Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
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Abstract |
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Key words: cytokine/microenvironment/uterine lymphocytes
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Introduction |
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In blood, the circulating CD56bright subset of NK cells proliferates in response to interleukin-2 (IL-2) alone without antigenic stimulation (Caligiuri et al., 1990; Nagler et al., 1990
; Baume et al., 1992
). Decidual CD56 NK cells behave in a similar manner (King et al., 1992
). However, it is clear that, like T and B cells, NK cells require other cofactors besides IL-2 for optimal proliferation (Robertson et al., 1993
). For example, NK cell proliferation is enhanced when B lymphoblastoid cells are present (Perussia et al., 1987
) and NK cell maturation in the bone marrow is dependent on bone marrow stromal cells (Mrozek et al., 1996
). We have therefore investigated whether decidual stromal cells could act as a cofactor for uterine NK cell proliferation. As a corollary to proliferation we have also studied the expression of Bcl-2, a protein which prevents apoptotic cell death (Vaux, 1993
) in CD56+ cells throughout the non-pregnant endometrium and decidua. Our results show that decidual stromal cells do influence the proliferation and survival of uterine CD56+ cells.
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Materials and methods |
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T-cell depletion from lymphocyte preparations
CD3+ T cells were depleted from lymphocyte preparations using AIS MicroCELLector T-25 cell culture flasks (Applied Immune Sciences Inc., Labtech International Ltd, Uckfield, Sussex, UK) according to the manufacturer's instructions. Lymphocytes were introduced into the flask and incubated for 1 h at room temperature, after which time the non-adherent CD3 cells were collected. CD3+ T cells remained bound to the antibody-coated surface of the flask. The purity of these CD56+ cells was confirmed by flow cytometry (see later).
Co-culture of decidual lymphocytes with decidual stromal cells separated by permeable Cyclopore membrane
After isolation and culture for up to 7 days, decidual stromal cells were treated with trypsin and transferred into plastic 15 mm-diameter tissue culture wells at 3x105 cells/well in RPMI/10% FCS with or without 15 U/ml IL-2. After incubation for 4 h at 37°C (the time taken for the stromal cells to adhere firmly), 9 mm-diameter Cyclopore cell culture inserts incorporating a 0.45 µm pore size permeable membrane (Falcon, Oxford, UK) and containing 2x105 decidual lymphocytes were gently lowered into the wells above the adherent cells. Control wells were assembled in which decidual lymphocytes were co-cultured with themselves in both the well and insert. After 80 h of culture, 5 µCi [3H]thymidine was added to each well and decidual lymphocytes harvested from the inserts after a further 16 h. The lymphocytes were transferred to a 96-well plate to facilitate harvesting with the cell harvester, as described previously. The cpm of all filter discs relating to each Cyclopore insert were summed and compared.
Co-culture of decidual lymphocytes in direct contact with irradiated decidual stromal cells
Decidual stromal cells were treated with trypsin and plated at 4x104 cells/well in a flat-bottomed, 96-well plastic culture plate (Nunc) with or without 5 or 15 U/ml IL-2. The whole culture plate was irradiated with 700 rads X-irradiation. Triplicates of irradiated and non-irradiated cells were plated to check irradiation efficiency. Decidual lymphocytes were added to the irradiated decidual stromal cells at 2x104, 4x104, 8x104 and 1.6x105 cells/well in triplicate, with or without 5 or 15 U/ml IL-2. Decidual lymphocytes were also plated in the absence of stimulator cells to give an indication of basal responder cell proliferation. After 80 h of culture, each well was spiked with 1 µCi [3H]thymidine. The cells were harvested after a further 16 h and the amount of incorporated radioactivity determined as described. The average cpm between triplicates of responder cells cultured with stroma was compared with that of responder cells cultured alone for each ratio and each dose of exogenous IL-2. Satisfactory irradiation of stimulator cells was verified as giving <10% of the count of non-irradiated cells both in the absence and presence of IL-2. The potential of the decidual lymphocytes used in each experiment to proliferate was assessed by their response to 100 U/ml IL-2 alone. The response of CD3+-depleted decidual NK cells was assayed in a similar manner.
