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a Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, England
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proliferation of uterine NK cells has been shown to occur in vivo, by assessing mitoses of granulated cells [7] and more specifically by double immunohistology for CD45 or CD56 and the proliferation marker Ki-67 [1, 8]. The CD56+ cells were found to be actively proliferating both in the luteal phase and in decidua. These are phases when the other mucosal elements (epithelial glands and stromal cells) have ceased to proliferate and begin to differentiate, except for endothelial cells, which continue to proliferate during this time. However, the stimulus for the in vivo proliferation of NK cells is still unknown.
Interleukin (IL)-2 has been shown to be a powerful proliferative agent for decidual NK cells in vitro, as well as an augmentor of cytolytic activity [9–13]. This, coupled with the observation that decidual NK cells express the IL-2 receptor (R) ß (IL-2Rß) chain [3, 11] would suggest that IL-2 is responsible for maintaining NK cells in utero. However, subsequent work has failed to detect IL-2 protein in either first-trimester trophoblast or decidual cells or IL-2 mRNA even after two rounds of polymerase chain reaction (PCR) amplification [14–16]. It therefore appears that IL-2 is not produced anywhere in the normal implantation site. This study sets out to determine whether IL-15 could be the active cytokine responsible for proliferation of uterine NK cells in vivo.
IL-15 is a four 
-helical cytokine that has been shown to be essential for NK cell development in other sites, notably the bone marrow [17–21]. IL-15 and IL-2 share a number of biological activities: both stimulate the proliferation of activated peripheral blood NK cells [22–25]; both induce cytotoxic activity of peripheral blood NK cells [26]; and both stimulate cytokine production by peripheral blood NK cells. Moreover, to mediate its effects, IL-15 interacts with a heterotrimeric receptor that consists of the ß and 
subunits of IL-2R, as well as a specific, high-affinity IL-15 binding subunit, which is designated IL-15R [27, 28]. To determine whether there is a role for IL-15 in maintaining NK cells in utero, we investigated the expression of the IL-15R by decidual NK cells. We also examined two other issues. Although IL-15 mRNA has been demonstrated in the placenta [24], it is unclear whether the protein is present or which cell type is responsible for its production. In addition, it is not known what effects IL-15 may have on the phenotype and function of decidual NK cells. In this paper we present data that address these points.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Decidual and placental tissues were obtained from normal first-trimester pregnancies terminated for social reasons. Ethical approval to use these tissues was obtained from the Cambridge Local Research Ethics Committee. Cell preparations containing a mixture of all types of maternal decidual cells were isolated by collagenase enzymic disaggregation as previously described [29]. CD56+ CD3- decidual NK cells were purified by magnetic bead isolation. One x 108 decidual cells were suspended in 300 µl buffer (PBS/2 mM EDTA/1% human AB serum). One hundred microliters 0.5% human gamma globulins in Dulbecco's PBS with EDTA (calcium and magnesium free) and 100 µl CD56 MACS microbeads (Miltenyl Biotech, Bergisch Gladbach, Germany) were then added, and the suspension was incubated at 4°C for 20 min. The cells were then washed, resuspended in buffer, and applied to a VS+ column in a VarioMACS magnet (Miltenyl Biotech). The column was washed, and the CD56+ cells were eluted with buffer, washed, and resuspended in RPMI/10% fetal calf serum (FCS). The purity of the decidual NK cells isolated by this method (>97%) was confirmed by immunofluorescent labeling for the antigens CD56, CD16, and CD3, with subsequent flow cytometry (Fig. 1). Decidual stromal cells and macrophages were isolated as described [14, 29]. Macrophage-enriched cultures were obtained by allowing mixed decidual cell preparations to adhere to plastic for 20 min at 37°C and then removing nonadherent cells. Macrophages adhere more rapidly than other cell types. First-trimester placental tissue was isolated according to the method described previously [30]. Briefly, first-trimester placental tissue from elective terminations was incubated for 10 min with trypsin/EDTA and stirred with a magnetic stirrer. The cell suspension was filtered through gauze, and the resultant filtrate was resuspended in Ham's F-12 and then layered over Lymphoprep (ICN Biomedicals Ltd., Costa Mesa, CA). After centrifugation at 610 x g for 20 min, the band of cells at the interface was aspirated and plated out onto plastic dishes for 40 min at 37°C to allow contaminating macrophages to adhere. The nonadherent cells were then transferred onto 35-mm Petri dishes that had been precoated with laminin. The trophoblast preparations obtained in this manner routinely contain 80–90% trophoblast cells with characteristics of extravillous trophoblast [31, 32].
