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Regulation of NK cell cytotoxicity during pregnancy
RBMOnline - Vol 16 No 2. 2008 211-217 Reproductive BioMedicine Online; www.rbmonline.com/Article/3005 on web 25 October 2007
Symposium: Current knowledge on natural killer cells, pregnancy and pre-eclampsia Regulation of NK cell cytotoxicity during pregnancy Julia Szekeres-Bartho is a professor of Microbiology and Immunology at the Medical School, Pecs University, Hungary. Her research has focused on the role of immuno-endocrine interactions in the establishment and maintenance of pregnancy, with a special emphasis on progesterone. She is a member of EMBIC a European Network of Excellence within the 6th Framework Programme of the European Union.
Dr Julia Szekeres-Bartho Julia Szekeres-Bartho Department of Medical Microbiology, Medical School Pecs University, 12 Szigeti Str, H-7643 Pecs, Hungary Correspondence: e-mail: [email protected]
Abstract Uterine and peripheral natural killer (NK) cells represent phenotypically and functionally distinct populations. Decidual NK cells resemble the CD56bright peripheral NK subset in their CD56bright CD16neg phenotype but, unlike the peripheral population, they contain cytotoxic granules. The activity of NK cells is regulated by the expression of activating or inhibitory receptors on NK cells and by that of major histocompatibility complex class I molecules on target cells. The embryonic trophoblast that forms the interface between the maternal and fetal compartments expresses non-classical human leukocyte antigens that serve as ligands for NK receptors. Decidual NK cells fulfil dual tasks. During normal conditions they contribute to creating a favourable environment for placentation, but at the same time they are equipped with cytotoxic potential to fight intrauterine infections. Decidual NK activity is regulated by a complex, mutually interacting network of cytokines and hormones. Keywords: cytokines, natural killer cells, NK cell receptors, NK cytotoxicity, perforin, progesterone
Introduction Natural killer (NK) cells play diverse roles in the reproductive process. CD56bright CD16neg and CD3neg granulated decidual NK cells, which constitute a dominant lymphocyte population in the early decidua (King et al., 1998), are, despite their high perforin content, not cytotoxic (Rukavina et al., 1995; Crncic et al., 2006), but secrete an array of angiogenic factors and cytokines (Koopman et al., 2003). The dynamics of the appearance of uterine NK cells suggests that one of their functions might be the control of placentation. Potential cytotoxic mechanisms exerted by NK cells can damage the trophoblast and induce ablation of placenta; on the other hand, tumour necrosis factor (TNF) α, produced by NK cells in response to intrauterine infections, may induce uterine contractions by facilitating prostaglandin synthesis and initiate the induction of preterm labour. Decreased expression of human leukocyte antigen (HLA) G on the trophoblast may
result in inadequate trophoblast invasion leading to an abnormal interaction with decidual NK cells, which are believed to play a major role in these processes through the production of immunoregulatory cytokines and angiogenic factors (Sargent et al., 2006). Furthermore, recurrent miscarriage has been shown to be associated with an increased number of endometrial NK cells (Quenby and Farquharson, 2006). In mice, high peripheral NK activity was shown to be associated with deleterious effects on fetal development (De Fougerolles and Baines, 1987). Transfer of high NK-activity spleen cells from poly(I)poly(C)-treated mice to pregnant BALB/c mice induces abortion (Kinsky et al., 1990). In human pregnancy, such a direct relationship between high NK activity and spontaneous pregnancy termination cannot be demonstrated: however, normal human pregnancy is characterized by low peripheral NK activity (Aoki et al., 1995) and increased NK
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho activity seems be an attribute of spontaneous abortions of unknown aetiology (Fukui et al., 1999; Ntrivalas et al., 2001; Yamada et al., 2001; Shakhar et al., 2003; Putowski et al., 2004; Ntrivalas et al., 2005). This paper aims to review the information currently available on the factors that regulate NK cell cytotoxic activity during normal and pathological gestation.
Phenotypic and functional differences between peripheral and decidual NK cells Regarding the expression of receptors and adhesion molecules, as well as cytokine production and cytotoxic potential, uterine and peripheral NK cells represent two phenotypically and functionally distinct populations. While approximately 90% of human peripheral NK cells express low density of CD56 (CD56dim) and high concentrations of the FCγRIII (CD16), the majority of decidual NK cells express high density of the CD56 molecule (CD56bright) and no CD16. Peripheral CD56dim NK cells are granular and known to be cytotoxic, while CD56bright peripheral NK cells do not contain granules and are non-cytotoxic, but display an immunoregulatory role via cytokine production (Cooper et al., 2001). Functional differences between the two populations are also reflected by the expression pattern of adhesion molecules and chemokines, involved in cell–cell interactions, trafficking, and homing. CD56bright, but not CD56dim NK cells, express high levels of CCR7 and L-selectin, the latter mediating early interactions with vascular endothelium (Frey et al., 1998). On the other hand, CD56dim NK cells express high levels of leukocyte function-associated antigen 1 (Frey et al., 1998). Decidual NK cells resemble the CD56bright peripheral NK subset in their CD56brightCD16neg phenotype but, unlike the former, they contain cytotoxic granules (King et al., 1998), and gene expression profiling has identified decidual NK cells as a distinct subset (Koopman et al., 2003). Among the genes selectively over-expressed in decidual NK cells are secreted proteins with known immunosuppressive activity, suggesting that decidual NK might contribute to the generation of an immunosuppressive environment at the maternal–fetal interface. On the other hand, perforin and granzymes A and B are expressed by decidual NK cells to a similar or higher level than by CD56dim peripheral NK cells (Koopman et al., 2003; Bogovic et al., 2005), suggesting that decidual NK cells may have cytotoxic potential. The major differences between CD56bright and CD16neg cells are summarized in Table 1.
