Receptor expression by JEG-3 trophoblast cells in the presence of placenta secreted factors

Abstract

Preeclampsia still remains one of the most severe pregnancy complications and is an actual problem in the obstetrics practice. At present, the joint impact of cytokines and other placenta secreted factors on trophoblast cell functional activity during preeclampsia complicated pregnancy remains unclear. The aim of the study is to estimate the surface receptors expression by trophoblast cells in the presence of placenta secreted factors during physiological pregnancy and at preeclampsia. Trophoblast cells of the JEG-3 line were incubated in the presence of supernatants obtained by cultivation of placentas from women with physiological pregnancy and with preeclampsia. Surface receptors expression by trophoblast cells was estimated by FACS Canto II flow cytometer. It was established that in the third trimester both under normal and pathological conditions, the placenta secreted factors impact on the cytokine receptor expression by trophoblast differs while the trophoblast response capacity to the migration and proliferation stimulating and inhibiting signals remains stable. JEG-3 line cells enhanced the expression of CD186, CD140a, Integrin β6, VE-cadherin, CD29, and CD140a in the case of incubation in the presence of placenta supernatants from the third-trimester pregnancy complicated with preeclampsia compared to incubation in the presence of placenta supernatants form the third trimester of physiological pregnancy.

Keywords:

• Trophoblast
• receptors
• cytokines
• chemokines
• growth factors
• preeclampsia

Introduction

Successful pregnancy development is primarily dependent on placenta adequate formation and functioning. The placenta formation is determined by cooperation between placenta formation cells (trophoblast) and immune system cells (NK cells, macrophages, and lymphocytes). The decisive part in this kind of cell cooperation is played by cytokines. Any disturbance of cytokine balance production, growth factors in the placenta or in the utero-placenta complex may lead to obstetrical pathology, preeclampsia in particular.

Preeclampsia still remains one of the most severe pregnancy complications and is, therefore, a vital task for obstetrical practice. Recent investigations offer several hypotheses on preeclampsia origins, one of which is about trophoblast cell functional changes [1]. Preeclampsia causes the decrease of HLA-G locus molecules expression by trophoblast cells leading to decidual NK cells and cytotoxic T-lymphocyte hyperactivity. That, in turn, leads to IFNγ and TNFα production increase, negatively affecting the trophoblast invasion into the endometrium and its viability. The increased secretion of anti-inflammatory cytokines in placenta accompanied by IL-10 and IL-5 decrease leads to decidual and placenta macrophages activation [2] producing in their turn cytokines IL-1β, IL-6, and CXCL-8 [3]. The result is an uncontrolled inflammatory response leading also to trophoblast invasion disturbance, inducing apoptosis of endothelial, trophoblast, and other placenta cells [4].

Trophoblast cells express receptors for VEGF and PlGF – VEGFR-1. In course of pregnancy, the placenta is secreting both VEGF and PlGF, as well as soluble forms of VEGFR-1 (sFlt-1) which are capable of competitive binding to VEGF and PlGF ligands hindering signaling pathway activation by growth factors in endothelial cells (EC) and trophoblast [5]. Preeclampsia is marked by sFlt-1 placenta production increase and its elevated count in peripheral blood correlating with trophoblast invasion decrease [5]. Along with that, the placenta shows VEGF, bFGF cytokine expression decrease together with PDGF, TGFβ, MMP-2 expression growth [1]. The serum level of a soluble receptor sEng (endoglin membrane receptor – CD105) in women with preeclampsia is determined long before the clinical symptom appearance. This receptor binds to TGFβ suppressing its effects on trophoblast cells. Such kind of changes may lead to trophoblast migrating and proliferating activity decrease. Damaged trophoblast invasion into uterine endometrium may be connected with the disorder of adhesive molecule trophoblast expression during spiral arteries remodeling. Since preeclampsia usually shows α6β4, αvβ6, and E-Cadherin integrin expression increase by trophoblast cells together with expression decrease of α1β1 и αvβ3, VE-Cadherin, VCAM-1, and PECAM-1 expression it confirms cytotrophoblast low differentiation and correlates to its low invasiveness.

