Molecular Human Reproduction, Vol. 7, No. 1, 97-101,
January 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Expression profiles of interleukin-15 in early and late gestational human placenta and in pre-eclamptic placenta
Department of Obstetrics and Gynaecology, National University Hospital, National University of Singapore, Lower Kent Ridge Road, Singapore 119074
Abstract
The presence of interleukin-15 (IL-15) mRNA in human placenta has been demonstrated previously. The present study was undertaken to investigate the expression profiles of IL-15 mRNA and protein in early and late gestational placental tissues, and also the effect of labour on its production. Levels of placental IL-15 expression were also determined in patients presenting with pre-eclampsia. An explant culture system was used to study the release of immunoreactive IL-15 by the placental tissues. Enzyme-linked immunosorbent assays were employed to quantify concentrations in the culture medium. The results showed that placental tissues from all groups released immunoreactive IL-15 into the culture medium. Moreover, the level of secretion by the term placental tissues was much higher than that by first trimester tissues. The presence of labour at term resulted in a further increase in placental IL-15 production. Reverse transcription–polymerase chain reaction (RT–PCR) was used to demonstrate the expression of IL-15 mRNA in these tissues. The results confirmed the expression of IL-15 in placenta from all the groups and the mRNA levels in the samples was highly correlated with the respective protein concentrations. Levels of both IL-15 mRNA and protein were significantly reduced in the pre-eclamptic placental tissue compared with the normal controls. The present study suggests an important role for this novel cytokine in human pregnancy.
first trimester/IL-15/labour/placenta/pre-eclampsia
Introduction
A successful pregnancy requires the maternal immune system to accept the immunologically semiallograft fetus. Although the key factors and precise mechanisms involved in this process are not clearly understood, it has been proposed that cytokines are one of the mediators of maternal immune reactivity (Clark, 1989
; Mitchell et al., 1993; Wegmann et al., 1993; Robertson et al., 1994). Cytokines and growth factors are known to play a pivotal role, not only in modulating maternal immune responses, but also in regulating other processes, e.g. implantation (Stewart et al., 1992), trophoblast invasiveness and tissue remodelling during placental development (Librach et al., 1994), and finally in labour (Opsjon et al., 1993). Aberrations in the cytokine network within the feto–placental unit have been suggested to be involved in pregnancy associated disorders, e.g. pre-eclampsia (Vince et al., 1995; Cross, 1996). Production of several cytokines, e.g. interleukin (IL)-1, IL-6, IL-8, IL-10, transforming growth factor-ß (TGFß) and tumour necrosis factor-
(TNF), by the human placenta has been reported previously (Stallmach et al., 1995; Paradowska et al., 1997; Agarwal et al., 2000).
A recent study has indicated the expression of a novel cytokine IL-15 in the human placenta (Grabstein et al., 1994). IL-15 is a 14 kDa glycoprotein which, like IL-2, acts as a T-cell stimulant and plays a pivotal role in cell-mediated immunity by activating T-cell proliferation and B-cell antibody production (Armitage et al., 1995). It also promotes natural killer (NK) cell cytotoxicity (Carson et al., 1994). In spite of its functional similarities to IL-2, IL-15 is unique in its cell and tissue distribution. Unlike IL-2, IL-15 is not produced by T-cells and is produced by a variety of tissues, e.g. spleen, skeletal muscles and placenta suggesting that it may have activities beyond the immune system (Grabstein et al., 1994). A recent study (Zygmunt et al., 1998) has suggested a role for IL-15 in cytotrophoblastic invasion, possibly by regulating expression of cell adhesion molecules. Its expression in the human endometrium and decidua indicates a role for IL-15 in uterine functions during pregnancy (Okada et al., 2000). Reports on the presence of IL-15 in the amniotic fluid are, however, conflicting. A recent study has shown the presence of IL-15 in the amniotic fluid and also revealed that fetal membranes are a source of its production (Fortunato et al., 1998). Failure to detect IL-15 in the amniotic fluid by another recent study (Searle et al., 2000) has led to speculations that, despite the presence of IL-15 mRNA in placental and fetal tissues, the production of IL-15 peptide may be restricted to bone marrow stromal cells, monocytes and dendritic cells. However, the identification of IL-15 in the culture supernatants of decidual cells and trophoblast cells purified from first trimester placental tissues has been reported recently (Verma et al., 2000). In the present study, we used the explant culture system to study both the release of immunoreactive IL-15 by human placenta at early and late stages of gestation, and the effect of labour on its production. In addition, the expression profile of IL-15 was investigated in the placental tissues obtained from patients presenting with pre-eclampsia, a common pregnancy associated disorder affecting 7–10% of pregnant women. The levels of IL-15 mRNA in each of these tissues were compared using semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR). It is envisaged that the present study may provide clues to the role of this cytokine in placental functions during human pregnancy.