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HLA-E) that are less likely to be available for peripheral NK cells. Three major super families of NK receptors have been described on human NK cells: the killer immunoglobulinlike receptor (KIR) superfamily which recognizes classical major histocompatibility complex (MHC) class I molecules, the C-type lectin superfamily recognizing non-classical MHC class I or class I-like molecules, and the natural cytotoxicity receptors (Lanier, 2005). KIR are classified by the length of their cytoplasmic tails. The long-tail receptors KIR2DL and KIR3DL mediate an inhibitory signal via their immunoreceptor tyrosine-based inhibition motifs, while the short-tail receptors KIR2DS and KIR3DS are associated with adaptor proteins bearing immunoreceptor tyrosine-based activating motifs and mediate activating signals. KIR2DL1 and KIR2DL2, and their activating counterparts KIR2DS1 and KIR2DS2, recognize epitopes shared by alleles of the group 1 or group 2 HLA-C allotypes, respectively (Mandelboim et al., 1996). KIR2DL4 (a receptor without a short counterpart) recognizes the non-classical MHC class I allele HLA-G (Rajagopalan and Long, 1999). Ligation of this receptor induces interferon-γ production, but no lytic activity. C-type lectin receptors are composed of a common subunit (CD94) and a C-type lectin NKG2, which determines the functional specificity of the receptor (Carretero et al.1997). The inhibitory receptor CD94/NKG2A/B specifically binds the nonclassical class I molecule HLA-E (Lazetic et al., 1996; PerezVillar et al., 1996; Brooks et al., 1997; Lanier, 1998). HLA-E is also bound by the activating receptor CD94/NKG2C, although with a lower affinity (Lopez-Botet et al., 2000). NKG2D is an activating C-type lectin receptor, which does not associate with CD94 but is expressed as a homodimer (Wu et al., 1999), and signals through association with an adaptor protein DAP10 and phosphatidylinositol-3 kinase (Wu et al., 1999). Ligands for NKG2D include the polymorphic MHC class I chainrelated (MIC) peptides, MICA and MICB (Bauer et al., 1999; Bahram et al., 2000) and the human cytomegalovirus UL16 binding proteins (ULBPs) (Sutherland et al., 2001). These are not expressed on normal cells, but are induced by ‘stress’ or neoplastic transformation. The expression of these ligands may, therefore, be signals of ‘danger’ to the NK cells. The natural cytotoxicity receptors are immunoglobulin-like activating receptors that have been implicated in the recognition and lysis of tumour cells by human NK cells (Pessino et al., 1998; Pende et al., 1999). Three natural cytotoxicity receptors have been described, of which two (NKp46 and NKp30) are constitutively expressed on all peripheral blood NK cells but not other immune cells, while the third, NKp44, is expressed only on interleukin (IL) -2-activated NK cells. The ligands for these receptors are unknown.
Target cell recognition by NK cells
Regulation of NK cytotoxicity
NK cell cytotoxic activity is the result of inhibitory and activating signals, following the interaction of cell surface activating and inhibitory receptors with ligands expressed on the surface of the target cell (Ljunggren and Karre, 1990; Long, 1999; Moretta et al., 2000). CD56bright and CD56dim NK cell subsets differ in their pattern of expression of NK cell receptors, furthermore, decidual NK cells encounter ligands (HLA-G and
NK cells recognize and lyse target cells by two basic mechanisms: antibody-dependent cellular cytotoxicity and natural cytotoxicity. The first occurs through the FcγRIIIA molecule (absent from decidual NK cells) when, following target-cell recognition, the contents of the cytotoxic granule are released into the immunological synapse formed between the killer cell and its target. Cytotoxic granules contain two
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho Table 1. Functional difference between decidual and peripheral NK cells. Parameter
Decidual Peripheral Peripheral (CD56bright) (CD16posCD56dim) (CD16negCD56bright)
CD16 Granular Cytotoxic Cytokine production CXCR4 L-selectin CD69
– + – + + – +
membrane-perturbing proteins, perforin and granulysin and a group of serine proteases called granzymes (Russell and Ley, 2002; Lieberman, 2003; Lieberman and Fan, 2003; Lord et al., 2003). The second mechanism, when cell destruction takes place, is mediated via the perforin/granzyme-mediated secretory/necrotic killing and the TNF family ligand-mediated apoptotic killing. Perforin, a Ca2+-dependent pore-forming protein, shows homology with complement components. Perforin monomers are inserted into the plasma membrane of target cells and polymerize into pore-forming aggregates (Liu et al., 1995), which leads to granzyme entry. In the cytoplasm, granzymes activate a cascade of caspases, leading to activation of DNase, resulting in apoptosis. Granulysin inserts into cell membranes, inducing ion fluxes and the induction of apoptosis (Kaspar et al., 2001; Pardo et al., 2001). Two important apoptosis-inducing pathways include the TNFinduced and the Fas/Fas ligand-mediated mechanisms, both involving receptors of the TNF receptor family. Human NK cells constitutively express several of the TNF family ligands. TNF-related apoptosis-inducing ligand is a protein consisting of 281 amino acids (Wiley et al., 1995; Pitti et al., 1996) and two of the five receptors identified to date contain a death domain and induce apoptosis upon engagement, whereas three of them lack functional death domains and serve as ‘decoy’ receptors. The Fas/Fas ligand pathway is important in many cellular events including the induction of apoptotic cell death and inflammation. Fas ligand, a type II transmembrane protein, is a member of the TNF family. Fas ligand is stored in specialized secretory lysosomes of NK cells and only delivered to the cell surface upon recognition of Fas on the target cell. Membranebound Fas ligand then triggers apoptosis of the Fas-expressing cell (Krammer, 1999). The activity of NK cells is regulated by the expression of activating or inhibitory receptors and by that of MHC class I molecules on potential target cells (Long et al., 2001). The embryonic trophoblast that forms the interface between the maternal and fetal compartments expresses ligands for NK receptors. RBMOnline®
+ + + – – – –
– – – + + + –
On the transcriptional level, HLA-A, -B, -C, -E and -G are present on individual trophoblast populations, but only HLAC, -G and -E are translated to proteins (Le Bouteiller, 1994; Guillaudeux et al., 1995). The function of NK cells depends on the availability of the ligands for the inhibitory and activating receptors on the target cells. HLA-G might induce resistance to lysis by decidual NK cells expressing inhibitory receptors (Moretta et al., 1993). HLA-G presents antigens and plays a role in the regulation of HLA-E expression (Le Bouteiller et al., 2003; Sala et al., 2004). NK cells can interact with HLAE complexed with specific peptides on target cells and the interaction is mediated by the CD94/NKG2A inhibitory receptor (Braud et al., 1998; Lee et al., 1998; Llano et al., 1998). The balance between activating and inhibitory signalling also controls the formation of activating and inhibitory NK immunological synapses (Davis et al., 1999; Vyas et al., 2002; Orange et al., 2003). At the activating NK immunological synapses, surface molecules (including CD2 and leukocyte function-associated antigen 1) and intracellular molecules (e.g. filamentous actin) accumulate in a time-dependent manner (Vyas et al., 2002), followed by the mobilization of perforin-containing granules towards the target cell contact site (Orange et al., 2003). On the other hand, at the inhibitory NK immunological synapses, killer cell immunoglobulin-like receptors accumulate on the NK side and HLA-C accumulates on the target cell side of the cell–cell contact. (Davis et al., 1999; Vyas et al., 2002). In decidual NK cell–target cell activating immune synapses, Kopcow et al. (2005) demonstrated a failure of the perforin-containing granules to polarize toward the contact side, which might account for the lack of cytotoxicity by decidual NK cells. Decidual NK cells fulfil dual roles. During normal conditions, they contribute to creating a favourable environment for placentation, but at the same time they are fully armed to fight intrauterine infections if necessary. Decidual NK cells contain perforin and express NKG2D receptors that recognize stress signals, e.g. MIC peptides or human cytomegalovirus UL16 binding peptides. Certain pregnancy pathologies are associated with increased decidual and peripheral NK activity. Gulan et al. (1997) demonstrated decreased perforin content of decidual
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho lymphocytes from failed pregnancies compared with those from normal pregnancy deciduas, suggesting that an increased rate of degranulation had taken place in the former case. The expression of the KIR2DL1 inhibitory KIR (the ligand of which is HLA-C), as well as that of CD94, were shown to be significantly decreased while perforin content was increased in women with miscarriage with normal chromosomal content compared with those in controls (Yamada et al., 2001). In another study, women who miscarried have been shown to lack the appropriate inhibitory receptors to interact with the HLA-Cw molecules on trophoblast cells and to deliver signals to inhibit NK cell activation and protect the embryo (VarlaLeftherioti et al., 2005).