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Highly purified subsets of villous or extravillous trophoblast cells were obtained by fluorescence-activated cell sorting (FACS) as described previously [16]. Briefly, enzymatically disaggregated trophoblast cells from first-trimester placentae were incubated with a monoclonal antibody (mAb) to either the epidermal growth factor (EGF)R (Ab-1; Oncogene Science, Uniondale, NY) to label villous cytotrophoblast or to c-erbB2 (Ab-5; Oncogene Science) to label extravillous trophoblast. The cells were then stained with secondary fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG F(ab')2 fragments (Serotec, Inc., Raleigh, NC). At the same time, these cells were also labeled with a directly phycoerythrin (PE)-conjugated mAb to CD14 (Becton Dickinson, Cowley, UK) to stain macrophages. Flow cytometric sorting was done using a Becton Dickinson FACStar, and villous (EGFR+ c-erbB2- CD14-) or extravillous (e-erbB2+ EGFR- CD14-) trophoblast cells were obtained in excess of 99% purity as determined by subsequent FACS analysis.
Cell Lines
The JEG-3 and BeWo choriocarcinoma cell lines were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD). They were maintained in RPMI/10% FCS) (Gibco, Gaithersburg, MD), 2.0 mM L-glutamine, and antibiotics. The NK-derived cell lines, NK-92 [33] and NKL [34] were obtained from Immune Medicine (Vancouver, BC, Canada) and Dr. M.J. Robertson (Harvard University, Cambridge, MA), respectively. NK-92 was maintained in MyeloCult medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 150 IU/ml IL-2 (Chiron, St. Louis, MO), antibiotics, and L-glutamine. NKL was cultured in RPMI/15% FCS, supplemented with 50 IU/ml IL-2, 1% sodium pyruvate, 10-6 M hydrocortisone (Sigma, St. Louis, MO), antibiotics, and L-glutamine. K562 (ICN Flow), LCL 721.221-Parent and U937 (ECACC) cell lines were maintained by twice-weekly subculture in RPMI/10% FCS, antibiotics, and L-glutamine. LCL 721.221 cells transfected with human leukocyte antigen (HLA)-G [35] were maintained in RPMI/10% FCS supplemented with 100 µg/ml hygromycin B (Sigma), antibiotics and L-glutamine.
Flow Cytometry
Immunofluorescent labeling for cell surface antigens was carried out as previously described [30]. EB6, GL183, HP-3E4, DX9, and 5.133 are mAbs for killer immunoglobulin receptors (KIR) and were obtained from and used as previously described [36]. HP-3B1, reactive to CD94 [37] and HP-F1, reactive to immunoglobulin-like transcript-2 [38] were gifts from M. Lopez-Botet (Madrid, Spain). All analysis was carried out on a Becton Dickinson FACScan flow cytometer (with an argon laser set to 488 nm), interfaced with a Hewlett-Packard (Palo Alto, CA) 310 computer using Lysis II software (Becton Dickinson).
Analysis of IL-15 and IL-15R mRNA Expression
RNA extraction, cDNA synthesis, and RT-PCR amplification
RNA was prepared from tissue samples (from 2 to 6 individuals) of decidua parietalis, proliferative endometrium, secretory endometrium, menstrual endometrium, and first-trimester placental tissue using the CLONsep total RNA kit according to the manufacturer's instructions (Clontech, Palo Alto, CA). Decidual and peripheral blood lymphocyte subpopulations and first-trimester trophoblast cells labeled with mAbs to c-erbB2 or the EGFR were obtained to a purity in excess of 99% by flow cytometric sorting as described [16, 39]. Total cellular RNA was extracted using the guanidine-acid-phenol method from 1 to 2 x 105 cells to give 10 µl of RNA isolate [40]. First-strand cDNA was synthesized by using oligo(dT) primer and avian myeloblastosis virus reverse transcriptase (HT Biotechnology Ltd., Cambridge, UK). PCR was performed using 30 cycles of amplification and annealing temperatures of 55°C for ß-actin, 60°C for IL-15, and 59°C for IL-15R. For cases in which cell numbers were small, a nested PCR for IL-15R was used with a second PCR round of 20 cycles at an annealing temperature of 55°C. The primer sequences were as follows: ß–actin, 5' GTG GGG CGC CCC AGG CAC CA, 5' CTC CTT AAT GTC ACG CAC GAT TTC; IL-15, 5' GGC TTT GAG TAA TGA GAA TTT CGA, 5' ATC AAT TGC AAT CAA GAA GTG TTG; IL-15R 5' GGC GAC GCG GGG CAT CAC, 5' TCG CTG TGG CCC TGT GGA TA; and IL-15R primers for nested PCR, 5' GTC AAG AGC TAC AGC TTG TA, 5' GCT GTG TTG TTT GAG CTG G.