Due to relatively low chorion accessibility and laboriousness of trophoblast cell extraction techniques, we should qualify as vague the joint influence of cytokines and other placenta secreted factors during pregnancy complicated by preeclampsia on the trophoblast cells surface receptor expression and cytokine production. The aim of the study, therefore, was to estimate the surface receptor expression by trophoblast cells in the presence of placenta secreted factors during physiological pregnancy and at preeclampsia.

Materials and techniques

Patients

The placentas were either those obtained through artificial abortion from women on the 9th/11th week of physiological pregnancy (n = 19, group 1); or those on 38th and 39th weeks of normal pregnancy (n = 32, group2) after cesarean delivery before the labor start. Patients with type I and II diabetes mellitus, multiple pregnancies, polyhydramnios, urinary tract infection, acute infection or recrudescence of chronic infection, chronic kidney disease, chronic hypertension, and other cardiovascular diseases were excluded. All pregnant women were matched for age (31.6 ± 4.2 years), parity and obstetric history. All participants were Caucasian raсe coming from The North-West region of Russia. Preeclampsia was defined as pregnancy-induced hypertension (blood pressure ≥ 140/90 mm Hg), proteinuria (300 mg/24 h), and edema in the second half of pregnancy in women who were normotensive before pregnancy and had no underlying clinical problems such as renal disease.

Ethical approval

The research was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed Consent was obtained from all the examined patients. The study was approved by the ethical committee of D.O. Ott Research Institute of Obstetrics, Gynecology, and Reproductology.

Cell culture

We used JEG-3 line trophoblast cells (American Typical Culture Collection (ATCC), USA). The cells were cultured in DMEM medium with addition of 10% inactivated fetal calf serum (FCS), 100 unit/ml penicillin and 100 mkg/ml streptomycin, 0,5mM l-glutamine, 1 ml MEM and 1 mM sodium pyruvate (Sigma, St. Louis, MO). The cell monolayer was disintegrated by versene and trypsin solutions (Biolot, Russia), commixed in 1:1 proportion. Trypan blue solution was used to estimate the cell vitality, which was not less than 96%.

Placenta explant incubation

The placental tissue explants from the central part of the placentas were incubated in DMEM medium without FCS addition within 24 h. Then the supernatants were collected and frozen at a temperature of −20 °C until the investigation.

Surface receptor expression estimation

The JEG-3 line cells were incubated in a 24-well flat-bottom plate at a concentration of 1,7 × 10⁵ cells per well in 1 ml of DMEM/F12 medium with addition of 10% FCS (37 °С in a humid atmosphere, 5% Со₂) until the obtaining of the confluent monolayer. Then the cells were triply washed by warm Hanks solution and supernatants obtained after placenta incubation were imported, dissolved by DMEM\F12 medium in 1:1 proportion. The FCS concentration was raised to 10%. The cells were incubated for 22 h and then collected from the plate surface by versene solution. The cell viability was estimated by trypan blue solution (Sigma, St. Louis, MO) and it was up to 95–97%. Next cells were incubated with FcR blocking reagent (MACS, Frankfurt/Main, Germany) to reduce the nonspecific antibody binding and then treated with antibodies to CD118, CD119, IFNγ-R2, CD120a, CD120b, CD126, CD130, CD181, CD182, CD183, CD184, CD186, CD192, CCR1, CD140a, CD295, CD29, CD31, CD49a, CD49e, CD49f, CD51/61, CD54, CD95, CD104, CD106, Integrin β6 (BD, Сша), VEGFR1, VEGFR2, EGFR, TGFβ-R2, HGFR, E-Cadherin, VE-Cadherin, and HLA-G (R&D, Minneapolis, MN) according to manufacturer’s recommendations. Table 1 presents the groups including here analyzed receptors, their ligands and possible ligand placenta sources (Table 1). Isotype controls (BD, USA; R&D, USA) were used as negative controls. Cell incubation in the presence of TNFα (50 unit/ml) with further CD54 expression estimation was the positive control. We have clearly established the constitutive CD54 expression and its stimulating intensiveness by JEG line cells in the presence of TNFα (Figure 1) which corresponds to the former data. Each surface molecule expression was estimated individually in three repeats using FACSCantoII flow cytometer (BD, Franklin Lakes, NJ).

