THE EFFECT OF IL-6 ON THE TROPHOBLAST CELL LINE HTR-8/SVNEO

Embryonic development up to the blastocyst stage, implantation into the uterine wall and the development of the functional placenta are steps crucial for the establishment of normal pregnancy. Specific cells of the placenta, the trophoblast cells, invade the uterine stroma and spiral arteries, adapting them to pregnancy. Interleukin-6 is present in the human endometrium during the receptive phase and early pregnancy. Trophoblasts also produce IL-6, which was found to stimulate trophoblast invasion and migration in vitro. Here we show that the activity of MMP-9 may contribute to the observed increased invasion. In addition, in the HTR-8/SVneo trophoblast cell line IL-6 increases cell proliferation.


INTRODUCTION
Development of the embryo to the stage of blastocyst, implantation in to the uterine wall and the development of the functional placenta are crucial steps in the establishment of a normal pregnancy.Specific cells of the placenta, invasive trophoblast cells, colonize and invade the uterine stroma and spiral arteries, adapting them to pregnancy.The invasive properties of these trophoblastic cells, called extravillous trophoblast (EVT), are largely due to their ability to degrade the extracellular matrix (Fisher et al., 1985) by proteolytic enzymes.Matrix metalloproteinases 2 and 9 (MMP-2 and -9, respectively) have been shown to be particularly relevant to this process (Librach et al., 1991;Bishof et al., 1995).The process of implantation involves complex interactions between uterine and embryonic cells.
There is an ever-increasing wealth of data implicating various autocrine and paraine factors as modulators of trophoblast function.Some of the cytokines produced by trophoblasts and/or diverse decidual cell types relevant for the establishment of pregnancy have been reviewed recently (Salamonsen et al., 2007).Interleukin-6 (IL-6) is a pleiotro-pic cytokine that belongs to the family of gp130 cytokines, all of which share an accessory signal transducing unit gp130.Apart from IL-6, this family includes the leukemia inhibitory factor (LIF), IL-11, cardiotrophin 1, ciliary neurotropic factor, oncostatin M and cardiotrophin-like cytokine/cytokine-like factor (Dimitriadis et al., 2005).In addition to the involvement of IL-6 in various processes such as the immune response, the acute phase reaction and hematopoesis, a significant role in reproduction is supported by data showing that IL-6-deficient mice have reduced fertility and a decreased number of viable implantation sites (Robertson et al., 2000).IL-6 is expressed in the human endometrium throughout the menstrual cycle (Tabibzadeh and Sun, 1992;Vandermolen et al., 1996).In decidua, IL-6 is expressed in stromal cells in vivo and in vitro (Lockwood et al., 2008) but also in human trophoblast.Cultured placenta from the first trimester of pregnancy releases IL-6 to the supernatant (Kameda et al., 1990).Kauma et al. (1993) found that IL-6 is expressed in both trophoblast and villous core compartments.Jauniaux et al. (1996) showed that both syncytiotrophoblast and extravillous trophoblasts (EVT) express IL-6.The expression of IL-6 mRNA declined with differentiation to syncytiotrophoblast (Stephanou et al., 1995).A receptor protein for IL-6 (IL-6R) and gp130 have been detected in trophoblasts (Nishino et al., 1990;Sawai et al., 1995).
Interleukin-6 has previously been shown to increase cell migration and invasion in extravillous trophoblasts (Jovanović and Vićovac, 2009).In the HTR-8-SVneo cell line the integrins α 5 β 1 and α 1 β 1 were implicated.In this study the additional effector molecules in extravillous trophoblast were sought among the MMPs by gelatin zymography using HTR-8/SVneo cells as a model.We also demonstrate the effect of IL-6 on cell proliferation and the survival of HTR-8/SVneo cells.

