Elsevier

Human Immunology

Volume 66, Issue 11, November 2005, Pages 1146-1154
Human Immunology

Induction of Neutrophil Extracellular DNA Lattices by Placental Microparticles and IL-8 and Their Presence in Preeclampsia

https://doi.org/10.1016/j.humimm.2005.11.003Get rights and content

Abstract

Preeclampsia, a severe pregnancy-related disorder, involves an overt activation of the maternal innate immune system, proposed to result from the elevated release of inflammatory syncytiotrophoblast microparticles (STBM) and cytokines from underlying placental anomaly involving abnormal trophoblast differentiation. Activation of circulating neutrophils has recently been shown to lead to the generation of fibrous extracellular lattices containing DNA, termed NETs (neutrophil extracellular traps). Therefore, we examined whether placentally derived factors activated peripheral neutrophils to generate NETs, and whether NETs formation was increased in preeclamptic placenta. Activation of isolated circulatory neutrophils on treatment with placentally derived factors (interleukin-8 and STBM) was assessed by measuring CD11b expression using Flow cytometry. Furthermore, NETs generation by these activated neutrophils in vitro and in vivo in the placental tissue sections were examined using electron microscopy and fluorescence microscopy. We report that placentally-derived interleukin-8 and STBM efficiently activated neutrophils and triggered NETs formation. Large numbers of NETs were present directly in the intervillous space of preeclamptic placentae. NETs, therefore, appear to be an integral part of neutrophil activation, and their increased presence in preeclampsia suggests that NETs may play a role in the underlying pathology.

Introduction

Preeclampsia is a significant cause of fetal and maternal mortality worldwide [1, 2, 3]. This disorder, which is peculiar to human pregnancy, is typically characterized by elevated blood pressure and proteinuria in previously normotensive pregnant women [1, 2, 3]. Although the etiology of this enigmatic disorder is likely to be multifactorial, the placenta has been proposed to play a key role. This is largely thought to result from superficial placentation associated with a condition of oxidative stress [4, 5], which leads to the elevated release of inflammatory placentally derived microparticles and cytokines, [6] as well as an imbalance of angiogenic factors [7, 8]. Although it is likely that the latter represent a key event in the development of maternal hypertensive symptoms [7], the elevated shedding of syncytiotrophoblast microparticles (frequently termed STBM) in the maternal circulation during preeclampsia has been proposed to be instrumental in eliciting an overt activation of the innate arm of the maternal immune system, in particular of circulatory neutrophils [9, 10].

In addition, we and others have previously shown that preeclampsia is associated with increased transplacental trafficking of fetal cells [11], as well as elevated concentrations of cell-free fetal DNA [12, 13, 14], which may occur before onset of symptoms [15, 16, 17]. These studies, however, also indicated that cell-free DNA levels of maternal origin were also significantly elevated in preeclampsia, and that these levels correlated well with severity of the disorder [13]. While the results concerning fetal cells and cell-free fetal nucleic acids have been interpreted as further evidence of an underlying placental dysfunction in preeclampsia, those concerning the elevated presence of circulatory DNA of maternal origin have been more difficult to explain, because the source of this material is unclear [18].

For this reason, we were intrigued by the recent observation that, on activation by inflammatory signals, peripheral neutrophils generate extracellular DNA containing fibrous lattices, termed NETs (neutrophil extracellular traps) [19]. In this seminal report, the extracellular lattices exuded by activated peripheral neutrophils were found to trap bacteria and to possess bactericidal activities. Furthermore, it has also been demonstrated that interferon-γ priming and subsequent C5a stimulation resulted in NETs formation in mature but not in immature neutrophils [20]. This observation suggests that the NETs generation is a characteristic feature of mature neutrophils [20].

Because this feature may therefore link the independent observations concerning the activation of peripheral neutrophils and the elevated presence of maternal circulatory DNA during preeclampsia, we examined whether placentally derived inflammatory factors can trigger peripheral neutrophils to generate NETs. We also examined for the presence of such NETs in preeclamptic placentae.

Section snippets

Collection of Samples

The Cantonal Institutional Review Board of Basel, Switzerland, and Department of Obstetrics and Gynecology, University of Stellenbosch, South Africa, approved this study. Written informed consent was requested in all instances. Blood samples (20 ml each) for the isolation of peripheral neutrophils were obtained from healthy donors at the blood donation center of the Swiss Red Cross, University Hospital, Basel. Placentae were obtained from normal and preeclamptic pregnancies at the University

Placentally Derived IL-8 and STBM-Activated Neutrophils in Independent Manner

Although placentally derived microparticles can be detected in maternal blood samples, they cannot be reliably isolated for in vitro experiments [6]. For this reason, we made use of STBM generated from placental villous explants in vitro as described previously [21]. Because the placenta also produces a variety of inflammatory cytokines, we also prepared culture supernatants that had been extensively cleared of any microparticulate matter.

To assess the activation of isolated peripheral

Discussion

In this study we expand on the recent observations that, on activation with PMA, IL-8 and IFN γ neutrophils generated extracellular DNA containing NETs [19, 20], by demonstrating that these NETs can be induced by physiological signals, such as placentally derived inflammatory debris (STBM) and IL-8. Therefore, this phenomenon is not solely mediated by foreign objects, such as bacteria, but may be a part of normal physiology. Our observations further suggest that NETs formation may be a general

Acknowledgments

We would like to thank Mr. Daniel Mathys (ZMB, University of Basel) for his excellent technical assistance with the scanning electron microscopy and Mrs. Vivian Kiefer-Vargas and Mrs. Lisbeth Dudler for their kind help in the preparation of specimens for microscopic analysis. We would also like to thank Drs. Susanne Mergenthaler for her help with fluorescence microscopy and Carolyn Troeger for placentae collection. We thank Drs. Berthold Huppertz and Corinne Rusterholz for constructive

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