Materials and methods
Sample collection
Placental tissues were obtained immediately after delivery: (i) from normal women undergoing elective termination of pregnancy at 7–12 weeks of gestation (group 1, n = 8), or elective Caesarean section at 37–41 weeks gestation (group 2, n = 10), or having normal spontaneous vaginal deliveries at term (group 3, n = 8); and (ii) from pre-eclamptic patients undergoing Caesarean section at term before the onset of labour (group 4, n = 8). Pre-eclampsia is defined as a maternal blood pressure of 
160/110 mmHg on two separate readings at least 6 h apart with proteinuria >5 g/24 h. Women from all the groups had no other clinical disorders such as chronic hypertension or chorioamnionitis. Chorionic villi were dissected free from fetal membranes and processing was commenced within 15 min of delivery. Tissues were either snap-frozen in liquid nitrogen and stored at –75°C for RNA extraction, fixed for histological viewing or processed immediately for explant cultures.
Short-term placental explant cultures
Placental tissue explants collected from the above mentioned groups were cultured as described previously (Agarwal et al., 2000). Briefly, placental tissue fragments were washed thoroughly with saline and minced into 1–2 mm diameter pieces. These placental explants were cultured in 6-well 35 mm polystyrene tissue culture dishes (Corning Glass Works, Corning, NY, USA) in Dulbecco's Modified Eagle's Medium (DMEM; GibcoBRL, Rockville, MD, USA) containing 100 IU penicillin/ml, 100 µg streptomycin/ml and 10% heat inactivated calf serum at 37°C in a 5% CO2 incubator. Explant cultures in each experiment were incubated for 24, 48 and 72 h. Cultures were harvested and the conditioned media collected at the end of the incubation period and stored at –80°C until analysis. Structural integrity and biochemical viability of the explant culture system was monitored regularly by histological examination of the processed tissue samples and steady state release of human chorionic gonadotrophin (HCG) into the culture medium by explants respectively. Protein concentrations in the explant culture medium were measured by the Coomassie Blue dye-binding assay (Bradford, 1976) using the protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA).
Measurement of IL-15 by enzyme-linked immunosorbent assay (ELISA)
Concentrations of IL-15 in the explant culture media were quantified by a sandwich ELISA (Chemicon International Inc, Tennecula, CA, USA) using a monoclonal antibody specific to IL-15. The sensitivity of IL-15 ELISA was 2.7 pg/ml. The range of standard curve was 7.81–500 pg/ml. Serial dilutions of the samples yielded linear responses parallel to the standard curve. The samples were read at 490 nm in a 96-well microplate auto-reader. All samples were analysed in the same batch of assay.
Total RNA isolation
Total RNA was extracted from frozen chorionic villi using the TRIzolR reagent following the manufacturer's instructions (Gibco-BRL). RNA integrity was checked by 1% denaturing agarose gel electrophoresis and the concentration was determined by spectrophotometer. To determine the levels of IL-15 transcripts in the tissue, RT–PCR was carried out using IL-15 specific primers. Isolated total RNA was treated with DNase to eliminate genomic DNA contamination before using it for RT–PCR. Human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as the standard for quantification.
Semi-quantitative RT–PCR analysis
In order to compare the levels of expression of placental IL-15 mRNA between the various groups studied, a semi-quantitative RT–PCR technique was performed following established procedures described previously with slight modifications (Rinehart et al., 1999; Gyomorey et al., 2000). Reverse transcription was performed on the total RNA isolated using the SuperScriptTM Preamplification system for the first strand cDNA synthesis following the manufacturer's instructions (Gibco-BRL). Briefly, equal amounts of RNA (2 µg) were used for first strand cDNA synthesis in a total reaction volume of 25 µl containing 200 IU of reverse transcriptase, 1 IU/µl RNasin (Promega, Madison, WI, USA), 2.5 µmol/l oligo dT, 1 mmol/l dNTP, 1x reaction buffer and 5 mmol/l MgCl2. Reaction lacking the reverse transcriptase enzyme was used as the negative control. The reaction was carried out at 42°C for 1 h. The samples were then heated to 75°C to stop the reverse transcription reaction. Aliquots of the first strand cDNA were stored at 4°C until amplification.
One tenth of the cDNA generated was subsequently used for PCR amplification using Thermus aquaticus (Taq) DNA polymerase (Promega). IL-15 specific primers used were as described previously (Fortunato et al., 1998). The sequences of the primers were: 5'-GTCTTCATTTTGGGCTGTTTCAGT-3' (sense) and 5'-CCTCACATTCTTTGCATCCAGATTCT-3' (antisense). The primer sequences used for G3PDH were: 5'-ACCACAGTCCATGCCATCAC-3' (upstream primer) and 5'-TCCACCACCCTGTTGCTGTA-3' (downstream primer). The predicted sizes of PCR products for IL-15 and G3PDH were 315 and 452 bp respectively.