cells, and blocking of IL-15 abrogates this effect (Sotosek et al., 1999). Trophoblast that originally resists NK-mediated lysis is rendered susceptible in the presence of IL-2 (Zuckermann and Head, 1987). However, IL-2 is among the few cytokines that have never been detected in the decidua. Low concentrations of IL-15 augment the cytotoxicity of decidual NK cells to K562 but, in contrast to IL-2, not to trophoblast (Verma et al., 2000). These findings suggest that IL-15, while inducing activation and proliferation of decidual NK cells, by up-regulating CD94/ NKG2A expression on these cells, protects the trophoblast from potential cytotoxicity. On the other hand, IL-15 can also upregulate NKG2D on NK cells, thus enabling them to recognize infected or transformed cells (Sutherland et al., 2002).
The mechanisms that control cytotoxicity during pregnancy have not been completely elucidated. However, the unique milieu of the decidua certainly plays a role. In this setting, NK cells are exposed to a variety of locally acting cytokines and hormones.
Regulation of NK activity by progesterone
Regulation of NK activity by cytokines In spite of their high perforin content, freshly isolated decidual NK cells display strongly reduced lytic activity on classical MHC class I negative targets, but culturing the cells outside the decidual environment restores the cytotoxic potential (Kopcow et al., 2005). In women with recurrent miscarriage with normal chromosomal content, implantation sites contain decreased numbers of Th2 cells (Michimata et al., 2003), suggesting abnormal cytokine regulation. The classical Th1/Th2 concept cannot be directly applied to the decidual microenvironment where the action of cytokines is stage-specific (Chaouat et al., 2007). Transforming growth factor (TGF) β has been identified as one of the key molecules regulating NK activity in the decidua. During culture in TGFβ1-containing conditioned medium from decidual stromal cells, highly purified CD16+CD9– peripheral blood NK cells acquire characteristics (CD9 expression) of decidual NK cells. TGFβ1 exerts a similar effect (Keskin et al., 2007). After blocking the action of endogenous TGFβ, both the number of responding cells and interferon-γ production by stimulated human peripheral NK cells increase (Meadows et al., 2006). Endogenous TGFβ alters cytokine production of uterine NK cells in human endometrium in response to stimulation through Toll-like receptors; thus endometrial NK cell responses to microbial pathogens are also regulated by the amount of biologically active TGFβ present in the human endometrium (Eriksson et al., 2006). Taken together, these data suggest that TGFβ might be one of the factors responsible for the downregulated cytotoxic activity of decidual NK cells. The hormonal and cytokine environment can modulate the expression of both the receptors and the ligands. TGFβ possibly contributes to decreased NK cytotoxicity by modulating KIR2DL expression (Shin et al., 2004). On the other hand, IL-15 produced by uterine macrophages induces the expression of CD94/NKG2A (Mingari et al., 1997) or NKG2D (Sutherland et al., 2002).
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Decidual NK cells do not express the high-affinity IL-2R α chain, while the β and γ chains of the IL-2R are present and serve as subunits of the IL-15R. Supernatants of decidual macrophages up-regulate perforin expression of decidual NK
Pregnancy is characterized by profound alteration in the endocrine functions. Among the pregnancy-related hormones, the effect of progesterone on NK cell function is well documented. Early studies suggested a dose-dependent decrease of peripheral NK activity of pregnancy lymphocytes by low concentrations (40–500 nM) of progesterone (Szekeres-Bartho et al., 1985a,b). Experiments using lymphocytes of non-pregnant subjects failed to demonstrate such an effect at similar concentrations (Sulke et al., 1985; Uksila, 1985). These data might suggest the presence of a population with increased progesterone sensitivity among peripheral pregnancy lymphocytes. Indeed, progesterone receptors were demonstrated by immunocytochemistry in peripheral γ/δ T cells of pregnant women (Szekeres-Bartho et al., 2001). Furthermore, RU 486, a potent antiprogestin, has been shown to significantly augment NK cell cytolytic activity in vitro; this down-regulation is effectively blocked by progesterone at a concentration of 25 µmol/l (Hansen et al., 1992). At the progesterone concentrations (10–5 mol/l) believed to be present at the maternalfetal interface (Stites and Siiteri, 1983), studies consistently show inhibition of NK cytolytic activity in vitro (Hansen et al., 1992). Henderson et al. (2003) demonstrated the absence of progesterone receptors in purified decidual NK cells. Progesterone could act on these cells through glucocorticoid receptors (Henderson et al., 2003), putative membrane receptors (Zhu et al., 2003) or calcium-activated K+ channels (Chien et al., 2007), but the most likely possibility is that progesterone affects NK activity in an indirect way, by induction of cytokine production in the neighbouring cells, which would in turn influence NK activity. In response to progesterone, T cells produce progesterone-induced blocking factor (PIBF) (Szekeres-Bartho et al., 2001), that, by controlling NK activity, exerts an antiabortive effect in mice (Szekeres-Bartho et al.1997). PIBF has recently been cloned and has been shown to enhance IL-10 production and suppress IL-12 production by human peripheral lymphocytes (Polgar et al., 2003). Although decidual NK cells do not express the progesterone receptor, they appear to be affected by PIBF secreted by surrounding cells. PIBF blocks up-regulation of perforin expression in decidual lymphocytes cultured with decidual adherent cells and inhibits NK cell cytotoxicity by blocking granule exocytosis without interfering with target conjugation (Faust et al., 1999; Laskarin et al., 1999). Progesterone also contributes to the establishment of a Th2 environment by downregulating IL-12 and up-regulating IL-10, possibly through the intermediary production of PIBF (Raghupathy, 1997).
