Mol. Hum. Reprod. Advance Access originally published online on April 22, 2008
Molecular Human Reproduction 2008 14(5):281-289; doi:10.1093/molehr/gan018
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IFN-
-mediated extravillous trophoblast outgrowth inhibition in first trimester explant culture: a role for insulin-like growth factors
1Department of Obstetrics and Gynaecology, University of British Columbia, 2H30-4500 Oak Street, Vancouver, BC, Canada V6H 3N1 2Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada 3Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 4Department of Dermatology and Skin Science, University of British Columbia, Vancouver, Canada
5 Correspondence address. E-mail: pvd{at}cw.bc.ca
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Abstract |
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Pre-eclampsia is often associated with inadequate cytotrophoblast invasion and remodelling of the uterine spiral arteries. Examining a first trimester, 2D in vitro explant culture model which mimics in vivo placentation, including trophoblast column formation and extravillous cytotrophoblast (EVT) migration, we previously suggested that excessive maternal decidual natural killer cell interferon (IFN-
) limits EVT migration. Types-1 and -2 insulin-like growth factor (IGF-1, IGF-2) are trophic for EVT, act through their surface receptors, IGFR-1 and IGFR-2, and are regulated by the IGF-binding proteins (IGFBPs). Could the observed IFN--mediated inhibition of EVT outgrowth and migration be related to either expression changes of IGF-1 or IGF-2, their receptors, their binding proteins, or apoptosis? Using the 2D explant culture model, we examined the effect of IFN- exposure on IGF-1 and -2, IGFR-1 and -2, IGFBPs and apoptosis. IFN- relatively increased IGF-1 and -2 secretion. In EVT, IFN- decreased IGFR-2, but not IGFR-1 expression. IGBP-2, -3 and -4 production were not influenced by IFN-. IFN- induced trophoblast apoptosis measured by the highly sensitive M30 neo-epitope, but not caspase 3 activity, in conditioned medium and EVT cell lysates. The observed IFN--mediated EVT migration inhibition may occur through the down-regulation of IGFR-2 and subtle induction of EVT apoptosis.
Key words:
villous explant culture/IFN-/insulin-like growth factors/insulin-like growth factor receptors/insulin-like growth factor binding proteins
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Trophoblast proliferation, migration and invasion in situ are regulated by locally derived cytokines, growth factors, growth factor-binding proteins, extracellular matrix and adhesion molecules (Chakraborty et al., 2002). For example, interferon IFN-
is produced by decidual natural killer (dNK) cells (Haddad et al., 1997; Hu et al., 2006). IFN- has antiviral (Dallalio et al., 1996; Bocher et al., 1999), anti-proliferative (Martin et al., 1993) and differentiating effects (Martin et al., 1993), and can induce apoptosis of primary human trophoblast (Garcia-Lloret et al., 1996; Lash et al., 2006), and mouse embryonic stem cells (Zou et al., 2000) and placenta (Liu et al., 2002). In vitro, IFN- alters blastocyst attachment and trophoblast outgrowth (Hill, 1992), and increases miscarriage (Clark and Croitoru, 2001) and early embryo loss (Haddad et al., 1997) in rodents. Plasma IFN- is elevated in women with pre-eclampsia (Saito et al., 1999); lymphocytes from term pre-eclampsia placentae produce large amounts of IFN- (Wilczynski et al., 2003). These findings supported our hypothesis that excessive IFN- may be harmful to pregnancy and so to fetal growth. Type I insulin-like growth factor (IGF-1) and type II IGF-2 at the maternal–fetal interface have been reported in both human and other species (Hill et al., 1993; Han and Carter, 2000; Qiu et al., 2005). IGF-1, produced by a subpopulation of placental villous mesenchymal cells, provides paracrine stimulation of trophoblast proliferation and migration (Baker et al., 1993; Lacey et al., 2002); whereas IGF-2, largely produced by extravillous cytotrophoblast (EVT) throughout gestation (Han et al., 1996; McKinnon et al., 2001), provides autocrine stimulation of trophoblast proliferation and migration.