Flow cytometric analysis of responding lymphocyte populations
Depletion of T cells using anti-CD3 monoclonal antibodies results in internalization of CD3. Therefore, T cells were detected by CD4 and CD8 staining. Decidual lymphocytes were double-labelled with CD8FITC/CD56PE and CD4FITC/CD8PE conjugated antibodies (Becton Dickinson Ltd, Oxford, UK) immediately following T-cell depletion and then after co-culture for 4 days with decidual stroma + 15 U/ml IL-2. Cells were incubated with antibody at 1/10 dilution for 30 min at 4°C, washed to remove unbound antibody, and fixed with 1% paraformaldehyde. Fluorescence-activated cell sorting (FACS) analysis was performed immediately, or after overnight storage at 4°C. Simultest control (Becton Dickinson) was used as a negative.
Immunohistology
Frozen sections of non-pregnant endometrium, decidua basalis and decidua parietalis were obtained as described previously (King et al., 1989). Cytospin preparations of decidual lymphocytes were made with a Shandon cytocentrifuge.
Dual immunohistology was performed to localize Bcl-2 and CD56 reactivity as described previously (King et al., 1996). The mouse monoclonal antibody to Bcl-2, 124 (culture supernatant, 1:5 dilution) was kindly provided by Prof. D.Mason, Oxford (Pezzella et al., 1990
).
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Results |
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IL-2 time course and dose
The first significant proliferative effect of IL-2 on decidual lymphocytes was noted at 25 U/ml, reaching a maximum at 75100 U/ml. One unit of IL-2 is defined as the amount of IL-2 that is required to support half-maximal [3H]thymidine incorporation into CTLL-6 cells. Doses of IL-2 from 515 U/ml maintained approximately 70% cell viability (as shown by trypan blue exclusion) for 96 h, without inducing obvious proliferative effects. At 100 U/ml IL-2, [3H]thymidine incorporation peaked at 96 h of culture. This was followed by a second, similar peak at 144 h after the medium and IL-2 were replenished. Co-culture experiments were therefore centred around an expected 96-h response.
Depletion of T cells from lymphocytes
FACS analysis of CD3+-depleted lymphocytes revealed that NK cells comprised two distinct populations with respect to CD56 expression. Most were CD56bright and the remainder CD56dim (Figure 2A). CD56FITC/CD8PE double-labelling revealed that although 11.6% of the total cell population was CD8+, the vast majority of these cells were also CD56+ and therefore of NK cell phenotype. Only 0.4% of the total population were CD56/CD8+ T cells (Figure 2A
). CD8FITC/CD4PE double-labelling to separate T-cell subsets revealed that CD4+ T cells constituted only 0.5% of the total population (Figure 2B
). It can also be seen that the CD56bright cells are CD8dim, whereas the CD56dim and CD56 cells contain both CD8bright and CD8dim cells (Figure 2A
).
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Immunohistology for Bcl-2
We have previously investigated the proliferation of uterine CD56+ cells in vivo by performing double immunohistology for CD56 and the proliferation marker Ki-67 (King et al., 1991). To investigate the presence of Bcl-2 in these cells, similar sections and cytospin smears were double-stained with monoclonal antibodies to Bcl-2, (124), and to CD56, (Leu-19), which identifies uterine NK cells. Sections of decidua stained for Bcl-2 alone showed positive staining of nuclei in cells with the morphology and distribution typical of uterine NK cells (Figure 4A
). Dual immunohistology showed that there were numerous strongly staining CD56+ Bcl-2+ cells (Figure 4B
). Cytospin smears of decidual lymphocytes were also stained for Bcl-2, and cells with the characteristic reniform nuclear morphology of LGL were clearly positive. Most strongly stained were decidual NK cells which had been in culture for four days with 100 U/ml IL-2. Similar cells cultured in the absence of IL-2 were negative for Bcl-2 and showed features of apoptosis with nuclear disintegration and vacuolation (not shown).
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Discussion |
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The molecular nature of the human NK cell stimulatory response is not yet clear, but does not appear to involve receptors which can co-stimulate T-cell proliferation such as CD2, CD28 and CD11a (Robertson et al., 19967). Recently, it has been shown that ligation of the NK lectin receptor, CD94, on blood CD56bright cells and co-culture with IL-2 will augment the proliferative response (Voss et al., 1998
). Interestingly, this effect was not seen with the major CD56dim population; indeed, ligation of CD94 could even result in an inhibitory response which was variable among donors. Both blood and decidual CD56bright cells are all CD94bright Z199+, which correlates with the CD94/NKG2A phenotype (Verma et al., 1997
), whereas CD56dim cells have a much more heterogeneous expression of CD94/NKG2A which varies between donors (Perez-Villar et al., 1995
, 1996
). CD94 binds to HLA-E which is expressed on the cell surface if peptides derived from the signal sequence of some other classical HLA class I molecules are also present (Braud et al., 1997
, 1998
; X.López-Botet, personal communication), as would be expected to occur with decidual stromal cells.
