1 Reproductive Medicine and 2 Microbiology and Immunology, FUHS/The Chicago Medical School, N.Chicago, IL, USA
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Abstract |
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Key words: activation markers/flow cytometry/natural killer cells/spontaneous abortions/trophoblast
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Introduction |
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NK cells belong to the innate immune system, and closely resemble T cells; however, NK cells do not express the T-cell receptor (TCR)CD3 complex (Lanier, 1998). NK cells cause their cytotoxic effects against target cells either by release of their granular components (perforin, granzymes), inducing apoptosis to target cells, or by secretion of a wide range of cytokines (Mendes et al., 2000
). T helper (Th) 1- or Th 2-related cytokines, secreted by T cells or NK cells, have an immunoregulatory role on the activation of NK cells (Naume and Espevik, 1994
). Conversely, certain cytokines such as interleukin (IL) -2 and tumour necrosis factor
(TNF
) can induce apoptosis on NK cells (Ross and Caligiuri, 1997
), indicating the dual role of cytokines on these cells.
Human peripheral blood NK cells constitutively express CD56 antigen or neural cell-adhesion molecule (NCAM) and CD16 or FcRIII, which is responsible for antibody dependent cell-mediated cytotoxicity (ADCC). In addition, NK cells express C-type lectin receptors such as CD94 (an inhibitory receptor), and CD69 (which is an early activation marker; Yokoyama, 1999). CD128, which is the receptor for IL-8 chemokine (Chuntharapai et al., 1994
), and CD122 (IL-2Rß) and CD25 (IL-2R
), which are receptors for IL-2 cytokine (David et al., 1998
), are constitutively expressed on NK cells. Other receptors such as CD30 (Chiarle et al., 1999
) and CD154 (CD40L) (Carbone et al., 1997
) are also expressed on NK cells.
NK cells represent the most abundant population of lymphocytes in decidua of early pregnancy. These large granular lymphocytes (CD3/CD16/CD56bright) are phenotypically different from peripheral blood NK cells (Moller et al., 1998). Their physiological roles have been imposed to control excessive trophoblast invasion and prevent local infections. However, elevated numbers of peripheral blood NK cells and increased infiltration of endometrial NK cells have been reported to be related to pregnancy complications such as pre-eclampsia and miscarriages (Quenby et al., 1999
). In such cases, uterine NK cells appear to be activated with the expression of surface molecules that are found only on activated NK cells (Yokoyama, 1997
) and CD56+/CD69+ cells were found more commonly in the decidua from spontaneous abortions than from normal pregnancy (Kodama et al., 1998
).
NK cells originate from bone marrow. After differentiation, they enter into the peripheral circulation at a very early stage of maturation. It has been speculated that interaction between the integrin ß2L on peripheral NK cells and intracellular adhesion molecule-1 on the endothelium of decidual vessels induces the migration of NK cells out of the peripheral blood to the stroma of the endometrium or decidua (Kammerer et al., 1999
). Although immunophenotypes of the majority of peripheral blood NK cells are different from endometrial NK cells, peripheral blood NK cells seem to be keenly related with decidual NK cells, and may reflect the decidual NK cell functional status.
The aim of this study was to investigate the functional status and immunophenotypic characteristics of peripheral blood NK cells in women with recurrent spontaneous abortions (RSA) and implantation failures, by detecting the expression of surface receptors including activation and inhibitory molecules on NK cells and their subpopulations. Additionally, in-vitro co-cultures with ED27, a human trophoblast cell line, were used to investigate if trophoblast cell line induces expression of CD69 activation marker on NK cells.
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Materials and methods |
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Women with anatomical, hormonal or infectious causes of RSA were excluded from the study, as were women with two or more live-born infants. Women with known causes of infertility, including male factor, presence of endometriosis and tubal factors, were also excluded. None of the women had antiphospholipid, antithyroglobulin or antimicrosomal antibodies. Thirteen healthy non-pregnant multiparous women served as controls and also gave their informed consent. Age, gravidity and number of spontaneous abortions of each study group are listed in Table I.