For comparison of IL-15 mRNA levels, cDNA concentrations were normalized to yield equivalent ß-actin PCR products. IL-15 reverse transcription (RT)-PCR products were quantified by intensity analysis using a film documentation system (Ofoto; Apple, Cambridge, UK) and NIH (Bethesda, MD) Image software. Samples were normalized to the ß-actin PCR product according to the following equation after demonstration that both PCR reactions were linear over the range of cycles carried out for each one: Normalized IL-15 message = (Intensity of IL-15 PCR product/Intensity of ß-actin PCR product) x 100.
Southern blot analysis
To confirm the identity of the RT-PCR products, Southern blot analysis was performed. After separation of the PCR products by agarose gel electrophoresis and overnight transfer onto Hybond N+ membrane (Amersham, Arlington Heights, IL), the blots were hybridized with a biotin end-labeled internal oligonucleotide probe: IL-15, 5' GGG CTG TTT CAG TGC AGG GCT TCC T; IL-15R, 5' CCA GCT CAA ACA ACA CAG C. After hybridization at 42°C for 4 h, the IL-15 blots were washed twice with single-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) containing 0.1% SDS at 42°C for 15 min. The IL-15Ra blots were washed with double-strength SSC plus 0.1% SDS at 63°C for 15 min. The blots were visualized using an enhanced chemiluminescence (ECL) detection kit (RPN 3004; Amersham).
Supernatant Generation and IL-15 ELISA
In all experiments to collect cell culture supernatants, cells were cultured in triplicate in 48-well plates (Costar, Cambridge, MA), 5 x 105 cells/well, in a total volume of 500 µl of the appropriate culture medium, in a humidified incubator at 37°C in 95% air + 5% CO2. After culture for 48 h, the culture supernatants were aspirated, centrifuged at 400 x g to remove particulate matter, and transferred to fresh Eppendorf tubes (Hamburg, Germany). These were then frozen and stored at -70°C until analysis by ELISA. Aliquots of supernatants were also concentrated 10-fold using a Centriprep Concentrator (Amicon, Beverly, MA). Samples were assayed for human IL-15 using a commercially available ELISA (R&D Systems, Minneapolis, MN) with a sensitivity of 1 pg/mL. Each individual standard or sample was assayed in duplicate.
Cytotoxicity Assays
Cytotoxic activity was assessed by the lysis of 51Cr-labeled K562, JEG-3 choriocarcinoma, or trophoblast target cells, as previously described [41]. Spontaneous 51Cr release was <10% for K562 cells, <15% for JEG-3 cells, and <20% for trophoblast cells. Experiments were performed at least 3 times.
Proliferation Assays
Tritiated thymidine incorporation by purified CD56+ decidual NK cells was measured to give an indication of cellular proliferation in response to exogenous cytokines. Recombinant human (rh)IL-2 was obtained from Chiron, rhIL-15 from PeproTech (Rocky Hill, NJ), rh stem cell factor (SCF) from Genzyme (Cambridge, MA), and rhIL-12 and -18 from R&D Systems. Sheep anti-human IL-15 was obtained from Peprotech and mouse anti-human IL-2 from R&D Systems. CD56+ decidual NK cells were cultured in RPMI/10% FCS medium in round-bottomed 96-well plates (Becton Dickinson). Cells were cultured in a volume of 200 µl at a final concentration of 5 x 105 cells/ml, in a variety of culture conditions. All cultures were done in quadruplicate. Radiolabeled thymidine incorporation was measured at 48 and 96 h. Cells were spiked with 1 µCi/well of [3H]thymidine (Amersham) 14 h before cell harvesting. All cultures were harvested onto glass fiber filter paper (Skatron Instruments, Sterling, VA) using a Dynatech Automash 2000 cell harvester (Dynatech Labs., Alexandria, VA). The filter mats were dried and placed in scintillation vials (Pharmacia, Kalamazoo, MI) with 2 ml of OptiPhase "Hisafe" II scintillation fluid (Pharmacia), and radioactivity was measured on a Tri-Carb 1600 TR liquid scintillation counter (Packard, Pangbourne, UK). All experiments were performed at least 3 times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR and Southern blotting were used to investigate expression of transcripts for IL-15R in decidual NK cells. Two bands of 432 and 531 base pairs (bp) were detected in purified decidual NK cells and NK cell lines NK-92 and NKL as well as the choriocarcinoma cell line, JEG-3 (Fig. 2, a and b). These bands represent two splice variants of IL-15R derived from alternative splicing of exon 3. When the nested RT-PCR was performed with cDNA samples from CD56bright cells sorted to >99.5% purity, the expected 283- and 184-bp bands were detectable. After isolation of CD56+ decidual NK cells with two rounds of selection using MACS beads, there was relatively little change seen in receptor transcription on subsequent 48-h culture in the presence or absence of IL-2 or IL-15 (Fig. 2c).
