Role of Interleukin 8 in Uterine Natural Killer Cell Regulation of Extravillous Trophoblast Cell Invasion

Placenta 31 (2010) 595e601

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Role of Interleukin 8 in Uterine Natural Killer Cell Regulation of Extravillous Trophoblast Cell Invasion L.G. De Oliveira a, b,1, G.E. Lash a, *,1, C. Murray-Dunning a, J.N. Bulmer a, B.A. Innes a, R.F. Searle a, N. Sass b, S.C. Robson a a b

Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK Department of Obstetrics, Federal University of Sao Paulo, Sao Paulo, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 26 April 2010

Background: Extravillous trophoblast cell (EVT) invasion of maternal tissues is critical for successful pregnancy. Decidual factors, including uterine natural killer (uNK) and T cell derived cytokines play a role in regulating this process. Interleukin (IL) 8 has been implicated as a regulator of EVT invasion. Hypothesis: uNK cell stimulation of EVT invasion is associated with IL-8 levels. Methods: CD8þ, total decidual and CD56þ uNK cells (8e10 and 12e14 weeks gestational age) were cultured. IL-8 mRNA and protein levels were determined. IL-8 receptors (IL-8RA and IL-8RB) were localised in first trimester placental bed biopsies. The effect of IL-8  IL-8 neutralising antibodies and CD8þ T cell or uNK cell supernatants  IL-8 neutralising antibodies on EVT invasion was assessed. EVT secreted levels of MMP-2, MMP-9, uPA, PAI-1 and PAI-2 were assessed by substrate zymography or Western Blot. Results: High levels of IL-8 protein and mRNA were detected in all samples. IL-8RA and IL-8RB were expressed by EVT. Exogenous IL-8 stimulated EVT invasion in a paracrine manner. uNK cell supernatants, but not CD8þ cell supernatants, stimulated EVT invasion. IL-8 neutralising antibody partially abrogated this uNK cell stimulated invasion. IL-8 increased levels of secreted MMP-2, but did not alter any of the other proteases or protease inhibitors tested. Conclusion: uNK cell stimulation of EVT invasion is partially mediated by IL-8. Unstimulated CD8þ T cells do not alter EVT invasion despite secreting similar levels of IL-8 as uNK cells. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Uterine natural killer cells Extravillous trophoblast Invasion Interleukin 8 Proteases

1. Introduction Invasion of the uterine decidua and inner third of the myometrium by extravillous trophoblast (EVT) with resultant spiral artery remodelling, is the hallmark of successful pregnancy [1]. Failure of this process is associated with important clinical disorders such as second trimester miscarriage [2], pre-eclampsia [3] and fetal growth restriction [4]. The invasive capacity of EVT can be regulated by growth factors, cytokines and chemokines. There are several sources of secretion of these proteins at the maternalefetal interface, including epithelial cells, endothelial cells, decidual stromal cells, leukocytes and trophoblast cells themselves [5]. Uterine natural killer (uNK) CD56brightCD16dim cells, which constitute up to 70% of the decidual leukocyte population in the first * Corresponding author at: Institute of Cellular Medicine, 3rd Floor, William Leech Building, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. Tel.: þ44 191 222 8578; fax: þ44 191 222 5066. E-mail address: [email protected] (G.E. Lash). 1 LGDO and GEL contributed equally to the current study. 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.04.012

half of pregnancy, are considered to be an important source of regulatory growth factors and cytokines [6,7]. We and others have demonstrated that uNK cell supernatants stimulate EVT invasion, but only when taken from pregnancies at 12e14 weeks gestational age [8,9]. The uNK cell stimulation of EVT invasion was inhibited by a combination of neutralising antibodies to interleukin (IL)-8 and IP10 (CXCL10) [8]. In addition, the uNK cell stimulation of EVT invasion was associated with an increase in matrix metalloproteinase (MMP)9 [9]. CD3þ lymphocytes account for fewer than 20% of the decidual leukocyte population in early pregnancy and, in contrast to the peripheral circulation, the majority are CD8þCD3þ T cytotoxic cells rather than CD4þCD3þ T helper cells [10]. The role of CD3þ T lymphocytes in early pregnancy decidua remains unknown. We recently demonstrated that CD8þ T cells produce large amounts of cytokines when stimulated in vitro and supernatants stimulated trophoblast invasion, further supporting a non-immunological role for these cells in early pregnancy decidua [11]. IL-8 is classified as a CXC chemokine according to its structure and it can bind to two receptors, IL-8RA (CXCR1) and IL-8RB (CXCR2) [12]. These receptors are expressed by a variety of cell

