Do uterine natural killer (uNK) cells contribute to female reproductive disorders?

Journal of Reproductive Immunology 88 (2011) 156–164

Contents lists available at ScienceDirect

Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm

Review

Do uterine natural killer (uNK) cells contribute to female reproductive disorders? Gendie E. Lash ∗ , Judith N. Bulmer Reproductive and Vascular Biology Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom

a r t i c l e

i n f o

Article history: Received 23 September 2010 Received in revised form 23 December 2010 Accepted 12 January 2011

Keywords: Uterine natural killer cells Trophoblast invasion Spiral artery remodelling Miscarriage Preeclampsia

a b s t r a c t The most abundant immune cells in the uterine decidua around the time of implantation and early placental development are the uterine natural killer (uNK) cells. Altered numbers of uNK cells have been associated with several human reproductive disorders, including recurrent miscarriage, recurrent implantation failure, uterine fibroids, sporadic miscarriage, fetal growth restriction and preeclampsia. Understanding of the function of uNK cells in non-pregnant and pregnant endometrium is now increasing; the potential contribution of altered numbers and function of uNK cells to reproductive disorders is the focus of this review. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

2. Uterine natural killer cells

Immune cells are a key feature of non-pregnant endometrium and decidualised endometrium in pregnancy. One such immune cell type is the uterine natural killer (uNK) cells, altered numbers of which have been associated with recurrent miscarriage (Quenby et al., 1999), recurrent implantation failure (Tuckerman et al., 2010), uterine leiomyomata (fibroids) (Kitaya and Yasuo, 2010), sporadic miscarriage (Zenclussen et al., 2001), fetal growth restriction (Williams et al., 2009a) and preeclampsia (Williams et al., 2009a). Understanding of the function of these cells in non-pregnant and pregnant endometrium is now increasing and clarification of the potential contribution of altered numbers and function of uNK cells to reproductive disorders may therefore now be possible.

Uterine NK cells are the most abundant of all decidual leucocytes accounting for up to approximately 70% of decidual stromal CD45+ cells in the first trimester of human pregnancy. Uterine NK cells are brightly CD56positive but are CD16-negative; they therefore differ phenotypically from ‘usual’ peripheral blood NK cells which are CD56+ CD16+ . Although a small population of CD56bright CD16− NK cells are detectable in peripheral blood, these are usually agranular; in contrast uNK cells are CD56bright CD16− CD57− and are highly granulated.

∗ Corresponding author. Tel.: +44 191 222 8578; fax: +44 191 222 5066. E-mail address: [email protected] (G.E. Lash). 0165-0378/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2011.01.003

3. Distribution of uNK cells in non-pregnant and pregnant uterine endometrium Initially uNK cells were identified by the presence of phloxinophilic cytoplasmic granules using the phloxine tartrazine histochemical stain. They were reported to be absent in proliferative endometrium, with numbers increasing in the mid and late secretory phase of the

