Molecular Human Reproduction, Vol. 5, No. 11, 1055-1058,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of implantation |
Effects of interleukin-6 (IL-6) on cytotrophoblastic cells
Department of Obstetrics and Gynaecology, Geneva University Hospital and WHO Collaborating Centre in Human Reproduction, University of Geneva, Switzerland
Abstract
Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMP) and their inhibitors. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is stringently controlled both in tissue localization and developmental stage. The factors responsible for these important regulatory processes are unknown, but in-vitro studies point to endometrial cytokines and growth factors as possible candidates. Here we examined the possibility that interleukin-6 (IL-6), a trophoblastic and endometrial cytokine, represents such a regulatory factor. Purified first trimester cytotrophoblastic cells (CTB) were cultured for 4 days in presence or absence of increasing concentrations of IL-6. MMP-2 and MMP-9 bioactivity (zymography) and immunoactivity were measured in the culture supernatants together with total human chorionic gonadotrophin (HCG), fetal fibronectin (FFN) and leptin. IL-6 did not change the cytotrophoblastic secretion of FFN or total HCG. In contrast, this cytokine induced a dose-dependent stimulation of the leptin secretion and increased the activity, but not the immunoreactivity, of MMP-9 and MMP-2. These results indicate that IL-6 could be considered as an endometrio–trophoblastic regulator of cytotrophoblastic gelatinases.
interleukin-6/leptin/MMP/trophoblastic invasion
Introduction
In haemochorial mammals, e.g. humans, massive trophoblast invasion is required for blastocyst implantation and placentation (Cross et al., 1994
; Bischof and Campana, 1996). The transiently invasive properties of trophoblastic cells are related to their capacity of secreting proteolytic enzymes such as the matrix metalloproteinases (MMP) and the serine proteinases. Gelatinase B is also called MMP-9 and is considered to be the rate-limiting factor in extracellular matrix remodelling that occurs in the endometrium during trophoblastic invasion (Librach et al., 1991; Shimonovitz et al., 1994; Bischof et al., 1995a). In contrast to tumour invasion of a host tissue, trophoblastic invasion is stringently controlled both in location (it is limited to the endometrium and the proximal myometrium) and in developmental stage (it ends by about mid-gestation). The factors responsible for these important regulatory processes are unknown but in-vitro studies point to endometrial cytokines and growth factors as possible candidates. Cytokines, e.g. transforming growth factor ß (TGFß; Graham and Lala, 1991), leukaemia inhibitory factor (LIF; Bischof et al., 1995b), epidermal growth factors (EGF; Bass et al., 1994), tumour necrosis factor (TNF; Meisser et al., 1999), interleukin-1ß (IL-1ß; Simón et al., 1994a; Meisser et al., 1999) and insulin-like growth factor binding protein-1 (IGFBP-1; Bischof et al., 1998) have been described as potential regulators of trophoblastic invasion. There is no reason to suppose that other cytokines are not involved in this regulatory process. Indeed, interleukin-6 (IL-6) is suitably positioned at the feto–maternal interphase to suggest a modulatory role for this cytokine in trophoblast invasion.
IL-6 is a multifunctional protein that plays important roles in host defence, acute phase reactions, immune responses and haematopoiesis. It has a four helix-bundle type tertiary structure found in several other cytokines, e.g. LIF, granulocyte colony stimulating factor and interleukin-11 (Bazan, 1990). IL-6 exerts its activity through binding to a high affinity receptor complex consisting of two membrane proteins: an 80 kDa IL-6-binding receptor protein (Yamasaki et al., 1988) and a 130 kDa signal-transducing protein (gp 130, Hibi et al., 1990). It is interesting to note that gp 130 is also associated with the LIF receptor (Gearing et al., 1991).
IL-6 mRNA and protein have been localized in human endometrium where their expression was maximal during the mid-secretory phase, thus at the time of implantation (Tabibzadeh et al., 1995; Vandermolen and Gu, 1996). Furthermore, IL-6 secretion by endometrial stromal cells is enhanced by interferon-
(Nasu et al., 1998). In first trimester trophoblasts, IL-6 protein (Jauniaux et al., 1996) and mRNA (Stephanou et al., 1995) are present in both the cytotrophoblastic cells (CTB) and the syncytiotrophoblast (STB). It must be noted that the expression of this cytokine decreases significantly during differentiation of CTB into STB (Jauniaux et al., 1996) and that IL-6 concentrations are higher in decidual than in placental tissue (Jauniaux et al., 1996). IL-6R and gp 130 have been immunolocalized in endometrial epithelial cells (Tabibzadeh et al., 1995) and gp 130 mRNA has been detected in first and third trimester decidua and trophoblast (Kojima et al., 1995) as well as in human blastocysts (Sharkey et al., 1995). Thus IL-6 and its receptor proteins are present simultaneously on maternal and fetal tissues during implantation and placentation. These observations prompted us to examine the potential role of IL-6 on several parameters involved in the invasive behaviour of CTB particularly MMP and fetal fibronectin (FFN) since these reflect matrix degradation and deposition respectively. We also examined the effect of IL-6 on trophoblastic leptin secretion since leptin is secreted by CTB and up-regulated by IL-1 (Chardonnens et al., 1999). This last cytokine is known to exert similar effects to IL-6 on human chorionic gonadotrophin (HCG) secretion (Masuhiro et al., 1991).