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IL-15 mRNA Was Present in Decidua and Placenta
RT-PCR for IL-15 mRNA revealed that both the 513-bp and 632-bp isoforms were relatively ubiquitous (Fig. 3). IL-15 mRNA was found in pregnant and nonpregnant endometrial samples. Semiquantification of the PCR reaction was performed by normalizing the results relative to ß-actin. There was little variation in levels of IL-15 mRNA throughout the proliferative, secretory, and premenstrual phases of the nonpregnant cycle. However, IL-15 message was increased in pregnancy, with placenta and decidua expressing high levels of IL-15 mRNA (Fig. 4a). Analysis of highly purified cDNA, obtained by flow cytometric sorting of placental cells, indicated that IL-15 mRNA was present in decidual macrophages and NK cells, as well as villous and extravillous trophoblast (Fig. 4b). It was not, however, detected in decidual T cells.
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IL-15 but Not IL-12 Protein Was Produced by Cells at the Maternal-Fetal Interface
The presence of IL-15 was evaluated by ELISA in culture supernatants from decidual, extravillous trophoblast, macrophage-enriched, and stromal cell cultures (Fig. 5). Both decidual and extravillous trophoblast cell cultures contained detectable amounts of IL-15, and production by decidua was enhanced by coculture with extravillous trophoblast and culture in 100 IU/ml IL-2. Relatively high levels of IL-15 were detected in supernatants from macrophage-enriched cultures, and this production was reduced by culture in decidualization medium containing prostaglandin (PG) E2 and progesterone. In contrast, production of IL-15 by stromal cells was enhanced by PGE2 and progesterone.
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The presence of IL-12 was also sought in the same culture supernatants using a high-sensitivity ELISA assay capable of detecting 0.7 pg/ml. No IL-12 was detected in culture supernatants from a variety of cells isolated from the utero-placental interface even if the supernatants were concentrated (data not shown).
Proliferation of Decidual NK Cells in the Presence of IL-15 Alone or Together with Stem Cell Factor (SCF), IL-12, IL-18, or Decidual Stromal Cells
Decidual NK cells responded in a dose-dependent manner to IL-15, with optimal proliferation at 5 ng/ml IL-15 (Fig. 6). This was generally equivalent to the response seen with 100 IU/ml IL-2. A further experiment indicated that the proliferative response was more marked after culture for 96 h than for 48 h. The proliferative response to IL-15 was also seen to vary with cell number and was blocked by neutralizing doses of polyclonal anti-IL-15 (200 µg/ml), but not by anti-IL-2 (100 µg/ml). Cells cultured in the optimal dose of IL-2 (100 IU/ml) acted as positive controls, and showed the anti-IL-15 blocking to be specific (Fig. 7). SCF alone had no effect on decidual NK cell proliferation, but when both IL-15 and SCF were present, NK cell proliferation was substantially increased compared to that seen with IL-15 alone and also reached a level greater than that exhibited by IL-2-stimulated cells (Fig. 8). Similar experiments were also performed with decidual NK cells exposed to IL-15 either alone or cocultured with an irradiated layer of decidual stromal cells. There was a synergistic response when the decidual NK cells were cultured with IL-15 and in contact with stromal cells. This response was seen even when suboptimal doses of IL-15 were used, which would not initiate NK cell proliferation alone in the absence of the irradiated stromal cell monolayer (data not shown). IL-18 had no synergistic effect on decidual NK cell proliferation either in the presence or absence of stromal cells. In contrast, the addition of IL-12 resulted in partial inhibition of the IL-15-induced response, especially when the cells were in contact with stromal cells (data not shown).
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IL-15 Augmented Decidual NK Cell Cytotoxicity Against K562 and JEG-3 Cells, but Normal Trophoblast Remained Resistant to Lysis
Preliminary experiments established that the optimal concentration of IL-15 for augmenting decidual NK cell cytotoxicity against the NK-susceptible cell line K562 was 5 ng/ml after 96 h. CD56+ decidual NK cells cultured in IL-15 killed K562 generally as well as cells cultured in optimal doses of IL-2, and the effect was abolished by the addition of anti-IL-15 (Fig. 9a). IL-15 was able to induce decidual NK cell cytotoxicity against the choriocarcinoma cell line JEG-3, but always to a lesser degree than that seen in response to IL-2. Overall levels of killing were lower than those seen against K562 (Fig. 9b). Finally, in contrast to cells cultured in IL-2, decidual NK cells cultured in IL-15 exhibited minimal levels of cytotoxicity against extravillous trophoblast (Fig. 9c).