Cell culture
For immunocytochemical analysis HTR-8/SVneo cells were cultured on glass coverslips, with or without IL-6 (5 ng/ml) until approximately 80% confluence was reached, at 37 °C in a moist atmosphere with 5% CO 2 .The coverslips were then rinsed twice with PBS and fixed with ice-cold acetone-methanol (1:1) for 10 min.The coverslips were kept frozen until staining.
For gelatin zymography, 100 000 HTR-8/SVneo cells were seeded in 300 μl RPMI 1640 with 5% FCS per well in 24-well plates.Cells were incubated at 37°C in a moist atmosphere with 5% CO 2 until close to confluence.The cells were then carefully rinsed with warm s-PBS, and 200 μl of serum-free RPMI 1640 with or without IL-6 (5 ng/ml).The cells were then incubated for another 24 h.Culture media were collected and centrifuged for 5 min at 2 000 rpm to remove cells.Protein concentrations were determined in supernatants using a BCA protein assay kit (Pierce, USA).

Flow cytometry
Flow cytometry was used to evaluate the expression of IL-6R on HTR-8/SVneo cells.Dynabeads -M-280 sheep anti-mouse IgG (Invitrogen, USA) were coated with mouse monoclonal anti-CD45 antibody (1:100) according to the manufacturer's instructions.The coated beads were stored in PBS/0.1% BSA at 4°C until use.The HTR-8/SVneo cells (8 x 10 5 cells/tube) were washed twice with cold PBS 2 (PBS, 2% FCS and 0.01% sodium azide) and permeabilized with Fixation/Permeabilization Concentrate diluted in Fixation/Permeabilization diluent, 1:4 (eBioscience, USA) overnight at 4°C.The cells were washed twice in permeabilization buffer (PB), diluted in deionized water (1:10) and incubated with an anti-IL-6R (1:10) antibody for 45 min at 4°C.After incubation and subsequent washing with PB, the cells were stained for 30 min with Alexa488 ® goat anti-mouse IgG diluted 1:1000 in PB with 5% goat serum.Control cells were incubated with the secondary antibody alone.Labeled cells were fixed with 4% formalin and analyzed on an EPICS XL-MCL flow cytometer (Coulter, Krefeld, Germany).

Determination of viable cell number
The MTT test was used as an indicator of cell number (Hanish et al., 1993).HTR-8/SVneo cells were seeded in 96-well plates in 100 μl of medium, at 20 000 cells/well.The cells were allowed to adhere overnight in a humidified chamber with 5% CO 2 at 37°C.The cells were then carefully rinsed with warm s-PBS and challenged with IL-6 (5 ng/ml) dissolved in medium so that the total culture volume was 200 μl.After 24 or 48 h, the cells were carefully rinsed with warm s-PBS twice.100 μl of MTT in 10% FCS/PBS (1 mg/ml) was added to each well.After incubation for 2 h at 37 °C, the medium was replaced by 1-propanol (100 μl/well) and the plates were vigorously shaken to ensure complete solubilization of blue formazan.Absorbance was measured at 540 nm using a Microplate reader (LKB) and cell numbers were determined using a standard curve obtained with 5x10 3 , 1x10 4 , 2x10 4 , 4x10 4 , 6x10 4 or 8x10 4 cells/well.The experiment was repeated three times, n=12.

Immunocytochemistry
The cells on the coverslips were rehydrated in PBS for 5 min.Non-specific staining was blocked by quenching endogenous peroxidase activity with 1% hydrogen-peroxide for 30 min, and for non-specific binding of proteins by incubation in 1% casein in PBS for 10 min at room temperature (RT).The cells were then incubated either with the primary anti-Ki67 antibody (1: 75) or with anti-M30 (1: 50) antibody for 2 h at RT.This was followed by incubation with biotinylated horse anti-mouse IgG secondary antibody (1: 200) for 30 min and the avidin-biotinylated peroxidase complex (ABC) for another 30 min.Between steps, the cells were washed with PBS.Reaction was visualized using DAB as a chromogen.The nuclei were visualized by hematoxylin staining.Negative controls were performed routinely.Omission of the primary antibody and use of non-immune serum in place of a specific antibody resulted in the complete absence of staining.The slides were then mounted and examined using a Reichert-Jung microscope with a Leica DC150 Digital Camera System (Wetzlar, Germany).Ki-67 and M30-positive cells out of 2 000 total cells were counted on coverslips from three different experiments.The results are expressed as percentages relative to the control.

