Early pregnancy decidual lymphocytes beside perforin use Fas ligand (FasL) mediated cytotoxicity

Journal of Reproductive Immunology 73 (2007) 108–117

Early pregnancy decidual lymphocytes beside perforin use Fas ligand (FasL) mediated cytotoxicity Tatjana Bogovic Crncic a , Gordana Laskarin a , Koraljka Juretic Frankovic a , Vlatka Sotosek Tokmadzic a , Natasa Strbo a , Ivica Bedenicki a , Philippe Le Bouteiller b , Julie Tabiasco b , Daniel Rukavina a,∗ a

Department of Physiology and Immunology, Medical Faculty, University of Rijeka, B. Branchetta 20, HR-51000 Rijeka, Croatia b INSERM U563, Hospital Purpan, Toulouse, France Received 4 January 2006; received in revised form 20 June 2006; accepted 11 July 2006

Abstract Decidual natural killer (NK) cells are the predominant lymphocytes at the maternal–fetal interface. They are involved in defense against virally infected, parasitized and transformed cells and may contribute to the control of trophoblast invasion. The presence of perforin and other possible cytolytic mediators suggests these functions. Cytolytic mechanisms of unstimulated and Th1 cytokine stimulated decidual lymphocytes (DL), as well as purified decidual CD56+ cells, were analyzed against NK sensitive and resistant targets. DL were isolated from decidual mononuclear cells (DMC) cultured in the medium only or in the presence of Th1 cytokines: IL-2, IL-12, IL-15, IL-18 and their combinations (IL-12/IL-18 or IL-15/IL-18). Fas ligand (FasL), perforin and granzyme B mRNAs expression and cytotoxicity were analyzed by flow cytometry and/or RT-PCR. DL (containing 72.19 ± 7.53% of CD56+ cells), obtained from 18 h-cultured DMC in the medium only, expressed perforin, FasL and granzyme B mRNAs and lysed the NK-sensitive K-562 cell line, and also the NK-resistant P815 and P815-Fas transfected cell lines. Concanamycin A, a blocker of granule exocytosis, decreased significantly K-562 lysis, but not P815 lysis. However, the addition of anti-FasL antibody diminished significantly P815 lysis as well. IL-2 and IL-15, known inducers of perforin and FasL mRNAs and protein expression, could not additionally increase P 815 cell lysis by DL cultured within DMC. These results suggest that DL cultured in DMC for 18 h, have the characteristics of lymphokine-activated killer (LAK) cells and are able to use efficiently both the perforin and the FasL cytolytic pathways. © 2006 Published by Elsevier Ireland Ltd. Keywords: Cytotoxicity; Decidual lymphocytes; Fas ligand; Perforin; Th1 cytokines

1. Introduction Lymphokine activated killer (LAK) cells were originally described by Grimm et al. (1982) as IL-2 activated peripheral blood effector cells that become able to lyse a variety of major histocompatibility complex

∗ Corresponding author. Tel.: +385 51 651 150; fax: +385 51 675 699. E-mail address: [email protected] (D. Rukavina).

0165-0378/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.jri.2006.07.001

(MHC) distinct tumor cells. Lymphokine activated cells of CD3− CD56+ natural killer (NK) phenotype play an important role in antitumor immunity (Hashimoto et al., 2003). Since the first trimester trophoblast cells show characteristic of invasive growth (Anin et al., 2004), the modulation of cytolytic activity of decidual lymphocytes (DL) and their role in the regulation of trophoblast invasion have been extensively studied (Zdravkovic et al., 1999). NK cytolytic action is regulated by a dynamic maturation and activation dependent expression of NK cell inhibitory and activating receptors that bind to MHC

