Serum Levels of TNF-α and IL-6 Are Associated With Pregnancy-Induced Hypertension

Abstract

Tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) are proinflammatory cytokines and known to be involved in many pathological processes. However, the association between serum levels of TNF-α, IL-6, and pregnancy-induced hypertension (PIH) is unclear. The aim of the present study was to determine the serum levels of TNF-α and IL-6 and to investigate their potential correlation with PIH. In this study, the serum concentrations of TNF-α and IL-6 in pregnant women who developed PIH and normal pregnant women were measured. We found that the serum concentrations of TNF-α and IL-6 were significantly increased in the patients with PIH compared to the normal pregnant women. In addition, elevated TNF-α and IL-6 concentrations were associated with pathological complications. Moreover, in a hypoxia-induced PIH mice model, animals from the PIH group demonstrated higher TNF-α and IL-6 levels when compared to control, and serum TNF-α and IL-6 levels were positively correlated with right ventricular systolic blood pressure. Furthermore, TNF-α and IL-6 levels were decreased when the PIH mice were treated with remodulin compared to control group. In conclusion, our results suggested that high serum TNF-α and IL-6 levels are associated with PIH, and TNF-α and IL-6 might be potential predictors in the prognosis of PIH.

Keywords

IL-6 pregnancy-induced hypertension serum tumor necrosis factor α

Introduction

Pregnancy-induced hypertension (PIH) is characterized by generalized vasoconstriction and impaired uteroplacental perfusion, and PIH is one of the commonest medical problems encountered during pregnancy.¹ Previous studies demonstrated that hypertensive pregnant women are at an increased risk of later cardiovascular disease (CVD) and mortality² regardless of their proteinuric status.³ This can be explained by the fact that metabolic syndrome is an important risk factor for CVD in women.⁴ Pregnancy-induced hypertension includes a spectrum of disorders, such as preeclampsia, gestational hypertension, and eclampsia, and if not diagnosed in a timely manner, the most severe form, eclampsia, may develop. Recently, a variety of biological, biochemical, and biophysical markers implicated in the pathophysiology of preeclampsia have been proposed to predict its development.⁵ However, more reliable, economic, and reproducible screening tests are still needed. Recent evidence suggests that the causes of pre-eclampsia are related to inadequate delivery of blood to the developing uteroplacental unit, which leads the placenta to become hypoxic and ischemic. In response, the placenta releases pathogenic factors, which enter the maternal blood stream and are responsible for the clinical manifestations of the disorder.^6,7 It was already reported that preeclampsia is an excessive maternal inflammatory response to normal pregnancy.⁸ Supporting evidence includes leukocyte and platelet activation and elevated levels of endothelial adhesion molecules and P-selectin in the maternal circulation in women with preeclampsia.⁹ Moreover, proinflammatory cytokine levels, such as tumor necrosis factor α (TNF-α) and interleukin (IL)-6, are also changed during pre-eclampsia.¹⁰ The identities of soluble factors released from placenta have been a major area of investigation. One of the most consistent findings is the activation of the innate immune response, in particular inflammatory cytokines, such as TNF-α and IL-6.¹¹ Tumor necrosis factor α is produced largely by monocytes and macrophages in response to inflammatory stimuli¹² and increases the release of other cytokines, chemokines, growth factors, and acute phase proteins.¹³ As a pleiotropic cytokine, TNF-α exerts multiple effects, and increased production of TNF-α can also produce oxidative stress.^13,14 Interleukin 6 is a multifunctional cytokine, produced by various cell types, such as macrophages, endothelial cells (ECs), vascular smooth muscle cells, and fibroblasts.¹⁵ It has been reported that IL-6 trans-signaling pathway is considered to exert mainly proinflammatory actions.¹⁶ The increase in TNF-α and IL-6 has been observed in animal models of experimental placental ischemia.¹⁷ Furthermore, IL-6 and TNF-α are associated with quality of life-related symptoms in pulmonary arterial hypertension.¹⁸ However, the association between serum levels of TNF-α, IL-6, and PIH remains unclear.

In the present study, we aimed to determine the serum levels of TNF-α and IL-6 in patients with PIH and PIH rat model and to investigate its possible association with various disease parameters and severity.