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho Progesterone has been shown to increase IL-15 production by endometrial stromal cells (Okada et al., 2000). Human decidual NK cells express the IL-15 receptor and, in response to IL-15, proliferate and augment their cytolytic activity against K562, but not against the trophoblast (Verma et al., 2000). Progesterone up-regulates HLA-G gene expression through a novel progesterone response element (Yie et al., 2006). Increased expression of HLA-G (the ligand for NK inhibitory receptors) might play a role in controlling antitrophoblast NK activity. Although KIR2DL4 also recognizes HLA-G, engagement of this receptor induces interferon-γ production, rather than lytic activity. Some argue that, since there is no evidence for the association between uterine and peripheral NK cells, investigating NK cell count or activity during normal or pathological pregnancy is irrelevant (Moffet et al., 2004; Rai et al., 2005). There are only a few studies comparing peripheral and decidual NK cells from the same patient (Gulan et al., 1997; Lewis et al., 2002; Olivares et al., 2002; Higuma-Myojo et al., 2005). It is hard to tell how measurable changes at the periphery relate to local events. Currently available data suggest that recurrent aborters demonstrate an increased peripheral NK activity. (Aoki et al., 1995; Emmer et al., 2000; Ntrivalas et al., 2001, 2005; Yamada et al., 2003). Whether or not these peripheral changes reflect local events needs to be further investigated, all the more so, because studies on decidual NK activity have their inherent limitations. The availability of normal human placentas is restricted to the first trimester and labour. Placentas from spontaneous miscarriages are likely to contain an altered cytokine milieu, as well as toxic products of necrotic tissue, and it is not clear whether the observed changes are the cause or the consequence of miscarriage. Furthermore, there is no evidence at present that the high decidual NK activity is due to activation of the resident population and not due to migration of activated peripheral NK cells to the decidua.
Conclusions Human decidual lymphocytes contain a high amount of perforin that enables them to fight intrauterine infections, yet these cells are not cytotoxic in the absence of a danger signal. Decidual NK cell cytotoxicity is regulated by the balance of activating or inhibitory receptors and by expression of ligands on the target cells. The trophoblast expresses HLA-C, HLA-G and HLA-E, recognized by NK cell receptors. Cytotoxicity is controlled by precisely coordinated action of locally produced cytokines and hormones. Progesterone up-regulates HLA-G expression, while IL-15 up-regulates the expression of inhibitory receptors on decidual NK cells. Progesterone affects NK activity in an indirect way, by inducing Th2-type cytokine production that, in turn, would act on NK cells. TGFβ controls NK cytotoxicity via several pathways, whereas IL-15 activates NK cells. Disturbance of the delicate balance established among players of decidual homeostasis may result in increased NK cytotoxicity and the cessation of pregnancy.
Acknowledgements This work was supported by grants from the Hungarian National Research Fund (OTKA T031737), Hungarian Ministry of
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Health (ETT 045/2003), National Research and Development Program (NKFP 1A-057–2004), Economic Competitiveness Operative Program (GVOP-3.1.1.-2004–05–0329/3.0), and the Hungarian Academy of Sciences. The author is the member of a European Network of Excellence on Embryo Implantation Control supported by EU Contr. No. 512040. The authors report no financial or commercial conflicts of interest.
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho involved in natural cytotoxicity mediated by human natural killer cells. Journal of Experimental Medicine 190, 1505–1516. Perez-Villar JJ, Carretero M, Navarro F et al. 1996 Biochemical and serologic evidence for the existence of functionally distinct forms of the CD94 NK cell receptor. Journal of Immunology 157, 5367–5374. Pessino A, Sivori S, Bottino C et al. 1998 Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity. Journal of Experimental Medicine 188, 953–960. Pitti RM, Marsters SA, Ruppert S et al. 1996 Induction of apoptosis by Apo-2 ligand, a new member of the tumour necrosis factor cytokine family. Journal of Biological Chemistry 271, 12687– 12690. Polgar B, Kispal G, Lachmann M et al. 2003 Molecular cloning and immunologic characterization of a novel cDNA coding for progesterone-induced blocking factor. Journal of Immunology 171, 5956–5963. Putowski L, Darmochwal-Kolarz D, Rolinski J et al. 2004 The immunological profile of infertile women after repeated IVF failure (preliminary study). European Journal of Obstetrics and Gynecology and Reproductive Biology 112, 192–196. Quenby S, Farquharson R 2006 Uterine natural killer cells, implantation failure and recurrent miscarriage. Reproductive BioMedicine Online 13, 24–28. Raghupathy R 1997 Th1-type immunity is incompatible with successful pregnancy. Immunology Today 18, 478–482. Rai R, Sacks G, Trew G 2005 Natural killer cells and reproductive failure-theory, practice and prejudice. Human Reproduction 20, 1123–1126. Rajagopalan S, Long EO 1999 A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells. Journal of Experimental Medicine 189, 1093–1100. Rukavina D, Rubesa G, Gudelj L et al. 1995 Characteristics of perforin expressing lymphocytes within the first trimester of human pregnancy. American Journal of Reproductive Immunology 33, 394–404. Russell JH, Ley TJ 2002 Lymphocyte-mediated cytotoxicity. Annual Reviews of Immunology 20, 323–370. Sala FG, Moral PM, Pizzato N et al. 2004 The HLA-G*0105N null allele induces cell surface expression of HLA-E molecule and promotes CD94/NKG2A-mediated recognition in JAR choriocarcinoma cell line. Immunogenetics 56, 617–624. Sargent IL, Borzychowski AM, Redman CW 2006 Immunoregulation in normal pregnancy and pre-eclampsia: an overview. Reproductive BioMedicine Online 13, 80–86. Shakhar K, Ben Eliyahu S, Loewenthal R et al. 2003 Differences in number and activity of peripheral natural killer cells in primary versus secondary recurrent miscarriage. Fertility and Sterility 80, 368–375. Shin EC, Choi KD, Kim JS, Shin JS 2004 Modulation of the surface expression of CD158 killer cell Ig-like receptor by interleukin-2 and transforming growth factor-beta. Yonsei Medical Journal 45, 510–514. Sotosek V, Laskarin G, Strbo N et al. 1999 Decidual macrophages are the population of decidual adherent cells which regulates perforin expression in cytolytic cells. American Journal of Reproductive Immunology 42, 76–82. Stites DP, Siiteri PK 1983 Steroids as immunosuppressants in pregnancy. Immunological Reviews 75, 117–138. Sulke AN, Jones DB, Wood PJ 1985 Hormonal modulation of human natural killer cell activity in vitro. Journal of Reproductive Immunology 7, 105–110. Sutherland CL, Chalupny NJ, Schooley K et al. 2002 UL16-binding proteins, novel MHC class I-related proteins, bind to NKG2D and activate multiple signaling pathways in primary NK cells. Journal of Immunology, 168, 671–679. Sutherland CL, Chalupny NJ, Cosman D 2001 The UL16-binding proteins, a novel family of MHC class I-related ligands for NKG2D, activate natural killer cell functions. Immunological Reviews 181, 185–192.
