Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome

Abstract

Antiphospholipid syndrome (APS) is defined by recurrent pregnancy loss and thrombosis in the presence of antiphospholipid (aPL) Ab's. Currently, therapy for pregnant women with APS is focused on preventing thrombosis, but anticoagulation is only partially successful in averting miscarriage. We hypothesized that complement activation is a central mechanism of pregnancy loss in APS and tested this in a model in which pregnant mice receive human IgG containing aPL Ab's. Here we identify complement component C5 (and particularly its cleavage product C5a) and neutrophils as key mediators of fetal injury, and we show that Ab's or peptides that block C5a-C5a receptor interactions prevent pregnancy complications. The fact that F(ab)'2 fragments of aPL Ab's do not mediate fetal injury and that C4-deficient mice are protected from fetal injury suggests that activation of the complement cascade is initiated via the classical pathway. Studies in factor B-deficient mice, however, indicate that alternative pathway activation is required and amplifies complement activation. In contrast, activating Fc gamma Rs do not play an important role in mediating aPL Ab-induced fetal injury. Our findings identify the key innate immune effectors engaged by pathogenic autoantibodies that mediate poor pregnancy outcomes in APS and provide novel and important targets for prevention of pregnancy loss in APS.

Figures

Figure 1

Activating FcγRs are not required for aPL Ab–mediated pregnancy loss. Pregnant FcRγ+/+ and FcRγ–/– mice were treated with IgG from a healthy non-autoimmune individual (NH-IgG), three different patients with APS (aPL-IgG1, aPL-IgG2, aPL-IgG3), F(ab)′2 fragments from a pool of aPL-IgG from patients 2 and 3 [aPL-F(ab)′2], or human monoclonal aPL Ab (aPL mAb) on days 8 and 12 of pregnancy. Mice were sacrificed on day 15 of pregnancy, fetuses were weighed, and frequency of fetal resorption calculated (n = 4–7 mice/group). (a) Treatment with all intact aPL-IgG preparations and aPL mAb caused an increase in fetal resorptions in FcRγ+/+. *P < 0.05 versus NH-IgG, Student’s t test. Administration of aPL-F(ab)′2 did not affect pregnancy outcome. FcRγ–/– mice were not protected from fetal loss induced by intact aPL-IgG. *P < 0.05 versus NH-IgG, Student’s t test). In surviving fetuses from FcRγ –/– mice there was 36% decrease in weight. (b–e) Immunohistochemical analysis of decidual tissue from day 8 of pregnancy. Sections were stained with goat anti-human IgG, the chromogen was DAB (brown), and the counterstain was hematoxylin. Human IgG was deposited in deciduas from FcRγ+/+ mice within 60 minutes of administration of aPL-IgG (b) or aPL-F(ab)′2 (c), whereas no IgG was detected in deciduas from FcRγ+/+ mice treated with NH-IgG (d). Deposition of human IgG was similar after treatment with aPL-IgG in FcRγ–/– (e) and FcRγ+/+ mice (b). Data are representative of observations from three to six decidua from mice in each experimental group. Original magnification was ×200.

Figure 2

C4 or C5 deficiency prevents aPL Ab–induced fetal loss and growth restriction. (a) Pregnant C4+/+ and C4–/– mice were treated with aPL-IgG (aPL) (10 mg, intraperitoneally) or NH-IgG on days 8 and 12 of pregnancy, and fetal resorption frequencies were determined on day 15 (n = 5 mice/group). *P < 0.001, aPL versus control. (b–d) Pregnant C5+/+ and C5–/– mice were treated intraperitoneally with aPL-IgG (10 mg), monoclonal human aPL Ab (1 mg), monoclonal human anti-DNA (α-DNA; 1 mg), or their respective controls (NH-IgG or monoclonal human anti-rabies Ab, α-R) on days 8 and 12 of pregnancy. Fetal resorption frequencies and fetal weights were determined on day 15 of pregnancy (n = 5–11 mice/group). (b and c) C5–/– mice were protected from fetal loss (b) and growth restriction (c), whereas in the C5+/+ mice background strain aPL-IgG or aPL mAb caused pregnancy complications. *P < 0.01, aPL versus control. (d) Day 15 fetuses from C5–/– and C5+/+ mice treated with aPL-IgG. Scale bar: 1 cm.