Materials and Methods

Study Population

Study participants were prospectively enrolled from Shandong Provincial Hospital Affiliated to Shandong University (between May 2013 and May 2014). According to a report of the National High Blood Pressure Education Program Working Group,¹⁹ PIH was defined as the development of hypertension due to pregnancy in a previously normotensive woman, as demonstrated by proteinuria or more than 2 measurements of systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg after 20 weeks of gestation, with measurements taken at least 4 hours apart. The current study included 59 participants, stratified into 2 groups. One group consisted of 27 women who developed PIH and delivered at the hospital (PIH group). The other group consisted of 32 women who passed all gestations without any complications and delivered a normal baby were defined as normal controls. The PIH and control groups were matched for gestational age and maternal age. Blood samples used in this study were collected during the third (28-40 gestational weeks) trimester. All cases were uncomplicated singleton pregnancies. Pregnant women had secondary forms of hypertension, coronary heart disease, kidney disease, or type 1 or 2 diabetes were excluded in this study. This study was approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University. All participants provided written informed consent.

Collection of Blood Samples and Case Materials

Blood samples were collected at the prenatal clinic, centrifuged for 20 minutes at 2000 rpm, and stored at −80°C until analysis. The concentrations of TNF-α and IL-6 were determined using enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). Case materials were obtained from the medical records room of the hospital and from prenatal and postpartum questionnaires. Maternal demographic, medical, and obstetric data were collected under permission. Biological sample data were used to explore putative associations between plasma markers in the control and PIH groups. Double-blind assay was used in our study.

Experimental Animals

All procedures and experiments involving animals in this study were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. The study protocol was approved by the Animal Ethics Committee at Shandong Provincial Hospital Affiliated to Shandong University, China. Experimental mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and were housed in the Animal Resource Facility. Three groups of female C57BL/6 mice (10 mice per group) were used: (1) normoxic control time-dated pregnant group; (2) in the PIH group, the pregnant mice were continuously exposed to normobaric hypoxia (10% O₂), as described previously;²⁰ (3) hypoxic (10% O₂) pregnant mice treated with remodulin (treprostinil) infusions at a dose 60 ng/kg/min for 1 week. The remodulin treatment was started 2 days prior to the initiation of 10% O₂ treatment to allow the recovery of animals from the surgical implantation of osmotic pumps before the hypoxia treatment. Blood taken from the heart was stored at −80°C for TNF-α and IL-6 analysis. Animals also underwent hemodynamic measurements, evaluation of right ventricular systolic blood pressure (RVSP) as previously described.²⁰ PE-50 polyethylene tubing was inserted into the right ventricle, and the RVSP was recorded using a Power-Lab data acquisition system.

RNA Isolation and qRT-PCR

Total RNA was extracted from the mice lung tissues by Trizol Reagent (Invitrogen, Carlsbad, California). For messenger RNA (mRNA) analyses, complementary DNA was synthesized using Moloney murine leukaemia virus reverse transcriptase (Promega, Madison, Wisconsin). Quantitative real time polymerase chain reaction (qRT-PCR) was performed with SYBR Premix ExTaq with the Stratagene Mx3000P real-time PCR system. 18S was used as internal controls for mRNA quantification. The relative expression ratio of mRNA was calculated by the 2^−ΔΔCT method. Polymerase chain reaction reactions for each gene were repeated 3 times. Independent experiments were done in triplicate. The sequences of the PCR primers used in this study are as follows: sense strand TNF-α primer, 5′-ATG AGC ACA GAA AGC ATG ATC-3′; antisense strand TNF-α primer, 5′-TAC AGG CTT GTC ACT CGA ATT-3′; sense strand IL-6 primer, 5′-GAC AAA GCC AGA GTC CTT CAG AGA G-3′; antisense strand IL-6 primer, 5′-CTA GGT TTG CCG AGT AGA TCT C-3′.

Immunohistochemistry

Paraffin sections (4 μm thickness) from samples were deparaffinized in 100% xylene and rehydrated in descending ethanol series and water according to standard protocols. Heat-induced antigen retrieval was performed in 10 mmol/L citrate buffer for 2 minutes at 100 °C. Endogenous peroxidase activity and nospecific antigens were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum, followed by incubation with TNF-α or IL-6 antibody (Abcam, Cambridge, Massachusetts) overnight at 4 °C. After washing, the sections were incubated with biotin-labeled rabbit anti-goat antibody for 15 minutes at room temperature and subsequently were incubated with streptavidin-conjugated horseradish peroxidase (Maixin, Fuzhou, China). The peroxidase reaction was developed using 3,3-diaminobenzidine (DAB) chromogen solution in DAB buffer substrate. Sections were visualized with DAB and counterstained with hematoxylin, mounted in neutral gum and analyzed using a bright field microscope. Each sample was examined separately and scored by 2 blinded pathologists.

