- Email: [email protected]
Decreased expression of complement 3a receptor (C3aR) in human placentas from severe preeclamptic pregnancies
European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Decreased expression of complement 3a receptor (C3aR) in human placentas from severe preeclamptic pregnancies Ratana Lim a,b, Martha Lappas a,b,* a b
Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 March 2012 Received in revised form 11 July 2012 Accepted 1 August 2012
Objectives: The aim of this study was to determine the expression of the anaphylatoxin receptors complement C3a receptor (C3aR) and C5a receptor (C5aR) in the placentas of pregnancies complicated by severe early onset preeclampsia. Study design: We recruited women with pregnancies complicated by severe early-onset preeclampsia (n = 19, 11 of which were further complicated with IUGR) and women with preterm pregnancies not affected by preeclampsia (n = 8). Gene and protein expression of C3aR and C5aR was analysed by quantitative RT-PCR and Western blotting, respectively. Results: C3aR was detected in the Hofbauer cells in the villous stroma of the placenta. C5aR staining was detected in the syncytiotrophoblast and endothelial cells. We found significantly decreased expression of C3aR mRNA and protein expression in placentas with preeclampsia compared to controls. However, C5aR expression was not significantly different between preeclamptic and control placentas at either the mRNA or protein level. Conclusions: Decreased C3aR expression indicates a dysregulation of the complement system in the placentas of preeclamptic women. Further studies would elucidate the exact mechanisms that complement has in preeclampsia. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: C3aR C5aR Placenta Preeclampsia
1. Introduction There is an important role of innate immunity and inflammation in the pathogenesis of complications of pregnancy. In preeclampsia, excessive inflammation is central to hypotheses that attempt to explain its aetiology; inflammation may enhance the pathology induced by the imbalance in angiogenic factors [1,2]. The complement pathway is involved in production of inflammatory cytokines, chemokines and angiogenic factors [3–8] associated with adverse pregnancy outcomes. Complement is one of the major first lines of defence in innate immunity and recognizes microbes and unwanted host molecules to augment phagocytosis and clearance [9], a process tightly controlled to prevent immunopathology. Complement is a system comprising over 35 proteins, participating in three main pathways of activation: the classical, the lectin and the alternative pathway, which can be stimulated by various triggers [9]. Complement activation in turn activates pro-inflammatory mediators, generates anaphylactic peptides, recruits effector cells and induces effector
* Corresponding author at: Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg 3084, Victoria, Australia. Tel.: +61 3 8458 4370; fax: +61 3 8458 4380. E-mail address: [email protected] (M. Lappas). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.08.003
responses [10,11]. Two known effectors generated by complement activation are the anaphylatoxins C3a and C5a, small peptide fragments released by the cleavage of C3 and C5, named after their role in the induction of anaphylaxis [12]. These fragments can enhance vascular permeability and induce smooth muscle contraction [13], and attract leukocytes [14]. Abnormal or excessive production of anaphylatoxins has been implicated in inflammatory disease, including pregnancy loss [15] and preterm labour [16]. Women with preeclampsia have significantly higher levels of circulating C3a and/or C5a than healthy pregnant women [17–20]. In a recent report, elevated C3a, as early as the first trimester of pregnancy, was found in women who developed hypertensive diseases of pregnancy [21]. Cord plasma or amniotic fluid concentrations of C3a and C5a in preeclampsia have not been reported. The actions of C3a and C5a are elicited via their receptors, C3aR and C5aR, respectively. C3aR and C5aR are widely distributed in many tissue types [22,23], where they have been associated with innate immunity and inflammation [24,25]. Their localization in human placenta and their expression in placental pathologies, however, are not known. Thus, the aims of this paper are to determine the localization of C3aR and C5aR in the placenta and to examine whether there is differential expression of C3aR and C5aR in pregnancies complicated by preeclampsia.
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
195
2. Materials and methods
2.5. Western blotting
2.1. Study population
Western blotting was performed according to our previously published studies [26]. Mouse monoclonal anti-C3aR and mouse monoclonal anti-C5aR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at 1 mg/ml for 24 h. Protein expression was identified by comparison with the mobility of protein standard and using two cell lines known two express C3aR and C5aR (data not shown). Membranes were viewed and analysed using the ChemiDoc system (Bio-Rad). Quantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad). Data were corrected for background, and expressed as peak density, normalized with bactin expression.
The Research Ethics Committee of the Mercy Hospital for Women approved this study. Written, informed consent was obtained from all participating women. Placenta was collected at caesarean section prior to labour onset (to account for any effects of labour on the levels of the endpoints) and from singleton gestations. Placenta was obtained from normotensive preterm pregnancies (n = 8) and those complicated by severe early-onset preeclampsia (n = 19). Severe early-onset preeclampsia was defined as preeclampsia prior to 34 weeks’ gestational age in association with either blood pressure greater than 160 mmHg systolic, 110 mmHg diastolic or proteinuria greater than 5 g per day. HELLP (haemolysis, elevated liver enzymes and low platelets) syndrome was defined as haematological evidence of haemolysis, thrombocytopenia and elevated liver enzymes. Babies were classified as IUGR when birthweight was below the 5th percentile for gestational age calculated using www.gestation.net, which uses the principles of the Gestation Related Optimal Weight (GROW) programme. All preeclampsia (n = 8) and preeclampsia-IUGR (n = 11) cases were matched to control patients who also delivered preterm (n = 8). Indications for preterm delivery (in the absence of preeclampsia) were prolonged pre-labour rupture of the foetal membranes (PROM), placenta praevia or antepartum haemorrhage (APH). All placentas collected from preterm pregnancies were swabbed for microbiological culture investigations and histopathological examination, and patients with chorioamnionitis were excluded from analyses. 2.2. Preparation of placental tissue Placental lobules (cotyledons) were obtained from various regions of the placenta. The basal plate and chorionic surface were removed from the cotyledon, and villous tissue was obtained from the middle cross-section. Placental tissue was blunt-dissected to remove visible connective tissue and calcium deposits. Tissue samples were fixed and paraffin embedded for immunohistochemical analysis, or snap frozen in liquid nitrogen and immediately stored at 80 8C for RNA and protein analysis. 2.3. Immunohistochemistry Immunohistochemistry was performed according to our previously published methods [26]. Mouse monoclonal antiC3aR was used at 4 mg/ml, and mouse monoclonal anti-C5aR was used at 2 mg/ml (purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA)). Positive controls, which were composite slides with tonsil, breast tumour and ovarian tumour, were included in each run. Negative control slides, where primary antibody was replaced with normal mouse IgG serum, were also included. 2.4. RNA extraction and quantitative RT-PCR (qRT-PCR) RNA extractions and qRT-PCR was performed according to our previously published methods [26]. We used pre-designed primers from Qiagen (QuantiTect Primer Assays, Qiagen, Germantown, MD, USA). The C3aR (catalogue number QT01676941) gene ID is 719. The C5aR (catalogue number QT00997766) gene ID is 728. Average gene CT values were normalized to GAPDH of same cDNA sample. Fold differences were determined using the comparative CT method.