Gelatin zymography
HTR-8/SVneo cells were incubated as described above.Gelatinase activities in the serum-free conditioned media of the HTR-8/SVneo cells were determined using SDS-polyacrylamide gel zymography.Samples were electrophoresed on SDS-polyacrylamide gels containing 1 mg/ml of gelatin under non-reducing conditions.All samples to be compared were always run on the same gel.Twenty five μg of protein were loaded per lane.Following electrophoresis, the gels were washed 2 times for 15 min in 2.5% Triton X-100 to remove SDS, and then washed in deionized H 2 O.After overnight incubation in a reaction buffer (50 mM Tris-HCl, pH 7, containing 5 mM CaCl 2 ) at 37° C, the gels were stained with Coomassie brilliant blue R-250 for 30 min RT and destained in 30% methanol and 10% glacial acetic acid.Proteinase activity was observed as a clear band of digested gelatin.Gelatinase activity was semi-quantiated by densitometric analysis of zymograms using the ImageMaster TotalLab v2.01 program (Amersham Biosciences, UK).
All zymography experiments were repeated at least three times in duplicate.

Statistical analysis
Statistical analysis of data was carried out with the Statistical Software Program, ver.5.0 (Primer of Biostatistic, McGraw-Hill Companies, Inc., New York, NY, USA) using Student's T-test with values considered significantly different when p <0.05.

Determination of viable cell number
One of the aims of the study was to investigate the effect of IL-6 on HTR-8/SVneo cell proliferation.
To that end viable cell numbers were determined by the MTT test in IL-6-treated vs. untreated cultures.As we have previously shown (Jovanović and Vićovac, 2009), IL-6 (5 ng/ml) did not significantly change the HTR-8/SVneo cell number in treated cultures after 24 h of culture, while after 48 h there was a slight but significant increase in cell viability (106% relative to the control, p<0,01; Figure 2).

The effect of IL-6 on Ki 67 and M30 expression
To further investigate the possibility that IL-6 might change HTR-8/SVneo cell proliferation, the expression of the proliferation marker Ki67 was studied by immunocytochemistry.Cells were cultured on coverslips until 80% confluence was reached (~72h) as described above, and stained for the Ki67 antigen (Figure 3A).Interleukin-6 had a stimulatory effect on HTR-8/SVneo cell proliferation, and the number of Ki67-positive cells in IL-6 treated cultures was 142% of the control (p<0.05, Figure 3B).The effect of IL-6 on HTR-8/SVneo cell apoptosis was studied by immunocytochemistry for the expression of M30, a caspase-cleaved cytokeratin fragment (Figure 4A).The number of apoptotic cells was low in the control cultures (less than 1%).Treatment with IL-6 decreased the number of M30-positive cells to 50% of the control, which, however, was not significant (Figure 4B).

Gelatin zymography
To test a possibility that IL-6 exerts its effect via MMP-2 and MMP-9, their activity was studied using gelatin zymography.The activity was quantified by densitometric analysis.The data obtained demonstrated that the activity of both proteases was increased.IL-6 (5 ng/ml) increased the activity of MMP-2 to 115% of the control (Figure 5A), which was not statistically significant, while MMP-9 activity was stimulated to 140% of control (p<0.05, Figure 5B).