Materials and methods
Preparation of cytotrophoblastic cells (CTB) and culture conditions
CTB were isolated, purified and cultured as previously described (Bischof et al., 1991). Briefly, trophoblastic villi obtained from induced abortions (6–12 weeks of pregnancy) were digested by trypsin. CTB were separated from blood cells and syncytia on a discontinuous Percoll gradient and the contaminating leukocytes removed by immunopurification with an antibody to CD45 coupled to magnetic particles. These CTB were counted in a Neubauer cell in presence of Trypan Blue and diluted to 106cells /ml.
Cells (2x105/well) were cultured overnight in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Basle, Switzerland) containing 2 mmol/l L-glutamine, 4.2 mmol/l magnesium sulphate, 2.5 mmol/l HEPES, 1% gentamycin, 1% Amphoptericin-B, 100 µg/ml streptomycin (Grünenthal, Stolberg, Germany) and 100 U/ml penicillin (Hoechst-Pharma, Zürich, Switzerland) in presence of 10% FCS (Life Technologies). The next morning (day 0), medium was changed to serum-free DMEM and the cells were incubated in the presence or the absence of increasing concentrations of IL-6 (0.1–10 ng/ml; R&D Systems, Bühlmann, Basle, Switzerland). Incubation was performed under a 5% CO2 and 95% air atmosphere in a humidified incubator at 37°C. Medium was harvested on days 2 and 4 and the culture was stopped on day 4. The supernatants from days 2 and 4 were divided into aliquots and stored at –20°C until assayed. The cells were lysed with 200 µl Triton X-100 (25% in water) and stored at –20°C for total cell protein measurements.
Gelatinolytic assays
Zymography was performed as previously described (Martelli et al., 1993). Zymograms were scanned in an `Apple Onescanner' and the surface of the digestion bands measured by the NIH Image 1.60 program on a Power Macintosh 7100/66 computer. All zymograms were evaluated using the same pre-set standards with a coefficient of variation of 18.6%.
Hormone and protein assays
Total HCG (HCG + free ß-HCG) was measured in the supernatants by a microparticle enzyme immunoassay with a sensitivity of 1 mIU/ml and a coefficient of variation of 3.6% (Abbott, Abbott Park, IL, USA). FFN was measured by a commercially available enzyme immunoassay with a sensitivity of 50 ng/ml and a coefficient of variation of 7.5% (Adeza Biochemical, Sunnyvale, CA, USA). MMP-2 and MMP-9 immunoreactivities (MMP-2i and MMP-9i) were measured in the supernatants using our own enzyme immunoassays as described elsewhere (Meisser et al., 1999). Leptin was measured by a commercially available enzyme immunoassay (DRG Instruments, CIS-Medipro, Vernier, Switzerland) with a sensitivity of 0.2 ng/ml and an inter-assay coefficient of variation of <7%. Total cell proteins were measured in the cell lysate with the Bio-Rad protein assay according to the manufacturer's instructions and using bovine serum albumin as the standard (Bio-Rad, München, Germany).
Statistical analysis
To evaluate the effects of IL-6 on the different trophoblastic parameters, the individual values were transformed into values per mg cell proteins and per day in culture [(conc.day2/mg Prot)+(conc.day4/mg Prot)]/4 and expressed as percentage of the respective controls (CTB in absence of IL-6). All experiments were run in duplicates and repeated with three different preparations of CTB. Statistical analyses were performed by analysis of variance using the StatView 4.5 program on the Power Macintosh 7100/66 computer and P values corrected for small sample size.
Results
The effects described below were similar on days 2 and 4 of culture in the presence of IL-6. As described in the Materials and methods section, the production rate/day was calculated for each parameter, taking into account the day 2 and day 4 results.