2.6. Statistical analysis Statistical analyses were performed using a commercially available statistical software package (Statgraphics Plus version 3.1, Statistical Graphics Corp., Rockville, MD, USA). Two sample comparisons were analysed by Student’s t-test or Mann–Whitney (Wilcoxon) test. Statistical significance was ascribed to P value <0.05. Data were expressed as mean standard error of the mean (SEM). 3. Results 3.1. Expression and localization of C3aR and C5aR in human placenta The expression of C3aR and C5aR in placental sections was determined by immunohistochemistry. Human placenta was obtained at term caesarean section from normal uncomplicated pregnancies. C3aR was detected in the Hofbauer cells in the villous stroma of the placenta (Fig. 1A). No staining was apparent in syncytiotrophoblast or endothelial cells. C5aR staining was detected in the syncytiotrophoblast and endothelial cells, but not the villous stroma (Fig. 1B). No staining for C3aR or C5aR was seen in the negative control (Fig. 1C). 3.2. Expression of C3aR and C5aR in preeclamptic placenta We recruited 19 women with pregnancies complicated by severe early-onset preeclampsia and 8 women with preterm pregnancies not affected by preeclampsia. Baseline characteristics for these populations are outlined in Table 1. Placental weight, foetal birthweight and parity in the preeclamptic-IUGR cohort were significantly lower than the preterm controls. Of note, two of the patients were on aspirin due to previous preterm preeclamptic deliveries. We examined C3aR and C5aR expression in placentas obtained from preterm normotensive pregnancies (n = 8) and those complicated by severe preterm preeclampsia (delivery < 34 weeks gestational age) with (n = 11) and without (n = 8) IUGR. C3aR mRNA expression was significantly lower in the preeclampsia and preeclampsia-IUGR groups compared to the control preterm group (Fig. 2A). Additionally, by Western analysis, C3aR protein expression was significantly decreased in the preeclampsia and preeclampsia-IUGR groups compared to the preterm control group (Fig. 2B). No significant differences were detected, however, for C5aR mRNA (Fig. 2A) and C5aR protein expression (Fig. 2B) between groups. 4. Comment We have shown in human placenta a decrease in C3aR expression at both mRNA and protein levels in the preeclamptic
196
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
Fig. 1. Immunohistochemical localization of (A) C3aR and (B) C5aR in human placenta obtained at term Caesarean section from normal uncomplicated pregnancy. (A) C3aR staining was detected in the Hofbauer cells in the villous stroma of the placenta. No C3aR staining was detected in the syncytiotrophoblast and endothelial cells. (B) C5aR staining was detected in the syncytiotrophoblast and endothelial cells. There was no C5aR staining in the villous stroma. (C) No specific staining for C3aR or C5aR is seen in the negative control for placenta. Magnification 250. hc, Hofbauer cells; sy, syncytiotrophoblast cells; vs, villous stroma; ec, endothelial cells. Red arrows indicate no staining in cell types. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
group with and without IUGR compared to controls. On the other hand, there was no significant difference in C5aR mRNA and protein levels between the two groups. In human placenta, C3aR expression was present in the Hofbauer cells in the villous stroma but not in syncytiotrophoblast or endothelial cells. C5aR expression was present in the syncytiotrophoblast and endothelial cells. Our study is the first to describe C3aR and C5aR expression in the human placenta. Several membrane proteins have evolved to provide host cells with protection against the consequences of complement attack; decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 [27], all of which have been found in abundance in the human placenta [28,29]. Dysregulation of complement activation
has been linked to pathological events, including preeclampsia, which will be discussed below. The complement system is activated through the classical and/ or lectin pathways with increased terminal complex formation during normal pregnancy, and further in preeclampsia [17–20]. While C3a and C5a are both generated by complement activation, C3aR and C5aR use different signal transduction cascades [30]. While no other studies have described C3aR and C5aR in preeclampsia patients, there are data on other aspects of the complement pathway. Patients with preeclampsia have higher levels of plasma C3a, which is the ligand for C3aR [18–20]. Soto et al. reported that plasma C5a concentrations were increased in preeclamptic women, but there was no change in C3a or C4a
Table 1 Relevant characteristics of the preeclampsia study group.