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types and ligand binding leads to distinct functional responses [13]. IL-8 has been reported to be present in the human reproductive tract. IL-8 levels were found to be higher in late secretory and early to mid proliferative phase endometrium compared to early secretory phase, suggesting a role in endometrial degradation and menstruation or in endometrial neovascularization [14]. Saito et al. [15] demonstrated IL-8 mRNA in uNK cells in decidua during early pregnancy. Hanna et al. [8] reported expression of IL-8RA, but not IL-8RB, by invasive EVT and that IL-8 (100ng/ml) stimulated invasion of these cells, although the mechanism of action was not investigated. In addition, Jovanovic et al. [16] demonstrated that IL-8 (5ng/ml) stimulated invasiveness of the HTR-8/SVneo trophoblast-like cell line and isolated cytotrophoblast cells. The stimulation of invasion of the HTR-8/SVneo cell line was associated with an increase in MMP-2 and MMP-9 [16]. A key aspect of cellular invasion is breakdown of the extracellular matrix by proteases with movement of the invading cell into the cleared space [17]. EVT employ several different protease systems, including the gelatinases MMP-2 and MMP-9 [17] and the urokinase plasminogen activator (uPA) system, which includes uPA receptor (uPAR) and plasminogen activator inhibitors (PAI-1 and PAI-2) [18]. IL-8 has been shown to stimulate MMP-2 [19], MMP-9 [20] and uPA [21] in various different cancer cell types. We hypothesised that IL-8 secreted by decidual cells in early human pregnancy stimulates EVT invasion in vitro via a mechanism dependent on increased protease activity. In addition, we also hypothesised that CD8þ T lymphocytes and uNK cells stimulate trophoblast invasion via mechanisms involving IL-8 production. 2. Methods 2.1. Sample collection Placental and decidual samples were obtained from women undergoing elective surgical termination of pregnancy at the Royal Victoria Infirmary, Newcastle upon Tyne, UK. The study was approved by the Joint Ethics Committee of Newcastle and North Tyneside Health Authority and Newcastle University and all women gave informed written consent. Placenta was obtained from pregnancies at 8e10 and 12e14 weeks gestational age (n ¼ 20 each gestational age). Decidua was obtained from pregnancies at 8e10 and 12e14 weeks gestational age (n ¼ 20 each gestational age). Gestational ages were determined by ultrasound measurement of crown rump length or biparietal diameter at the time of termination. Following collection, tissues were immediately suspended in sterile saline, transported to the laboratory and washed 2e3 times in sterile phosphate-buffered saline (PBS) to remove excess blood. Placental bed biopsies were obtained from women undergoing elective surgical termination of early pregnancy (8e10 weeks (n¼9), 12e14 weeks (n ¼ 8) and 15e20 weeks (n ¼ 8) gestational age) as described previously [22]. 2.2. Positive selection of CD56þ uterine natural killer cells and CD8þ T lymphocytes Decidua was subjected to enzymatic disaggregation with collagenase/DNase and total decidual cell isolates, CD56þ uNK cell enriched isolates and CD8þ T lymphocyte enriched isolates were prepared by positive immunomagnetic selection (MACS) as described previously [23]. Using this method the CD56þ uNK and CD8þ T lymphocytes enriched isolates were both consistently >95% pure [23,24]. Isolates were periodically checked for purity by immunostaining of cell smears. After isolation 25% of the total decidual suspension, CD56þ and unstimulated CD8þ cells were snap frozen and stored at 80 C for RNA isolation. The remaining isolated cells (1  105 cells/well) were incubated for 24 h in RPMI1640 (100 ml) containing 10% fetal bovine serum (FBS), penicillin/streptomycin, amphotericin B and glutamine (all from Sigma Chemical Co., Poole, UK; complete medium). CD8þ T lymphocytes were cultured in the presence (stimulated) and absence (unstimulated) of phytohaemagglutinin (PHA)-P (10 mg/ml) (Sigma Chemical Co.) for 24 h in complete medium. Conditioned medium was stored at 20  C.

instructions (ELISA Development System, human IL-8, R&D Systems Inc, Minneapolis, USA), with a dynamic range of 31.25 pg/mLe2000 pg/mL. Samples were diluted 1:100 in reagent diluent (0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline) for use in the assay.