IL-6 did not change the cytotrophoblastic secretion of FFN or total HCG (Figure 1A,C). In contrast, this cytokine induced a dose-dependent increase in the secretion of leptin (P = 0.05 to P = 0.038 for 1–10 ng/ml, Figure 1B). Under basal conditions (without IL-6), CTB secreted 2.9 + 1.6 ng/day/2x105cells of leptin (mean + SD). Concentrations of IL-6 of 0.5–10 ng/ml significantly stimulated MMP-9 activity (Figure 1D, P = 0.0064 to P = 0.0017 respectively) but not MMP-9 immunoreactivity. Similarly, IL-6 significantly increased the activity of proMMP-2 (Figure 1F, P = 0.0015 to P < 0.0008 for concentrations of IL-6 from 0.1–10 ng/ml), the activity of MMP-2 (Figure1G, P = 0.0007 and P = 0.012 for 0.5 and 1 ng/ml of IL-6) but was without effects on the immunoreactivity of MMP-2.
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Discussion
Under our experimental conditions with primary trophoblastic cells, IL-6 remains without effects on HCG secretion. This is in contrast to previously published data showing a marked stimulation of HCG by IL-6 via its own receptor (Nishino et al., 1998). This discrepancy might be due to the fact that these authors incubated IL-6 with primary CTB for only 2.5 h whereas under our procedure, IL-6 remained in contact with the cells for 2–4 days. This last protocol might have favoured an internalization of the IL-6R leading to a lack of effects. Another possible explanation could be that IL-6 stimulates HCG release (as measured by Nishino et al., 1998), rather than HCG synthesis, as measured in our experiments.
The obese gene product, leptin, a 167 amino acid peptide (Zhang et al., 1994), has provided a potential link between food intake, energy balance and reproductive events (Chehab et al., 1996). A well-recognized site of leptin production is the adipocyte. Consequently, the amount of body fat appears as the strongest determinant of plasma leptin values. Recently (Hardie et al., 1997; Schubring et al., 1997), it was demonstrated that circulating leptin values were elevated during pregnancy, reaching a peak during the second trimester. However, fat deposition during pregnancy or pregnancy-induced endocrine changes could hardly explain such dramatic changes implying that the feto–placental unit could be another important source of leptin (Masuzaki et al., 1997). Here we show that leptin is indeed secreted by primary cytotrophoblast and that this secretion is stimulated by IL-6. It is interesting to note in this respect that leptin and IL-6 share structural similarities and that the full-length leptin receptor has IL-6 signalling capabilities (Baumann et al., 1996; Tartaglia 1997). The cross-reactivity with IL-6 in the leptin assay has not been tested by the manufacturer but it is probably insignificant since rat leptin, which is closer to human leptin than IL-6, has a cross-reactivity <0.2%. This IL-6 signalling capability might explain our recent results that recombinant human leptin activates MMP-2 and MMP-9 in our primary trophoblastic cell cultures (M.Castellucci, R.De Matteis, A.Meisser et al., unpublished observations).
In the present study, IL-6 induced the activation of trophoblastic MMP-2 and MMP-9 but not their synthesis since this cytokine did not change the concentration of immunoreactive MMP-2 and MMP-9 in the cell culture supernatants. Taking these data together, one could speculate that IL-6 activates trophoblastic metalloproteinases either through an increased secretion of leptin or a mechanism independent of leptin secretion. IL-6 has been shown to enhance the invasiveness of head and neck cancer cell lines in vitro (Nishino et al., 1998) and of human ovarian cancer cell lines (Obata et al., 1997). However, in both models, IL-6 did not affect MMP-2 and MMP-9 activation (Mann et al., 1995; Obata et al., 1997). The contrasting effects of IL-6 on cancer cells and CTB is probably due to the nature of transformed and non-transformed cells.
Since LIF and IL-6 share the same signal transducer (gp 130, Gearing et al., 1991) one would expect IL-6 and LIF to act in a similar way on trophoblastic cells. This is, however, not the case since we have shown that LIF, in contrast to IL-6, exerts potent inhibitory effects on trophoblastic MMP-2 and MMP-9 (Bischof et al., 1995b). More research is necessary into the molecular mechanisms governing signal transduction from the LIF and the IL-6 receptors.
Acknowledgments
The skilful technical help of L.Haenggeli and C.Wuillemin is acknowledged. This work was supported by the Swiss National Science Foundation grant no 32–39307.93
Notes
1 To whom correspondence should be addressed at: Laboratoire d'Hormonologie Maternité, 1211 Geneva 14, Switzerland 
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Submitted on January 18, 1999; accepted on August 12, 1999.
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