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class I or MHC class I like molecules (Ishitani et al., 2003). Th1 type cytokines can induce highly cytotoxic peripheral blood (PB) killer cells, particularly interleukin (IL)-2 (Verma et al., 2000b), IL-12, IL-15 and IL-18 (Sotiriadou et al., 2005). An increased expression of adhesion molecules (Liebau et al., 2002) and cytolytic mediators, perforin and TNF␣ in PB lymphocytes (Ozdemir et al., 2005) or perforin and Fas ligand (FasL) in interstitial intraepithelial lymphocytes (Ebert, 2004) on the mRNA and protein level is attributed to the increased cytolytic potential of LAK cells and can be induced by Th1 type cytokines. Pregnant uterine tissue harbors the highest content of perforin in any physiological or pathological condition (Rukavina et al., 1995). This primary mediator of short-term cytotoxicity, together with serine esterases (Gudelj et al., 1997), is stored primarily within granules of a unique subset of CD3− CD16− CD56bright+ NK cells, which make up 70–80% of DL (Strbo et al., 2006). Even though DL express high number of cytolytic molecules, they have poor cytolytic activity against trophoblast cells (Verma et al., 2000a). The particular combination of non-classical MHC class I (HLA-G and HLA-E) and classical MHC class I (HLA-C) molecule expression on human extravillous trophoblast cells, together with full distribution of NK cell receptors on the surface of decidual NK cells, is at least partially responsible for decreased cytotoxicity at the maternal–fetal interface (King et al., 2000). It has been shown recently that the functions of antigen presenting cells (APC) and NK cells are interrelated (Degli-Esposti and Smyth, 2005). Decidual NK cells are in a close contact with APC comprising numerous CD14+ macrophages (Laskarin et al., 2005) and less frequent mature myeloid CD83+ dendritic cells (Juretic et al., 2004). Dendritic cells could trigger innate NK cell mediated anti-tumor responses in mice (van den Broeke et al., 2003) and human (Degli-Esposti and Smyth, 2005), and both of them determine the final outcome of the immune response. At the maternal–fetal interface the Th2 biased cytokine pattern is considered beneficial for maintaining of pregnancy (Chaouat et al., 1999), although a mild inflammatory response mediated by IL-15, IL-18, IFN-␥ and low concentrations of IL-12, supports tissue remodeling events during implantation (Chaouat et al., 2004). On the other hand, recurrent spontaneous abortions (RSA) are associated with high levels of proinflammatory Th1 type cytokines (Kwak-Kim et al., 2003) and/or the absence of Th2 type cytokines (Michimata et al., 2003). Injection of IL-2 to normally pregnant mice leads to fetal loss (Shiraishi et al., 1996). IL-12, when co-injected with IL-18 in mice, also triggers abortion (Chaouat et al., 2004). Strong IL-12 and/or IL-

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18 expression, as well as marked local depletion of both cytokines in endometrium of implantation failure, were correlated with vascular disorders (Ledee-Bataille et al., 2004). The aim of this study was to investigate whether close contact with decidual APC could induce LAK activity in DL, particularly in CD56+ cells and enable them to lyse NK resistant but LAK sensitive P815 cell line by short term cytotoxicity mediators—perforin and FasL. Potential enhancing effects of the Th1 cytokines (IL-2, IL-12, IL-15, IL-18 and their combinations) were investigated as well. 2. Materials and methods 2.1. Patients and tissues specimen The study was approved by the Ethics Committee of the Medical Faculty, University of Rijeka. Samples of decidual tissues were obtained from 17 healthy women that underwent vaginal elective termination of normal 6–10 weeks old pregnancy. All women were in the reproductive age (range 20–35 years) and had at least one liveborn child. 2.2. Isolation of decidual mononuclear cells Isolation of decidual mononuclear cells (DMC) from decidual tissue was described elsewhere (Rukavina et al., 1995). Briefly, decidual tissue was cut into pieces, exposed to collagenase IV digestion (equal volume of tissue and 0.5% collagenase type IV, Sigma, Munchen, Germany) at 37 ◦ C for 60 min with gentle stirring on the magnetic stirrer. Cell suspension was passed twice through 100 ␮m nylon mesh (Becton Dickinson, Franklin Lakes, NJ, USA) for tissue debris elimination and centrifuged at 600 × g for 10 min. The pellet was resuspended in RPMI 1640 (Institute of Immunology, Zagreb, Croatia) and overlaid on Lymphoprep (Nycomed Pharma AS, Oslo, Norway) and centrifuged at 800 g for 20 min. DMC were collected from the interface, washed twice in RPMI 1640, resuspended 106 /mL of cell culture medium [RPMI 1640 supplemented with l-glutamine (2 mM), penicillin (1 × 10−5 U/l), streptomycin sulphate (0.05 g/l) and 10% Fetal Calf Serum (all from GIBCO, Gaithersburg, MD, USA)]. 2.3. Cell cultures and isolation of decidual lymphocytes Freshly isolated DL were obtained as non-adherent cell fraction of approximately 20 × 106 DMC cultured