2.4. Real time reverse transcriptase polymerase chain reaction for IL-8 Total RNA was isolated from total decidual, CD56þ and unstimulated CD8þ cells (t ¼ 0, n ¼ 5 8e10 and 12e14 weeks gestational age) using TRI-reagent according to the manufacturer’s instructions (TRIZOL, Sigma, Poole, UK). The quantity of RNA was assessed at 260 nm using a 96 well spectrophotometer (Molecular Devices, Workingham, UK) and UV visible 96 well plates (Greiner, Gloucestershire, UK). 500 ng total RNA was reverse transcribed using Superscript III according to the manufacturer’s instructions (InVitrogen, Paisley, UK). Real time RT-PCR was performed using an ABI7000 (Applied Biosystems, Foster City, CA, USA) PCR machine and probes labelled with the fluorophore 6-Fam and quencher TAMRA. In each run a standard curve was produced for each set of primers and probe based on serial dilutions of a standard amount of RNA that was reverse transcribed. For each sample, RT-PCR was performed for GAPDH (GAPDH Assay on Demand, Applied Biosystems) and IL-8 (IL-8 Assay on Demand, Applied Biosystems). Data are presented as the ratio of the amount of IL-8 to GAPDH.

2.5. Immunohistochemistry Immunohistochemistry was performed on 3 mm sections of formalin fixed placental bed biopsies from 8 to 10 weeks (n ¼ 9), 12e14 weeks (n ¼ 8) and 15e20 weeks (n ¼ 8) gestational age, using a streptavidin-biotin peroxidase method to localise expression of IL-8 receptors (IL-8RA and IL-8RB) by EVT and IL-8 protein in uNK cells and CD8þ T lymphocytes. Details of primary and secondary antibodies are shown in Table 1. Where possible (anti-IL-8 and anti-CD56) sections were double labeled allowing easier identification of uNK cells. Where double labeling was not possible due to incompatibility in pretreatments 3 mm serial sections were immunostained (anti-CD8 and anti-IL-8; cytokeratin/PAS and either anti-IL-8RA or anti-IL-8RB). The reaction was developed with diaminobenzidine (DAB) for single labeling or the first label of double labeling, and with the Vector VIP kit (Vector Laboratories) for the second stain of double labeling to give a purple reaction product. Washes between each step were performed in 0.15 M Tris buffered 0.05 M saline (TBS, pH7.6). Single labeled sections were lightly counterstained with Mayer’s haematoxylin (BDH, Poole, UK) and mounted in DPX synthetic resin (Raymond Lamb, London. UK). Double labeled sections were not counterstained. Positive control sections of tonsil were included for IL-8RA and IL-8RB. For all antibodies, negative controls were performed in the absence of primary antibody which was replaced by non-immune serum. Immunohistochemistry for IL-8, IL-8RA and IL-8RB was not quantified.

2.6. Invasion assay Placental explants were prepared as previously described [25]. Briefly, chorionic villous tips were dissected, minced to approximately 0.5 mm3 and resuspended in culture medium (DMEM:F12 containing 10% FBS, penicillin/streptomycin and amphotericin B [all from Sigma Chemical Co.]) such that 15 ml of the suspension constituted approximately 10 mg of tissue. Matrigel invasion assays were performed as previously described [25].

Table 1 Antibodies used for immunohistochemistry. Antibody

Species Pretreatment

Dilution

Incubation time

Secondary Kit

IL-8a

Goat

none

1/10

IL-8RA IL-8RBa CD8b

Goat Goat Mouse

1/100 1/75 1/100

Vector APC goatc Impressc Impressc Vector APC mousec

CD56b

Mouse

none none pressure cook in EDTA buffer pH 8.0, 1 min pressure cook in citrate buffer pH 6.0, 1 min trypsin pH 7.8, 10 min

37  C, overnight RT, 60 min. RT, 60 min. RT, 30 min

a

2.3. IL-8 ELISA IL-8 protein levels in 24 h cell culture supernatants of total decidual cells (n ¼ 5 8e10 and 12e14 weeks gestational age), CD56þ uNK cell enriched isolates (n ¼ 5 8e10 and 12e14 weeks gestational age) and both unstimulated (n ¼ 5 8e10 weeks gestational age, n ¼ 4 12e14 weeks gestational age) and stimulated CD8þ T lymphocyte isolates (n ¼ 5, 8e10 and 12e14 weeks gestational age) were determined using a commercially available ELISA kit according to the manufacturer’s

Cytokeratinb Mouse

1/50

RT, 30 min. Vector APC mousec

LP34 1/80 RT, 30 min. Vector þ 5D3 1/20 APC mousec

RT ¼ room temperature. Source: aR&D Systems. b Novocastra Laboratories, Newcastle upon Tyne, U.K. c Vector Laboratories, Peterborough, UK.