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Flow cytometry
Isolated lymphocytes were gated on a forward-versus-side scatter histogram (Figure 1), and CD56-PC5 fluorescence of this population was then displayed on a one-parameter histogram. In this histogram it was possible to gate on total CD56 cells, CD56dim and CD56bright cells, and then to analyse these subpopulations for the expression of the other surface markers.
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ED27 cells were cultured in Dulbecco's Modified Eagle's Medium (D-MEM)/F-12 medium (Gibco, Rockville, MD, USA) supplemented with 15% FBS, 1 mmol/l sodium pyruvate, 2 mmol/l glutamine and 50 µg/ml gentamicin. Subsequently, 1x106 cells in 2 ml medium were placed in 6-well plate tissue culture and incubated in a 5% CO2 humidified incubator. After 24 h incubation of ED27 cells, 1x106 PBMC from study subjects were added to the culture and incubated for another 24 h. After 24 h co-culture, lymphocytes were harvested by gentle aspiration. For negative control, freshly isolated PBMC were cultured for 24 h without ED27 cells under the same conditions. Immunophenotype studies were carried out after staining harvested lymphocytes using RD1-anti-CD56 (clone N901; Coulter) and PC5-anti-CD69 (clone TP1.55.3; Immunotech, Marseille, France) mAb. Propidium iodide (PI) was added to stain dead cells. Cells that were PI-positive were excluded from the analysis. Expression of CD69 on CD56 cells was determined by flow cytometric analysis. The results were analysed using a three-colour flow cytometric protocol, as described above.
In order to detect if there was any difference in the expression of CD69 on NK cells after co-culture with other cell lines, PBMC of two study subjects were co-cultured with non-trophoblast cell lines, human colorectal cancer cell line HT-29, and human breast adenocarcinoma cell line HTB-26. Preparation methods were the same as in the ED27 co-culture experiment.
Statistical analysis
A two-tailed t-test was used to analyse the results between study subjects and controls. The results were reported to be statistically significant if the P value was < 0.05.
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Results |
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The CD56bright/16 subpopulation of peripheral blood NK cells was gated, since these cells are phenotypically similar to the large granular lymphocytes that circulate in human decidua around the implantation time and in early pregnancy. The expression of CD69 on these cells was significantly different between women with a history of RSA and controls (3.35% versus 1.78% respectively, P = 0.04). However, there was no difference between infertile women and controls.
Co-culture experiment and CD69 expression
CD69 expression on CD56+ NK cells was statistically significantly increased when PBMC were co-cultured with ED27 (P < 0.00005) and HT-29 (P < 0.05) cell lines in all of the study population. However, there was no statistically significant increase in the expression of CD69 on NK cells when they were co-cultured with HTB-26 cell line (Figure 5).
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CD128 is the IL-8 receptor and is constitutively expressed on many other cells, including NK cells. Although in the current study CD128 was constitutively expressed on peripheral blood NK cells, there was no difference in CD128 expression between the study groups and controls.
CD30 and CD154 are members of the TNF receptor superfamily. There was no significant difference in the expression of these receptors on freshly isolated NK cells and their subpopulations between study groups and controls.
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Discussion |
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Peripheral blood NK cells circulate around the implantation site and are in direct contact with trophoblast cells. The latter lack classical HLA class I and II molecules, but express HLA-G1, which renders them potentially susceptible to NK cell lysis. Decidual NK cells express receptors, which belong to both the Killer Inhibitory Receptor and CD94 families (King et al., 1998). Compared with decidual NK cells, only a fraction of peripheral blood NK cells express the HLA-G1-specific CD94/NKG2A and/or lymphocyte inhibitory receptor (LIR)-1 receptors, which suppress NK cell cytotoxicity. In addition, P49, an HLA-G1-specific inhibitory receptor expression in peripheral blood NK cell is undetectable (Ponte et al., 1999
). How potentially NK susceptible trophoblast cells escape from peripheral blood NK cells has been questioned.