Maternal age (years) Maternal BMI (kg/m2)* Gestational age (wks) Placental weight (g) Foetal birthweight (g) Gravida Parity Ethinicity – no. (%) Caucasian Asian African Middle Eastern Pacific Islander
Preterm controls (n = 8)
Preeclampsia (n = 8)
Preeclampsia-IUGR (n = 11)
27.6 (1.6) 27.4 (2.5) 29+5 (1.0) 437 (51) 1492 (204) 2.5 (0.4) 2.4 (0.3)
30.0 (2.2) 29.5 (3.6) 30+0 (1.0) 353 (32) 1310 (179) 2.1 (0.4) 1.6 (0.4)
37.6 (1.6) 32.4 (2.7) 30+0 (0.9) 279 (26)** 1040 (125)** 1.9 (0.3) 1.5 (0.2)**
5 1 0 1 1
(62.5) (12.5) (12.5) (12.5)
Values represent mean (SEM). * Based on first antenatal visit at approximately 12 weeks. ** P < 0.05 vs. preterm control group.
4 2 0 1 1
(50) (25) (0) (12.5) (12.5)
7 1 2 1 0
(64) (9) (18) (9) (0)
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
197
Fig. 2. (A) C3aR and C5aR mRNA expression in placenta from preterm control (n = 8) and severe preeclampsia with (n = 11) and without (n = 8) IUGR. Gene expression is displayed as mean SEM. (B) Western blot of C3aR and C5aR protein expression in human placenta from control (n = 8) and severe preeclampsia with (n = 11) and without (n = 8) IUGR. Data are displayed as the mean SEM. *P < 0.05 vs. preterm control.
concentrations [17]. Preeclamptic cases with foetal growth restriction are characterized by extreme reductions in placental perfusion [31]: increased release of apoptotic trophoblast debris into the maternal circulation may account for the increase in complement activation, or conversely, complement activation may contribute to the development of foetal growth restriction. In this study, we report a decrease in C3aR in placenta in our preeclamptic group. C3aR was localized to the Hofbauer cells in the villous stroma; Hofbauer cells are placental macrophages that are localized close to foetal vessels. Foetal synthesis of complement factors has been reported; C3a has been detected in amniotic fluid [18,32], cord plasma [33,34] and placenta [35]. Although there are no reports on the effect of preeclampsia on amniotic fluid, cord plasma or placental C3a levels, excessive complement activation has been documented in the placentas of abnormal pregnancies [36,37]. One of the major roles of the complement system is to dispose of immune complexes and the products of inflammatory injury. Thus, decreased C3aR may be indicative of an inability of preeclamptic placentas to clear the excess of activated complement, whereby immune complexes accumulate, inciting inflammation and disease ensues [38]. Of note, we found that C3aR was also decreased in preeclamptic pregnancies that were also associated with foetal growth restriction. Future studies are required to determine if IUGR alone is also associated with alteration in the expression of the complement receptors in the placenta. Recent studies in mice have helped us to gain a better understanding of complement pathways in preeclampsia. Preeclampsia is associated with vascular constriction, most probably due to endothelial dysfunction, and with proteinuria: renal dysfunction can also follow [39]. In mouse models of systemic lupus erythematosus (MRL strain), an autoimmune disease strongly associated with complement activation and deposition, C3aRdeficient MRL mice developed accelerated onset of renal disease and a trend towards decreased survival [40]. On the other hand, C5aR-deficient mice have attenuated disease compared to control MRL mice [41]. The complement component C1q plays a crucial role in placental development [42]; it was further shown that mice
deficient in C1q developed the characteristic features of preeclampsia [43], demonstrating the requirement of C1q on trophoblast cells for healthy pregnancy outcomes. Collectively, these data demonstrated both the importance and complexity of the complement pathway in preeclampsia. Further studies are required to elucidate the role of C3aR in placenta and its relevance to preeclampsia. In conclusion, preeclampsia is not associated with changes in C5aR gene and protein expression, but we report a decrease in C3aR mRNA and protein expression in preeclamptic placentas, with and without IUGR, when compared to gestational agematched controls. This decrease indicates some dysregulation in the complement system in the placentas of women with preeclampsia, but further studies are required to fully elucidate the underlying mechanisms. Conflict of interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding Dr Martha Lappas was a recipient of a National Health and Medical Research Council (NHMRC) RD Wright Fellowship (grant no. 454777). The work described in this manuscript was funded by a project grant from the NHMRC (grant no. 454310). Funding for ChemiDoc XRS and xMark Microplate Absorbance Spectrophotometer was provided by the Medical Research Foundation for Women and Babies. Acknowledgements The authors gratefully acknowledge the assistance of the Clinical Research Midwives Gabrielle Fleming, Astrid Tiefholz, Lyndall Paolucci, Debra Jinks, Asha Ferguson; and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their co-operation.