L.G. De Oliveira et al. / Placenta 31 (2010) 595e601

Zymographic analysis was performed on supernatants collected from placental explant cultures as described previously [25,26]. Briefly, 20 mg total protein was resolved in a 12% SDS-PAGE containing either 2 mg/ml gelatin for MMP-2 and MMP-9 or 2 mg/ml casein and 0.025 units/ml plasminogen (American Diagnostica Inc., Greenwich, CT) for uPA. After electrophoresis, the gels were washed in 2.5% Triton X-100 to remove the SDS, rinsed with water and incubated overnight at 37  C in a solution of 50 mM Tris and 5 mM CaCl2 (pH 7.0) to allow substrate digestion. After incubation, gels were stained with 0.4% Coomassie Brilliant Blue R250 in 45% methanol/10% acetic acid, destained with 30% methanol/10% acetic acid, preserved and dried. The dried gels were scanned, digitized and densitometry was performed (Unscan-It, Silk Scientific Co., Orem, UT). In order to remove variability between samples from different subjects, all results were normalized to their respective controls. 2.8. Western blot analysis 30 mg total protein from invasion assay cell culture supernatants was resolved in a 12.5% SDS-PAGE under reducing conditions. The resolved proteins were transferred to an Hybond-P membrane (Amersham Biosciences, GE Healthcare Ltd, Chalfont St.Giles, Bucks., UK) using a wet transfer apparatus (Bio-Rad Labaoratories). The membranes were blocked overnight at 4  C in phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and 5% dry milk powder. Membranes were probed with rabbit anti-human PAI-1 antibody (AbCam Ltd., Cambridge, UK; 1:500, 4 h) or mouse anti-human PAI-2 antibody (American Diagnostica; 1:250, 4 h) in PBS-T containing 5% skimmed milk at room temperature. Membranes were incubated with goat anti-mouse (1:3000, Bio-Rad) or goat anti-rabbit (1:3000, Sigma) horseradish peroxidase conjugated secondary antibody, developed using an ECL-plus kit (Amersham Biosciences) and X-OMAT film (Eastman Kodak Co., Rochester, NY). Membranes were stained with Ponceau S (Sigma Chemical Co.) to ensure equal protein loading. 2.9. Statistical analysis Data are presented as means with standard errors. Statistical calculations were performed using the StatView statistical software package (Abacus Concepts Inc., Berkley, CA). Statistical significance was determined by use of one-way ANOVA followed by Fisher’s post hoc analysis unless otherwise stated. Student’s t-test was used when two sets of normally-distributed data were compared. All statistical tests were two-sided and differences were considered statistically significant at P < 0.05.

3. Results 3.1. Expression of IL-8 by total decidual cells, CD56þ uNK cells and CD8þ T lymphocytes Total decidual cell isolates secreted large amounts of IL-8 during early pregnancy (8e10 weeks gestation, 117.76  66.2 ng/ml; 12e14 weeks gestation, 219.7  120.6 ng/ml) as determined by ELISA (Fig. 1A). CD56þ uNK cell enriched isolates also secreted IL-8, levels

3.2. Localisation of IL-8 and its receptors, IL-8RA and IL-8RB, in the placental bed Immunohistochemistry demonstrated that both IL-8RA and IL-8RB were localized to extravillous trophoblast, including endovascular, intramural and interstitial EVT (Fig. 2A, B, C). The majority of EVT were immunopositive for IL-8RA. Immunoreactivity for IL-

A

400 350 300

IL8 (ng/ml)

2.7. Substrate gel zymography

being greater from isolates at 12e14 weeks’ gestation (40.6  25.9ng/ ml) than from isolates at 8e10 weeks gestation (11.7  3.3ng/ml) (P < 0.05, Fig. 1A). However uNK cells produced less IL-8 than from total decidual cell isolates at both gestational ages (8e10 weeks gestation, P ¼ 0.0002; 12e14 weeks gestation, P ¼ 0.02). CD8þ T lymphocytes also secreted high levels of IL-8 that increased after stimulation with PHA-P (8e10 weeks gestation, unstimulated 22.5  9.2 ng/ml, stimulated 160.3  72.1 ng/ml; 12e14 weeks gestation, unstimulated 58.1  18.7 ng/ml, stimulated 195.3  130.4 ng/ml; Fig. 1A). In contrast to stimulated CD8þ cells, levels of IL-8 produced by unstimulated CD8þ T lymphocytes were lower than those from total decidual cell isolates at both gestational ages (8e10 weeks gestation, P ¼ 0.02; 12e14 weeks gestation, P ¼ 0.05). IL-8 mRNA was detected in freshly isolated total decidual cells, CD56þ uNK cells and unstimulated CD8þ T lymphocytes by real time RT-PCR (Fig. 1B). There were no gestational age differences in levels of IL-8 mRNA for any of the cell types investigated (Fig. 1B).