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per 100 mm × 20 mm tissue culture Petri dish (TPP, Switzerland) at the concentration 106 /ml of cell culture medium in humidified 5% CO2 incubator at 37 ◦ C, after the short adherence procedure (2 h). DL repeatedly account for approximately 55% of DMC, while 45% of decidual adherent cells remain attached to the plastic surface of Petri dish, as it was found in our previous experiments (Sotosek et al., 1999). DL were also collected as non-adherent cell fraction from the suspensions of DMC cultured for 18 h with Th1 cytokines in above mentioned conditions. Since the effects of cytokines are dose, time and cell dependent, we decided to use 5 ng/ml of IL15 and 10 ng/ml of IL-18 (both from R&D Systems, Mineapolis, USA) and 1000 ng/ml of IL-2 (HoffmanLa Roche, Nutley, NJ, USA) according to our previous titrations for the effective doses of the cytokines on cytolytic mediators’ expression and cytolytic activity of DL (Strbo et al., 2006; Tokmadzic et al., 2002). Since IL12 is reported to be a potent inducer of peripheral blood, as well as decidual LAK activity against choriocarcinoma cell lines and primary culture human placental trophoblasts (Hayakawa et al., 1999), the concentration of 50 pg/ml of IL-12 (R&D Systems), representing the lowest value of its commercially branded biological activity (ED50 0.05–0.2 ng/ml) was used. Collected cultured DL were resuspended in 1 ml of culture medium, counted with assessing viability and used for further experiments. Fas ligand (FasL) protein expression in CD56 positive cells was detected by using indirect immunofluorescence labeling procedure and flow cytometer analysis in the samples cultured in the presence of 10 nM of metalloproteinase inhibitor KB8301 (provided by Prof. S. Saito, Dept. Obst. and Gyn., Toyama Medical and Pharmaceutical University, Toyama, Japan), while DL cultured without KB8301 were used for cytotoxicity assays, RTPCR or CD56 positive cell purification. 2.4. Purification of CD56 positive cells Freshly isolated or short term cultured DL were used for CD56 positive magnetic selection using VarioMACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions. Briefly, DL were filtrated through 100 ␮m nylon mash, washed and resuspended in ice-cold fluorescence-activated cell sorter (FACS) buffer [NaCl (140 mM), KH2 PO4 (1.9 mM), Na2 HPO4 (16.5 mM), KCl (3.75 mM), all from Kemika, Zagreb, Croatia; Na2 EDTA (0.96 mM), Fluka, Buchs, Switzerland; NaN3 (1.5 mM), Difco, Detroit, Michigan, USA; 2% Newborn Calf Serum, GIBCO, Gaithersburg, MD]. Primary mouse anti-human IgG1 anti-CD56 monoclonal antibody (anti-CD56 mAb)