198
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
References [1] Ramma W, Ahmed A. Is inflammation the cause of pre-eclampsia? Biochemical Society Transactions 2011;39:1619–27. [2] Lockwood CJ, Huang SJ, Krikun G, et al. Decidual hemostasis, inflammation, and angiogenesis in pre-eclampsia. Seminars in Thrombosis and Hemostasis 2011;37:158–64. [3] Conroy A, Serghides L, Finney C, et al. C5a enhances dysregulated inflammatory and angiogenic responses to malaria in vitro: potential implications for placental malaria. PLoS One 2009;4:e4953. [4] Langer HF, Chung K-J, Orlova VV, et al. Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis. Blood 2010;116:4395–403. [5] Monsinjon T, Gasque P, Chan P, Ischenko A, Brady JJ, Fontaine M. Regulation by complement C3a and C5a anaphylatoxins of cytokine production in human umbilical vein endothelial cells. The FASEB Journal 2003;17:1003–14. [6] Kahr WH. Complement halts angiogenesis gone wild. Blood 2010;116:4393–4. [7] Fischer WH, Jagels MA, Hugli TE. Regulation of IL-6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg). Journal of Immunology 1999;162:453–9. [8] Zhang XH, Kimura Y, Fang CY, et al. Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood 2007;110:228–36. [9] Sjoberg AP, Trouw LA, Blom AM. Complement activation and inhibition: a delicate balance. Trends in Immunology 2009;30:83–90. [10] Zipfel PF, Skerka C. Complement regulators and inhibitory proteins. Nature Reviews Immunology 2009;9:729–40. [11] Carroll MC. The complement system in regulation of adaptive immunity. Nature Immunology 2004;5:981–6. [12] Sacks SH. Complement fragments C3a and C5a: the salt and pepper of the immune response. European Journal of Immunology 2010;40:668–70. [13] Cochrane CG, Muller-Eberhard HJ. The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. Journal of Experimental Medicine 1968;127:371–86. [14] Hartmann K, Henz BM, Kruger-Krasagakes S, et al. C3a and C5a stimulate chemotaxis of human mast cells. Blood 1997;89:2863–70. [15] Girardi G, Berman J, Redecha P, et al. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. Journal of Clinical Investigation 2003;112:1644–54. [16] Soto E, Romero R, Richani K, et al. Anaphylatoxins in preterm and term labor. Journal of Perinatal Medicine 2005;33:306–13. [17] Soto E, Romero R, Richani K, et al. Preeclampsia and pregnancies with smallfor-gestational age neonates have different profiles of complement split products. Journal of Maternal–Fetal and Neonatal Medicine 2010;23:646–57. [18] Haeger M, Bengtson A, Karlsson K, Heideman M. Complement activation and anaphylatoxin (C3a and C5a) formation in preeclampsia and by amniotic fluid. Obstetrics and Gynecology 1989;73:551–6. [19] Haeger M, Unander M, Bengtsson A. Complement activation in relation to development of preeclampsia. Obstetrics and Gynecology 1991;78:46–9. [20] Derzsy Z, Proha´szka Z, Rigo´ Jr J, Fu¨st G, Molvarec A. Activation of the complement system in normal pregnancy and preeclampsia. Molecular Immunology 2010;47:1500–6. [21] Lynch AM, Gibbs RS, Murphy JR, Giclas PC, Salmon JE, Holers VM. Early elevations of the complement activation fragment C3a and adverse pregnancy outcomes. Obstetrics and Gynecology 2011;117:75–83. [22] Ames RS, Li Y, Sarau HM, et al. Molecular cloning and characterization of the human anaphylatoxin C3a receptor. Journal of Biological Chemistry 1996;271:20231–34.
[23] Nataf S, Stahel PF, Davoust N, Barnum SR. Complement anaphylatoxin receptors on neurons: new tricks for old receptors? Trends in Neurosciences 1999;22:397–402. [24] Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nature Immunology 2010;11:785–97. [25] Ward PA. Functions of C5a receptors. Journal of Molecular Medicine (Berlin) 2009;87:375–8. [26] Lappas M, Mitton A, Lim R, Barker G, Riley C, Permezel M. SIRT1 is a novel regulator of key pathways of human labor. Biology of Reproduction 2011;84:167–78. [27] Morgan BP, Holmes CH. Immunology of reproduction: protecting the placenta. Current Biology 2000;10:R381–3. [28] Holmes CH, Simpson KL. Complement and pregnancy: new insights into the immunobiology of the fetomaternal relationship. Baillieres Clinical Obstetrics and Gynaecology 1992;6:439–60. [29] Holmes CH, Simpson KL, Wainwright SD, et al. Preferential expression of the complement regulatory protein decay accelerating factor at the fetomaternal interface during human pregnancy. Journal of Immunology 1990;144: 3099–105. [30] Schraufstatter IU, Trieu K, Sikora L, Sriramarao P, DiScipio R. Complement C3a and C5a induce different signal transduction cascades in endothelial cells. Journal of Immunology 2002;169:2102–10. [31] Roberts JM, Gammill HS. Preeclampsia: recent insights. Hypertension 2005;46:1243–9. [32] Soto E, Romero R, Richani K, et al. Evidence for complement activation in the amniotic fluid of women with spontaneous preterm labor and intra-amniotic infection. Journal of Maternal–Fetal and Neonatal Medicine 2009;22:983–92. [33] Lappas M. Lower circulating levels of complement split proteins C3a and C4a in maternal plasma of women with gestational diabetes mellitus. Diabetic Medicine 2011;28:906–11. [34] Sonntag J, Brandenburg U, Polzehl D, et al. Complement system in healthy term newborns: reference values in umbilical cord blood. Pediatric and Developmental Pathology 1998;1:131–5. [35] Kohler PF. Maturation of the human complement system. I. Onset time and sites of fetal C1q, C4, C3, and C5 synthesis. Journal of Clinical Investigation 1973;52:671–7. [36] Girardi G, Yarilin D, Thurman JM, Holers VM, Salmon JE. Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction. Journal of Experimental Medicine 2006;203:2165–75. [37] Holers VM, Girardi G, Mo L, et al. Complement C3 activation is required for antiphospholipid antibody-induced fetal loss. Journal of Experimental Medicine 2002;195:211–20. [38] Redman CW, Sargent IL. Placental stress and pre-eclampsia: a revised view. Placenta 2009;30(Suppl. A):S38–42. [39] Karumanchi SA, Maynard SE, Stillman IE, Epstein FH, Sukhatme VP. Preeclampsia: a renal perspective. Kidney International 2005;67:2101–13. [40] Wenderfer SE, Wang H, Ke B, Wetsel RA, Braun MC. C3a receptor deficiency accelerates the onset of renal injury in the MRL/lpr mouse. Molecular Immunology 2009;46:1397–404. [41] Wenderfer SE, Ke B, Hollmann TJ, Wetsel RA, Lan HY, Braun MC. C5a receptor deficiency attenuates T cell function and renal disease in MRLlpr mice. Journal of the American Society of Nephrology 2005;16:3572–82. [42] Agostinis C, Bulla R, Tripodo C, et al. An alternative role of C1q in cell migration and tissue remodeling: contribution to trophoblast invasion and placental development. Journal of Immunology 2010;185:4420–9. [43] Singh J, Ahmed A, Girardi G. Role of complement component C1q in the onset of preeclampsia in mice. Hypertension 2011;58:716–24.