8-10wk 12-14wk

250 200 150 100

P<0.05

50 0 Total

B

CD56

CD8 (Unstimulated) CD8 (Stimulated)

4000

3500

IL8:GAPDH mRNA

In order to investigate the effects of IL-8 on trophoblast invasion, exogenous IL-8 (1 and 10 ng/ml, R&D Systems) was added to both the Matrigel and culture medium. A specific anti-IL-8 neutralizing antibody (goat anti-human IL-8 nAB, 0.1 mg/ml, R&D Systems) was added to inhibit the effect of endogenous IL-8. The specificity of the neutralizing antibody was verified by adding combined cytokine and neutralizing antibody (10 ng/ml IL-8 þ 0.1 mg/ml IL-8 nAB). Each experiment was performed in triplicate and repeated on ten separate occasions using placental explants from 8 to 10 weeks gestational age. Neutralizing antibody concentrations were chosen based on manufacturer’s specifications to give a minimum of 50% neutralization. To assess whether CD8þ T cells or CD56þ uNK cells promote trophoblast invasion by means of IL-8 production, invasion assays using CD8þ or uNK cell supernatants were performed using placental explants and cell culture supernatants from 8 to 10 and 12e14 weeks gestational age. Placental explants and cell culture supernatants from the same gestational age were used. Anti-IL-8 neutralizing antibody (0.5 mg/ml, n ¼ 10 each condition) was added to 33% v/v CD8þ or uNK cell culture supernatants to inhibit IL-8 activity. Each experiment was performed in duplicate. Placental explants were then cultured at 37  C in a 5% CO2 incubator for 6 days. After 6 days incubation supernatants were harvested and stored at 20  C until required for substrate gel zymography or Western blot analysis. The matrigel and explants were removed and the upper side of the membrane cleaned with a cotton wool bud. In order to visualize and quantify the number of invasive cells, the filters were stained with haematoxylin and eosin and mounted on glass microscope slides using DPX. Data are expressed as the mean invasion index (SEM) where the level of invasion was normalized to the control within each experiment.

597

3000

2500 8-10 wk 12-14 wk

2000

1500

1000

500

0 Total

CD56

CD8 (unstimulated)

Fig. 1. A) Levels of IL-8 protein secreted by total decidual cell isolates, uNK cells and unstimulated and stimulated CD8þ T lymphocytes as measured by ELISA (mean ng/ ml  SEM). N ¼ 10 each group. B) Levels of IL-8 mRNA in freshly isolated total decidual cells, CD56þ uNK cells and unstimulated CD8þ T lymphocytes as measured by real time RT-PCR (mean  SEM; arbitrary units). N ¼ 10 each group. Student’s t-test, P < 0.05 considered statistically significant.

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8RB was very weak in both placental bed (Fig. 2C) and tonsil (positive control, Fig. 2I), and only a few EVT appeared to be immunopositive for this receptor. Double immunostaining for CD56 and IL-8 demonstrated that some CD56þ cells in the placental bed were also immunopositive for IL-8 (Fig. 2D). In contrast, only weak IL-8 immunostaining was observed in CD8þ T lymphocytes in the placental bed biopsies (Fig. 2E, F). IL-8 was also localized to other cell types including EVT, glandular epithelium and decidual stromal cells (Fig. 2D, E).

3.3. IL-8 stimulates extravillous trophoblast invasion Recombinant human IL-8 stimulated EVT invasion at a concentration of 10 ng/ml (P ¼ 0.006, Fig. 3), but IL-8 did not increase EVT invasion from placental villous explants at a concentration of 1 ng/ml. The effect of IL-8 (10 ng/ml) was abrogated with coadministration of an IL-8 specific neutralizing antibody (0.1 mg/ml). Administration of the neutralizing antibody alone had no effect on invasion (Fig. 3).