The biological effects of IGF-1 and IGF-2 are mediated through specific cell surface receptors, IGFR-1 and IGFR-2 (mannose-6-phosphate receptor) (Jones and Clemmons, 1995). IGFR-1 possesses tyrosine kinase activity and binds both IGF-1 and IGF-2 with equal affinity (Jones and Clemmons, 1995; Stewart and Rotwein, 1996). IGFR-2 has greater affinity for IGF-2 than for IGF-1 (Hamilton et al., 1998). In addition to binding to cell surface receptors, IGFs can also bind to a family of binding proteins present in blood and at the maternal–fetal interface. IGF-binding proteins (IGFBPs) have been shown to influence IGF action in a positive or a negative manner (Hamilton et al., 1998). IGFBPs may enhance IGF-mediated biological actions by retarding the degradation and clearance of IGFs or facilitating the binding to their receptors (Hamilton et al., 1998). IGFBPs may reduce IGF biological actions by sequestering the ligands away from their receptors (Hamilton et al., 1998).
IGF-1 expression and production can be down-regulated by IFN- in mouse macrophages (Arkins et al., 1995; Wynes and Riches, 2003), and IGF-2 down-regulation by IFN- has also been reported in human neuroblastoma (Martin et al., 1993) and fetal adrenal cell cultures (Ilvesmaki et al., 1993). IFN- down-regulates membrane interleukin-6 (IL-6)-binding GP80 protein in myeloma cells (Portier et al., 1993), and stem cell factor and erythropoietin receptors in human haematogenous cell lines (Taniguchi et al., 1997). The production and expression of IGFBPs is increased by pro-inflammatory cytokines such as tumour necrosis factor TNF-
and IL-1β (Price et al., 2002). However, it remains unclear whether or not IFN- can regulate the expression of IGFs, their receptors and their binding proteins in human trophoblast explants.
Using a concordant human first trimester explant and dNK co-culturing model system, we have shown that dNK cells inhibit EVT migration and that dNK-dependent EVT migration inhibition is mediated by dNK-produced soluble factors (Hu et al., 2006). Neutralizing antibody against IFN- can partially reverse this effect (Hu et al., 2006).
In this study, we have extended our first trimester explant model observations, investigating the effect of exogenous IFN- on (i) IGF-1 and IGF-2 expression and secretion; (ii) IGFR-1 and IGFR-2 expression in EVT, (iii) IGFBP-2, -3 and -4 expression and secretion, to determine whether or not changes in IGFs, IGFRs and IGFBPs contribute to the observed IFN--mediated effect on trophoblast proliferation and migration and (iv) whether or not the observed IFN- effect on EVT outgrowth and migration might be modulated in part by trophoblast apoptosis.
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Explant culture
We prepared first-trimester placental explant cultures on rat tail collagen type-I, as described (Hu et al., 2006). The gestational age of the specimens was between 6 and 10 weeks. For each assay, explants were from the same placental specimen. After an initial period of 24 h, during which cytotrophoblast proliferation occurs at the villous tips, the explant cultures were incubated with 0.2–5.0 ng/ml human recombinant IFN-
(R&D Systems, Minneapolis, MI, USA), generally for a total of 96 h. To test if neutralizing antibody to IFN- (R&D Systems) could reverse any IFN- effect, IFN- was pre-incubated with neutralizing antibody (10 µg/ml) for 30 min on ice. To reduce the influence of varying villous tip size on the data, each assay group usually contained 4–6 explants for each placental sample. EVT outgrowth occurs largely across the surface of the collagen gel. The observations were made on EVT column formation, EVT outgrowth pattern and outgrowth distance on individual sites at villous tips as described in our previous publication (Hu et al., 2006). Briefly, the distance of the cell outgrowth was measured from the villous tips to the distal edge of the outgrowth, using the arbitrary scale provided by the microscope aperture grid. The distances were then corrected to the mean distance in the control villous tips for each placental sample. This was to reduce the effect of growth potential variation between the 10 individual samples of reproductive tissues, providing observed/expected data for analysis.