Figure 1. CD54 molecules expression intensity by JEG-3 line cells. (A) JEG-3 line trophoblast cell suspension without antibodies to CD54. (B) JEG-3 line trophoblast cell suspension processed with isotype antibodies. (C) Spontaneous CD54 expression by JEG-3 line trophoblast cells (white diagram) compared to non-stained cells (red diagram). (D) TNFα affected CD54 expression by JEG-3 line trophoblast cells (white diagram) compared to spontaneous CD54 expression (red diagram).

Table 1. Trophoblast cell surface receptors and their ligands.

Receptor	Ligand	Placenta ligand source
Cytokine receptors
CD118 (LIF R)	LIF	Endometrium [12], trophoblast [9]
CD119 (IFNγ-R1)	IFNγ	dNK cells, decidual CD8+ T-cells [13]
IFNγ-R2
CD120a (TNFα-R1)	TNFα	Mph, trophoblast, decidual CD8+ T-cells [13]
CD120b (TNFα-R2)	TNFα, lympotoxin	Mph
CD126 (IL-6αR)	IL-6	Cytotrophoblast, endometrium [12], decidual and placental Mph, endothelial cell (EC) [3], decidual CD8+ T-cells
CD130 (IL-6βR)
Chemokine receptors
CD181 (CXCR1)	IL-8	Endometrium, placental and decidual Mph [14], EC [15], decidual CD8+ T-cells
CD182 (CXCR2)
CD183 (CXCR3)	IP-10, Mig
CD184 (CXCR4)	SDF-1	Trophoblast
CD186 (CXCR6)	CXCL16	NK cells, trophoblast [16]
CD192 (CCR2)	MCP-1	Placental and decidual Mph, trophoblast
CCR1	RANTES, MIP-1α	T-lymphocytes, monocytes
Growth factor receptors
VEGFR1	VEGF, PlGF	Decidual Mph, placental Mph [3], trophoblast, EC
VEGFR2
CD140a (PDGFR1)	PDGF	EC, monocytes/macrophages
EGFR	EGF (epidermal growth factor)	Trophoblast, decidual membrane
TGFβ-R2	TGFβ	dNK cells, trophoblast [8]
HGFR	HGF (hepatocyte growth factor)	trophoblast
IGFR2	IGF (insulin-like growth factor)	Fibroblasts, cytotrophoblasts [8]
Integrins and adhesive molecules
CD29 (integrin β1)	CD106 (VCAM-1)	EC
CD49a (integrin α1)	Collagen	Extracellular matrix [17]
CD49e (integrin α5)	Fibronectin	Extracellular matrix [17]
CD49f (integrin α6)	Laminin	Extracellular matrix [17]
CD51/61 (integrin αvβ3)	Fibronectin, fibrinogen, vitronectin, thrombospondin, osteopontin, collagen
CD54 (ICAM-1)	LFA-1 CD11a\CD18	Leukocytes
CD104 (Integrin β4)	Collagen XVII	Extracellular matrix [17]
CD106 (VCAM-1)	VLA-4 (CD49d\CD29)	Leukocytes
Integrin β6	Fibronectin, vitronectin, fibrinogen	Extracellular matrix [17]
CD31 (PECAM-1)	CD31 (PECAM-1)	EC
Other receptors
HLA-G	KIR2DL4, ILT-2, ILT4, CD160	NK cells, CD8+ T-lymphocytes, Mph and dendritic cells, EC [18]
CD295 (LEPR)	Leptin	Trophoblast, peripheral blood fetal-maternal transport [19] fetus fetal-maternal transport [7]
CD95 (Fas)	CD95L (FasL)	NK cells, Mph, endometrium
E-Cadherin	E-Cadherin	trophoblast
VE-Cadherin (CD144)	VE-Cadherin (CD144	EC, trophoblast [20]

Statistical analysis

Statistical analysis was performed using Statistica 10 software (STAT Inc., Cary, NC). The descriptive statistics was used to characterize patient groups. The descriptive statistics and Mann-Whitney criteria was used to compare the values obtained by Flow cytometry method in spontaneous cultivation and with the addition of an inductor (supernatants or TNFα). The differences under p < .05 were considered to be significant.