Fig. 2. AeC) Serial sections of placental bed biopsy (13 weeks gestational age) immunostained for A) Cytokeratin 7 with periodic acid Schiff counterstain, B) IL-8RA, C) IL-8RB. D) Double immunolabeling for CD56 (purple) and IL-8 (brown), arrows denote double positive cells (16 weeks gestational age). Inset shows higher magnification of the central CD56þ cells where some double labelling can be more clearly seen. E, F) Serial sections of placental bed biopsy (20 weeks gestational age) immunostained for E) IL-8, arrow denotes CD8 cells positive for IL-8, F) CD8. G) negative control, no primary antibody, H) positive control for IL-8RA (tonsil), I) positive control for IL-8RB (tonsil). EV ¼ endovascular EVT, IM ¼ intramural EVT, IS ¼ interstitial EVT. Original magnification 400. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

L.G. De Oliveira et al. / Placenta 31 (2010) 595e601

3.4. IL-8 increases MMP-2 secretion Levels of MMP-2 secreted by EVT were increased in the presence of 10 ng/ml IL-8 (P ¼ 0.01) compared to controls (n ¼ 10). Addition of 1 ng/ml IL-8 or anti-IL-8 (0.1 mg/ml) did not alter secreted MMP-2 levels. Addition of exogenous IL-8 did not alter MMP-9 secretion by EVT (n ¼ 10) (Fig. 5A). There was also no difference, relative to controls, in levels of secreted uPA (Fig. 5B) or secreted PAI-1 or PAI-2 (Fig. 5C) in the presence of IL-8 (0, 1, 10 ng/ml) or anti-IL-8 (0.1 mg/ml) (n ¼ 5 each). 4. Discussion The current study demonstrates that IL-8 is secreted in high levels by decidual cells during early pregnancy and that CD56þ uNK cells constitute one source. Furthermore, both IL-8 receptors (IL-8RA and IL-8RB) are localized to extravillous trophoblast. We also show that IL-8 stimulation of EVT invasion is associated with increased secretion of MMP-2. Finally, we confirm that uNK cell supernatants stimulate trophoblast invasion and this effect is partially dependent on IL-8 secretion. In contrast, we report for the first time that unstimulated CD8þ T cell supernatants had no effect on trophoblast invasion. Engert et al. [27] recently reported IL-8 production by decidual stromal cells. In addition, Vacca et al. [28] reported significant IL-8 production by uNK cells, although they did not investigate samples from different gestational ages. Similarly, we demonstrated IL-8 production by decidual cells and showed that uNK and CD8þ T cells are two of the decidual sources of this chemokine. The substantially higher levels of IL-8 at 12e14 weeks gestational age infer a greater functional role at this gestation. Levels of IL-8 in normal adult tissues have been reported to be low [29]. The functional significance of

Invasion Index (mean +/- SEM)

2.5

*

2 1.5 1 0.5 0 Control

1ng/ml IL8

10ng/ml IL8

0.1µg/ml IL8 nAB

10ng/ml IL8 + 0.1 µg/ml IL8 nAB

Fig. 3. Effect of IL-8 on EVT invasion from placental chorionic villous explants (8e10 weeks gestational age) through Matrigel (n ¼ 10 each group). * denotes statistical significance compared with controls. ANOVA with Fisher’s post-hoc test, P < 0.05 considered statistically significant.

2.5 Invasion Idex (mean +/- SEM)

In contrast to our previous results with PHA-P stimulated CD8þ decidual T cells [11], unstimulated CD8þ T cell supernatants from either 8e10 or 12e14 weeks gestational age did not alter EVT invasion from placental villous explants at the same gestational age (Fig. 4). There was no further effect of IL-8 nAB in combination with the cell culture supernatant (Fig. 4). CD56þ uNK cell supernatants from 8 to 10 weeks gestational age did not alter invasion of EVT cells from placental explants of the same gestational age. In contrast, CD56þ uNK cell supernatants (12e14 weeks gestation) stimulated EVT invasion (P ¼ 0.001, Fig. 4). The addition of 0.5 mg/ml IL-8 specific neutralizing antibody partially abrogated this stimulatory effect (Fig. 4). The uNK cell supernatant þ IL-8 neutralizing antibody group was not significantly different from control group or the uNK cell supernatant group.