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Decreased expression of complement 3a receptor (C3aR) in human placentas from severe preeclamptic pregnancies Ratana Lim a,b, Martha Lappas a,b,* a b
Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 March 2012 Received in revised form 11 July 2012 Accepted 1 August 2012
Objectives: The aim of this study was to determine the expression of the anaphylatoxin receptors complement C3a receptor (C3aR) and C5a receptor (C5aR) in the placentas of pregnancies complicated by severe early onset preeclampsia. Study design: We recruited women with pregnancies complicated by severe early-onset preeclampsia (n = 19, 11 of which were further complicated with IUGR) and women with preterm pregnancies not affected by preeclampsia (n = 8). Gene and protein expression of C3aR and C5aR was analysed by quantitative RT-PCR and Western blotting, respectively. Results: C3aR was detected in the Hofbauer cells in the villous stroma of the placenta. C5aR staining was detected in the syncytiotrophoblast and endothelial cells. We found significantly decreased expression of C3aR mRNA and protein expression in placentas with preeclampsia compared to controls. However, C5aR expression was not significantly different between preeclamptic and control placentas at either the mRNA or protein level. Conclusions: Decreased C3aR expression indicates a dysregulation of the complement system in the placentas of preeclamptic women. Further studies would elucidate the exact mechanisms that complement has in preeclampsia. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: C3aR C5aR Placenta Preeclampsia
1. Introduction There is an important role of innate immunity and inflammation in the pathogenesis of complications of pregnancy. In preeclampsia, excessive inflammation is central to hypotheses that attempt to explain its aetiology; inflammation may enhance the pathology induced by the imbalance in angiogenic factors [1,2]. The complement pathway is involved in production of inflammatory cytokines, chemokines and angiogenic factors [3–8] associated with adverse pregnancy outcomes. Complement is one of the major first lines of defence in innate immunity and recognizes microbes and unwanted host molecules to augment phagocytosis and clearance [9], a process tightly controlled to prevent immunopathology. Complement is a system comprising over 35 proteins, participating in three main pathways of activation: the classical, the lectin and the alternative pathway, which can be stimulated by various triggers [9]. Complement activation in turn activates pro-inflammatory mediators, generates anaphylactic peptides, recruits effector cells and induces effector
* Corresponding author at: Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg 3084, Victoria, Australia. Tel.: +61 3 8458 4370; fax: +61 3 8458 4380. E-mail address: [email protected] (M. Lappas). 0301-2115/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.08.003
responses [10,11]. Two known effectors generated by complement activation are the anaphylatoxins C3a and C5a, small peptide fragments released by the cleavage of C3 and C5, named after their role in the induction of anaphylaxis [12]. These fragments can enhance vascular permeability and induce smooth muscle contraction [13], and attract leukocytes [14]. Abnormal or excessive production of anaphylatoxins has been implicated in inflammatory disease, including pregnancy loss [15] and preterm labour [16]. Women with preeclampsia have significantly higher levels of circulating C3a and/or C5a than healthy pregnant women [17–20]. In a recent report, elevated C3a, as early as the first trimester of pregnancy, was found in women who developed hypertensive diseases of pregnancy [21]. Cord plasma or amniotic fluid concentrations of C3a and C5a in preeclampsia have not been reported. The actions of C3a and C5a are elicited via their receptors, C3aR and C5aR, respectively. C3aR and C5aR are widely distributed in many tissue types [22,23], where they have been associated with innate immunity and inflammation [24,25]. Their localization in human placenta and their expression in placental pathologies, however, are not known. Thus, the aims of this paper are to determine the localization of C3aR and C5aR in the placenta and to examine whether there is differential expression of C3aR and C5aR in pregnancies complicated by preeclampsia.
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
195
2. Materials and methods
2.5. Western blotting
2.1. Study population
Western blotting was performed according to our previously published studies [26]. Mouse monoclonal anti-C3aR and mouse monoclonal anti-C5aR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at 1 mg/ml for 24 h. Protein expression was identified by comparison with the mobility of protein standard and using two cell lines known two express C3aR and C5aR (data not shown). Membranes were viewed and analysed using the ChemiDoc system (Bio-Rad). Quantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad). Data were corrected for background, and expressed as peak density, normalized with bactin expression.
The Research Ethics Committee of the Mercy Hospital for Women approved this study. Written, informed consent was obtained from all participating women. Placenta was collected at caesarean section prior to labour onset (to account for any effects of labour on the levels of the endpoints) and from singleton gestations. Placenta was obtained from normotensive preterm pregnancies (n = 8) and those complicated by severe early-onset preeclampsia (n = 19). Severe early-onset preeclampsia was defined as preeclampsia prior to 34 weeks’ gestational age in association with either blood pressure greater than 160 mmHg systolic, 110 mmHg diastolic or proteinuria greater than 5 g per day. HELLP (haemolysis, elevated liver enzymes and low platelets) syndrome was defined as haematological evidence of haemolysis, thrombocytopenia and elevated liver enzymes. Babies were classified as IUGR when birthweight was below the 5th percentile for gestational age calculated using www.gestation.net, which uses the principles of the Gestation Related Optimal Weight (GROW) programme. All preeclampsia (n = 8) and preeclampsia-IUGR (n = 11) cases were matched to control patients who also delivered preterm (n = 8). Indications for preterm delivery (in the absence of preeclampsia) were prolonged pre-labour rupture of the foetal membranes (PROM), placenta praevia or antepartum haemorrhage (APH). All placentas collected from preterm pregnancies were swabbed for microbiological culture investigations and histopathological examination, and patients with chorioamnionitis were excluded from analyses. 2.2. Preparation of placental tissue Placental lobules (cotyledons) were obtained from various regions of the placenta. The basal plate and chorionic surface were removed from the cotyledon, and villous tissue was obtained from the middle cross-section. Placental tissue was blunt-dissected to remove visible connective tissue and calcium deposits. Tissue samples were fixed and paraffin embedded for immunohistochemical analysis, or snap frozen in liquid nitrogen and immediately stored at 80 8C for RNA and protein analysis. 2.3. Immunohistochemistry Immunohistochemistry was performed according to our previously published methods [26]. Mouse monoclonal antiC3aR was used at 4 mg/ml, and mouse monoclonal anti-C5aR was used at 2 mg/ml (purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA)). Positive controls, which were composite slides with tonsil, breast tumour and ovarian tumour, were included in each run. Negative control slides, where primary antibody was replaced with normal mouse IgG serum, were also included. 2.4. RNA extraction and quantitative RT-PCR (qRT-PCR) RNA extractions and qRT-PCR was performed according to our previously published methods [26]. We used pre-designed primers from Qiagen (QuantiTect Primer Assays, Qiagen, Germantown, MD, USA). The C3aR (catalogue number QT01676941) gene ID is 719. The C5aR (catalogue number QT00997766) gene ID is 728. Average gene CT values were normalized to GAPDH of same cDNA sample. Fold differences were determined using the comparative CT method.