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Szekeres-Bartho J, Barakonyi A, Miko E et al. 2001 The role of γ/δ T cells in the feto-maternal relationship. Seminars in Immunology 13, 229–233. Szekeres-Bartho J, Par G, Dombay G et al. 1997 The antiabortive effect of progesterone-induced blocking factor in mice is manifested by modulating NK activity. Cellular Immunology 177, 194–199. Szekeres-Bartho J, Hadnagy J, Pacsa AS 1985a The suppressive effect of progesterone on lymphocyte cytotoxicity: unique progesterone sensitivity of pregnancy lymphocytes. Journal of Reproductive Immunology 7, 121–128. Szekeres-Bartho J, Kilar F, Falkay G et al. 1985b Progesterone-treated lymphocytes of healthy pregnant women release a factor inhibiting cytotoxicity and prostaglandin synthesis. American Journal of Reproductive Immunology 9, 15–19. Uksila J 1985 Human NK cell activity is not inhibited by pregnancy and cord serum factors and female steroid hormones in vitro. Journal of Reproductive Immunology 7, 111–120. Varla-Leftherioti M, Spyropoulu-Vlachou M, Keramitsoglu T et al. 2005 Lack of the appropriate natural killer cell inhibitory receptors in women with spontaneous abortion. Human Immunology 66, 65–71. Verma S, Hiby SE, Loke YW, King A 2000 Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15. Biology of Reproduction 62, 959–968. Vyas YM, Maniar H, Dupont B 2002 Visualization of signaling pathways and cortical cytoskeleton in cytolytic and noncytolytic natural killer cell immune synapses. Immunological Reviews 189, 161–178. Wiley SR, Schooley K, Smolak PJ et al. 1995 Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673–682. Wu J, Song Y, Bakker AB et al. 1999 An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285, 730–732. Yamada H, Morikawa M, Kato EH et al. 2003 Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype. American Journal of Reproductive Immunology 50, 351–354. Yamada H, Kato EH, Kobashi G et al. 2001 High NK cell activity in early pregnancy correlates with subsequent abortion with normal chromosomes in women with recurrent abortion. American Journal of Reproductive Immunology 46, 132–136. Yie SM, Xiao R, Librach CL 2006 Progesterone regulates HLA-G gene expression through a novel progesterone response element. Human Reproduction 21, 2538–2544. Zhu Y, Rice CD, Pang Y et al. 2003 Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proceedings of the National Academy of Sciences of the United States of America 100, 2231–2236. Zuckermann FA, Head JR 1987 Possible mechanism of non-rejection of the feto-placental allograft: trophoblast resistance to lysis by cellular immune effectors. Transplantation Proceedings 1, 554–556. Received 3 July 2007; refereed 2 August 2007; accepted 14 August 2007.
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Symposium: Current knowledge on natural killer cells, pregnancy and pre-eclampsia Regulation of NK cell cytotoxicity during pregnancy Julia Szekeres-Bartho is a professor of Microbiology and Immunology at the Medical School, Pecs University, Hungary. Her research has focused on the role of immuno-endocrine interactions in the establishment and maintenance of pregnancy, with a special emphasis on progesterone. She is a member of EMBIC a European Network of Excellence within the 6th Framework Programme of the European Union.
Dr Julia Szekeres-Bartho Julia Szekeres-Bartho Department of Medical Microbiology, Medical School Pecs University, 12 Szigeti Str, H-7643 Pecs, Hungary Correspondence: e-mail: [email protected]
Abstract Uterine and peripheral natural killer (NK) cells represent phenotypically and functionally distinct populations. Decidual NK cells resemble the CD56bright peripheral NK subset in their CD56bright CD16neg phenotype but, unlike the peripheral population, they contain cytotoxic granules. The activity of NK cells is regulated by the expression of activating or inhibitory receptors on NK cells and by that of major histocompatibility complex class I molecules on target cells. The embryonic trophoblast that forms the interface between the maternal and fetal compartments expresses non-classical human leukocyte antigens that serve as ligands for NK receptors. Decidual NK cells fulfil dual tasks. During normal conditions they contribute to creating a favourable environment for placentation, but at the same time they are equipped with cytotoxic potential to fight intrauterine infections. Decidual NK activity is regulated by a complex, mutually interacting network of cytokines and hormones. Keywords: cytokines, natural killer cells, NK cell receptors, NK cytotoxicity, perforin, progesterone
Introduction Natural killer (NK) cells play diverse roles in the reproductive process. CD56bright CD16neg and CD3neg granulated decidual NK cells, which constitute a dominant lymphocyte population in the early decidua (King et al., 1998), are, despite their high perforin content, not cytotoxic (Rukavina et al., 1995; Crncic et al., 2006), but secrete an array of angiogenic factors and cytokines (Koopman et al., 2003). The dynamics of the appearance of uterine NK cells suggests that one of their functions might be the control of placentation. Potential cytotoxic mechanisms exerted by NK cells can damage the trophoblast and induce ablation of placenta; on the other hand, tumour necrosis factor (TNF) α, produced by NK cells in response to intrauterine infections, may induce uterine contractions by facilitating prostaglandin synthesis and initiate the induction of preterm labour. Decreased expression of human leukocyte antigen (HLA) G on the trophoblast may
result in inadequate trophoblast invasion leading to an abnormal interaction with decidual NK cells, which are believed to play a major role in these processes through the production of immunoregulatory cytokines and angiogenic factors (Sargent et al., 2006). Furthermore, recurrent miscarriage has been shown to be associated with an increased number of endometrial NK cells (Quenby and Farquharson, 2006). In mice, high peripheral NK activity was shown to be associated with deleterious effects on fetal development (De Fougerolles and Baines, 1987). Transfer of high NK-activity spleen cells from poly(I)poly(C)-treated mice to pregnant BALB/c mice induces abortion (Kinsky et al., 1990). In human pregnancy, such a direct relationship between high NK activity and spontaneous pregnancy termination cannot be demonstrated: however, normal human pregnancy is characterized by low peripheral NK activity (Aoki et al., 1995) and increased NK
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho activity seems be an attribute of spontaneous abortions of unknown aetiology (Fukui et al., 1999; Ntrivalas et al., 2001; Yamada et al., 2001; Shakhar et al., 2003; Putowski et al., 2004; Ntrivalas et al., 2005). This paper aims to review the information currently available on the factors that regulate NK cell cytotoxic activity during normal and pathological gestation.