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

menstrual cycle and in pregnancy until the third month of gestation; thereafter they were reported to decline to be virtually absent at term (Hamperl and Hellweg, 1958; Dallenbach-Hellweg and Nette, 1964). This distribution was confirmed for pregnancy using phloxine tartrazine staining of pregnancy hysterectomies from 8 weeks to term (Bulmer and Lash, 2005); granulated leucocytes were rare after 20 weeks of gestation. With the identification of CD56 as a key marker for human uNK cells, immunostaining for CD56 largely confirmed the distribution of uNK cells based on phloxine tartrazine staining. CD56+ cells are, however, present in proliferative and early secretory phase endometrium, albeit in small numbers (Bulmer et al., 1991; King et al., 1989), with numbers increasing in the late secretory phase of the menstrual cycle and increasing further into early pregnancy. In normal human placental bed during the first half of pregnancy CD56+ uNK cell numbers do not alter between first (8–12 weeks’ gestation) and early second (13–20 weeks’ gestation) trimester decidua (Williams et al., 2009b), although the number of cells immunostaining for perforin and granzyme B and showing phloxinophilic granules in phloxine/tartrazine are reduced across this same time period (Bulmer et al., 2010). A decline in CD56+ cell numbers is observed at term, although substantial numbers of CD56+ uNK cells still remain in both decidua basalis and decidua parietalis (Williams et al., 2009b). The discrepancy between these and early findings may be due to uNK cell loss of cytoplasmic granules as pregnancy progresses, although this has not been examined in detail. The loss of cytoplasmic granules may reflect functional differences between uNK cells in early and late pregnancy. Uterine NK cells are detected predominantly in the stratum functionalis of late secretory phase endometrium and early pregnancy decidua, often forming aggregates around spiral arterioles/arteries and glands (Bulmer et al., 1991) (Fig. 1). In addition, in early pregnancy decidua uNK cells are also found associated with invading interstitial extravillous trophoblast (EVT) cells (Fig. 1). Several studies have compared the distribution and number of uNK cells between decidua basalis and decidua parietalis in pregnancy with varying results (reviewed in Bulmer and Lash, 2005; Bulmer et al., 2010); some groups have noted no difference between the two decidual areas, whereas others have reported increased numbers of uNK cells in decidua basalis in association with extravillous trophoblast. The origin of uNK cells in endometrium and decidua remains the subject of debate; there is evidence supporting both trafficking of differentiated uNK cells from the peripheral circulation and/or alternatively in situ differentiation and proliferation of precursor cells locally in endometrium. Several recent reviews cover this subject in detail (Manaster and Mandelboim, 2008; Yagel, 2009; Bulmer et al., 2010). 4. Functional investigations The precise in vivo functions of uNK cells are still not clear. However, in vitro studies are providing clues to their functions in early human pregnancy, although further stud-

157

ies are required to determine the function of uNK cells in non-pregnant endometrium. 4.1. Cytotoxicity Uterine NK cells isolated from non-pregnant endometrium (Jones et al., 1997) and early pregnancy decidua (Ritson and Bulmer, 1989) exhibit cytotoxic activity against the NK cell target K562, although this cytotoxic activity is consistently lower than that of peripheral blood NK cells (Kopcow et al., 2005). Uterine NK cells are non-cytolytic to extravillous trophoblast (EVT) cells, due in part to EVT expression of HLA-G and uNK cell expression of inhibitory receptors (Chumbly et al., 1994; Rouas-Freiss et al., 1997; Chen et al., 2010). In addition, uNK cell secreted vascular endothelial growth factor-C (VEGFC) up-regulates EVT expression of TAP-1, which plays a role in peptide loading for MHC class I assembly and antigen presentation in EVT cells, protecting them from cytolytic attack by uNK cells (Kalkunte et al., 2009). 4.2. Cytokine, growth factor and protease secretion Uterine NK cells are a rich source of many cytokines and growth factors, including tumour necrosis factor-␣ (TNF␣), interleukin-10 (IL-10), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1␤, transforming growth factor-␤1 (TGF␤1), macrophage colony stimulating factor (M-CSF, CSF1), leukemia inhibitor factor (LIF) and interferon-␥ (IFN␥) (Saito et al., 1993; Jokhi et al., 1994; Lash et al., 2010a). Using multiplex cytokine/growth factor analysis and ELISA we have shown that uNK cell secretion of IL-1␤, GM-CSF (Lash et al., 2010a), IL-6 (Champion et al., 2007), IL-8 (De Oliveira et al., 2010) and IFN␥ (Lash et al., 2006a) increases with gestational age from 8–10 to 12–14 weeks gestational age. We have also demonstrated that uNK cells are a major decidual source of angiogenic growth factors, including angiopoietin (Ang)-1, Ang-2, VEGF-C, placental growth factor (PlGF) and TGF␤1 (Lash et al., 2006b). In contrast with cytokine production, uNK cell secretion of Ang-2 and VEGF-C was reduced at 12–14 weeks gestation compared with 8–10 weeks gestation (Lash et al., 2006b). Li et al. (2001) also reported localisation of angiogenic growth factor mRNA to uNK cells in non-pregnant endometrium by in situ hybridisation. In addition, differences in mRNA expression profiles between uNK cells isolated from nonpregnant endometrium and early pregnancy decidua have been demonstrated using gene array technology (Kopcow et al., 2010), as well as between NK cells in peripheral blood and early pregnancy decidua (Hanna et al., 2006; Kopcow et al., 2010). These data suggest that at 8–10 weeks gestational age uNK cells are a major producer of angiogenic growth factors, some of which decrease with gestational age. In contrast, at 12–14 weeks gestational age uNK cells are a major producer of cytokines, some of which increase with gestational age. The switch that alters the angiogenic growth factor/cytokine secretion profile of uNK cells with gestational age is not clear, although alterations in secretion profile may impact on their in vivo function. The change from angiogenic growth factor expression to