Statistical Analysis

Data are presented as mean ± standard deviation unless otherwise indicated. The statistical significance of the difference between the values of control and treatment groups was determined by either Student t test or simple 1-way analysis of variance followed by Tukey post hoc test for multiple comparisons using Prism version 5 (GraphPad Software, Inc, San Diego, CA). Correlations between variables were assessed using Pearson correlation. Values of P < .05 were considered statistically significant.

Results

The Clinical Characteristics of Participants

The clinical characteristics of the study groups are summarized in Table 1 according to pregnancy outcome. There were no significant differences between the groups in terms of marital status, childbearing history, ethnicity, or education level. Compared to gestational age and maternal age-matched controls, women in the PIH group had a higher prpregnancy body mass index and a greater incidence of anemia.

Table 1.

Clinical Characteristics of 59 Pregnancies.

Characteristics	PIH Group, n = 27	Control Group, n = 32
Maternal average age, year	27.3	28.2
Pre-pregnancy BMI, kg/m²	25.2	17.8
Anemia	9/27 (33.3%)	1/32 (3.1%)
Asian ethnicity	27/27 (100.0%)	32/32 (100.0%)
Childbearing history
History of spontaneous abortion (>3)	3/27 (11.1%)	4/32 (12.5%)
Gravidity (>3)	7/27 (25.9%)	9/32 (28.1%)

Abbreviations: PIH, pregnancy-induced hypertension; BMI, body mass index.

Serum TNF-α and IL-6 Levels Are Increased in Patients With PIH

The serum concentrations of TNF-α and IL-6 were determined using enzyme-linked immunosorbent assay kit. The data showed that the serum concentrations of TNF-α and IL-6 were significantly higher in patients with PIH compared to normal pregnant women (Figure 1A). In addition, since the pathophysiology of pulmonary arterial hypertension exacerbated by pregnancy confers both high maternal and fetal mortality,²¹ we further determined the TNF-α and IL-6 levels of patients having PIH with pulmonary hypertension, which may be defined as a mean pulmonary artery pressure. We found that in the 14 patients with PIH for whom we had hemodynamic data available, the serum TNF-α and IL-6 levels of patients with mean pulmonary arterial pressure (mPAP) of ≥50 mm Hg were significantly higher than in those with mPAP of <50 mm Hg (Figure 1B). These results indicated that serum TNF-α and IL-6 levels were elevated in patients with PIH and associated with pathological complications.

Tumor Necrosis Factor α and IL-6 Levels Are Upregulated in a Hypoxia-Induced PIH Mice Model

To determine the TNF-α and IL-6 levels in hypoxia-induced PIH mice (constant exposure to 10% O₂), the serum levels of TNF-α and IL-6 were measured. As shown in Figure 2A, the serum TNF-α and IL-6 levels were significantly increased after hypoxic exposure. Additionally, we detected an overall increase in TNF-α and IL-6 expression in lung tissues in response to hypoxic exposure by qRT-PCR and immunohistochemistry assay (Figure 2B and C). Moreover, we found that serum TNF-α and IL-6 levels are positively correlated with RVSP (Figure 3A and B). These results suggest that TNF-α and IL-6 levels could be associated with hypoxia-induced PIH in mice.

Medical Treatment Decreases Serum TNF-α and IL-6 Levels in PIH Mice Model

To investigate whether serum levels of TNF-α and IL-6 could be the biomarker for the effectiveness of PIH therapy in mice, the serum TNF-α and IL-6 levels in hypoxia-induced PIH mice were monitored during remodulin (treprostinil) treatment. Remodulin is a vasodilator that works by dilating/widening the arteries used to treat pulmonary arterial hypertension. We chose remodulin to treat hypoxia-induced PIH mice as recent study showed that intravenous remodulin is an effective therapeutic protocol for the treatment of pulmonary hypertension in pregnancy.²² After hypoxic exposure, a significantly higher RVSP was detected in hypoxic mice compared with normoxic mice (Figure 4A). We found that remodulin treatment significantly inhibited the progression of hypoxia-induced PIH (Figure 4A). Notably, the serum levels of TNF-α and IL-6 were significantly decreased by remodulin treatment compared to the control group (Figure 4B). Moreover, the mRNA levels of TNF-α and IL-6 in lung tissues were reduced in response to medical treatment as compared with control group (Figure 4C). These data suggest that TNF-α and IL-6 could be the potential biomarkers for the effectiveness of PIH treatment in mice.