conjugated with MACS MicroBeads (20 ␮l; Miltenyi Biotec) and diluted in FACS buffer 1:5 was added per 107 total cells. After 15 min of incubation at +4 ◦ C, cells were washed and up to 107 cells per 600 ␮l of FACS buffer were applied onto the LS column (Miltenyi Biotec). After the cells ran through, column was rinsed with 3 × 3 ml of FACS buffer and removed from the separator. FACS buffer (5 ml) was applied to the reservoir of the LS column and CD56+ cells were flushed out with the plunger and used for cytotoxicity assays. The purity of cells was measured by direct immunofluorescence using anti-CD56 mAb conjugated with CyChrome or IgG1 isotype matched control (both from Becton Dickinson, Erembodegen, Belgium). 2.5. Cytotoxicity assay Lytic assays were performed with NK sensitive K562, NK resistant P815 or Fas transfected P815 (P815Fas) target cells (provided by Prof. E.R. Podack, Dept. Immunol Mycrobiol, School of Medicine, University of Miami, Florida, USA) labeled with PKH-26 (a lipophilic dye that binds to the cell membrane and fluoresces orange), following manufacturer’s instructions (Sigma Biosciences, St. Louis, MO, USA; PKH-26 Red Fluorescent Cell Linker Kit). Decidual lymphocytes or purified CD56+ cells were incubated in various ratios (50:1, 25:1, 12.5:1, 6:1) with 1 × 104 PKH-26 prestained targets in the volume of 200 ␮l for 2 h at 37 ◦ C, in a humidified atmosphere with 5% CO2 . After washing in FACS buffer, 200 ␮l of propidium iodide (PI, 10 ␮g/ml, Sigma) and 300 ␮l of FACS buffer were added. Some samples of CD56+ cells obtained from cultured DL were pretreated with an inhibitor of vesicular transport, concanamycin A (Malchow et al., 2004) (100 nM, Sigma) for 2 h at 37 ◦ C or with concanamycin A and mouse IgG1 anti-Fas ligand (anti-FasL mAb, 10 ␮g/ml, Becton Dickinson) for 30 min at +4 ◦ C, before cytolytic activity against K-562 or P815 targets was measured. Killed cells were enumerated by FACS CaliburTM (Becton Dickinson) using CellQuestPro software (Becton Dickinson) and detected as double labeled PKH-26 (orange, fluorescence 2) and PI (red, fluorescence 3) positive cells. 2.6. Flow cytometry Intracellular FasL protein detection within CD56+ population was measured after short term culture of stimulated or unstimulated DMC by cell permeabilization method, following the indirect immunofluorescence procedure described previously for perforin

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detection (Rukavina et al., 1995). Briefly, samples were fixed with 4% paraformaldehyde (Kemika), pH 7.4 for 10 min at room temperature and the cells were permeabilized by incubation in saponin buffer [0.1% saponin (Sigma, Poole, Dorset, USA), 2% new born calf serum in phosphate buffered saline (PBS) (NaCl 136.8 mM, Na2 HPO4 × 12H2 O 33.9 mM and KH2 PO4 3 mM, all from Kemika)] for 20 min at room temperature. AntiFasL mAb or mouse IgG1 (1 ␮g diluted in 100 ␮l of saponin buffer) was added to the cell sample and incubated for 30 min at +4 ◦ C. After two washes in saponin buffer, secondary fluorescein-conjugated goat anti-mouse IgG (Becton Dickinson) was added (30 min at +4 ◦ C). Samples were washed twice in saponin buffer and resuspended in 1 ml of FACS buffer to restore the membrane integrity and labeled with CyChrome conjugated anti-CD56 mAb (30 min at +4 ◦ C). FasL was analyzed by flow cytometry at least on 104 cells belonging to lymphocytes and CD56 positive subpopulation gates. 2.7. RT-PCR for cytolytic mediators’ expression Using the guanidine-acid-phenol method (Chomczynski and Sacchi, 1987), total cellular RNA was extracted from 5 × 106 of freshly isolated DL, cultured for further 18 h in the medium only and DL obtained from the suspensions of DMC cultured with IL-2 (100 U/ml), IL-15 (2 ng/ml) or in the medium only for 18 h. Reverse transcription was performed with 10 ␮l of RNA isolate under the following conditions: 0.175 ␮g of total RNA, 0.5 pmol/␮l of oligo (dT) primer (GIBCO-Life Technologies), 1 U/␮l of RNasin (Promega), 0.5 mM dNTP, 10 mM DTT, 10 U/␮l of Superscript II reverse transcriptase (GIBCO-Life Technologies). The reaction was allowed to proceed for 10 min at 65 ◦ C. The reaction mix was stored at −20 ◦ C until usage. PCR was performed under the following conditions: 0.8 pmol/␮l of each primer, 0.2 mM dNTPs, 1.5 mM MgCl2 , 0.1% Triton X-100, 50 mM KCl, 10 mM Tris–HCl, pH 9.0 and 3 U of Taq polymerase (all from GIBCO-Life Technologies). Primers used for amplification had the following sequences: 5 -CAGTACAGCTTCAGCACTGAC3 and 3 -ATGAAGTGGGTGCCG TAGTTG-5 for perforin, 5 -TCGAGGAAGATCGAAAGTGCG-3 and 3 -GAGGCATGCCATTGTTTCGTC-5 for granzyme B or 5 -AAATAGGCCACCCCAGTCCAC-3 and 3 TTCCCCTCCATCATCACCAGA-5 for FasL. The sizes of products were 295, 180 or 220 bp for perforin, granzyme B or FasL, respectively. Primers used for amplification of human glyceraldehyde-3phosphate dehydrogenase (GAPDH) had the fol-

