mice that do not express TF on myeloid cells allowed us to distinguish the role of trophoblasts TF from that of myeloid cells TF. The protection from aPL-induced pregnancy loss observed in these mice emphasises the crucial role of TF in maternal myeloid cells.45
Moreover, knowing that monocytes are not required for aPL-induced pregnancy loss and that neutrophils from aPL-treated TFfloxed/floxed
/LysM-Cre mice do not express TF allow us to conclude that TF expression on maternal neutrophils plays a causative and crucial role in aPL-induced fetal injury. TFfloxed/floxed
/LysMCre mice treated with aPL showed normal pregnancies and diminished decidual inflammation compared with wild type mice, suggesting that TF expression on neutrophils modulates the ability of the neutrophil to induce tissue injury. Indeed, neutrophils from TFfloxed/floxed
/LysMCre mice treated with aPL showed a lower generation of oxidants and less free radical-mediated lipid peroxidation in deciduas () than TFfloxed/floxed
control mice () that express TF, suggesting that TF modulates oxidative burst in neutrophils.45
Figure 3 TF expression by myeloids cells but not fetal-derived cells contributes to aPL-induced decidual oxidative stress and fetal loss. (A) Treatment with aPL-IgG caused an increase in fetal resorptions in TFfloxed/floxed mice (*P < 0.001 versus NH-IgG). (more ...)
Anticoagulants hirudin and fondaparinux did not prevent pregnancy loss in this mouse model of APS,51
suggesting that anticoagulation is not sufficient to prevent miscarriages. These anticoagulants inhibit coagulation at the level of thrombin and FXa, respectively, but they do not inhibit the formation of the TF:FVIIa and TF:FVIIa:FXa complexes. These complexes can activate G-protein-coupled PARs and induce inflammation.3
Agonists of PARs, notably of PAR-2, induce inflammation in many diseases associated with neutrophil infiltration and alterations in epithelial permeability.1,3,52
Therefore, TF-mediated signaling through PARs may promote inflammation leading to trophoblast injury and pregnancy loss.
Collectively, our study shows that maternal TF on neutrophils is essential in the pathogenesis of aPL-induced fetal loss and shows a functional linkage between complement components, TF and neutrophils. We propose that after aPL-IgG binding to trophoblasts, complement activation occurs leading to generation of the anaphylatoxin C5a, which attracts and activates neutrophils through C5aR (). Trophoblasts do not express complement receptors. C5a-C5aR interaction on neutrophils results in TF expression. Activated neutrophils release reactive oxygen species and proteolytic enzymes leading to decidual damage. TF expression on neutrophils appears to lead to the formation of complexes that amplify inflammation, cell injury and fetal death. The identification of TF, acting as an important pro-inflammatory mediator in aPL-induced fetal injury, provides a new target for therapy to prevent pregnancy loss in the APS.
Figure 4 Mechanisms of aPL-induced TF increase and fetal death. aPL are preferentially targeted to the placenta where they activate complement leading to the generation of potent anaphylatoxin C5a. C5a attracts and activates neutrophils. As a result of C5a-C5aR (more ...)