Optimal PCR conditions for both IL-15 and G3PDH were determined in preliminary experiments, so that the number of cycles selected was within the linear range of amplification. IL-15 and G3PDH were amplified in the same round of PCR. Cycling regime was 94°C for 2 min followed by 30 cycles of 94°C for 1 min, 54°C for 45 s and 72°C for 1 min, and a final extension at 72°C for 10 min. Amplified products were fractionated by 1% agarose gel electrophoresis and the bands visualized under UV light after staining with ethidium bromide. The relative intensities of the bands were analysed by densitometric scanning using a commercially available software package (Gel-ProTM Analyzer, version 3.0; Media Cybernetics, Silverspring, MD, USA). The intensity of IL-15 band in each sample was normalized to the intensity of the G3PDH band of the same sample.
Cloning and sequencing of the IL-15 PCR product
The PCR products were electrophoresed on 1.5% agarose gel to fractionate the amplified bands for gel extraction. The 315 bp band corresponding to IL-15 (absent in no RT control) was gel extracted and purified using the QIAquick gel purification kit (Qiagen GmbH, Hilden, Germany). For PCR cloning, the pGEMT vector (Promega) was used. Ligation was performed using T4 DNA ligase (NEB, USA). Sequencing was carried out using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (The Perkin-Elmer corporation, Foster City, CA, USA). DNA sequences obtained were analysed using DNASIS version 2.5 (Hitachi Software Engineering Co. Ltd, Yokohama, Japan) software.
Statistical analysis
Values for concentrations of IL-15 are expressed as mean ± SEM/mg of the protein secreted unless otherwise indicated. Statistical analysis was performed with Student's t-test. P < 0.05 was considered to be statistically significant.
Results
IL-15 concentrations in the explant culture medium
Immunoreactive IL-15 was detected in the placental conditioned medium of all samples from the four groups of tissues. The secretion profile of IL-15 by placental explants of the various groups is illustrated in Figure 1. In order to study the production of IL-15 by early and late gestational placental tissues, IL-15 levels in groups 1 and 2 were compared. Mean ± SEM levels of IL-15 produced by group 1 placental villi were 38.71 ± 4.23, 57.7 ± 5.43, 90.3 ± 6.11 pg/mg protein at 24, 48 and 72 h respectively. These concentrations were significantly lower (P < 0.005) than the corresponding concentrations of IL-15 in conditioned medium of group 2 samples, which were 62.39 ± 5.76, 101.17 ± 6.52 and 140.4 ± 13.25 pg/mg protein, indicating that term placental explants produced higher amounts of IL-15 compared with the first trimester tissues. A comparison was also made of secreted IL-15 concentrations between patients undergoing spontaneous vaginal deliveries (group 3) and women having elective Caesarean section (group 2) at term. The concentrations of IL-15 in group 3 were 105.28 ± 5.88, 170.45 ± 12.51, 244.44 ± 25.32 pg/mg protein which were significantly higher (P < 0.005) than those of group 2, indicating that placental tissues in active labour produce enhanced concentrations of IL-15 compared with those from Caesarean section. Pre-eclamptic placental tissues (group 4) produced significantly lower concentrations (P < 0.001) of IL-15 compared with the normal controls (group 2). The mean ± SEM concentrations of IL-15 produced by explants from patients presenting with pre-eclampsia (group 4) were 19.36 ± 2.9, 33.87 ± 3.34, 52.29 ± 4.99 pg/mg protein at 24, 48 and 72 h respectively. Since the tissues in the pre-eclamptic group were collected after Caesaerean section at term, group 2 samples which were also obtained following the similar procedure were taken as the control for comparison.
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IL-15 expression in the placental tissues
IL-15 mRNA expression as detected by RT–PCR in placental tissues of the four groups studied is illustrated in Figure 2
. An IL-15-specific 315 bp PCR product was amplified in placental villi of all the groups. The sequence of the PCR product was found to be identical to the published sequence (Grabstein et al., 1994). Negative control samples (without RT reaction) revealed no amplification product. G3PDH, the housekeeping gene used for this study, was abundantly expressed in all the samples. To compare the levels of IL-15 transcripts between the various groups, densitometric scanning of the bands was performed. Levels of IL-15 mRNA were expressed as a ratio of IL-15 band intensity to the G3PDH band intensity as shown in Figure 3. Results indicated that the level of IL-15 mRNA in the term placental villi (group 2) was ~2.4-fold higher (P < 0.01) than in the first trimester (group 1). It was further elevated in placental tissues from women with normal vaginal delivery at term (group 3), the levels being 1.77-fold higher than those undergoing Caesarean section (group 2; P < 0.005). IL-15 mRNA expression was observed to be significantly reduced in the pre-eclamptic placenta (group 4), the level of which was ~3.5-fold lower than the normal controls (group 2; P < 0.01).