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lowing sequence: 5 -CCAAATTCGTTGTCATAC-3 , 5 -CTGCCGTCTAGAAAA ACC-3 and the size of the product was 176 bp. PCR was performed with 35 cycles of amplification and annealing temperature at 57 ◦ C (15 cycles were performed at 94 ◦ C for 40 s, at 57 ◦ C for 45 s and at 72 ◦ C for 1 min 15 s, whereas final 20 cycles were performed at 94 ◦ C for 40 s, at 56 ◦ C for 1 min and at 72 ◦ C for 1 min 45 s). PCR products were electrophoresed on 1% agarose gel (Sigma), containing ethidium bromide (Sigma) and visualized by UV illumination. 2.8. Statistical analyses The data are presented as mean ± standard deviation (S.D.) using Sigma Plot for Windows, Version 1.02 (Jandel Scientific Software, Chicago, IL, USA). The Mann–Whitney U-test was used to establish the differences between two groups of interest. The difference was considered significant when p < 0.05. Statistic were carried out with Statistica for Windows, Kernel release 5.5 A (StatSoft, Inc., Tulsa, OK, USA). 3. Results 3.1. The frequency of CD56+ cells and the cytolytic activity of decidual lymphocytes against NK sensitive (K-562) and NK resistant (P815 and P815-Fas) cell lines Short term cultured DL in the medium only comprised 72.19 ± 7.53% (mean ± S.D.) of CD56+ cells, which was observed in 10 experiments. Representative experiment is illustrated by the histogram on the Fig. 1A. Decidual lymphocytes lysed NK sensitive K-562 cells within the range of 20–50%, depending on the effector versus target ratio (6:1–50:1) (Fig. 1B). These DL also lysed NK resistant P815 cell line within the range of 10–30% at the effector versus target ratios of 6:1–50:1, which is significantly lower compared to lysis of K-562 targets (p < 0.05, Fig. 1B). However, cytolytic activity of DL against Fas transfected P815 cell line was significantly higher, compared to P815 cell lysis (p < 0.05 at all effector versus target ratios) and quite equal to K-562 cell lysis (Fig. 1B). To answer to the question whether their cytotoxicity can be ascribed to decidual NK cells, positive magnetic separation of CD56+ cells was performed from freshly isolated or short term cultured DL in the medium only. The purity of isolated CD56+ cells observed in 10 experiments was 94.48 ± 3.28% (mean ± S.D.), which is illustrated by the histogram of the representative sample (Fig. 2A). Freshly isolated

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Fig. 1. Short term cytotoxicity of decidual lymphocytes (DL) obtained from the suspension of decidual mononuclear cells after 18 hour-culture at 37 ◦ C against K-562 (), P815 (䊉) or P815Fas () targets in indicated effector vs. target ratios (B). Representative histogram shows the percentage of anti-CD56 mAb labelled cells (solid line), compared to the isotype matched IgG1 (dashed line) (A).