599

2

*

8-10 weeks 12-14 weeks

1.5 1 0.5 0 Control

CD8

CD8 + 0.5µg/ml IL8 nAB

CD56

CD56 + 0.5µg/ml IL8 nAB

Fig. 4. Effect of unstimulated CD8þ T lymphocyte and uNK CD56þ cell supernatants (8e10 and 12e14 weeks gestation) on EVT invasion from placental chorionic villi explants (8e10 and 12e14 weeks gestation) through Matrigel alone (n ¼ 10) and in combination with 0.5 mg/ml (n ¼ 10) IL-8 neutralising antibody. * denotes statistical significance compared with controls. ANOVA with Fisher’s post-hoc test, P < 0.05 considered statistically significant.

such high IL-8 levels in early pregnant decidua is not yet clear, but a role in regulating trophoblast invasion has been explored in the current study. CD3þ T lymphocytes comprise fewer than 20% of the decidual leukocyte population with a predominance of CD8þCD3þ T cytotoxic cells [10]. We previously reported that PHA-P stimulated decidual CD8þ T lymphocytes produce high levels of IL-8 in vitro [11]. Similar results were obtained in the current study with PHAPstimulated CD8þ T lymphocytes. Significantly, IL-8 protein and mRNA levels comparable to those found in CD56þ uNK cells were also detected in unstimulated CD8þ T lymphocytes although CD8þ cells in the placental bed were only weakly immunopositive for IL-8. This apparent discrepancy may be explained by the sparse distribution of CD8þ T lymphocytes in the placental bed, particularly compared to CD56þ uNK cells. In addition, it is difficult to immunolocalise proteins in cells with minimal cytoplasm, such as CD8þ T lymphocytes. Alternatively, CD8þ T lymphocytes may rapidly secrete IL-8 making immunolocalisation difficult but allowing detection of secreted protein by ELISA. From these findings it is not clear whether CD8þ T lymphocytes are an important source of IL-8 in normal early pregnant decidua. Hanna et al. [8] reported IL-8RA expression by EVT, although the subtype of EVT was not identified. Jovanovic et al. [16] also reported expression of IL-8RA by isolated cytotrophoblast cells, although they did not investigate the expression of IL-8RB. In the current study both IL-8RA and IL-8RB were immunolocalized to intramural, endovascular and interstitial EVT. Subjectively intramural and endovascular EVT immunostaining of IL-8RB was greater than interstitial EVT. Different functional responses have been observed when IL-8 is bound to IL-8RA and IL-8RB; phospholipase D activation only occurs when IL-8 binds to IL-8RA [14,30]. The signaling events following ligand binding to each receptor are not fully elucidated, although this may involve a G protein and tyrosine kinase [31,32], there have been no studies of these signaling events in EVT. IL-8RB also has high affinity for other CXC chemokines, for example CXCL1 (GROa) [31], and it remains to be determined if these chemokines influence trophoblast function. The control of trophoblast invasion by cytokines and chemokines is likely dependent on a balance between stimulatory and inhibitory molecules. We previously demonstrated that IFN-g, TNF-a and TGF-b1 inhibit EVT invasion in vitro [25,33,34]. In the current study we demonstrate that IL-8 (10ng/ml) stimulates EVT

600

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A

Control

1.0ng/ml IL8

10ng/ml 0.1µg/ml IL8 IL8 nAB

92kDa 82kDa

MMP9

Relative Light Units (Mean +/- SEM)

72kDa 62kDa

B

MMP2

1.6

*

1.4 1.2 1 0.8

MMP-9 MMP-2

0.6 0.4 0.2 0 Control

Control

1ng/ml IL-8

1.0ng/ml IL8

10ng/ml IL-8

10ng/ml IL8

0.1µg/ml IL-8 nAB 0.1µg/ml IL8 nAB

uPA

C

Control

1.0ng/ml IL-8

10ng/ml IL-8

0.1µg/ml IL-8 nAB

PAI-1

PAI-2

Fig. 5. A) Representative gelatin zymogram for MMP-2 and MMP-9 in cell culture supernatants of placental chorionic villi explants treated with IL-8 (0, 1, 10 ng/ml) and anti-IL-8 (0.1 mg/ml). Histogram of densitometry (n ¼ 10), * P ¼ 0.01. B) Representative casein/plasminogen zymogram for uPA in cell culture supernatants of placental chorionic villi explants treated with IL-8 (0, 1, 10 ng/ml) and anti-IL-8 (0.1 mg/ml). C) Western blot analysis for PAI-1 and PAI-2 in cell culture supernatants of placental chorionic villi explants treated with IL-8 (0, 1, 10 ng/ml) and anti-IL-8 (0.1 mg/ml). ANOVA with Fisher’s post-hoc test, P < 0.05 considered statistically significant.