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IL-15 Had No Effect on the Decidual NK Cell Receptor Repertoire
Three samples were analyzed (of 8, 9, and 9 wk gestation). Whole decidual cell preparations were obtained, and some cells stained immediately and were analyzed for their NK repertoire using flow cytometry and a panel of mAbs comprising EB6, GL183, HP-3E4, DX9, 5.133, HP-3B1, and HP-F1. The remaining cells were then cultured for 72 h with or without 5 ng/ml IL-15 and stained with the same mAbs again. In all cases, uncultured cells and cells cultured in the presence or absence of IL-15 expressed an identical repertoire of NK receptors, indicating that IL-15 had no effect on the NK repertoire of mature decidual NK and T cells (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biological effects of IL-15 are mediated through interaction with the ß and subunits of the IL-2R [21, 27]. A specific subunit, IL-15R, is required for high-affinity binding of IL-15 [44]. Since there are no antibodies available to IL-15R protein, we looked for IL-15R message in decidual cells. IL-15R message was found to be abundant in the NK-derived, macrophage-derived, and choriocarcinoma cell lines used in this study. This was consistent with previous observations that reported a widespread tissue distribution of IL-15R message that included liver, heart, spleen, lung, skeletal muscle, and activated vascular endothelial cells [45]. IL-15R message has also been previously demonstrated in placenta [24]. IL-15R message was also present at low levels in purified CD56+ decidual NK cells. Since the expression of the IL-2Rß by decidual NK cells has been previously reported by many investigators [3, 9–11] and we have recently observed the expression of the IL-2R by these cells (unpublished), it would appear that all three of the trimeric components of the IL-15R are present. This would suggest that decidual NK cells are potentially able to respond to IL-15. This conclusion is supported by subsequent functional studies.
IL-15 induced the proliferation of decidual CD56+ NK cells in a dose-dependent manner, showing that these tissue NK cells readily respond to this cytokine. IL-15 was first identified as a T-cell growth factor designated IL-T [24, 26] and was subsequently found also to act on NK cells. A dose of 5 ng/ml produced a maximal proliferative response, which is similar to results reported for blood CD56bright cells. In contrast, the circulating CD56dim cells are reported to respond poorly to IL-15. We also found that there was a synergistic response when decidual NK cells were cultured with IL-15 (even at suboptimal levels) in contact with a monolayer of irradiated decidual stromal cells, indicating that other factors are responsible for the proliferation in vivo. We have previously reported similar results using IL-2 [29]. Other studies support the importance of stromal cells in proliferation of CD56bright cells. From the pool of CD56+, CD3- blood NK cells, only the CD56bright subset was responsible for the expansion mediated by contact with an irradiated murine fibroblast cell line [46]. The close physical association of NK cells to the uterine mucosal stromal cells also suggests a mutual interdependence in vivo. IL-15 has also been shown to induce expression of the NK cell receptor CD94/NKG2A, but not of killer immunoglobulin receptors (KIR), in the process of NK cell maturation from immature thymocytes [20]. Interestingly, in peripheral blood, CD56bright cells are CD94/NKG2A+ but negative for KIR. In contrast, in decidua, a large proportion express KIR specific for HLA-C class I molecules [36, 39]. We were unable to detect any further change in expression of NK receptors after culture of decidual NK cells with IL-15, suggesting that these tissue NK cells have already fully differentiated in the decidual microenvironment.
SCF has been found in the endometrium and decidua, and it may be produced by stromal cells or smooth muscle medial cells [47, 48]. In the present study, we found that addition of SCF augmented the IL-15-induced proliferative response of decidual NK cells. Similar findings have been reported for IL-2 and SCF in both blood and decidual NK cells [49, 50]. The SCF receptor, c-kit, is expressed by blood CD56bright cells [23, 51, 52] and also by a proportion of decidual CD56bright cells [50]. This subpopulation may represent the undifferentiated cells capable of a vigorous proliferative response. CD56bright cells increase in peripheral blood of pregnant women in the first trimester [36]. It would be of interest to determine whether it is these CD56bright, CD16-, and c-kit+ cells that home to the uterine mucosa and then undergo proliferation and differentiation in response to SCF and IL-15. CD56+ CD3- cells develop from 21-day cultures of CD34+ hematopoietic progenitor cells exposed to IL-15 or IL-15 and SCF [17]. Whereas IL-15 alone was able to induce the expression of CD56 on these CD34+ cells, SCF and IL-15 together were required for the differentiation and expansion of the CD56+ CD3- cells. More recently, NK cell differentiation has been induced by the culture of cord blood, bone marrow, or peripheral blood CD34+ cells with a combination of cytokines including IL-15, SCF, IL-2, and IL-7 [18, 20, 53]. A combination of IL-15 and SCF induced NK maturation from immature (CD34+ CD7-) populations into CD56+ CD3- cells. Taken together, these observations strongly suggest an important role for IL-15 with SCF in NK development in vivo from both adult and fetal precursors.