Phenotypic and functional differences between peripheral and decidual NK cells Regarding the expression of receptors and adhesion molecules, as well as cytokine production and cytotoxic potential, uterine and peripheral NK cells represent two phenotypically and functionally distinct populations. While approximately 90% of human peripheral NK cells express low density of CD56 (CD56dim) and high concentrations of the FCγRIII (CD16), the majority of decidual NK cells express high density of the CD56 molecule (CD56bright) and no CD16. Peripheral CD56dim NK cells are granular and known to be cytotoxic, while CD56bright peripheral NK cells do not contain granules and are non-cytotoxic, but display an immunoregulatory role via cytokine production (Cooper et al., 2001). Functional differences between the two populations are also reflected by the expression pattern of adhesion molecules and chemokines, involved in cell–cell interactions, trafficking, and homing. CD56bright, but not CD56dim NK cells, express high levels of CCR7 and L-selectin, the latter mediating early interactions with vascular endothelium (Frey et al., 1998). On the other hand, CD56dim NK cells express high levels of leukocyte function-associated antigen 1 (Frey et al., 1998). Decidual NK cells resemble the CD56bright peripheral NK subset in their CD56brightCD16neg phenotype but, unlike the former, they contain cytotoxic granules (King et al., 1998), and gene expression profiling has identified decidual NK cells as a distinct subset (Koopman et al., 2003). Among the genes selectively over-expressed in decidual NK cells are secreted proteins with known immunosuppressive activity, suggesting that decidual NK might contribute to the generation of an immunosuppressive environment at the maternal–fetal interface. On the other hand, perforin and granzymes A and B are expressed by decidual NK cells to a similar or higher level than by CD56dim peripheral NK cells (Koopman et al., 2003; Bogovic et al., 2005), suggesting that decidual NK cells may have cytotoxic potential. The major differences between CD56bright and CD16neg cells are summarized in Table 1.
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HLA-E) that are less likely to be available for peripheral NK cells. Three major super families of NK receptors have been described on human NK cells: the killer immunoglobulinlike receptor (KIR) superfamily which recognizes classical major histocompatibility complex (MHC) class I molecules, the C-type lectin superfamily recognizing non-classical MHC class I or class I-like molecules, and the natural cytotoxicity receptors (Lanier, 2005). KIR are classified by the length of their cytoplasmic tails. The long-tail receptors KIR2DL and KIR3DL mediate an inhibitory signal via their immunoreceptor tyrosine-based inhibition motifs, while the short-tail receptors KIR2DS and KIR3DS are associated with adaptor proteins bearing immunoreceptor tyrosine-based activating motifs and mediate activating signals. KIR2DL1 and KIR2DL2, and their activating counterparts KIR2DS1 and KIR2DS2, recognize epitopes shared by alleles of the group 1 or group 2 HLA-C allotypes, respectively (Mandelboim et al., 1996). KIR2DL4 (a receptor without a short counterpart) recognizes the non-classical MHC class I allele HLA-G (Rajagopalan and Long, 1999). Ligation of this receptor induces interferon-γ production, but no lytic activity. C-type lectin receptors are composed of a common subunit (CD94) and a C-type lectin NKG2, which determines the functional specificity of the receptor (Carretero et al.1997). The inhibitory receptor CD94/NKG2A/B specifically binds the nonclassical class I molecule HLA-E (Lazetic et al., 1996; PerezVillar et al., 1996; Brooks et al., 1997; Lanier, 1998). HLA-E is also bound by the activating receptor CD94/NKG2C, although with a lower affinity (Lopez-Botet et al., 2000). NKG2D is an activating C-type lectin receptor, which does not associate with CD94 but is expressed as a homodimer (Wu et al., 1999), and signals through association with an adaptor protein DAP10 and phosphatidylinositol-3 kinase (Wu et al., 1999). Ligands for NKG2D include the polymorphic MHC class I chainrelated (MIC) peptides, MICA and MICB (Bauer et al., 1999; Bahram et al., 2000) and the human cytomegalovirus UL16 binding proteins (ULBPs) (Sutherland et al., 2001). These are not expressed on normal cells, but are induced by ‘stress’ or neoplastic transformation. The expression of these ligands may, therefore, be signals of ‘danger’ to the NK cells. The natural cytotoxicity receptors are immunoglobulin-like activating receptors that have been implicated in the recognition and lysis of tumour cells by human NK cells (Pessino et al., 1998; Pende et al., 1999). Three natural cytotoxicity receptors have been described, of which two (NKp46 and NKp30) are constitutively expressed on all peripheral blood NK cells but not other immune cells, while the third, NKp44, is expressed only on interleukin (IL) -2-activated NK cells. The ligands for these receptors are unknown.