DISCUSSION
The HTR-8/SVneo cell line has been shown to produce IL-6, as reported in previous studies (Svinarich et al., 1996), including our group (Jovanović and Vićovac, 2009).The latter study by our group has shown by immunocytochemistry that IL-6R is expressed in these cells.In the present work this finding was confirmed using flow cytometry which has shown that 52% of HTR-8/SVneo cells expressed this receptor.In our previous study, the short-term effects of IL-6 were studied on cell migration and cell invasion.Hence, cell viability  and cell numbers were reported for the same 24 h interval.Since IL-6 is known to induce proliferation in many cell types, in this study the HTR-8/SVneo cell proliferation in IL-6-treated cultures was studied in more detail.After 24 h of culture, IL-6 had no significant effect on cell viability in the MTT test (Jovanović and Vićovac, 2009).After 48 h of culture, however, there was a slight but significant increase in viable cell numbers (106% of the control).In order to investigate whether further culture with IL-6 would have an effect on cell proliferation and/or apoptosis, cells were cultured until close to confluence for approximately 72 h.The data obtained show that IL-6 increased Ki67positive cell numbers to 142% of the control, suggesting that IL-6 has a stimulatory effect on HTR-8/SVneo cell proliferation.IL-6 has been shown to stimulate the proliferation of other cell types such as the prostate cancer cell line PC-3m (Yifei et al., 2001), myeloma cell lines (Keller and Ershler., 1995;Hov et al., 2009), neuroblastoma cells (Ara et al., 2009), and vascular smooth muscle cells (Morimoto et al., 1991).On the other hand, IL-6 was also reported to reduce endometrial stromal cell proliferation (Zarmakoupis et al., 1995).In our study, treatment with IL-6 decreased the number of M30-positive cells to 50% of the control, suggesting that IL-6 may also inhibit caspase-dependent apoptosis.However, the overall percentage of M30-positive cells was very low (less than 1%) in both treated and control cells and the data were not significant.IL-6 inhibited apoptosis of various cell types, such as gastric cancer AGS cells (Lin et al., 2001), advanced prostate cancer cells (Cavarretta et al., 2007), B-chronic lymphocytic leukaemia cells (Reittie i sar., 1996), neutrophils (Asensi et al., 2004), multiple myeloma Cells (Chauhan et al., 1997), etc.A previous study by another group has reported that IL-6 stimulates the activity of matrix metaloproteinases-2 and -9 in the cytotrophoblast but not their expression (Meisser et al., 1999).Since it was shown that this cytokine has a stimulatory effect on trophoblast invasion and migration, and that this increase can be partly attributed to an increase in integrin subunit expression (Jovanović and Vićovac, 2009), the possible involvement of MMPs was considered.We have investigated whether IL-6 had the same effect on MMP-2 and MMP-9 activity in HTR-8/SVneo cells using a gelatin zymography assay.The data obtained showed stimulation of both MMP-2 (115% of control, not significant) and MMP-9 (140%, p<0.05) activity by IL-6.Our data show for the first time that IL-6 has a stimulatory effects on MMPs in HTR-8/SVneo cells and that the effect is similar to that shown for isolated trophoblasts (Meisser et al., 1999).
The data reported here, taken together with our previous findings (Jovanovic and Vicovac, 2009), support the relevance of IL-6 for human trophoblasts.They show that it exerts multiple effects on the trophoblast cell line HTR-8/SVneo as a model for extravillous trophoblasts.Other authors have also shown that IL-6 may be important for the process of implantation, since the expression of IL-6 and IL-6 receptor was altered in placentas from pre-eclamptic pregnancies, a pathological condition characterized by reduced trophoblast invasion among other symptoms (Zhao et al., 2008).In addition, IL-6-deficient mice have reduced fertility and fewer viable implantation sites (Robertson et al., 2000).Based on the data reported here we propose that this cytokine is relevant for trophoblast survival and is an important mediator of trophoblast invasion in vitro.This could bear relevance for the process of embryo implantation in vivo.

Figure 1 .
Figure 1.Expression of IL-6R in HTR-8/SVneo cells detected cytofluorometrically. Specific staining is shown in grey, and non-specific staining in white histogram.

Figure 2 .
Figure 2. The effect of IL-6 (5 ng/ml) on HTR-8 SV/neo viable cell number in MTT test.Data are expressed as percent of untreated control, values given as mean±SEM.