Complement activation and regulation in preeclamptic placenta

Abstract

Preeclampsia (PE) is a common disorder of pregnancy originating in the placenta. We examined whether excessive activation or poor regulation of the complement system at the maternal-fetal interface could contribute to the development of PE. Location and occurrence of complement components and regulators in placentae were analyzed. Cryostat sections of placentae were processed from 7 early-onset PE (diagnosis <34 weeks of gestation), 5 late-onset PE, 10 control pregnancies, and immunostained for 6 complement activators and 6 inhibitors. Fluorescence was quantified and compared between PE and control placentae. Gene copy numbers of complement components C4A and C4B were assessed by a quantitative PCR method. Maternal C4 deficiencies (≥1 missing or non-functional C4) were most common in the early-onset PE group (71%), and more frequent in late-onset PE compared to healthy controls (60 vs. 38%). Complement C1q deposition differed significantly between control and patient groups: controls and early-onset PE patients had more C1q than late-onset PE patients (mean p = 0.01 and p = 0.005, respectively). C3 activation was analyzed by staining for C3b/iC3b and C3d. C3d was mostly specific to the basal syncytium and C3b/iC3b diffuse in other structures, but there were no clear differences between the study groups. Activated C4 and membrane-bound regulators CD55, CD46, and CD59 were observed abundantly in the syncytiotrophoblast. Syncytial knots, structures enriched in PE, stained specifically for the classical pathway inhibitor C4bp, whereas the key regulator alternative pathway, factor H (FH) showed a wider distribution in the placenta. Differences in C1q deposition between late- and early-onset PE groups may be indicative of the different etiology of PE symptoms in these patients. Irregular distribution of the complement regulators C4bp and FH in the PE placenta and a higher frequency of C4A deficiencies suggest a disturbed balance between complement activation and regulation in PE.

Keywords: complement; immunohistochemistry; innate immunity; placenta; preeclampsia; pregnancy.

Figure 1

A model of innate immunity incompatibility between maternal and fetal cells in preeclampsia and the maternal immune system. Failure of complement regulation on fetal tissue or excessive activation of the maternal complement system could result in complement attack against 1) invading trophoblast cells or 2) placental syncytiotrophoblast that represent the discordant interfaces. Accordingly, an imbalance between complement activation and regulation could contribute to the pathogenesis of preeclampsia. Specific foci for complement to attach could include syncytial bodies (apoptotic syncytial knots and syncytial sprouts), which are observed more often in preeclamptic placentae than in healthy controls.

Figure 2

High-intensity analysis workflow of C4bp staining of an early-onset preeclamptic placenta using ImageJ 1.46 software. The image is processed through steps (A–D) to produce a quantification of the high-intensity fluorescence areas, which correspond to the structures where (C) deposition/expression is most conspicuous. (A) The original image. (B) Black and white rendering of the image in (A). (C) Threshold set at 75% positive fluorescence (calculated to be value 77 for this image). (D) Area (in pixels) of positive signal (in green), % area is given in output and compared across and between the patient groups.

Figure 3

Summary of expression patterns of complement components in the placenta. Our findings are highlighted in black arrows (PE: preeclampsia, LO-PE: late-onset preeclampsia). Pictured are the components that we studied in their respective position in the activation cascade (green solid lines and shapes) or regulatory network (red dotted lines and shapes). The C system is composed of approximately 20 plasma proteins, which can be activated in a stepwise cascade via the classical (CP, via Ag-Ig: immunocomplexes or CRP: C-reactive protein), lectin (LP), or the alternative pathway (AP). In addition, there are approximately 15 components that act as receptors or protective molecules on cell membranes. All three pathways result in the activation of the main complement component C3 and thereafter of the terminal pathway causing the formation of membrane attack complexes (MACs) and ultimately target cell damage. C5b (not studied, in gray) is the activated component at the onset of terminal pathway. FH is a regulator of the alternative pathway, where its main role is to act as a cofactor for Factor I in the cleavage of C3b into iC3b. Similarly in the classical pathway, cleavage of C4 to activated form C4b is inhibited by a potent regulator C4bp. C3b and C4b are both generated by activation of not only the classical, but of the lectin pathway as well. C1q is a potent activator of the classical pathway. Binding of the complex (C1qr₂s₂) activates the C4 step. Together with decay accelerating factor (DAF; CD55) and membrane cofactor protein (MCP, CD46), C4bp regulates the progression of the classical C pathway by controlling the formation and function of the classical pathway C3 convertase, C4b2a. Like FH, MCP can also act as a cofactor in C3b inactivation. In the classical pathway, DAF accelerates the disassembly of C4b2a and in the alternative pathway that of C3bBb. DAF is a glycosylphosphatidylinositol-anchored membrane molecule. Complement receptor type 1(CR1, CD35) is a membrane-bound regulator expressed primarily by bone-marrow derived cells.

Figure 4

Side-by-side comparison of C1q, C9, and s-Eng staining of the same individual placentas. C1q is absent from large villi of the late-onset PE control. (B) There is a negative correlation between high-intensity areas of C1q and C9. s-Endoglin is deposited on the syncytiotrophoblast of PE placentae (I,J) and absent from the control placenta (K). Top row (A,E,I): early-onset PE. Middle top row (B,F,J): late-onset PE. Middle bottom row (C,G,K): control. Bottom row (D,H,L): negative control (antibody I omitted). 20× magnification.

Figure 5

Side-by-side comparison of C4 and C4bp stainings in the same placental samples. C4 is deposited in syncytial knots, visible as bright clusters but also in the syncytia of selected villi. C4bp is observed in syncytial bodies. C4bp is not observed in a circumferential pattern of the syncytium. C4 and C4bp are both observed in fibrinoid structures [here in control panels (G,C)]. Top row (A,E): early-onset PE. Middle top row (B,F): late-onset PE. Middle bottom row (C,G): control. Bottom row (D,H): negative control (antibody I omitted). 20× magnification.

Figure 6

Side-by-side comparison of C3b/iC3b, C3d, and FH staining patterns in the same placentas. C3b/iC3b and FH are observed in stromas of large stem villi [early-onset preeclampsia (PE): (A,I) and control: (C)] and in fibrinoid structures (J). C3d is typically observed in circumferential pattern of basal membrane of the syncytium (E–G). The circumferential pattern is often interrupted in PE (E,F) where physical damage to the syncytium causes increased rate of syncytial shedding. Top row (A,E,I): early-onset PE. Middle top row (B,F,J): late-onset PE. Middle bottom row (C,G,K): control. Bottom row (D,H,L): negative control (antibody I omitted). 20× magnification.