Measurement of expanded numbers of EVT in outgrowths by MTS staining
The expanding numbers of EVT were measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) tetrazolium assay (Promega, Madison, WI, USA). At the end of the explant culture (96 h incubation), villi were carefully dissected off from villous tips in IFN--treated or -untreated groups. Collagen gels with attached EVT were then transferred into 96-well plates and the volume made up to 100 µl. Twenty-five microlitre of MTS was added to each well. MTS changes colour as reduced intracellularly in proportion to the number of cells and incubation time (usually 4–6 h after adding MTS). The plate was read at 490 nM wavelength. To account for the variation between villous tips, at least 4–6 explants were evaluated in each assay group, the data were standardized to the mean control per explant and the normalized data were then averaged from three different placental samples.
Measurement of IGFS and IGFBPS in explant culture conditioned medium
To determine if EVT outgrowth and migration inhibition in the IFN--treated group were mediated through production changes of IGFs and IGFBPs, the explant culture conditioned media were collected after 24, 48, 72 and 96 h incubation. Conditioned media were pooled from the same assay group and used for the measurement. IGF concentrations from different time points and IGFBP concentrations at 96 h incubation were measured by enzyme-linked immunoabsorbent assay (ELISA) following the manufacturers’ instructions (IGF kits, Antigenix America Inc, Huntington Station, NY, USA; IGFBP kits from R&D Systems). To minimize the potential effect of villous tip size on variation in IGF and IGFBP secretion, the conditioned media from five individual placental specimens were evaluated. The concentrations of IGFs and IGFBPs were corrected by total protein concentrations (Dc protein method, Bio-Rad Laboratories, Mississauga, ON, Canada) and then averaged from five individual specimens.
Total RNA preparation and first-strand cDNA synthesis
At the end of 24, 48, 72 and 96 h incubation of explant culture, villi were carefully dissected from outgrowth sheets, pooled from the same assay group and kept for RNA extraction. In parallel, EVT were lysed and pooled from each assay group for RNA extraction. RNA was extracted according to the manufacturer’s protocol (RNeasy mini kit, Qiagen Inc., Mississauga, ON, Canada). cDNA synthesis was followed as described by the manufacturer (ThermoScript RT–PCR System, Invitrogen Corp, Burlington, ON, Canada). There were four clinical samples evaluated, with 
4 tips per experimental group per sample.
Real-time quantitative PCR
SYBR dye-based real-time quantitative PCR was performed to quantify the mRNA expression using the ABI prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). After 2 min at 50°C and 10 min at 95°C the samples were cycled 40 times at 95°C for 15 s and 60°C for 60 s. The relative quantification of gene expression was calculated using 18S gene expression as endogenous control as described (Fink et al., 1998) and presented as fold changes compared with the medium control group.
IGF-1 and IGF-2 and their receptor IGFR-1 and IGFR-2 protein mRNA expression were evaluated. The primer sequences with Genebank accession number and amplicon size are summarized in Table I.
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Immunocytochemistry staining for IGFR-1 and IGFR-2 expression
To assess the time course of any IFN-
-mediated effect on IGFR-1 and -2 expression in EVT, explants were treated for 24, 48, 72 or 96 h, fixed with 70% methanol at each time point and stored at –20°C for batch immunocytochemistry. Briefly, stored explants were permeabilized with 0.5% Triton-100 for 5 min and then incubated with rabbit anti-human IGFR-1 and -2 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), overnight at 4°C and then washed extensively with phosphate-buffered saline (PBS). Biotinylated link universal secondary antibody was added and incubated for 1 h at room temperature, followed by incubation with streptavidin–horse-radish peroxidase (DakoCytomation, Mississauga, ON, Canada). After washing with PBS, AEC substrate was applied. Images were taken at x4 magnification with a digital NIKO camera. Samples from three women were evaluated.