Results

The incubation of JEG-3 line cells in the presence of third trimester physiological pregnancy placenta supernatants led to a relative increase of cell count, expressing CD118, CD119, IFNγ-R2, CD120b, CD181, CD183, CD186, CD192, CCR1, CD295, EGFR, TGFβ-R2, and Integrin β6 and decrease of cells expressing E-cadherin compared to a spontaneous level (Figure 2). Also an increased expression intensity of CD118, CD119, IFNγ-R2, CD181, CD183, CD186, CD192, CD49e, and Integrin β6 was seen together with IGFR2, CD104, and E-cadherin expression intensity reduction in the presence of third trimester physiological pregnancy placenta supernatants compared to the spontaneous level for JEG-3 line cells (Figure 2).

Figure 2. Relative count (A–D) and receptor expression intensity (E–H) changes by JEG-3 line cells in the presence of placenta supernatants of women with physiological pregnancy on the 38th and 39th weeks (group 1) or women with preeclampsia on the 38th and 39th weeks (group 2). The significance of differences between the groups: *p < .05; **p < .01; ***p < .001.

The JEG-3 line cells incubation in the presence of supernatants from third trimester preeclampsia complicated pregnancy placenta led to a relative increase of cell count expressing CD119, CD181, CD183, CD186, VEGFR1, CD140a, TGFβ-R2, Integrin β6, and VE-cadherin and fall of cell count expressing CD130, IGFR2, and E-cadherin compared to spontaneous level (Figure 2). Along with that, the presence of third-trimester preeclampsia complicated pregnancy placenta supernatants enhanced JEG-3 line cells expression intensity of CD118, CD119, IFNγ-R2, CD181, CD186, VEGFR1, CD140a, CD29, CD49e, and Integrin β6 and reduced expression intensity of IGFR2 and E-cadherin (Figure 2).

The relative JEG-3 line cell number expressing CD118, CD119, IFNγ-R2, CD120b, CD130, CD192, CCR1, CD295, EGFR as well as their CD130, CD183, IGFR2 expression intensity were lower in case of incubation in the presence of third trimester preeclampsia complicated pregnancy placenta supernatants compared to incubation in the presence of third trimester physiological pregnancy placenta supernatants (Figure 2).

No spontaneous or induced receptor expression for CD31, CD49a, CD51/61, CD95, CD106, CD120a, CD126, CD182 CD184, and VEGFR2 by JEG-3 line cells could be detected.

Discussion

The presented trophoblast receptor expression comparative analysis reveals that placenta secreted factors within the third trimester, irrespective of preeclampsia complication, raise the trophoblast sensitivity to cytokines LIF, IFNγ, IL-8, IP-10, MIG, CXCL16, and TGFβ (Figure 3) and increase its adhesion ability due to Integrin β6 expression growth (Table 1). Along with that JEG-3 line trophoblast cells reduce the IGF sensitivity and inter-cellular contact effectivity by suppressing E-cadherin expression (Figure 3). We also established the difference in placenta secreted factors effects on trophoblast in normal and pathology cases. Thus, in the presence of factors secreted in physiologically forming placenta, the trophoblast raises the expression of receptors to TNFα, lymphotoxin, MCP-1, RANTES, MIP-1α, Leptin, and EGF (Figure 3 and Table 1). The parallel decrease of CD104 expression, the extracellular matrix component receptor (Table 1), along with E-cadherin reduced expression may increase the trophoblast invasiveness [6]. However, HGFR, CD49f, CD54, and HLA-G expression remains quite high and does not change in the presence of placenta secreted factors neither in normal nor in pathology case (Figure 3).

Figure 3. JEG-3 line trophoblast receptor expression in the presence of third trimester physiological pregnancy placenta supernatants (left circle) and those of preeclampsia complicated pregnancy (right circle) compared to spontaneous level of the same receptor expression by intact JEG-3 line cells.