Fig. 2. Short term cytotoxicity of CD56+ cells separated from the suspension of decidual mononuclear cells immediately after isolation () or after 18 hour-culture in the medium only () against K-562 (B) or P815 (C) targets. Some cultured CD56+ cells were pretreated with 100 nM of Concanamycin A () or Concanamycin A and 10 mg/ml of anti-FasL mAb () and their cytotoxicity was measured against K-562 (D) or P815 (E) targets. Representative histogram shows anti-CD56 mAb labelled cells after CD56 positive VarioMACS separation (solid line) compared to the isotype matched IgG1 (dashed line) (A). Each point represents a mean of at least three experiments and level of statistical significance was p < 0.05 for two particular groups compared.

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CD56+ cells lysed K-562 targets with equal efficiency to CD56+ cells purified from cultured DL in the medium only (5–30% at effector versus target ratios of 6:1–50:1, Fig. 2B). Contrary to K-562 targets, freshly isolated CD56+ cells lysed P815 targets only within the range of 0–10% (ratios of 6:1–50:1), which was significantly less compared to CD56+ cells purified from DL cultured within the suspension of DMC in medium only (p < 0.05 at all effector versus target ratios, Fig. 2C). Perforin was mostly involved in killing of K-562 targets, since concanamycin A pretreated CD56+ cells significantly reduced cytotoxicity (p < 0.05 at an effector versus target ratios of 50:1) and subsequent treatment with antiFasL mAb did not induce further reduction (Fig. 2D). Concanamycin A only slightly decreased cytotoxicity against P815, but the pretreatment of CD56+ cells with both concanamycin A and anti-FasL mAb caused signif-

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icant decrease of cytotoxicity in all effector versus target ratios (p < 0.05) (Fig. 2E). 3.2. The influence of Th1 cytokines on cytolytic activity of decidual lymphocytes Cytolytic potential of DL obtained from the shortterm culture of the suspension of DMC in the medium or in the presence of Th1 cytokines, was tested against NK resistant cell lines (Fig. 3). IL-2 did not significantly change the cytolytic effect of DL against P815 and P815-Fas cells, compared to the cytotoxicity of DL cultured in the medium only (Fig. 3A). Almost identical pattern of cytolytic activity was observed for other cytokines tested: IL-12 (Fig. 3B), IL-15 (Fig. 3C) and IL18 (Fig. 3D). Since IL-2, IL-12, IL-15 and IL-18 were not able to increase the cytotoxicity of DL against P815

Fig. 3. Short term cytotoxicity of decidual lymphocytes (DL), obtained from the suspension of decidual mononuclear cells stimulated for 18 hours with indicated concentrations of IL-2 (A), IL-12 (B), IL-15 (C), IL-18 (D) or cultured in the medium only (A, B, C, D), against P815 or P815Fas target cells. Three to five experiments were performed in each group and level of statistical significance was p < 0.05 for two particular groups compared.