Measurement of IGFR-1 and IGFR-2 expression in EVT by flow cytometry
Villous tissues were dissected from explant cultures at the end of 96 h incubation, and then EVT were collected following detachment with 0.25% EDTA, and pooled from the same assay group. The EVT were fixed with Cell Fixation Buffer (Biosource International, Camarillo, CA, USA) for 10 min, and then washed with Cell Permeabilization Buffer (Biosource). EVT were incubated with rabbit anti-human IGFR-1 antibody or rabbit anti-human IGFR-2 antibody (Santa Cruz Biotechnology, Inc) diluted in Cell Permeabilization Buffer to 2 µg/ml. Rabbit IgG was used as isotype control. After 1 h incubation on ice, EVT were washed and stained with FITC-conjugated anti-rabbit secondary antibody which was also diluted in Cell Permeabilization Buffer. After 30 min incubation on ice, EVT were washed again and analysed by flow cytometry (FACSscan, Becton Dickinson, Oakville, ON, Canada). Samples from three women were evaluated.
Measurement of trophoblast apoptosis by M30-Apoptosense ELISA and Caspase-3 activity
To investigate any influence of IFN- exposure on trophoblast (villous and EVT) apoptosis, cell extracts from EVT and villous tissues were prepared and explant conditioned medium reserved at the completion of 96 h incubation. Following the manufacturer’s instructions, M30-Apoptosense assays of these extracts and media were performed (Peviva, Brömma, Sweden). The concentration of the M30 neo-epitope of CK18Asp396 fragments was corrected by total protein concentration, measured by the DC protein method (Bio-Rad Laboratories), and standardized to the mean control result per explant. Caspase-3 activity in the lysates of EVT and villi was measured as previously described (Hu et al., 2006). Staurosporine was used as a positive apoptosis inducer in these two assays. Samples from three women were evaluated, with 4 tips per experimental group examined per clinical sample.
Statistical analysis
Groups were compared using either parametric one-way analysis of variance (ANOVA), with Bonferroni’s multiple comparison tests, or non-parametric Kruskal–Wallis ANOVA, with Dunn’s post-test, as appropriate, using GraphPad Prism 4.0 software (San Diego, CA, USA). Statistical significance was set at P < 0.05.
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IFN-
-mediated inhibition of EVT outgrowth and migration, but not integrin expressionExplant attachment on collagen gel I and EVT outgrowth and migration over the gel surface have been described in detail previously (Hu et al., 2006). After overnight attachment, villous explants were either exposed to culture medium or to various concentrations of IFN-
. In the medium control group, EVT formed columns and multilayered sheets of cells in 24–48 h, and migrated out in a radially oriented direction, especially from column edges, usually in 48–96 h. In the presence of exogenous IFN-, especially with concentrations 1 ng/ml (up to 50 ng/ml has been tested), EVT outgrowth and EVT outgrowth expansion occurred over several days, but the majority of EVT remained tightly packed at the edge of the columns; migration of EVT away from the columns was not observed (Fig. 1A). Both expanding numbers of EVT in each outgrowth (MTS staining; Fig. 1B) and outgrowth distance (Fig. 1C) were inhibited in the IFN--treated groups (Hu et al., 2006). IFN- concentrations ranging 0.2–5 ng/ml were chosen for the remaining experiments. Neutralizing antibody to IFN- reversed the IFN--induced inhibitory effect on EVT migration and numerical expansion (Fig. 1A–C).
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Although EVT outgrowth and proliferation seemed to be inhibited by exposure to IFN-
, integrin-1 and -5 expressions in EVT were not affected (data not shown).
We have shown that dNK could inhibit EVT outgrowth and migration in a first trimester collagen gel model (Hu et al., 2006). dNK-mediated EVT migration and outgrowth inhibition was associated with changes in the cytotrophoblast expression of metalloproteases-2, -9 and plasminogen activator inhibitor-1. We have also demonstrated that dNK-dependent EVT migration inhibition is mediated by dNK produced soluble factors, and that neutralizing antibody against IFN- can partially reverse this effect (Hu et al., 2006). IFN- is one of the major cytokines produced by dNK cells. IFN- regulates the expression and production of many growth factors. Because IGFs are critical growth factors in cytotrophoblast proliferation and migration, we examined whether or not IFN- influenced the expression of IGFs in this ex vivo model of human placentation.