Target cell recognition by NK cells
Regulation of NK cytotoxicity
NK cell cytotoxic activity is the result of inhibitory and activating signals, following the interaction of cell surface activating and inhibitory receptors with ligands expressed on the surface of the target cell (Ljunggren and Karre, 1990; Long, 1999; Moretta et al., 2000). CD56bright and CD56dim NK cell subsets differ in their pattern of expression of NK cell receptors, furthermore, decidual NK cells encounter ligands (HLA-G and
NK cells recognize and lyse target cells by two basic mechanisms: antibody-dependent cellular cytotoxicity and natural cytotoxicity. The first occurs through the FcγRIIIA molecule (absent from decidual NK cells) when, following target-cell recognition, the contents of the cytotoxic granule are released into the immunological synapse formed between the killer cell and its target. Cytotoxic granules contain two
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho Table 1. Functional difference between decidual and peripheral NK cells. Parameter
Decidual Peripheral Peripheral (CD56bright) (CD16posCD56dim) (CD16negCD56bright)
CD16 Granular Cytotoxic Cytokine production CXCR4 L-selectin CD69
– + – + + – +
membrane-perturbing proteins, perforin and granulysin and a group of serine proteases called granzymes (Russell and Ley, 2002; Lieberman, 2003; Lieberman and Fan, 2003; Lord et al., 2003). The second mechanism, when cell destruction takes place, is mediated via the perforin/granzyme-mediated secretory/necrotic killing and the TNF family ligand-mediated apoptotic killing. Perforin, a Ca2+-dependent pore-forming protein, shows homology with complement components. Perforin monomers are inserted into the plasma membrane of target cells and polymerize into pore-forming aggregates (Liu et al., 1995), which leads to granzyme entry. In the cytoplasm, granzymes activate a cascade of caspases, leading to activation of DNase, resulting in apoptosis. Granulysin inserts into cell membranes, inducing ion fluxes and the induction of apoptosis (Kaspar et al., 2001; Pardo et al., 2001). Two important apoptosis-inducing pathways include the TNFinduced and the Fas/Fas ligand-mediated mechanisms, both involving receptors of the TNF receptor family. Human NK cells constitutively express several of the TNF family ligands. TNF-related apoptosis-inducing ligand is a protein consisting of 281 amino acids (Wiley et al., 1995; Pitti et al., 1996) and two of the five receptors identified to date contain a death domain and induce apoptosis upon engagement, whereas three of them lack functional death domains and serve as ‘decoy’ receptors. The Fas/Fas ligand pathway is important in many cellular events including the induction of apoptotic cell death and inflammation. Fas ligand, a type II transmembrane protein, is a member of the TNF family. Fas ligand is stored in specialized secretory lysosomes of NK cells and only delivered to the cell surface upon recognition of Fas on the target cell. Membranebound Fas ligand then triggers apoptosis of the Fas-expressing cell (Krammer, 1999). The activity of NK cells is regulated by the expression of activating or inhibitory receptors and by that of MHC class I molecules on potential target cells (Long et al., 2001). The embryonic trophoblast that forms the interface between the maternal and fetal compartments expresses ligands for NK receptors. RBMOnline®
+ + + – – – –
– – – + + + –
On the transcriptional level, HLA-A, -B, -C, -E and -G are present on individual trophoblast populations, but only HLAC, -G and -E are translated to proteins (Le Bouteiller, 1994; Guillaudeux et al., 1995). The function of NK cells depends on the availability of the ligands for the inhibitory and activating receptors on the target cells. HLA-G might induce resistance to lysis by decidual NK cells expressing inhibitory receptors (Moretta et al., 1993). HLA-G presents antigens and plays a role in the regulation of HLA-E expression (Le Bouteiller et al., 2003; Sala et al., 2004). NK cells can interact with HLAE complexed with specific peptides on target cells and the interaction is mediated by the CD94/NKG2A inhibitory receptor (Braud et al., 1998; Lee et al., 1998; Llano et al., 1998). The balance between activating and inhibitory signalling also controls the formation of activating and inhibitory NK immunological synapses (Davis et al., 1999; Vyas et al., 2002; Orange et al., 2003). At the activating NK immunological synapses, surface molecules (including CD2 and leukocyte function-associated antigen 1) and intracellular molecules (e.g. filamentous actin) accumulate in a time-dependent manner (Vyas et al., 2002), followed by the mobilization of perforin-containing granules towards the target cell contact site (Orange et al., 2003). On the other hand, at the inhibitory NK immunological synapses, killer cell immunoglobulin-like receptors accumulate on the NK side and HLA-C accumulates on the target cell side of the cell–cell contact. (Davis et al., 1999; Vyas et al., 2002). In decidual NK cell–target cell activating immune synapses, Kopcow et al. (2005) demonstrated a failure of the perforin-containing granules to polarize toward the contact side, which might account for the lack of cytotoxicity by decidual NK cells. Decidual NK cells fulfil dual roles. During normal conditions, they contribute to creating a favourable environment for placentation, but at the same time they are fully armed to fight intrauterine infections if necessary. Decidual NK cells contain perforin and express NKG2D receptors that recognize stress signals, e.g. MIC peptides or human cytomegalovirus UL16 binding peptides. Certain pregnancy pathologies are associated with increased decidual and peripheral NK activity. Gulan et al. (1997) demonstrated decreased perforin content of decidual
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho lymphocytes from failed pregnancies compared with those from normal pregnancy deciduas, suggesting that an increased rate of degranulation had taken place in the former case. The expression of the KIR2DL1 inhibitory KIR (the ligand of which is HLA-C), as well as that of CD94, were shown to be significantly decreased while perforin content was increased in women with miscarriage with normal chromosomal content compared with those in controls (Yamada et al., 2001). In another study, women who miscarried have been shown to lack the appropriate inhibitory receptors to interact with the HLA-Cw molecules on trophoblast cells and to deliver signals to inhibit NK cell activation and protect the embryo (VarlaLeftherioti et al., 2005).
cells, and blocking of IL-15 abrogates this effect (Sotosek et al., 1999). Trophoblast that originally resists NK-mediated lysis is rendered susceptible in the presence of IL-2 (Zuckermann and Head, 1987). However, IL-2 is among the few cytokines that have never been detected in the decidua. Low concentrations of IL-15 augment the cytotoxicity of decidual NK cells to K562 but, in contrast to IL-2, not to trophoblast (Verma et al., 2000). These findings suggest that IL-15, while inducing activation and proliferation of decidual NK cells, by up-regulating CD94/ NKG2A expression on these cells, protects the trophoblast from potential cytotoxicity. On the other hand, IL-15 can also upregulate NKG2D on NK cells, thus enabling them to recognize infected or transformed cells (Sutherland et al., 2002).
The mechanisms that control cytotoxicity during pregnancy have not been completely elucidated. However, the unique milieu of the decidua certainly plays a role. In this setting, NK cells are exposed to a variety of locally acting cytokines and hormones.
Regulation of NK activity by progesterone
Regulation of NK activity by cytokines In spite of their high perforin content, freshly isolated decidual NK cells display strongly reduced lytic activity on classical MHC class I negative targets, but culturing the cells outside the decidual environment restores the cytotoxic potential (Kopcow et al., 2005). In women with recurrent miscarriage with normal chromosomal content, implantation sites contain decreased numbers of Th2 cells (Michimata et al., 2003), suggesting abnormal cytokine regulation. The classical Th1/Th2 concept cannot be directly applied to the decidual microenvironment where the action of cytokines is stage-specific (Chaouat et al., 2007). Transforming growth factor (TGF) β has been identified as one of the key molecules regulating NK activity in the decidua. During culture in TGFβ1-containing conditioned medium from decidual stromal cells, highly purified CD16+CD9– peripheral blood NK cells acquire characteristics (CD9 expression) of decidual NK cells. TGFβ1 exerts a similar effect (Keskin et al., 2007). After blocking the action of endogenous TGFβ, both the number of responding cells and interferon-γ production by stimulated human peripheral NK cells increase (Meadows et al., 2006). Endogenous TGFβ alters cytokine production of uterine NK cells in human endometrium in response to stimulation through Toll-like receptors; thus endometrial NK cell responses to microbial pathogens are also regulated by the amount of biologically active TGFβ present in the human endometrium (Eriksson et al., 2006). Taken together, these data suggest that TGFβ might be one of the factors responsible for the downregulated cytotoxic activity of decidual NK cells. The hormonal and cytokine environment can modulate the expression of both the receptors and the ligands. TGFβ possibly contributes to decreased NK cytotoxicity by modulating KIR2DL expression (Shin et al., 2004). On the other hand, IL-15 produced by uterine macrophages induces the expression of CD94/NKG2A (Mingari et al., 1997) or NKG2D (Sutherland et al., 2002).