2.6. Statistical analysis Statistical analyses were performed using a commercially available statistical software package (Statgraphics Plus version 3.1, Statistical Graphics Corp., Rockville, MD, USA). Two sample comparisons were analysed by Student’s t-test or Mann–Whitney (Wilcoxon) test. Statistical significance was ascribed to P value <0.05. Data were expressed as mean standard error of the mean (SEM). 3. Results 3.1. Expression and localization of C3aR and C5aR in human placenta The expression of C3aR and C5aR in placental sections was determined by immunohistochemistry. Human placenta was obtained at term caesarean section from normal uncomplicated pregnancies. C3aR was detected in the Hofbauer cells in the villous stroma of the placenta (Fig. 1A). No staining was apparent in syncytiotrophoblast or endothelial cells. C5aR staining was detected in the syncytiotrophoblast and endothelial cells, but not the villous stroma (Fig. 1B). No staining for C3aR or C5aR was seen in the negative control (Fig. 1C). 3.2. Expression of C3aR and C5aR in preeclamptic placenta We recruited 19 women with pregnancies complicated by severe early-onset preeclampsia and 8 women with preterm pregnancies not affected by preeclampsia. Baseline characteristics for these populations are outlined in Table 1. Placental weight, foetal birthweight and parity in the preeclamptic-IUGR cohort were significantly lower than the preterm controls. Of note, two of the patients were on aspirin due to previous preterm preeclamptic deliveries. We examined C3aR and C5aR expression in placentas obtained from preterm normotensive pregnancies (n = 8) and those complicated by severe preterm preeclampsia (delivery < 34 weeks gestational age) with (n = 11) and without (n = 8) IUGR. C3aR mRNA expression was significantly lower in the preeclampsia and preeclampsia-IUGR groups compared to the control preterm group (Fig. 2A). Additionally, by Western analysis, C3aR protein expression was significantly decreased in the preeclampsia and preeclampsia-IUGR groups compared to the preterm control group (Fig. 2B). No significant differences were detected, however, for C5aR mRNA (Fig. 2A) and C5aR protein expression (Fig. 2B) between groups. 4. Comment We have shown in human placenta a decrease in C3aR expression at both mRNA and protein levels in the preeclamptic
196
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
Fig. 1. Immunohistochemical localization of (A) C3aR and (B) C5aR in human placenta obtained at term Caesarean section from normal uncomplicated pregnancy. (A) C3aR staining was detected in the Hofbauer cells in the villous stroma of the placenta. No C3aR staining was detected in the syncytiotrophoblast and endothelial cells. (B) C5aR staining was detected in the syncytiotrophoblast and endothelial cells. There was no C5aR staining in the villous stroma. (C) No specific staining for C3aR or C5aR is seen in the negative control for placenta. Magnification 250. hc, Hofbauer cells; sy, syncytiotrophoblast cells; vs, villous stroma; ec, endothelial cells. Red arrows indicate no staining in cell types. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
group with and without IUGR compared to controls. On the other hand, there was no significant difference in C5aR mRNA and protein levels between the two groups. In human placenta, C3aR expression was present in the Hofbauer cells in the villous stroma but not in syncytiotrophoblast or endothelial cells. C5aR expression was present in the syncytiotrophoblast and endothelial cells. Our study is the first to describe C3aR and C5aR expression in the human placenta. Several membrane proteins have evolved to provide host cells with protection against the consequences of complement attack; decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 [27], all of which have been found in abundance in the human placenta [28,29]. Dysregulation of complement activation
has been linked to pathological events, including preeclampsia, which will be discussed below. The complement system is activated through the classical and/ or lectin pathways with increased terminal complex formation during normal pregnancy, and further in preeclampsia [17–20]. While C3a and C5a are both generated by complement activation, C3aR and C5aR use different signal transduction cascades [30]. While no other studies have described C3aR and C5aR in preeclampsia patients, there are data on other aspects of the complement pathway. Patients with preeclampsia have higher levels of plasma C3a, which is the ligand for C3aR [18–20]. Soto et al. reported that plasma C5a concentrations were increased in preeclamptic women, but there was no change in C3a or C4a
Table 1 Relevant characteristics of the preeclampsia study group.
Maternal age (years) Maternal BMI (kg/m2)* Gestational age (wks) Placental weight (g) Foetal birthweight (g) Gravida Parity Ethinicity – no. (%) Caucasian Asian African Middle Eastern Pacific Islander
Preterm controls (n = 8)
Preeclampsia (n = 8)
Preeclampsia-IUGR (n = 11)
27.6 (1.6) 27.4 (2.5) 29+5 (1.0) 437 (51) 1492 (204) 2.5 (0.4) 2.4 (0.3)
30.0 (2.2) 29.5 (3.6) 30+0 (1.0) 353 (32) 1310 (179) 2.1 (0.4) 1.6 (0.4)
37.6 (1.6) 32.4 (2.7) 30+0 (0.9) 279 (26)** 1040 (125)** 1.9 (0.3) 1.5 (0.2)**
5 1 0 1 1
(62.5) (12.5) (12.5) (12.5)
Values represent mean (SEM). * Based on first antenatal visit at approximately 12 weeks. ** P < 0.05 vs. preterm control group.