This is the first study to explore functional status of NK cells by assessing constitutive, activation and inhibitory receptors on peripheral blood NK cells. In this study, it is reported that women with RSA and primary infertility of unknown aetiology have a significantly higher proportion of activated NK cells in peripheral blood, and express up-regulated CD69 as compared with those of normal fertile controls. Women with two or more pregnancy losses were included in the study, and this may have led to some of the negative results obtained. A total of five women among 22 recurrent aborters had a history of two spontaneous abortions.
CD69 is a type II integral membrane glycoprotein that is expressed on many activated cells of haematopoietic origin (Testi et al., 1994). Human CD69 gene is mapped in the short arm of chromosome 12 within the NK gene complex (Lopez-Cabrera et al., 1993
). The role of CD69 is to initiate cell activation. It represents a functional triggering molecule on activated NK and T cells, and cross-linking of CD69 induces cytotoxic activity and cytokine production (Marzio et al., 1999
). CD69 has been detected in 5% of resting NK cells, and was found also to be induced on NK cells by IL-2, interferon (IFN)
, anti-CD16, PMA and K562 cells (Borrego et al., 1993
). Conversely, CD69 is involved in regulating NK functions such as proliferation and TNF
production (Borrego et al., 1999
). Previously, reports of significantly elevated Th 1 cytokine secretion by CD3+/CD4+ T cells were made in women with RSA and infertility of implantation failures (H.Chung-Bang et al., unpublished results). Certain Th 1 cytokines, secreted by T cells, have immunoregulatory roles on NK cells, and can activate them (Naume and Espevik, 1994
). It is speculated that markedly activated peripheral blood NK cells in the current study, by means of the increased expression of CD69, may result from dominant Th 1 cytokine expression (TNF
and IFN
) over Th 2 cytokines (IL-4 and IL-10) in these women.
This appears to be the first study to detect elevated expression of CD69 on freshly isolated peripheral blood NK cells in women who experience recurrent miscarriages or implantation failures. It has been reported that women who miscarried spontaneously demonstrated significantly increased numbers of CD69- and CD25-positive T cells in their decidua compared with those of women with therapeutic abortions (Vassiliadou et al., 1999). It was also reported (Kodama et al., 1998
) that increased numbers of CD56+/CD69+ NK cells were found in the decidua in women with spontaneous abortions compared with normal pregnancies. The results presented here of CD69 expression on peripheral blood NK cells are compatible with these studies. Interestingly, CD69 expression on CD56bright/16 NK cell subpopulation is up-regulated in women with RSA as compared with that of normal controls. These cells have the same immunophenotypic characteristics as decidual NK cells.
A co-culture experiment was conducted in this study to investigate if trophoblast cells can induce CD69 expression, using a human first-trimester trophoblast cell line, ED27. CD69 expression on NK cells of study groups after ED27 co-culture was the same, regardless of their obstetric histories. It is clear that decidual and peripheral blood NK cells up-regulate CD69 expression and become activated when they encounter trophoblast cells. However, in the current study excessive NK activation, as indicated by CD69 expression, was not present in the normal multiparous controls, although their NK cells had the capacity to express CD69 after ED27 cell co-culture stimulation. This raises speculation that dysregulation of NK cell functional status may result from either defective presence of inhibitory receptors on NK cells or lack of recognition of HLA-G on trophoblasts by inhibitory receptor such as CD94. CD94 expression after ED27 cell co-culture was not included in this report; these data will be reported elsewhere.
CD94 is a receptor that forms a heterodimer with NKG2A and recognizes HLA-G expressed on trophoblast cells. This recognition leads to inhibition of NK cytotoxicity, and may provide a mechanism to render maternal NK cells tolerant of the fetus. It has been found that CD94/NKG2A expressed by decidual and peripheral blood NK cells is the predominant inhibitory receptor for HLA-G (Soderstrom et al., 1997). Here, we report a statistically significant decrease in the expression of CD94 on peripheral blood NK cells, especially CD56dim population, in women with RSA and infertility of unknown aetiology. This suggests that down-regulation of CD94 expression in these women results in difficulties in recognizing HLA-G and failure to inhibit NK cytotoxicity against trophoblast cells. This may result in a higher proportion of activated NK cells after a pregnancy is lost in women with RSA.