invasion. Addition of an IL-8 neutralizing antibody had no effect on baseline EVT invasiveness but was able to attenuate the stimulating effect of exogenous IL-8, suggesting IL-8 is not acting in an autocrine fashion to alter invasiveness. These data support that of Hanna et al. [8] who also demonstrated that IL-8 (100ng/ml) could stimulate invasion of isolated cytotrophoblast cells in an in vitro invasion assay. Jovanovic et al. [16] also demonstrated that IL-8 (5ng/ml) could stimulate the invasion of isolated cytotrophoblast cells and the HTR-8/SVneo trophoblast-like cell line. The stimulation of HTR-8/SVneo cell line invasion was associated with increased secretion of both MMP-2 and MMP-9. Hanna et al. [8] reported that uNK cell supernatants stimulated cytotrophoblast invasion and the combined addition of IL-8 and CXCL10 (IP-10) neutralizing antibodies significantly abrogated this effect. We have recently demonstrated that uNK cell supernatants from 12 to 14 weeks gestation, but not from 8 to 10 weeks, stimulated EVT invasion [9]. This finding was confirmed in the current study and high concentrations (0.5 mg/ml) of IL-8 neutralizing antibody were required to partially abrogate this effect, most likely due to the high levels of IL-8 secreted by uNK cells at the end of the first trimester. Interestingly, there was no effect on trophoblast

invasion when 0.1 mg/ml IL-8 neutralizing antibody was used in combination with uNK cell supernatants. This suggests that stimulation of EVT invasion by uNK cells at 12e14 weeks gestation is at least in part due to IL-8 production. Unstimulated CD8þ cells secrete similar levels of IL-8 to uNK cells but did not stimulate EVT invasion. This is in contrast to our previous study where we reported that PHA-P stimulated CD8þ cells increased EVT invasion [11]. This suggests that either uNK cells secrete other factors (e.g. IP-10 [8]) that work in concert with IL-8 to stimulate invasion or that CD8þ cells secrete another factor that inhibits the stimulatory activity of IL-8. IL-8, and other cytokines, can also be protolytically cleaved by MMP-9 enhancing its chemoactive activity [35]. uNK cells are a rich source of MMP-9 in the placental bed [36], but it is not known whether CD8þ T cells are also a source of proteases in the placental bed. This may also help to explain the differences between these two cell types. Due to the highly complex nature of the cytokine milieu of the decidua and its inherent leucocyte population it is unlikely that determining the exact nature of this potential cytokine cross talk will be determined. We explored the possible mechanism by which IL-8 may influence EVT invasion. EVT secrete several proteases, including MMP-2, MMP-9 and uPA that play a role in their intrinsic invasive profile [18,19]. IL-8 is known to stimulate MMP secretion by several cell types, including human umbilical vein endothelial cells [37], endometrial stromal cells [38], prostate cancer cell lines [39], bladder cancer lines [40] and melanoma [19]. In contrast, IL-8 does not affect MMP expression in oral squamous cell carcinoma lines [40,41] or ovarian cancer cells [42]. There are no reports regarding the effect of IL-8 on protease levels in trophoblast cells. IL-8 is also known to stimulate uPA secretion in organ-cultured skin [43] and, via this mechanism, IL-8 has been implicated in colorectal cancer progression [39] and breast cancer cell invasiveness [21]. In the current study we found that IL-8 increased levels of MMP-2 secreted by EVT. Interestingly, we have previously demonstrated that the uNK stimulation of invasion is associated with an increase in secreted levels of MMP-9 [9]. In conclusion, we have demonstrated that the decidua is a significant source of IL-8 during early pregnancy, and that CD8þ T lymphocytes and uNK cells are important contributors. IL-8 stimulates trophoblast invasion by a mechanism that appears to involve increased secretion of MMP-2 by EVT. IL-8 contributes to the uNK cell stimulation of trophoblast invasion, but is likely not the only player in this complex process and despite similar IL-8 production to uNK cells unstimulated CD8þ T lymphocytes did not affect EVT invasion. Therefore, IL-8 appears to play a role at the fetalematernal interface and dysregulation of this chemokine may contribute to some complications of pregnancy.

Acknowledgement The authors acknowledge the staff at the Royal Victoria Infirmary, Newcastle upon Tyne, for their assistance with sample collection. This project was supported in part by funding from BBSRC (S19967).

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