IFN--mediated IGF-1 and IGF-2 expression and production in explant culture
Only IGF-2 mRNA, but not IGF-1 mRNA, expression was detected in EVT harvested from explant cultures, as previously described (Lacey et al., 2002), and IFN- inhibited its expression in both whole-villous tissue and isolated EVT at 24, 48, 72 and 96 h of incubation (Fig. 2A and B). IGF-1 mRNA expression was only detected in villous tissue, and its expression was down-regulated by IFN- (Fig. 2C). IGF-1 and -2 secretions into explant culture conditioned medium were measured. Despite inhibition of IGF-1 and IGF-2 mRNA expression by IFN-, no inhibition was observed of either IGF-1 or IGF-2 secretion; rather IGF-1 and IGF-2 secretions were increased by IFN- treatment at all time points (Fig. 2D and E).
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The biological effects of IGF-1 and IGF-2 on EVT are largely mediated through their receptors IGFR-1 and IGFR-2, respectively. Therefore, we examined whether or not IFN-
treatment could regulate their receptor expression on EVT.
IFN--mediated IGFR-1 and IGFR-2 expression on EVT
Both IGFR-1 and IGFR-2 mRNA were detected in EVT, and their expressions were significantly down-regulated by IFN- exposure at the end of 96 h incubation (Fig. 3A). Both IGFR-1 and IGFR-2 protein level on EVT were detected by immunohistochemistry (IHC) and flow cytometry. The IHC results demonstrated that IGFR-2 expression in EVT was reduced by IFN- (particularly at concentrations 5 ng/ml) at 24, 48, 72 and 96 h (Fig. 3B). However, IGFR-1 expression level was not significantly down-regulated by IFN- at any time point (data not shown). Flow cytometry was only applied to EVT collected following 96 h incubation and confirmed that IFN- could slightly reduce IGFR-1 and greatly reduce IGFR-2 expression level. The expression of IGFR-1 and IGFR-2 was decreased by 
8.5 and 28%, respectively, when IFN- concentration reached 5 ng/ml; the reduction was by 3.2 and 14%, respectively, when IFN- was at 1 ng/ml.
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In addition to IGFR-1 and IGFR-2, the actions of IGF-1 and IGF-2 are influenced by their binding proteins; therefore, we investigated the effect of IFN-
on the IGFBPs.
IFN--mediated IGFBP-2, -3 and -4 expression and production in explant culture
IGFBP1-6 mRNA expression in both villous tissue and EVT were investigated in the study. Villous tissue expressed all six IGFBP mRNA; IGFBP-3 showed the highest expression, followed by IGFBP-5, -4 -2 -1 and -6. In EVT, IGFBP-3 mRNA level was also the highest, followed by IGFBP-2 and IGFBP-4; IGFBP-5 and -6 mRNA expressions were low. IGFBP-1 mRNA expression was not detected (data not shown). Since IGFBP-2 -3 and -4 showed relative high mRNA expression in EVT, they were chosen for further investigation of mRNA expression and protein production in conditioned medium after IFN- treatment. IGFBP-2, -3 and -4 mRNA expressions on both of villous tissue and EVT were not significantly affected by IFN- treatment (data not shown). Consistent with mRNA expression in EVT and villi, IGFBP-3 production was the highest, followed by IGFBP-2 and -4. The average concentrations of IGFBP-2, -3 and -4 from five specimens reached 7.5, 13.1 and 0.345 ng/ml, respectively; however, these concentrations were not significantly influenced by IFN- (Fig. 4).