Figure 3

C5 deficiency limits inflammation, necrosis, and activation of C3 by aPL Ab’s. Pregnant C5+/+ and C5–/– mice were treated with aPL-IgG (a, b, d, and e) or NH-IgG (c and f) as described in the legend to Figure 2, and immunohistochemical analysis was performed on decidual tissue from day 8 of pregnancy. (a–c) Detection of C3 in day-8 deciduas from aPL-IgG– and NH-IgG–treated mice. The deciduas were stained with anti-mouse C3, the chromogen was DAB (brown), and the counterstain was hematoxylin. Decidua from C5+/+ mice (a) had extensive C3 deposition (arrows), inflammatory cell infiltrates, and necrotic fetal debris, whereas embryos from C5–/– mice (b) treated with aPL-IgG appeared normal, and there was limited C3 deposition in deciduas at the maternal-fetal interface compared with that of C5+/+ treated with NH-IgG (c). Original magnification was ×50. (d–f) Detection of human IgG in deciduas. Sections were stained with goat anti-human IgG, the chromogen was DAB (brown), and the counterstain was hematoxylin. Within 60 minutes of administering aPL-IgG, human IgG was detectable in deciduas from C5+/+ mice (d) and C5–/– mice (e), whereas no IgG was detected in deciduas from C5+/+ mice treated with NH-IgG (f). Data are representative of observations from three to six mice in each experimental group. Original magnification was ×200.

Figure 4

Inhibition of C5 activation with anti-C5 mAb prevents aPL Ab–induced pregnancy complications. (a and b) Pregnant BALB/c mice were treated with aPL-IgG (10 mg, intraperitoneally) or NH-IgG (10 mg, intraperitoneally) at days 8 and 12 of pregnancy. Mice also received either anti-C5 mAb (1 mg, intraperitoneally) or control murine IgG (Ctrl-mAb; 1 mg, intraperitoneally) on days 8 and 10 (n = 5–11 mice/group). Pregnancies were assessed as described in the legend for Figure 1. Administration of anti-C5 mAb prevented fetal resorption (a) and growth restriction (b). *P < 0.05 versus NH-IgG plus Ctrl-mAb.

Figure 5

Blockade of C5a-C5aR interaction protects pregnancies from aPL Ab–associated injury. (a and b) Pregnant BALB/c mice were given aPL-IgG (10 mg, intraperitoneally) or NH-IgG (10 mg, intraperitoneally) on days 8 and 12, and some also received C5aR antagonist peptide (C5aR-AP) (50 μg, intraperitoneally) on day 8, 30 minutes before administration of aPL-IgG (n = 5–11 mice/group). Pregnancy outcomes were assessed as described in the legend for Figure 1. Treatment with C5aR-AP prevented fetal loss and growth inhibition. #P < 0.01, aPL versus aPL plus C5aR-AP. (b) Uteri from day 15 of pregnancy. There are two small amnion sacs and five resorptions (*) in the uterus from an aPL-IgG–treated mouse, while the uterus of a mouse that received aPL-IgG along with C5aR-AP contained normal size amnionic sacs and no resorptions, similar to that from a mouse treated with NH-IgG. Data are representative of observations in five to seven mice in each experimental group. Scale bar: 1 cm. (c) The effects of effects of aPL-IgG on pregnancy outcomes in C5aR–/– and C5aR+/+ mice were compared (n = 5–11 mice/group). Pregnant mice were treated with aPL-IgG or NH-IgG as described above. C5aR–/– mice were protected from aPL-IgG–induced fetal resorption and growth inhibition. †P < 0.05, aPL-IgG versus NH-IgG.