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Fig. 4. Short term cytotoxicity of decidual lymphocytes (DL), obtained from the suspension of decidual mononuclear cells stimulated for 18 hours with the combination of IL-12 and IL-18 against P815 (A) or P815Fas (B) targets and the combination of IL-15 and IL-18 against P815 (C) or P815Fas (D) targets in indicated concentrations. Three experiments were performed in each group.

and P815-Fas cell lines above the cytotoxicity of DL obtained from the suspensions of DMC cultured in the medium only, we were intrigued to investigate whether their combinations in short term cultures could additionally enhance cytolytic effect after the same period of stimulation. Combination of cytokines IL-12 and IL-18 (Fig. 4A and B) or IL-15 and IL-18 (Fig. 4C and D) did not significantly increase the cytolytic activity of DL neither against P815 nor against P815-Fas cell line. 3.3. Cytolytic mediator expression in decidual lymphocytes

tured in the medium only for further 18 h (Fig. 5A). Contrary to this, FasL mRNA, granzyme B mRNA and perforin mRNA was present in DL obtained from the suspension of DMC cultured 18 h in the medium only (Fig. 5Ba). Both cytokines, IL-15 (2 ng/ml) and IL-2 (100 U/ml) expressed cytolytic mediators during an 18 h culture (Fig. 5Bb and c). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a catalytic enzyme involved in glycolysis and expressed at high levels in almost all tissues, was used as a positive control of the RT-PCR reaction. 4. Discussion

None of mRNA of cytolytic or apoptotic molecules tested (perforin, granzyme B and FasL) was present in DL isolated after short adherence procedure and cul-

The abundant presence of perforin and serine esterases in vesicles of early pregnancy decidual NK

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Fig. 5. The expression of perforin, granzyme B and FasL mRNAs in freshly isolated decidual lymphocytes (DL) after 18 hour culture in the medium only (A) and (B) in DL cultured within the suspension of decidual mononuclear cells (DMC) for 18 hours: (a) in the medium only, or in the presence of (b) IL-2 (100 U/ml) or (c) IL-15 (2 ng/ml). The total RNA was extracted and subjected to RT-PCR.

cells of CD3− CD16− CD56 bright+ phenotype (Gudelj et al., 1997) suggests their powerful killing potential. In this study, CD56bright+ cells represent about 72% of DL, which corresponds to data of our previous investigations, confirming that more than 70% of DL are NK cells (Rukavina et al., 1995). It is likely that these cells are the main cytotoxic effectors of DL. Indeed, unstimulated DL and purified CD56+ cells that were obtained after shortterm culture of DMC substantially lysed NK sensitive MHC class I negative K-562 cells. Lysis of these targets was mediated by perforin, since decidual CD56+ cells pretreated with an inhibitor of vesicular transport concanamycin A (Malchow et al., 2004) showed significantly reduced cytotoxicity; anti-FasL antibodies were not able to provide further reduction. Quite the same perforin mediated cytotoxicity of decidual CD56+ cells, obtained both immediately after isolation and after 18 hculture in the suspension of DMC, against K-562 targets is probably due to the high content of perforin which these cells possess naturally. Our previous investigation showed that during an 18 h-culture, decidual HLA-DR+ cells from DAC (Sotosek et al., 1999) sustained perforin expression (Strbo et al., 1999) and perforin-mediated cytotoxicity by either cell–cell contact or cytokine production, including IL-15 (Strbo et al., 2006). Storage of potentially dangerous perforin and other cytolytic mediators in the vesicles emphasized the importance of DL cytolytic activity regulation by distinct mechanisms (Rukavina and Podack, 2000). Early pregnancy decidual CD56+ cells are not cytolytic to trophoblast cells unless they acquire LAK properties (King and Loke, 1990; Hayakawa et al., 1999). NK resistant P815 mouse mastocytoma cell line is usually used for in vitro testing