For the investigation of possible cytokine-related NK activation, IL-2 receptors were studied on NK cells. IL-2 receptors are comprised of three subunits, IL-2R (CD25), IL-2Rß (CD122) and IL-2R
(CD132). IL-2 binding to CD122 stimulates NK cytotoxicity, whereas binding to CD25 leads to proliferation of NK cells. It was found that CD56bright/16 NK cells in peripheral blood express IL-2R
(CD25), which is compatible with a previous report (Nagler et al., 1990
). CD56+/CD16 NK cells are postulated to represent a peripheral blood progenitor population for mature CD56+/CD16+ NK cells, and also comprise the majority of NK cell population in decidua. This suggests that CD56bright/16 NK cells have proliferative potential and are capable of being activated in an IL-2 milieu in peripheral blood, as well as at the implantation site. However, there were no differences in expression of CD25 and CD122 on peripheral blood NK cells between the study groups and controls, which disputes the hypothesis that IL-2 may be a major cytokine to induce excessive NK cell activation in women with RSA and infertility. CD25 and CD122 expressions tend to be more present in CD56bright NK cells than CD56dim population in all study and control groups. Hence, further studies are needed to define the major role of CD56bright subpopulation.
CD30 is a member of the TNF receptor (TNFR) superfamily, and has been found on activated lymphoid cells, such as CD8 and NK cells, endothelial cells and decidual cells (Chiarle et al., 1999). Although its function has not been fully recognized, it is postulated that CD30 activation may lead to an increase in cell proliferation. In the populations of the current study, expression of CD30 on CD56 cells was not different between patients and controls. Another member of the TNFR superfamily is CD154 (previously known as CD40L). IL-2-activated human NK cells can express CD40L, and recognition of CD40 on target cells can provide a further mechanism of cytotoxicity for NK cells (Carbone et al., 1997
). No differences were detected in the expression of CD154 between the study patients and the controls.
CD128 is the IL-8R and belongs to the superfamily of seven transmembrane, G protein-coupled receptors. It is postulated that the expression of CD128 on peripheral blood NK cells helps them migrate to the decidua, since IL-8 is a chemokine that can be chemotactic for NK cells (Chuntharapai et al., 1994). Nevertheless, no difference was found in CD128 expression between the study groups and normal controls in the present investigation.
Functional activation and down-regulation of NK cell cytotoxicity may play a major role in reproductive outcome. In the current study, increased activation receptor and decreased inhibitory receptor expression on peripheral blood NK cells was documented. Dysregulation of functional receptors on NK cells may contribute to an understanding of the underlying pathology of NK cell dysfunction in women with RSA and infertility of unknown aetiology. Altered Th 1/Th 2 cytokine expression may also contribute to NK cell functional activation in these women. Co-lateral study of intracellular cytokine expression of the same population clearly demonstrated up-regulated Th 1 cytokine in CD4+ T cells by flow cytometric analysis. Failure to demonstrate either up- or down-regulation of cytokine- and chemokine-related receptors on NK cells might be explained by a lack of their role in NK cell activation, or a relatively rapid turnover time of these receptors. Changes identified in peripheral blood NK cells may not reflect similar changes in the uterine NK cell population. Further studies are needed to investigate CD56bright NK cell population and their function in peripheral blood and decidual NK cells. The immunoregulatory mechanisms of activation and inhibitory receptors on NK cells, as well as cytokine action on NK cell activities, need to be further explored to define better the underlying pathology of NK cells in women with RSA or infertility of unknown aetiology.
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Acknowledgements |
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Notes |
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References |
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Submitted on April 3, 2000; accepted on January 15, 2001.