Discussion

Although PIH is one of the major causes of maternal and perinatal mortality and morbidity, the pathophysiology of PIH so far has not been completely understood. The initiating event of the disease is associated with abnormal cytotrophoblast invasion resulting in inadequate remodeling of the uterine spiral arteries and reduced blood flow to the uteroplacental unit.^19,23,24 The poorly perfused and hypoxic placenta is thought to release factors that result in vasoconstriction and hypertension in the mother. Pregnancy-induced hypertension is considered to have a multifactorial etiology associated with inflammatory dysfunction.²⁵ In the present study, we found that the serum concentrations of TNF-α and IL-6 were significantly increased in patients with PIH compared with normal pregnant women, and the elevated TNF-α and IL-6 levels were associated with pathological complications. Moreover, hypoxia-induced PIH mice showed higher TNF-α and IL-6 levels as compared to control, and the levels were decreased when the PIH mice were treated with remodulin. Our results suggested that high serum TNF-α and IL-6 levels are associated with PIH, and TNF-α and IL-6 could might be potential predictors in the prognosis of PIH.

It is well known that PIH is characterized by generalized vasoconstriction and impaired uteroplacental perfusion.¹ Endothelial cells maintain the homeostasis of vascular tone, the coagulation cascade, and the inflammatory process.^26
–28 Thus, EC activation or dysfunction affects vasoconstriction, leading to hypertension, thrombosis, and inflammation.^26
–28 The interaction between inflammatory cells, such as leukocytes, and ECs is believed to play an important role in the etiology of this disease because chronic vasculitis is significantly increased in the placenta of patients with pre-eclampsia.²⁹ Previous studies have demonstrated that immunoactive cells and circulating leukocytes including macrophages, monocytes, and lymphocytes produce TNF-α and IL-6.³⁰ A number of studies have been reported on maternal cytokine levels in pre-eclampsia.^31,32 Tumor necrosis factor α and IL-6 are important proinflammatory cytokines and are involved in many immune responses and activate granulocytes and ECs.³³ The increase in TNF-α and IL-6 has also been observed in animal models of experimental placental ischemia.^17,24 It has been reported that infusion of TNF-α and IL-6 individually during pregnancy in rodents has been shown to induce hypertension, though not to the degree seen in a placental ischemic model.^17,34 Interestingly, though antagonism of TNF-α by administration of a soluble receptor tended to decrease the hypertension associated with placental ischemia, this was not significant, suggesting that its hypertensive effects are only a part of a much broader response.²⁴ In agreement with published results, we observed that the serum TNF-α and IL-6 levels were significantly increased in patients with PIH as well as in a hypoxia-induced PIH mouse model and were correlated with the severity of PIH.

Previous studies have demonstrated that prostaglandin I2 (PGI₂) exerts its function through the G_s protein-coupled inositol trisphosphate (IP) and signaling through the IP leads to increased levels of intracellular cyclic adenosine monophosphate (cAMP).³⁵ Because PGI₂ is very unstable in aqueous solution, PGI₂ analogs with more chemical stability have commonly been used in laboratory research and clinical applications. Prostaglandin I2 analogs, such as iloprost and cicaprost, have been shown to inhibit production of proinflammatory cytokine TNF-α and granulocyte-macrophage colony-stimulating factor (GM-CSF), while increasing the production of an anti-inflammatory cytokine IL-10 by human peripheral mononuclear cells in vitro.^36,37 In another study, the PGI₂ analog treprostinil inhibited production of multiple cytokines including IL-6, TNF-α, GM-CSF, and IL-1 by human alveolar macrophages and blocked nuclear factor-κB nuclear translocation.³⁸ Moreover, PGI2 analogues can downregulate pro-inflammatory cytokine (TNF-α, IL-1, IL-6, and interferon γ) and chemokine production in monocytes³⁹, dendritic cells,⁴⁰ and T-lymphocytes.⁴¹ Further, previous report has demonstrated the effectiveness of an intensive therapeutic protocol, including intravenous treprostinil, for the treatment of PIH.²² In this study, our data demonstrated that TNF-α and IL-6 levels were decreased when the PIH mice were treated with treprostinil, indicating that TNF-α and IL-6 could be useful biomarkers for the effective therapy of PIH.