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Decidual NK cells do not express the high-affinity IL-2R α chain, while the β and γ chains of the IL-2R are present and serve as subunits of the IL-15R. Supernatants of decidual macrophages up-regulate perforin expression of decidual NK
Pregnancy is characterized by profound alteration in the endocrine functions. Among the pregnancy-related hormones, the effect of progesterone on NK cell function is well documented. Early studies suggested a dose-dependent decrease of peripheral NK activity of pregnancy lymphocytes by low concentrations (40–500 nM) of progesterone (Szekeres-Bartho et al., 1985a,b). Experiments using lymphocytes of non-pregnant subjects failed to demonstrate such an effect at similar concentrations (Sulke et al., 1985; Uksila, 1985). These data might suggest the presence of a population with increased progesterone sensitivity among peripheral pregnancy lymphocytes. Indeed, progesterone receptors were demonstrated by immunocytochemistry in peripheral γ/δ T cells of pregnant women (Szekeres-Bartho et al., 2001). Furthermore, RU 486, a potent antiprogestin, has been shown to significantly augment NK cell cytolytic activity in vitro; this down-regulation is effectively blocked by progesterone at a concentration of 25 µmol/l (Hansen et al., 1992). At the progesterone concentrations (10–5 mol/l) believed to be present at the maternalfetal interface (Stites and Siiteri, 1983), studies consistently show inhibition of NK cytolytic activity in vitro (Hansen et al., 1992). Henderson et al. (2003) demonstrated the absence of progesterone receptors in purified decidual NK cells. Progesterone could act on these cells through glucocorticoid receptors (Henderson et al., 2003), putative membrane receptors (Zhu et al., 2003) or calcium-activated K+ channels (Chien et al., 2007), but the most likely possibility is that progesterone affects NK activity in an indirect way, by induction of cytokine production in the neighbouring cells, which would in turn influence NK activity. In response to progesterone, T cells produce progesterone-induced blocking factor (PIBF) (Szekeres-Bartho et al., 2001), that, by controlling NK activity, exerts an antiabortive effect in mice (Szekeres-Bartho et al.1997). PIBF has recently been cloned and has been shown to enhance IL-10 production and suppress IL-12 production by human peripheral lymphocytes (Polgar et al., 2003). Although decidual NK cells do not express the progesterone receptor, they appear to be affected by PIBF secreted by surrounding cells. PIBF blocks up-regulation of perforin expression in decidual lymphocytes cultured with decidual adherent cells and inhibits NK cell cytotoxicity by blocking granule exocytosis without interfering with target conjugation (Faust et al., 1999; Laskarin et al., 1999). Progesterone also contributes to the establishment of a Th2 environment by downregulating IL-12 and up-regulating IL-10, possibly through the intermediary production of PIBF (Raghupathy, 1997).
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Symposium - NK cell cytotoxicity during pregnancy - J Szekeres-Bartho Progesterone has been shown to increase IL-15 production by endometrial stromal cells (Okada et al., 2000). Human decidual NK cells express the IL-15 receptor and, in response to IL-15, proliferate and augment their cytolytic activity against K562, but not against the trophoblast (Verma et al., 2000). Progesterone up-regulates HLA-G gene expression through a novel progesterone response element (Yie et al., 2006). Increased expression of HLA-G (the ligand for NK inhibitory receptors) might play a role in controlling antitrophoblast NK activity. Although KIR2DL4 also recognizes HLA-G, engagement of this receptor induces interferon-γ production, rather than lytic activity. Some argue that, since there is no evidence for the association between uterine and peripheral NK cells, investigating NK cell count or activity during normal or pathological pregnancy is irrelevant (Moffet et al., 2004; Rai et al., 2005). There are only a few studies comparing peripheral and decidual NK cells from the same patient (Gulan et al., 1997; Lewis et al., 2002; Olivares et al., 2002; Higuma-Myojo et al., 2005). It is hard to tell how measurable changes at the periphery relate to local events. Currently available data suggest that recurrent aborters demonstrate an increased peripheral NK activity. (Aoki et al., 1995; Emmer et al., 2000; Ntrivalas et al., 2001, 2005; Yamada et al., 2003). Whether or not these peripheral changes reflect local events needs to be further investigated, all the more so, because studies on decidual NK activity have their inherent limitations. The availability of normal human placentas is restricted to the first trimester and labour. Placentas from spontaneous miscarriages are likely to contain an altered cytokine milieu, as well as toxic products of necrotic tissue, and it is not clear whether the observed changes are the cause or the consequence of miscarriage. Furthermore, there is no evidence at present that the high decidual NK activity is due to activation of the resident population and not due to migration of activated peripheral NK cells to the decidua.
Conclusions Human decidual lymphocytes contain a high amount of perforin that enables them to fight intrauterine infections, yet these cells are not cytotoxic in the absence of a danger signal. Decidual NK cell cytotoxicity is regulated by the balance of activating or inhibitory receptors and by expression of ligands on the target cells. The trophoblast expresses HLA-C, HLA-G and HLA-E, recognized by NK cell receptors. Cytotoxicity is controlled by precisely coordinated action of locally produced cytokines and hormones. Progesterone up-regulates HLA-G expression, while IL-15 up-regulates the expression of inhibitory receptors on decidual NK cells. Progesterone affects NK activity in an indirect way, by inducing Th2-type cytokine production that, in turn, would act on NK cells. TGFβ controls NK cytotoxicity via several pathways, whereas IL-15 activates NK cells. Disturbance of the delicate balance established among players of decidual homeostasis may result in increased NK cytotoxicity and the cessation of pregnancy.
Acknowledgements This work was supported by grants from the Hungarian National Research Fund (OTKA T031737), Hungarian Ministry of
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Health (ETT 045/2003), National Research and Development Program (NKFP 1A-057–2004), Economic Competitiveness Operative Program (GVOP-3.1.1.-2004–05–0329/3.0), and the Hungarian Academy of Sciences. The author is the member of a European Network of Excellence on Embryo Implantation Control supported by EU Contr. No. 512040. The authors report no financial or commercial conflicts of interest.
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