Figure 6

Neutrophil depletion protects mice from aPL Ab–induced pregnancy complications and limits C3 deposition. BALB/c mice received anti-mouse granulocyte RB6-8C5 mAb (anti-Gr) (100 μg, intraperitoneally) or IgG2b isotype control mAb on day 7 of pregnancy. On days 8 and 12, mice were treated with aPL-IgG or NH-IgG (n = 5–11 mice/group). (a and b) Neutrophil depletion protected mice from (a) fetal resorption (*P < 0.01, aPL-IgG plus anti-Gr versus aPL-IgG plus IgG2b) and (b) growth restriction (*P < 0.01, aPL-IgG plus anti-Gr versus aPL-IgG plus IgG2b). (c and d) Histologic sections of deciduas from day 8 of pregnancy were stained with H&E. In deciduas from mice treated with anti-Gr plus aPL-IgG (c) there were intact embryos (E) and no inflammatory infiltrates, while in deciduas from mice treated with aPL-IgG plus IgG2b (d) there was extensive neutrophilic infiltration (stained with anti-Gr shown in e) surrounding embryonic debris (ED). Original magnification was ×400. (e) Immunohistochemistry to detect infiltrating granulocytes in decidua from an aPL-IgG plus IgG2b–treated mouse. Original magnification was ×1,000. (f and g) Immunohistochemistry for C3 deposition in decidual tissue. Staining for C3 (arrows) was less intense and limited to the fetal-maternal interface in deciduas from mice that had received anti-Gr before treatment with aPL-IgG (f), compared with that of mice treated with aPL-IgG plus IgG2b (g). In the presence of infiltrating neutrophils, C3 deposits were present throughout decidual tissue (g), with particularly intense staining at the fetal-maternal interface surrounding the necrotic residual embryonic debris (arrows). Original magnification was 0400.

Figure 7

The absence of fB protects mice from aPL Ab–induced fetal loss and extensive C3 deposition within deciduas. fB+/+ and fB–/– mice were treated with aPL-IgG (10 mg, intraperitoneally) or NH-IgG (10 mg, intraperitoneally) on days 8 and 12 of pregnancy. Fetal resorption frequencies and fetal weights were determined on day 15 of pregnancy (n = 4–8 mice/group). (a and b) In contrast to fB+/+ mice, those deficient in fB were protected from fetal resorption (*P < 0.05, fB+/+ aPL-IgG versus NH-IgG) and growth restriction (P < 0.001, fB+/+ aPL-IgG versus NH-IgG). (c–e) Immunohistochemistry for C3 deposition in decidual tissue from day 8 of pregnancy following aPL-IgG administration. In deciduas from fB+/+ mice treated with NH-IgG (c), there was minimal C3 deposition and an intact embryo (E). In fB+/+ mice treated with aPL-IgG (d), C3 deposits were present throughout decidual tissue surrounding the necrotic residual embryonic debris (arrows). In contrast, in fB–/– mice treated with aPL-IgG (e), C3 deposition was limited (arrows) and the embryos remained intact (E). (f) Detection of C3 by Western blotting. Lysates from deciduas of fB+/+ mice and fB–/– mice were resolved by electrophoresis and blotted with anti-murine C3 Ab. C3 deposition was greater in deciduas from aPL-IgG–treated fB+/+ mice than in fB–/– mice, as evidenced by the presence of the cleaved C3-α′ chain.

Figure 8

Mechanism of aPL Ab–induced fetal damage. APL Ab’s are preferentially targeted to the placenta where they activate complement via the classical pathway leading to the generation of potent anaphylatoxins and mediators of effector cell activation, particularly C5a. C5a attracts and activates neutrophils, monocytes, and platelets and stimulates the release of inflammatory mediators, including reactive oxidants, proteolytic enzymes, chemokines, cytokines, and complement factors C3 and properdin. Secretion of C3 and properdin by neutrophils, as well as the presence of apoptotic and necrotic decidual tissue, may accelerate alternative pathway activation (dashed line), creating a proinflammatory amplification loop at sites of leukocyte infiltration that enhances C3 activation and deposition and generates additional C5a. This results in further influx of neutrophils, inflammation within the placenta, and, ultimately, fetal injury. Depending on the extent of damage, either death in utero or fetal growth restriction ensues.PMN, neutrophil; Mθ, monocyte/macrophage.