4 2 0 1 1
(50) (25) (0) (12.5) (12.5)
7 1 2 1 0
(64) (9) (18) (9) (0)
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
197
Fig. 2. (A) C3aR and C5aR mRNA expression in placenta from preterm control (n = 8) and severe preeclampsia with (n = 11) and without (n = 8) IUGR. Gene expression is displayed as mean SEM. (B) Western blot of C3aR and C5aR protein expression in human placenta from control (n = 8) and severe preeclampsia with (n = 11) and without (n = 8) IUGR. Data are displayed as the mean SEM. *P < 0.05 vs. preterm control.
concentrations [17]. Preeclamptic cases with foetal growth restriction are characterized by extreme reductions in placental perfusion [31]: increased release of apoptotic trophoblast debris into the maternal circulation may account for the increase in complement activation, or conversely, complement activation may contribute to the development of foetal growth restriction. In this study, we report a decrease in C3aR in placenta in our preeclamptic group. C3aR was localized to the Hofbauer cells in the villous stroma; Hofbauer cells are placental macrophages that are localized close to foetal vessels. Foetal synthesis of complement factors has been reported; C3a has been detected in amniotic fluid [18,32], cord plasma [33,34] and placenta [35]. Although there are no reports on the effect of preeclampsia on amniotic fluid, cord plasma or placental C3a levels, excessive complement activation has been documented in the placentas of abnormal pregnancies [36,37]. One of the major roles of the complement system is to dispose of immune complexes and the products of inflammatory injury. Thus, decreased C3aR may be indicative of an inability of preeclamptic placentas to clear the excess of activated complement, whereby immune complexes accumulate, inciting inflammation and disease ensues [38]. Of note, we found that C3aR was also decreased in preeclamptic pregnancies that were also associated with foetal growth restriction. Future studies are required to determine if IUGR alone is also associated with alteration in the expression of the complement receptors in the placenta. Recent studies in mice have helped us to gain a better understanding of complement pathways in preeclampsia. Preeclampsia is associated with vascular constriction, most probably due to endothelial dysfunction, and with proteinuria: renal dysfunction can also follow [39]. In mouse models of systemic lupus erythematosus (MRL strain), an autoimmune disease strongly associated with complement activation and deposition, C3aRdeficient MRL mice developed accelerated onset of renal disease and a trend towards decreased survival [40]. On the other hand, C5aR-deficient mice have attenuated disease compared to control MRL mice [41]. The complement component C1q plays a crucial role in placental development [42]; it was further shown that mice
deficient in C1q developed the characteristic features of preeclampsia [43], demonstrating the requirement of C1q on trophoblast cells for healthy pregnancy outcomes. Collectively, these data demonstrated both the importance and complexity of the complement pathway in preeclampsia. Further studies are required to elucidate the role of C3aR in placenta and its relevance to preeclampsia. In conclusion, preeclampsia is not associated with changes in C5aR gene and protein expression, but we report a decrease in C3aR mRNA and protein expression in preeclamptic placentas, with and without IUGR, when compared to gestational agematched controls. This decrease indicates some dysregulation in the complement system in the placentas of women with preeclampsia, but further studies are required to fully elucidate the underlying mechanisms. Conflict of interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding Dr Martha Lappas was a recipient of a National Health and Medical Research Council (NHMRC) RD Wright Fellowship (grant no. 454777). The work described in this manuscript was funded by a project grant from the NHMRC (grant no. 454310). Funding for ChemiDoc XRS and xMark Microplate Absorbance Spectrophotometer was provided by the Medical Research Foundation for Women and Babies. Acknowledgements The authors gratefully acknowledge the assistance of the Clinical Research Midwives Gabrielle Fleming, Astrid Tiefholz, Lyndall Paolucci, Debra Jinks, Asha Ferguson; and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their co-operation.