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IFN-
-induced trophoblast apoptosis had been previously reported (Garcia-Lloret et al., 1996; Zou et al., 2000; Liu et al., 2002; Lash et al., 2006). We examined whether or not trophoblast apoptosis might be involved in the observed IFN--mediated EVT outgrowth and migration inhibition.
Trophoblast apoptosis
At the end of the 96 h incubation time, the M30 neo-epitope, the cleavage site for cytokeratin 18, was detectable in explant culture conditioned medium, in EVT cell extracts, and cell extracts from villi, and increased in the staurosporine-treated group in all experiments; following IFN- treatment, M30 generation was increased in explant culture conditioned medium compared with control medium (Fig. 5). Similar differences were noted in the EVT cell extracts, but were not significant, compared with control medium, by Dunn’s post-test which corrects for multiple comparisons [Mann–Whitney U test P = 0.002 (control medium versus IFN- treatment)]. The differences in M30 neo-epitope were not observed in cell extracts from villi. No differences were noted in caspase-3 protein activity in the lysates of EVT and villous tissue between control and IFN--treated groups; staurosporine acted as a positive control (data not shown).
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IFN-
can be deleterious to pregnancy, as determined by in vivo animal model studies (Haddad et al., 1997; Clark and Croitoru, 2001; Liu et al., 2002). In this, and our previous (Hu et al., 2006), study, we provide direct evidence to show that IFN- can alter EVT outgrowth and outgrowth pattern using a first trimester explant culture model.
Under normal culture conditions, EVT grew out, and after 48–72 h, EVT started to break off from the edge of the column and migrate out; this process mimics in vivo placentation. In the presence of IFN-, even at 50 ng/ml, EVT still formed cell columns which expanded over several days of incubation. However, the majority of EVT remained tightly packed within the column. The whole outgrowth pattern was totally different from control group (Fig. 1A). The expanded EVT number and outgrowth distance were significantly affected (Fig. 1B and C). EVT have been described as both proliferative (Genbacev et al., 1992) and non-proliferative (Lash et al., 2006). Using BrdU incorporation, Aplin et al. (1999) found that cytotrophoblast of the proximal column maintained its proliferative potential for 24 h but ceased proliferation by 48 h of culture. MTS staining measures the number of cells, not their proliferation. Our own Ki (cell nuclear marker of proliferation) staining suggested that EVT from explant cultures are non-proliferative (data not shown). Therefore, the decreased MTS staining in IFN--treated groups implies that IFN-g inhibited either trophoblast proliferation at the level of the villous tips or EVT movement from the tips into the outgrowths.
Croy and her group indicated that dNK cells were essential for the initiation of pregnancy-associated spiral arterial modification through their production of IFN- in the mouse model (Monk et al., 2005). We do not believe this to be inconsistent with our current findings. Physiological amounts of IFN- may play regulatory roles in trophoblast invasion and differentiation. However, under pathological circumstances, IFN- may be deleterious to pregnancy. It has been reported that IFN- is elevated in pre-eclampsia (Saito et al., 1999), and decidual lymphocytes produce large amounts of IFN- (Wilczynski et al., 2003). Hanna et al. (2006) demonstrated that dNK may stimulate trophoblast invasion, a finding at odds with our own; they used dispersed and isolated trophoblasts, whereas we have used explants. It may be that trophoblast behaviour ex vivo is influenced by interactions with either other cell lines or other trophoblast cell types present in villi that are not present in isolated trophoblast cultures. Another difference in experimental design was their use of a 3D Matrigel model, whereas we used a 2D collagen model. Trophoblast cells change from integrin-5 to integrin-1 expression as they migrate into Matrigel (Caniggia et al., 1997); this ‘integrin switch’ does not occur with cytotrophoblast migrating in the collagen model (Aplin et al., 1999; Hu et al., 2006). Resolving these differences may unravel further details of the control of EVT invasion and differentiation.