of LAK activity, since it could be lysed by LAK cells only (Lee et al., 1996). In the experiments presented, we found that unstimulated DL and decidual CD56+ cells, both isolated after short term culture of DMC in the medium only, lysed efficiently P815 targets, contrary to freshly isolated CD56+ cells. Contact of decidual CD56+ cells with HLA-DR+ macrophages and mature myeloid CD83+ dendritic cells in the suspension of DMC during 18 h of culture (Juretic et al., 2004; Sotosek et al., 1999) could have a pivotal role in activation of these effectors. It is likely that the beginning of P815 killing is mediated by exocytosis of preformed perforin molecules, since concanamycin A partially decreased lysis of P815. A significant reduction of P815 cell lysis by anti-FasL antibodies and concanamycin A pretreated CD56+ cells in all effector versus target ratios indicates the importance of Fas/FasL mediated killing. This was supported by the presence of FasL mRNA in DL obtained from the suspension of DMC cultured in the medium only (Fig. 5) and the fact that Fas transfected P815 cells, which express ten times higher concentration of Fas on the surface (Lee et al., 1996), were always killed more efficiently than P815 targets. Th1 cytokines (IL2, IL-12, IL-15 and IL-18) added to the suspensions of DMC in the concentrations which efficiently caused LAK activity of peripheral blood lymphocytes of pregnant women (Juretic et al., 2001) could not additionally increase cytototoxicity of DL against P815/P815-Fas targets already stimulated in short term culture in the suspension of DMC. The presence of IL-15 specific ␣ chain and IL-2/IL-15 shared ␤ and ␥ signaling receptors (Verma et al., 2000b) probably allows better stimulation of decidual NK cells with IL-15 abundantly present in

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decidua (Kitaya et al., 2000) than with IL-2. The IL-15mediated up-regulation of CD94/NKG2A receptors on NK cells (Mingari et al., 1997) and the recognition by decidual CD56+ cells of HLA-G and HLA-E molecules on the trophoblast surface by this inhibitory receptor (Ishitani et al., 2003) could protect trophoblast from FasL mediated killing. FasL-mediated cytotoxicity, which is shown as a key cytolytic mechanism in our experiments, is down-regulated in CD94/NKG2A expressing LAK cells in response to the HLA-E bearing targets (Chua et al., 2004). FasL is unable to induce apoptosis of Fas+ trophoblast cells due to the lack of signaling pathway (Aschkenazi et al., 2002). Similarly, IL-18, which is increased in premature rupture of membranes, fails to turn on Fas/FasL mediated apoptosis (Menon et al., 2001). It goes along with our findings that IL-18 neither in combination with IL-15 nor with IL-12 can increase LAK activity and that FasL protein expression in decidual CD56+ cells, although these cytokines are required for perforin up-regulation and perforin mediated killing of NK sensitive targets (Tokmadzic et al., 2002). All these observations indicate immunoregulatory microenvironmental requirements at the maternal–fetal interface, including both cells and soluble factors, selectively dictate the expression of cytokines and cytokine receptors, as well as cytolytic mediators and NK receptors on decidual CD56+ cells and “fine tune” their activation. Acknowledgements This investigation was partially supported by the grants from Croatian Ministry of Science (Grant No. 0062029) and European Commission FP6 Project (EMBIC No. 512040, NoE). We are grateful to Mrs. Ivana Godnic for secretarial assistance. References Anin, S.A., Vince, G., Quenby, S., 2004. Trophoblast invasion. Hum. Fertil. 7, 169–174. Aschkenazi, S., Straszewski, S., Verwer, K.M., Foellmer, H., Rutherford, T., Mor, G., 2002. Differential regulation and function of the Fas/Fas ligand system in human trophoblast cells. Biol. Reprod. 66, 1853–1861. Chaouat, G., Cayol, V., Mairovitz, V., Dubanchet, S., 1999. Localization of the Th2 cytokines IL-3, IL-4, IL-10 at the fetomaternal interface during human and murine pregnancy and lack of requirement for Fas/Fas ligand interaction for a successful allogeneic pregnancy. Am. J. Reprod. Immunol. 42, 1–13. Chaouat, G., Ledee-Bataille, N., Dubanchet, S., Zourbas, S., Sandra, O., Martal, J., 2004. TH1/TH2 paradigm in pregnancy: paradigm lost? Cytokines in pregnancy/early abortion: reexamining the TH1/TH2 paradigm. Int. Arch. Allergy Immunol. 134, 93–119.

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