We have also found that IL-15 augmented decidual NK cell cytotoxicity against K562 in the same way as IL-2. However, while IL-15 also stimulated decidual NK cell killing against JEG-3, there was little or no cytotoxicity against trophoblast. One possibility for this finding is that the expression of triggering receptors such as NKp44 and NKp46 may differ after culture with either IL-2 or IL-15 [54, 55]. Cytolytic activity of human peripheral blood mononuclear cells has also been shown to be enhanced by IL-15 in HIV infection [56] and Herpes virus infection [57, 58]. Other effects of IL-15 on decidual NK cells can also be envisaged. When progesterone levels drop either premenstrually or as a result of a failing pregnancy, the first obvious morphological manifestation is apoptosis of the NK cells [6]. IL-15 has been shown to promote survival of blood NK cells with an increase in bcl-2 expression [59, 60]. It will therefore be of interest to see how IL-15 secretion is modified by progesterone.
Although IL-2 and IL-15 have numerous overlapping activities on cells of the immune system, the differential expression of these cytokines within tissues and by various cell types suggests that they may perform at least partially distinct physiological functions. In the present study, while exogenous IL-2 stimulated decidual NK cytotoxicity against trophoblast, as well as promoting NK proliferation, IL-15 is remarkable in that it could produce proliferation of decidual NK cells without transforming them into potent cytolytic cells capable of destroying trophoblast. This would be a more appropriate physiological function for a cytokine that is present at the materno-fetal interface. It is therefore reasonable to propose that in utero IL-15 may have a role to play in promoting decidual NK cell survival and expansion. A similar role has been proposed in mice [61].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 S.V. was in receipt of a Wellcome MB/PhD studentship. S.E.H. was supported by Tommy's Campaign and A.K. is the Medical Fellow at King's College, Cambridge. 
2 Correspondence: Ashley King, Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, England. FAX: 44 1223 333727; akk27{at}cam.ac.uk
Accepted: November 5, 1999.
Received: July 12, 1999.
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A. van der Meer, H.G.M. Lukassen, B. van Cranenbroek, E.H. Weiss, D.D.M. Braat, M.J. van Lierop, and I. Joosten Soluble HLA-G promotes Th1-type cytokine production by cytokine-activated uterine and peripheral natural killer cells Mol. Hum. Reprod., February 1, 2007; 13(2): 123 - 133. [Abstract] [Full Text] [PDF]
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Y. Hu, J. P. Dutz, C. D. MacCalman, P. Yong, R. Tan, and P. von Dadelszen Decidual NK Cells Alter In Vitro First Trimester Extravillous Cytotrophoblast Migration: A Role for IFN-{gamma} J. Immunol., December 15, 2006; 177(12): 8522 - 8530. [Abstract] [Full Text] [PDF]
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P. Vacca, G. Pietra, M. Falco, E. Romeo, C. Bottino, F. Bellora, F. Prefumo, E. Fulcheri, P. L. Venturini, M. Costa, et al. Analysis of natural killer cells isolated from human decidua: evidence that 2B4 (CD244) functions as an inhibitory receptor and blocks NK-cell function Blood, December 15, 2006; 108(13): 4078 - 4085. [Abstract] [Full Text] [PDF]
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 R. L Jones, C. Stoikos, J. K Findlay, and L. A Salamonsen TGF-{beta} superfamily expression and actions in the endometrium and placenta. Reproduction, August 1, 2006; 132(2): 217 - 232. [Abstract] [Full Text] [PDF]
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 E. Mavoungou Interactions between natural killer cells, cortisol and prolactin in malaria during pregnancy. Clin. Med. Res., March 1, 2006; 4(1): 33 - 41. [Abstract] [Full Text] [PDF]
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 A. Sun, H. Wei, R. Sun, W. Xiao, Y. Yang, and Z. Tian Human Interleukin-15 Improves Engraftment of Human T Cells in NOD-SCID Mice Clin. Vaccine Immunol., February 1, 2006; 13(2): 227 - 234. [Abstract] [Full Text] [PDF]
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 H. N. Jabbour, R. W. Kelly, H. M. Fraser, and H. O. D. Critchley Endocrine Regulation of Menstruation Endocr. Rev., February 1, 2006; 27(1): 17 - 46. [Abstract] [Full Text] [PDF]