We have previously established that IL-4, IL-10, IP-10, MCP-1, and RANTES secretion in the third trimester is higher than in the first one [4]. Thus, trophoblast migration and proliferation stimulators in the third trimester may appear IL-4, IL-10 cytokines and IP-10, MCP-1, and RANTES chemokines, which are significant for trophoblast viability support at term pregnancy. Reduced expression of receptors for growth factors IGF and HGF (Figure 3 and Table 1) may be the basis for migration, proliferation, and trophoblast invasion limits. However, the increased expression of receptors for trophoblast migration stimulating chemokines IP-10 and MCP-1 by JEG-3 line cells (Figure 3 and Table 1) coincides with their previously established third-trimester placenta production [4]. On the other hand, the increased expression of receptors for IFNγ, TNFα, and TGFβ (Figure 3 and Table 1) by JEG-3 line cells in the same case witnessed the trophoblast cell availability to receive a migration inhibiting signal from microenvironment cells. Along with that under the condition of reduced third-trimester placenta production of VEGF, PDGF, bFGF, the elevated expression of receptors for LIF, EGF, and Leptin by trophoblast should ensure its viability. Thus, for example, Leptin stimulates proliferation and migration inhibiting trophoblast apoptosis [7]; EGF stimulates proliferation [8], invasion, differentiation [8], migration, and inhibits trophoblast apoptosis; LIF also stimulates proliferation and migration [9].

In the presence of placenta secreted factors under preeclampsia, the trophoblast enhances expression of receptors for VEGF, PlGF и PDGF (Figure 2 and Table 1), together with reducing expression of a receptor for IL-6 (CD130). At preeclampsia, compared to physiological pregnancy the enhanced placental secretion for angiogenin IL-8, IL-6 [10], PDGF и TGFβ [4] had been previously established along with reduced bFGF, IP-10, MCP-1, RANTES, and MIG secretion [10]. The receptor expression analysis showed lower JEG-3 line trophoblast cells expression for nearly all receptors in the presence of placenta secreted factors under preeclampsia compared to physiological pregnancy (Figure 3). Still, the trophoblast expression of CD29, CD140a (PDGFR), CD186 (CXCR6), Integrin β6, and VE-cadherin was higher in the presence of placenta secreted factors at preeclampsia compared to physiological pregnancy. Hence, JEG-3 line cells in the presence of placenta secreted factors at preeclampsia reduced the sensitivity to leptin, cytokines IFNγ, TNFα, LIF, chemokines IP-10, MCP-1, RANTES, and EGF growth factor. All the mentioned factors except TNFα and IFNγ have stimulating influence on trophoblast cells migration process in vitro. The results obtained are in complete accordance with data, establishing trophoblast cells damaged invasiveness under preeclampsia. Hence, under preeclampsia apart from reduced placenta production of factors stimulating trophoblast migration, such as MMP-2, VEGF growth factors, bFGF, chemokines IP-10, MCP-1, and RANTES [4], one can observe also the reduced receptor expression to the majority of these ligands. However, under preeclampsia, there are some compensatory mechanisms aimed at localization and inhibition of pathology processes in the placenta. Examples may include reduced trophoblast expression of IFNγ and TNFα, the production of which under preeclampsia is enhanced. PDGF is a factor stimulating the proliferation of trophoblast, endothelial cells, pericytes, and vascular smooth muscle cells. Thus, enhanced PDGF placenta production during pregnancy at preeclampsia [4] combined with CD140a and CXCR6 receptor expression increase in trophoblast cells may be also considered as a compensatory mechanism, strengthening trophoblast viability under preeclampsia in injured areas of the placenta. On the contrary, CD29 and Integrin β6 trophoblast expression increase may also suppress migration and in the case of endovascular trophoblast even ensure tighter junction with endothelial cells by VE-cadherin higher expression [11].

To summarize, the third-trimester placenta secreted factors both in health and in disease variably changed the trophoblast receptor expression of cytokines, preserving its availability to stimulating and inhibiting signals of proliferation and migration. The support of trophoblast viability together with its inhibited invasiveness is ensured not only by balanced cytokine production [4], but also through trophoblast expression variability, its availability to proliferation and migration signals. Despite the preferential placenta production of pro-inflammatory and anti-angiogenic factors under preeclampsia there are also compensatory mechanisms strengthening trophoblast viability: enhanced PDGF placenta production [4], increased CD140a and CXCR6 receptor expression by trophoblast together with lower trophoblast expression of a receptor for IFNγ and TNFα.

Disclosure statement

All of the authors have read the journal's policy on disclosure of potential conflicts of interest. The authors have no conflicts of interest to declare.

Additional information

Funding

This work was supported by Russian Federation State program аааа-а19-119021290116–1.