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Discussion
IL-15, a proinflammatory cytokine, which shares a number of biological activities with IL-2, plays a role in regulation of T, B and NK cell proliferation, and acts as a growth promoter (Quinn et al., 1995; Azzarone et al., 1996). It also has a unique action on non-lymphoid cells, e.g. muscle, brain microglia and mast cells (Quinn et al., 1995; Tagaya et al., 1996). Although IL-15 mRNA was shown to be expressed constitutively by a number of cell and tissue types (Grabstein et al., 1994), it has been difficult to demonstrate the presence of IL-15 peptide in the supernatants from many cells that express mRNA for this cytokine.
The results of the present study confirm previous reports that IL-15 mRNA is expressed by the human placenta (Grabstein et al., 1994) which is also the source of its immunoreactive peptide (Verma et al., 2000). In addition, our data for the first time demonstrate that both expression of IL-15 mRNA and release of the immunoreactive peptide are significantly higher in the term placental tissues than that of the first trimester, suggesting that IL-15 expression in the placenta may be modulated according to gestational age. Moreover, it was also observed that placental tissues obtained from women delivering spontaneously at term produced significantly higher IL-15 concentrations when compared with those undergoing Caesarean section.
The process of labour is known to be associated with an increased expression of mRNA for the inflammatory cytokines in both maternal and fetal tissues (Opsjon et al., 1993; Saito et al., 1993; Dudley et al., 1996). Cytokines can induce the biosynthesis of prostanoids and other uterotonins in gestational tissues (Hansen et al., 1999). The increased production of IL-15 towards term may be consistent with the view that this cytokine may also have a role in labour (Fortunato et al., 1998). IL-2 has been shown to inhibit progesterone secretion and induce prostaglandin E2 synthesis (Ohno et al., 1994). Since IL-15 is known to share a number of functional activities with IL-2, both cytokines may have synergistic effects on prostaglandin synthesis. In addition, the stress of labour may further potentiate placental IL-15 expression since it has been suggested that IL-15 can be induced by stress and its expression is regulated by stress-related proteins, e.g. heat shock proteins (Mohamadzadeh et al., 1995).
During human pregnancy, cytokines play a role not only in modulation of the maternal immune system, but also in regulatory processes associated with placental growth and development (Graham and Lala, 1991; Guilbert et al., 1993). Invasion of trophoblast cells into the uterine wall and the remodelling of the utero–placental vessels occur during the development of the human placenta. Abnormally shallow cytotrophoblast invasion resulting in failure of physiological remodelling of decidual vessels with concomitant structural defects in the spiral arterioles contributes to the pathogenesis of pre-eclampsia (Meekins et al., 1994; Genbacev et al., 1999). Cytokines have been implicated in the regulation of trophoblast invasion (Graham and Lala, 1991). A recent study has suggested a role for IL-15 in trophoblast invasion and migration (Zygmunt et al., 1998). It has also been speculated that IL-15 might be influencing the changes in cell adhesion molecule phenotype during the process of invasion. Thus, IL-15 produced during early pregnancy may be involved in the process of trophoblastic cell invasion and tissue remodelling, a process vital to normal placental development.
Our data also demonstrate, for the first time, a significant decrease in both IL-15 mRNA and protein concentrations in pre-eclamptic placenta compared with normal controls which could be implicated in defective placentation. In addition, IL-15 is reported to be a potent inhibitor of apoptosis (Bulfone-Paus et al., 1997) and may also influence the placental growth by this pathway as well. Apoptosis plays an important role in placental remodelling (Smith et al., 1997) and previous studies have indicated a role for cytokines in this process (Lea et al., 1999). IL-15 may also affect placental development through its role as a promoter of angiogenesis, as shown in the murine system (Angiolillo et al., 1997).
IL-15 regulation is known to be multifaceted, controlled at the level of transcription and also post-transcriptionally at the levels of translation and intracellular trafficking (Waldmann and Tagaya, 1999). Results of the present study show that changes in the placental IL-15 protein concentrations coincide with changes in the mRNA level, suggesting that the expression of IL-15 in the placenta could be primarily regulated at the transcriptional level. Thus, from this and other studies it may be concluded that IL-15 has an important role of its own in human pregnancy.
Acknowledgments
This work was supported by a grant from the Lee Foundation, Singapore and the research fund of our Department. R.A. is a recipient of the post-graduate scholarship from the National University of Singapore.
Notes
1 To whom correspondence should be addressed. E-mail: obgannam{at}nus.edu.sg 
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Submitted on August 8, 2000; accepted on November 2, 2000.
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