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F Arcuri, M Cintorino, A Carducci, S Papa, M G Riparbelli, S Mangioni, A M Di Blasio, P Tosi, and P Vigano Human decidual natural killer cells as a source and target of macrophage migration inhibitory factor Reproduction, January 1, 2006; 131(1): 175 - 182. [Abstract] [Full Text] [PDF]
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 E. Dimitriadis, C.A. White, R.L. Jones, and L.A. Salamonsen Cytokines, chemokines and growth factors in endometrium related to implantation Hum. Reprod. Update, November 1, 2005; 11(6): 613 - 630. [Abstract] [Full Text] [PDF]
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H. D. Kopcow, D. S. J. Allan, X. Chen, B. Rybalov, M. M. Andzelm, B. Ge, and J. L. Strominger Human decidual NK cells form immature activating synapses and are not cytotoxic PNAS, October 25, 2005; 102(43): 15563 - 15568. [Abstract] [Full Text] [PDF]
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M. Roberts, X. Luo, and N. Chegini Differential regulation of interleukins IL-13 and IL-15 by ovarian steroids, TNF-{alpha} and TGF-{beta} in human endometrial epithelial and stromal cells Mol. Hum. Reprod., October 1, 2005; 11(10): 751 - 760. [Abstract] [Full Text] [PDF]
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 K. Kitaya, T. Yamaguchi, and H. Honjo Central Role of Interleukin-15 in Postovulatory Recruitment of Peripheral Blood CD16(-) Natural Killer Cells into Human Endometrium J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2932 - 2940. [Abstract] [Full Text] [PDF]
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M. J. van den Heuvel, J. Horrocks, S. Bashar, S. Taylor, S. Burke, K. Hatta, J. E. Lewis, and B. A. Croy Menstrual Cycle Hormones Induce Changes in Functional Interactions between Lymphocytes and Decidual Vascular Endothelial Cells J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2835 - 2842. [Abstract] [Full Text] [PDF]
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G. Laskarin, K. Cupurdija, V. S. Tokmadzic, D. Dorcic, J. Dupor, K. Juretic, N. Strbo, T. B. Crncic, F. Marchezi, P. Allavena, et al. The presence of functional mannose receptor on macrophages at the maternal-fetal interface Hum. Reprod., April 1, 2005; 20(4): 1057 - 1066. [Abstract] [Full Text] [PDF]
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C. Dosiou and L. C. Giudice Natural Killer Cells in Pregnancy and Recurrent Pregnancy Loss: Endocrine and Immunologic Perspectives Endocr. Rev., February 1, 2005; 26(1): 44 - 62. [Abstract] [Full Text] [PDF]
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M. Eriksson, S. K. Meadows, C. R. Wira, and C. L. Sentman Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-{beta} J. Leukoc. Biol., September 1, 2004; 76(3): 667 - 675. [Abstract] [Full Text] [PDF]
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K. Kitaya, T. Nakayama, N. Daikoku, S. Fushiki, and H. Honjo Spatial and Temporal Expression of Ligands for CXCR3 and CXCR4 in Human Endometrium J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2470 - 2476. [Abstract] [Full Text] [PDF]
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A. van der Meer, H.G.M. Lukassen, M.J.C. van Lierop, F. Wijnands, S. Mosselman, D.D.M. Braat, and I. Joosten Membrane-bound HLA-G activates proliferation and interferon-{gamma} production by uterine natural killer cells Mol. Hum. Reprod., March 1, 2004; 10(3): 189 - 195. [Abstract] [Full Text] [PDF]
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B. Wang and A. K. Goff Interferon-{tau} Stimulates Secretion of Macrophage Migration Inhibitory Factor from Bovine Endometrial Epithelial Cells Biol Reprod, November 1, 2003; 69(5): 1690 - 1696. [Abstract] [Full Text] [PDF]
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L. Gardner and A. Moffett Dendritic Cells in the Human Decidua Biol Reprod, October 1, 2003; 69(4): 1438 - 1446. [Abstract] [Full Text] [PDF]
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A. A. Ashkar, G. P. Black, Q. Wei, H. He, L. Liang, J. R. Head, and B. A. Croy Assessment of Requirements for IL-15 and IFN Regulatory Factors in Uterine NK Cell Differentiation and Function During Pregnancy J. Immunol., September 15, 2003; 171(6): 2937 - 2944. [Abstract] [Full Text] [PDF]