There are limitations in this present study. First, our findings are biomarker dependent, not assay dependent, and additional markers are needed to achieve clinical utility. Second, because this was a single-center study with a small sample size, other risk factors for PIH may exist that were not identified in this study. Future studies are necessary to include more patients at different stages of disease, so that serum levels of TNF-α and IL-6 in response to different treatments can be evaluated and also to predict the PIH progression.

In conclusion, our data demonstrate that the serum TNF-α and IL-6 levels are higher in patients with PIH than in normal pregnant women, indicating that high serum TNF-α and IL-6 levels are associated with the pathophysiology of PIH. Moreover, the measurement of serum TNF-α and IL-6 levels might enable clinicians to predict the onset of PIH.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Yang

Wang

. Central and peripheral hemodynamics in severe preeclampsia. Acta Obstet Gynecol Scand. 1996;75(2):120–126.

Valdiviezo

Garovic

Ouyang

. Preeclampsia and hypertensive disease in pregnancy: their contributions to cardiovascular risk. Clin Cardiol. 2012;35(3):160–165.

Mannisto

Mendola

Vaarasmaki

. Elevated blood pressure in pregnancy and subsequent chronic disease risk. Circulation. 2013;127(6):681–690.

Galassi

Reynolds

. Metabolic syndrome and risk of cardiovascular disease: a meta-analysis. Am J Med. 2006;119(10):812–819.

Yadav

Aggarwal

Verma

. Serum betahCG and lipid profile in early second trimester as predictors of pregnancy-induced hypertension. J Obstet Gynaecol India. 2014;64(3):169–174.

George

Granger

. Linking placental ischemia and hypertension in preeclampsia: role of endothelin 1. Hypertension. 2012;60(2):507–511.

Lim

Zhou

Janatpour

. Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia. Am J Pathol. 1997;151(6):1809–1818.

Redman

Sacks

Sargent

. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol. 1999;180(2 pt 1):499–506.

Beckmann

Visser

Struijk

van Dooren

Glavimans

Wallenburg

. Circulating bioactive tumor necrosis factor-alpha, tumor necrosis factor-alpha receptors, fibronectin, and tumor necrosis factor-alpha inducible cell adhesion molecule VCAM-1 in uncomplicated pregnancy. Am J Obstet Gynecol. 1997;177(5):1247–1252.

10.

Zhang

Lucas

Wang

. Increased endothelial monolayer permeability is induced by serum from women with preeclampsia but not by serum from women with normal pregnancy or that are not pregnant. Hypertens Pregnancy. 2003;22(1):99–108.

11.

Vince

Starkey

Austgulen

Kwiatkowski

Redman

. Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia. Br J Obstet Gynaecol. 1995;102(1):20–25.

12.

Gao

Zhang

Belmadani

. Role of TNF-alpha-induced reactive oxygen species in endothelial dysfunction during reperfusion injury. Am J Physiol Heart Circ Physiol. 2008;295(6):H2242–H2249.

13.

Navarro-Gonzalez

Mora-Fernandez

Muros de Fuentes

García-Pérez

. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol. 2011;7(6):327–340.

14.

Navarro

Mora

Gomez

Muros

López-Aguilar

García

. Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor-alpha and interleukin-6 in type 2 diabetic patients. Nephrol Dial Transplant. 2008;23(3):919–926.

15.

Khong

Brosens

. Defective deep placentation. Best Pract Res Clin Obstet Gynaecol. 2011;25(3):301–311.

16.

Rajakumar

Doty

Daftary

Harger

Conrad

. Impaired oxygen-dependent reduction of HIF-1alpha and -2alpha proteins in pre-eclamptic placentae. Placenta. 2003;24(2-3):199–208.

17.

Gadonski

LaMarca

Sullivan

Bennett

Chandler

Granger

. Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of interleukin 6. Hypertension. 2006;48(4):711–716.

18.

Matura

Ventetuolo

Palevsky

. Interleukin-6 and tumor necrosis factor-alpha are associated with quality of life-related symptoms in pulmonary arterial hypertension. Ann Am Thorac Soc. 2015;12(3):370–375.

19.

Roberts

Pearson

Cutler

Lindheimer

; National Heart Lung and Blood Institute. Summary of the NHLBI working group on research on hypertension during pregnancy. Hypertens Pregnancy. 2003;22(2):109–127.