Another possibility is that the unusual extracellular matrix, which is secreted under hormonal regulation by decidual stromal cells (Aplin et al., 1988; Loke et al., 1989
; Zhu et al., 1992
), influences NK cell proliferation. Decidual NK cells express integrins which are involved in binding to fibronectin and collagen (Burrows et al., 1993
, 1995
). Fibronectin has been observed to augment T-cell proliferation via binding to
4ß1 and
5ß1 receptors (Shimizu et al., 1990
; Klingemann and Kohn, 1991
).
The present study has clearly shown that IL-2 is important in decidual NK cell proliferation. However, the role of IL-2 in vivo is less clear because this cytokine has not been demonstrated at the implantation site (Saito et al., 1993; Jokhi et al., 1994a
; King et al., 1995
), nor in the non-pregnant endometrium (Lim et al., 1998
). Furthermore, NK cells can proliferate during an early stage of a viral infection in vivo before the IL-2-producing, antigen-specific T cells are activated, indicating that this cytokine is not the primary stimulus for NK cells. It is now thought that IL-15 may substitute for IL-2 in vivo (Carson et al., 1994
, 1995
). IL-15 is produced by a range of non-T cells and, like IL-2, is capable of inducing NK cell cytotoxicity, proliferation and cytokine production. In addition, the IL-15R is a heterotrimeric molecule of which only the
subunit is specific to IL-15, while the ß and
subunits are common to the IL-2 receptor (Giri et al., 1995
; Kennedy and Park, 1996
). We have shown recently that IL-15 is produced by cells of the decidua and will induce proliferation and augment cytotoxic activity in decidual NK cells in vitro (S.Verma, unpublished results). Investigations are in progress in our laboratory to assess whether similar co-stimulatory responses occur with IL-15 as are shown for IL-2 in the present study.
The regulation of cell numbers in a tissue is a balance between proliferation and cell death. Morphological features of apoptosis were observed in uterine NK cells at two to three days premenstrually, giving rise to the misnomer `endometrial granulocytes' (Hamperl and Hellweg, 1958). In the late-secretory endometrium of a pregnant woman, the uterine NK cells do not undergo apoptosis. Similarly, apoptosis is only seen in decidua in a failed pregnancy. Thus, pregnancy is associated with survival of uterine NK cells. In this study we demonstrate that CD56+ cells co-express Bcl-2 in decidua and proliferative phase endometrium; this confirms previous reports (Koh et al., 1995
). Positive Bcl-2 staining was also seen in the secretory phase, but this appeared to become weaker premenstrually, although quantification is difficult using immunohistology. There is thus a balance in the uterine mucosa between proliferation and apoptosis. The reason that apoptosis of NK cells is triggered premenstrually and with pregnancy failure is unknown, but there is obviously a correlation with falling progesterone concentrations. Uterine NK cells do not express the progesterone receptor, so the effect is likely to be indirect, acting via stromal cells (Inoue et al., 1996
; King et al., 1996
). We have shown that decidual NK cells undergo apoptosis rapidly when cultured in medium alone, but survive with IL-2, and these surviving cells stain strongly for Bcl-2. IL-15 can also sustain NK survival at low concentrations, and this is associated with Bcl-2 expression (Carson et al., 1997
).
In conclusion, proliferation and survival of uterine NK cells are influenced by contact with the decidual stromal cells. Further experiments are required to determine which factors mediate this interaction. However, stromal cells may influence NK cell biology in other ways. We have observed that co-culture with stromal cells increased the secretion of cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF) and leukaemia inhibitory factor (LIF) by decidual NK cells (Jokhi et al., 1994b). Thus, stromal cells may not act merely as a stimulus for NK cell proliferation but could also affect their biological function. It is of interest that the uterine mucosa is the only tissue where NK cells proliferate and survive in such large numbers. There is evidence that bone marrow stromal cells play an important role in NK cell development (Sitnicka and Hansson, 1992
). Stromal cells in the decidua could provide a similar microenvironment for NK cells in the uterus.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 15, 1998; accepted on December 10, 1998.