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 R. Feinmesser, B. Hardy, R. Sadov, A. Shwartz, P. Chretien, and M. Feinmesser Report of a Clinical Trial in 12 Patients With Head and Neck Cancer Treated Intratumorally and Peritumorally With Multikine Arch Otolaryngol Head Neck Surg, August 1, 2003; 129(8): 874 - 881. [Abstract] [Full Text] [PDF]
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E. M. Barber and J. W. Pollard The Uterine NK Cell Population Requires IL-15 but These Cells Are Not Required for Pregnancy nor the Resolution of a Listeria monocytogenes Infection J. Immunol., July 1, 2003; 171(1): 37 - 46. [Abstract] [Full Text] [PDF]
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K. Kitaya, T. Nakayama, T. Okubo, H. Kuroboshi, S. Fushiki, and H. Honjo Expression of Macrophage Inflammatory Protein-1{beta} in Human Endometrium: Its Role in Endometrial Recruitment of Natural Killer Cells J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1809 - 1814. [Abstract] [Full Text] [PDF]
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T. A. Henderson, P. T. K. Saunders, A. Moffett-King, N. P. Groome, and H. O. D. Critchley Steroid Receptor Expression in Uterine Natural Killer Cells J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 440 - 449. [Abstract] [Full Text] [PDF]
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 L. C. Kao, S. Tulac, S. Lobo, B. Imani, J. P. Yang, A. Germeyer, K. Osteen, R. N. Taylor, B. A. Lessey, and L. C. Giudice Global Gene Profiling in Human Endometrium during the Window of Implantation Endocrinology, June 1, 2002; 143(6): 2119 - 2138. [Abstract] [Full Text] [PDF]
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H. Engelhardt, B. A. Croy, and G. J. King Conceptus Influences the Distribution of Uterine Leukocytes During Early Porcine Pregnancy Biol Reprod, June 1, 2002; 66(6): 1875 - 1880. [Abstract] [Full Text] [PDF]
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O. Gubbay, H. O. D. Critchley, J. M. Bowen, A. King, and H. N. Jabbour Prolactin Induces ERK Phosphorylation in Epithelial and CD56+ Natural Killer Cells of the Human Endometrium J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2329 - 2335. [Abstract] [Full Text] [PDF]
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 S. E. Hiby, M. Lough, E. B. Keverne, M. A. Surani, Y. W. Loke, and A. King Paternal monoallelic expression of PEG3 in the human placenta Hum. Mol. Genet., May 1, 2001; 10(10): 1093 - 1100. [Abstract] [Full Text] [PDF]
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X. F. Li, D. S. Charnock-Jones, E. Zhang, S. Hiby, S. Malik, K. Day, D. Licence, J. M. Bowen, L. Gardner, A. King, et al. Angiogenic Growth Factor Messenger Ribonucleic Acids in Uterine Natural Killer Cells J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1823 - 1834. [Abstract] [Full Text]
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F. Arcuri, C. Ricci, F. Ietta, M. Cintorino, S. A. Tripodi, I. Cetin, E. Garzia, F. Schatz, P. Klemi, R. Santopietro, et al. Macrophage Migration Inhibitory Factor in the Human Endometrium: Expression and Localization During the Menstrual Cycle and Early Pregnancy Biol Reprod, April 1, 2001; 64(4): 1200 - 1205. [Abstract] [Full Text]
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S. Wongweragiat, R. F. Searle, and J. N. Bulmer Expression of Fas/Fas Ligand by Decidual Leukocytes in Hydatidiform Mole Biol Reprod, March 1, 2001; 64(3): 784 - 789. [Abstract] [Full Text]
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R. Agarwal, A. Loganath, A.C. Roy, Y.C. Wong, and S.C. Ng Expression profiles of interleukin-15 in early and late gestational human placenta and in pre-eclamptic placenta Mol. Hum. Reprod., January 1, 2001; 7(1): 97 - 101. [Abstract] [Full Text] [PDF]
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T. A. Fehniger and M. A. Caligiuri Interleukin 15: biology and relevance to human disease Blood, January 1, 2001; 97(1): 14 - 32. [Full Text] [PDF]
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H. Okada, T. Nakajima, M. Sanezumi, A. Ikuta, K. Yasuda, and H. Kanzaki Progesterone Enhances Interleukin-15 Production in Human Endometrial Stromal Cells in Vitro J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4765 - 4770. [Abstract] [Full Text]
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