198
R. Lim, M. Lappas / European Journal of Obstetrics & Gynecology and Reproductive Biology 165 (2012) 194–198
References [1] Ramma W, Ahmed A. Is inflammation the cause of pre-eclampsia? Biochemical Society Transactions 2011;39:1619–27. [2] Lockwood CJ, Huang SJ, Krikun G, et al. Decidual hemostasis, inflammation, and angiogenesis in pre-eclampsia. Seminars in Thrombosis and Hemostasis 2011;37:158–64. [3] Conroy A, Serghides L, Finney C, et al. C5a enhances dysregulated inflammatory and angiogenic responses to malaria in vitro: potential implications for placental malaria. PLoS One 2009;4:e4953. [4] Langer HF, Chung K-J, Orlova VV, et al. Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis. Blood 2010;116:4395–403. [5] Monsinjon T, Gasque P, Chan P, Ischenko A, Brady JJ, Fontaine M. Regulation by complement C3a and C5a anaphylatoxins of cytokine production in human umbilical vein endothelial cells. The FASEB Journal 2003;17:1003–14. [6] Kahr WH. Complement halts angiogenesis gone wild. Blood 2010;116:4393–4. [7] Fischer WH, Jagels MA, Hugli TE. Regulation of IL-6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg). Journal of Immunology 1999;162:453–9. [8] Zhang XH, Kimura Y, Fang CY, et al. Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood 2007;110:228–36. [9] Sjoberg AP, Trouw LA, Blom AM. Complement activation and inhibition: a delicate balance. Trends in Immunology 2009;30:83–90. [10] Zipfel PF, Skerka C. Complement regulators and inhibitory proteins. Nature Reviews Immunology 2009;9:729–40. [11] Carroll MC. The complement system in regulation of adaptive immunity. Nature Immunology 2004;5:981–6. [12] Sacks SH. Complement fragments C3a and C5a: the salt and pepper of the immune response. European Journal of Immunology 2010;40:668–70. [13] Cochrane CG, Muller-Eberhard HJ. The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. Journal of Experimental Medicine 1968;127:371–86. [14] Hartmann K, Henz BM, Kruger-Krasagakes S, et al. C3a and C5a stimulate chemotaxis of human mast cells. Blood 1997;89:2863–70. [15] Girardi G, Berman J, Redecha P, et al. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. Journal of Clinical Investigation 2003;112:1644–54. [16] Soto E, Romero R, Richani K, et al. Anaphylatoxins in preterm and term labor. Journal of Perinatal Medicine 2005;33:306–13. [17] Soto E, Romero R, Richani K, et al. Preeclampsia and pregnancies with smallfor-gestational age neonates have different profiles of complement split products. Journal of Maternal–Fetal and Neonatal Medicine 2010;23:646–57. [18] Haeger M, Bengtson A, Karlsson K, Heideman M. Complement activation and anaphylatoxin (C3a and C5a) formation in preeclampsia and by amniotic fluid. Obstetrics and Gynecology 1989;73:551–6. [19] Haeger M, Unander M, Bengtsson A. Complement activation in relation to development of preeclampsia. Obstetrics and Gynecology 1991;78:46–9. [20] Derzsy Z, Proha´szka Z, Rigo´ Jr J, Fu¨st G, Molvarec A. Activation of the complement system in normal pregnancy and preeclampsia. Molecular Immunology 2010;47:1500–6. [21] Lynch AM, Gibbs RS, Murphy JR, Giclas PC, Salmon JE, Holers VM. Early elevations of the complement activation fragment C3a and adverse pregnancy outcomes. Obstetrics and Gynecology 2011;117:75–83. [22] Ames RS, Li Y, Sarau HM, et al. Molecular cloning and characterization of the human anaphylatoxin C3a receptor. Journal of Biological Chemistry 1996;271:20231–34.
[23] Nataf S, Stahel PF, Davoust N, Barnum SR. Complement anaphylatoxin receptors on neurons: new tricks for old receptors? Trends in Neurosciences 1999;22:397–402. [24] Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nature Immunology 2010;11:785–97. [25] Ward PA. Functions of C5a receptors. Journal of Molecular Medicine (Berlin) 2009;87:375–8. [26] Lappas M, Mitton A, Lim R, Barker G, Riley C, Permezel M. SIRT1 is a novel regulator of key pathways of human labor. Biology of Reproduction 2011;84:167–78. [27] Morgan BP, Holmes CH. Immunology of reproduction: protecting the placenta. Current Biology 2000;10:R381–3. [28] Holmes CH, Simpson KL. Complement and pregnancy: new insights into the immunobiology of the fetomaternal relationship. Baillieres Clinical Obstetrics and Gynaecology 1992;6:439–60. [29] Holmes CH, Simpson KL, Wainwright SD, et al. Preferential expression of the complement regulatory protein decay accelerating factor at the fetomaternal interface during human pregnancy. Journal of Immunology 1990;144: 3099–105. [30] Schraufstatter IU, Trieu K, Sikora L, Sriramarao P, DiScipio R. Complement C3a and C5a induce different signal transduction cascades in endothelial cells. Journal of Immunology 2002;169:2102–10. [31] Roberts JM, Gammill HS. Preeclampsia: recent insights. Hypertension 2005;46:1243–9. [32] Soto E, Romero R, Richani K, et al. Evidence for complement activation in the amniotic fluid of women with spontaneous preterm labor and intra-amniotic infection. Journal of Maternal–Fetal and Neonatal Medicine 2009;22:983–92. [33] Lappas M. Lower circulating levels of complement split proteins C3a and C4a in maternal plasma of women with gestational diabetes mellitus. Diabetic Medicine 2011;28:906–11. [34] Sonntag J, Brandenburg U, Polzehl D, et al. Complement system in healthy term newborns: reference values in umbilical cord blood. Pediatric and Developmental Pathology 1998;1:131–5. [35] Kohler PF. Maturation of the human complement system. I. Onset time and sites of fetal C1q, C4, C3, and C5 synthesis. Journal of Clinical Investigation 1973;52:671–7. [36] Girardi G, Yarilin D, Thurman JM, Holers VM, Salmon JE. Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction. Journal of Experimental Medicine 2006;203:2165–75. [37] Holers VM, Girardi G, Mo L, et al. Complement C3 activation is required for antiphospholipid antibody-induced fetal loss. Journal of Experimental Medicine 2002;195:211–20. [38] Redman CW, Sargent IL. Placental stress and pre-eclampsia: a revised view. Placenta 2009;30(Suppl. A):S38–42. [39] Karumanchi SA, Maynard SE, Stillman IE, Epstein FH, Sukhatme VP. Preeclampsia: a renal perspective. Kidney International 2005;67:2101–13. [40] Wenderfer SE, Wang H, Ke B, Wetsel RA, Braun MC. C3a receptor deficiency accelerates the onset of renal injury in the MRL/lpr mouse. Molecular Immunology 2009;46:1397–404. [41] Wenderfer SE, Ke B, Hollmann TJ, Wetsel RA, Lan HY, Braun MC. C5a receptor deficiency attenuates T cell function and renal disease in MRLlpr mice. Journal of the American Society of Nephrology 2005;16:3572–82. [42] Agostinis C, Bulla R, Tripodo C, et al. An alternative role of C1q in cell migration and tissue remodeling: contribution to trophoblast invasion and placental development. Journal of Immunology 2010;185:4420–9. [43] Singh J, Ahmed A, Girardi G. Role of complement component C1q in the onset of preeclampsia in mice. Hypertension 2011;58:716–24.