20.

Liu

Yang

. Serum levels of tumor necrosis factor-related apoptosis-inducing ligand correlate with the severity of pulmonary hypertension. Pulm Pharmacol Ther. 2015;33:39–46.

21.

Obican

Cleary

. Pulmonary arterial hypertension in pregnancy. Semin Perinatol. 2014;38(5):289–294.

22.

Rosengarten

Kramer

. Pregnancy in a woman with pulmonary hypertension: favorable outcome with intravenous treprostinil. Clin Exp Obstet Gynecol. 2015;42(3):390–391.

23.

Granger

Alexander

Bennett

. Pathophysiology of pregnancy-induced hypertension. Am J Hypertens. 2001;14(6 pt 2):178S–185S.

24.

LaMarca

Speed

Fournier

. Hypertension in response to chronic reductions in uterine perfusion in pregnant rats: effect of tumor necrosis factor-alpha blockade. Hypertension. 2008;52(6):1161–1167.

25.

Bayram

Taskaya

Bagriacik

Ilhan

Yaman

. The effect of maternal serum sFAS/sFASL system on etiopathogenesis of preeclampsia and severe preeclampsia[published online August 22, 2012]. J Matern Fetal Neonatal Med. 2012.

26.

Drexler

Hornig

. Endothelial dysfunction in human disease. J Mol Cell Cardiol. 1999;31(1):51–60.

27.

Holvoet

Collen

. Thrombosis and atherosclerosis. Curr Opin Lipidol. 1997;8(5):320–328.

28.

Sellati

Burns

Ficazzola

Furie

. Borrelia burgdorferi upregulates expression of adhesion molecules on endothelial cells and promotes transendothelial migration of neutrophils in vitro. Infect Immun. 1995;63(11):4439–4447.

29.

Salafia

Pezzullo

Lopez-Zeno

Simmens

Minior

Vintzileos

. Placental pathologic features of preterm preeclampsia. Am J Obstet Gynecol. 1995;173(4):1097–1105.

30.

Belge

Dayyani

Horelt

. The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF. J Immunol. 2002;168(7):3536–3542.

31.

Conrad

Benyo

. Placental cytokines and the pathogenesis of preeclampsia. Am J Reprod Immunol. 1997;37(3):240–249.

32.

Conrad

Miles

Benyo

. Circulating levels of immunoreactive cytokines in women with preeclampsia. Am J Reprod Immunol. 1998;40(2):102–111.

33.

Espevik

Brockhaus

Loetscher

Nonstad

Shalaby

. Characterization of binding and biological effects of monoclonal antibodies against a human tumor necrosis factor receptor. J Exp Med. 1990;171(2):415–426.

34.

Alexander

Cockrell

Massey

Bennett

Granger

. Tumor necrosis factor-alpha-induced hypertension in pregnant rats results in decreased renal neuronal nitric oxide synthase expression. Am J Hypertens. 2002;15(2 pt 1):170–175.

35.

Boie

Rushmore

Darmon-Goodwin

. Cloning and expression of a cDNA for the human prostanoid IP receptor. J Biol Chem. 1994;269(16):12173–12178.

36.

Luttmann

Herzog

Matthys

Thierauch

Virchow

Kroegel

. Modulation of cytokine release from mononuclear cells by prostacyclin, IL-4 and IL-13. Cytokine. 1999;11(2):127–133.

37.

Meja

Barnes

Giembycz

. Characterization of the prostanoid receptor(s) on human blood monocytes at which prostaglandin E2 inhibits lipopolysaccharide-induced tumour necrosis factor-alpha generation. Br J Pharmacol. 1997;122(1):149–157.

38.

Raychaudhuri

Malur

Bonfield

. The prostacyclin analogue treprostinil blocks NFkappaB nuclear translocation in human alveolar macrophages. J Biol Chem. 2002;277(36):33344–33348.

39.

Wang

Kuo

Chu

. Prostaglandin I(2) analogues suppress TNF-alpha expression in human monocytes via mitogen-activated protein kinase pathway. Inflamm Res. 2011;60(7):655–663.

40.

Zhou

Hashimoto

Goleniewska

. Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells. J Immunol. 2007;178(2):702–710.

41.

Zhou

Blackwell

Goleniewska

. Prostaglandin I2 analogs inhibit Th1 and Th2 effector cytokine production by CD4 T cells. J Leukoc Biol. 2007;81(3):809–817.