IGF-1 and IGF-2 are important growth factors for trophoblast; they provide paracrine and autocrine stimulation of trophoblast migration and proliferation (Baker et al., 1993; Han et al., 1996; McKinnon et al., 2001; Lacey et al., 2002; Qiu et al., 2005). IFN- has been reported to influence IGF-1 and -2 gene expression in many cell types (Ilvesmaki et al., 1993; Martin et al., 1993; Arkins et al., 1995; Wynes and Riches, 2003). However, the effect of IFN- on IGF-1 and -2 gene expression has not previously been examined in a human explant culture model.
In our model system, we were able to demonstrate that IGF-1 and -2 protein concentrations in the conditioned medium of the villous tip cultures were increased by IFN- treatment (Fig. 2D and E). However, IGF-2 mRNA was reduced in both EVT and villi (Fig. 2A and B). While IGF-1 mRNA was not detectable in EVT, confirming the findings of Lacey et al. (2002), it was detectable in villi and down-regulated by IFN- (Fig. 2C). This suggests that IGF-2 production was probably regulated at the post-transcriptional or post-translational level. IGF-2 serum profiles of women carrying fetuses with growth restriction display greater levels of pro-IGF-2 when compared with controls, thus reflecting aberrant IGF-2 processing (Qiu et al., 2005). In addition, IFN- induced both IGF-1 and IGF-2 production in the villous stromal cells present in the villous explant model; in other mammals, placental stromal cells produce IGF-1 and -2 (Reynolds et al., 1997; Correia-da-Silva et al., 1999; Dalcik et al., 2001). Increased stromal production of IGF-1 and -2 may have resulted in compensatory down-regulation of the IGFR-1 and -2 on EVT, as well as down-regulation of IGF-2 production by the EVT. Increased IGF-2 in the conditioned media may also reflect reduced uptake or fewer IGFR-2 present on cells.
Pre-eclampsia is a condition associated with shallow trophoblast migration and invasion, especially when of early onset. IGF-2 concentrations may be increased prior to, and both IGF-1 and -2 with, the onset of clinical pre-eclampsia (Giudice et al., 1997; Hubinette et al., 2003) and fetal growth restriction (Qiu et al., 2005). Our model, using first trimester samples, suggests that locally increased levels of IFN- may participate in the elevation of these growth factor levels; however, it is possible that the changes noted at the time of clinical diagnosis (arising usually late in the second or during the third trimesters) are the consequence of, rather than precede, clinical disease.
We postulated that elevated IGF-1 and -2 might lead to enhanced placental function and thus, fetal growth. Variation of tip sizes could cause fluctuations in IGF-1 and -2 concentrations. We attempted to address this problem by preparing villous tips of as similar dimensions as possible, by culturing 4–6 explants for each study group from each placental specimen, and by then pooling the conditioned media from each group. In addition, IGF-1 and -2 concentrations were corrected by total protein concentrations and averaged from five different placental samples.
IFN- has been reported to down-regulate IL-6 receptor expression in IL-6-dependent myeloma cells (Portier et al., 1993) and stem cell factor and erythropoietin receptors, but not IGFR-1 in human erythroid colony-forming cells (Taniguchi et al., 1997). Our own data suggest that IGFR-2 expression on EVT was inhibited by exposure to IFN-, both at the mRNA (Fig. 3A) and protein levels at all time points by immunocytochemistry (Fig. 3B) and at 96 h by flow cytometry (Fig. 3C). Introducing a function-blocking antibody to IGFR-2 into the explant culture model will facilitate the understanding of the roles of IGF-2 and IGFR-2 in this biological system. However, we could not identify a functionally verified IGFR-2 blocking antibody. IGFR-1 mRNA expression in EVT was inhibited by IFN- (Fig. 3A), but inhibition was not significant at the protein level by either immunocytochemistry (data not shown) or flow cytometry (Fig. 3C). Therefore, IGF-2-dependent autocrine stimulation of EVT outgrowth and migration was probably down-regulated. IGF-2 acts mainly through IGFR-2, independent of IGFR-1 and its binding proteins (McKinnon et al., 2001). EVT had relatively high expression of IGFR-1 and its expression was not significantly reduced by IFN- exposure (Fig. 3C); however, it functioned poorly probably due to its low affinity. This observation was also made on HTR8 cells, an in vitro propagated normal EVT cell line that expresses the phenotypic and functional properties of EVT cells. HTR8 cells express IGFR-1 and -2 which respond well to IGF-2 but poorly to IGF-1 (Lee et al., 2001). Indeed, IGFR-1 down-regulation does not appear to occur in complicated pregnancies: placental IGFR-1 expression does not vary between appropriate for gestational age pregnancies, and those complicated by either fetal growth restriction (Holmes et al., 1999) or pre-eclampsia (Diaz et al., 2005).
The interaction between IGFs and their receptors is modulated both negatively and positively by a family of six IGFBPs (Han et al., 1996). The affinity of IGFs to IGFBPs is equal to or greater than to IGF receptors (Jones and Clemmons, 1995). IGFBP-2, -3 and -4 mRNA were expressed both in villi and EVT cells and their expressions in EVT were not significantly influenced by IFN-. EVT expression of IGFBP-3, but not the other IGFBPs, has been reported (Han et al., 2000). We extracted mRNA from an almost pure population of EVT—EVT in the explants were cytokeratin positive and vimentin negative (Hu et al., 2006). In addition, IGF-1 mRNA was undetectable in EVT, but present in villi; these data are consistent with previous findings (Lacey et al., 2002). IGFBP-2, -3 and -4 proteins were detected in culture conditioned media; among them IGFBP-3 showed the highest production. IGFBP-3 may both inhibit and potentiate IGF effects. Inhibition may be either through inhibition of IGF-1-mediated stimulation of DNA synthesis or through inhibition of a IGF-1-mediated insulin-like effect (Jones and Clemmons, 1995). IGF-1 potentiation occurs after preincubation of EVT with IGFBP-3 (Jones and Clemmons, 1995). However, none of these protein concentrations was significantly affected by IFN- (Fig. 4).
We confirmed IFN--mediated induction of trophoblast apoptosis, as has been reported by others (Garcia-Lloret et al., 1996; Zou et al., 2000; Liu et al., 2002; Lash et al., 2006); this was apparent with the more sensitive assay of early apoptosis, M30 neo-epitope generation, compared with the pro-apoptotic enzyme, caspase-3. We did not observed apoptosis induction by dNK in our previous experiments; however, in those experiments we assessed solely caspase-3 and Annexin-V expression (Hu et al., 2006). Therefore, the observed IFN--mediated inhibition of EVT outgrowth and migration may result from decreased EVT numbers through accelerated programmed cell death, as well as being related to IGFR-2 down-regulation.
As a multifunctional cytokine, IFN- regulates the expression and production of many other cytokines and growth factors; the regulatory effect observed in this study is but one example. Our ex vivo explant culture model, which mimics in vivo placentation and implantation, provides direct evidence that IFN- can influence EVT outgrowth and migration. The effect may be through regulation of IGF-1 and -2 gene expression and IGF-1 and -2 protein production, and expression of their respective receptors, especially IGFR-2, and a subtle induction of trophoblast apoptosis. However, it seems unrelated to either changes of expression and production of IGFBP-2, -3 and -4.
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Child and Family Research Institute (Infection and Immunity Program Pilot Grant; salary support: P.v.D. and C.D.M.); Canadian Institute for Health Research (salary support: Y.H. and P.v.D.); Michael Smith Foundation for Health Research (salary support: P.v.D., J.P.D. and R.T.); The University of British Columbia Clinical Investigator Program (G.E.); The APOG/CIHR Strategic Training Initiative for Research in the Reproductive Health Sciences (G.E. and S.Y.P.).
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Acknowledgements |
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We thank the CARE Program, BC Women’s Hospital and Health Centre, for their assistance in gaining access to reproductive tissue.
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References |
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Submitted on August 30, 2007; resubmitted on March 11, 2008; accepted on April 9, 2008.
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