158

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

Fig. 1. Photomicrographs of immunostained serial sections of non-transformed (A–D) and partially transformed (E–H) spiral arteries in a 10 weeks gestational age placental bed biopsy from an apparently normal pregnancy. (A and E) Cytokeratin 7 immunoreactivity in trophoblasts (brown) and counterstained with PAS (pink) showing fibrinoid deposition in the vessel wall. (B and F) H-caldesmon immune-reactivity in vascular smooth muscle cells (brown) with PAS (pink) counterstain showing fibrinoid deposition in the vessel wall. (C and G) CD56 immunoreactivity in uterine NK cells (brown) both within the wall and surrounding the vessel. Endovascular trophoblast within the vessel lumen is also CD56-positive. (D and H) CD14 immunoreactivity in macrophages (brown) both within the vessel wall and surrounding the vessel. Note that more uNK cells and macrophages are observed within the wall of the non-transformed vessel than the partially transformed vessel. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

cytokine expression could reflect alterations in the proportion of uNK cells undergoing in situ proliferation and differentiation and those that are trafficked in from the peripheral blood circulation. The function of uNK cells in third trimester decidua remains unknown. Trophoblast invasion and spiral artery remodelling both require breakdown of the extracellular matrix (ECM) by proteolytic enzymes such as matrix metalloproteinase-2 (MMP2) and MMP9, and the urokinase plasminogen activator (uPA) system. Uterine NK cells secrete MMP2, MMP9, tissue inhibitor of metalloproteinase-1 (TIMP1), TIMP2, TIMP3, uPA and uPA receptor (uPAR), although not plasminogen activator inhibitor-1 (PAI1) and PAI2 (Naruse et al., 2009a,b). Immunoreactivity of MMP7 and MMP9 by leucocytes surrounding spiral arteries during early pregnancy has also been reported (Smith et al., 2009).

of neutralising antibodies to IL-8 and IP-10 (Hanna et al., 2006). In contrast, Hu et al. (2006) demonstrated that IL15 stimulated uNK cell supernatants inhibited migration of EVT in a two dimensional migration assay via a mechanism dependent on IFN␥. We have demonstrated that uNK cell supernatants from 8–10 weeks’ gestational age have no effect on in vitro EVT invasion from placental explants at the same gestational age. However, when both uNK cells and placenta were from 12–14 weeks gestational age, uNK cell supernatants stimulated EVT invasion from placental explants. This uNK-mediated stimulation of EVT invasion was associated with increased MMP9 secretion and reduced EVT apoptosis (Lash et al., 2010b) and was partially abrogated in the presence of an IL-8 neutralising antibody (De Oliveira et al., 2010). 4.4. Angiogenesis and spiral artery remodelling

4.3. Regulation of trophoblast invasion Extravillous trophoblast (EVT) invasion is tightly regulated, and uNK cells have been proposed to play a role in control of this process. Hanna et al. (2006) demonstrated that IL-15 stimulated uNK cell supernatants stimulate invasion of isolated cytotrophoblast cells in vitro and that this stimulatory effect was partially abrogated in the presence

Spiral artery remodelling is a key feature of early placental development in human pregnancy and failure of this process has been linked to several serious pregnancy complications, including preeclampsia, fetal growth restriction and second trimester miscarriage (Pijnenborg et al., 2006). Although this process has largely been attributed to the effect of EVT on spiral arteries, with deficient EVT

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

invasion in pathological pregnancy, there is growing support for a ‘trophoblast independent’ phase of spiral artery remodelling (Pijnenborg et al., 2006) where the initial stages of spiral artery remodelling, including dilatation, some fibrinoid deposition, endothelial swelling and vascular smooth muscle cell separation occur in the absence of EVT (Craven et al., 1998; Kam et al., 1999). Uterine NK cells are frequently aggregated around the spiral arteries and arterioles in early human pregnancy and this distribution may reflect a role in mediating vascular changes in pregnancy (Fig. 1). Indeed, Smith et al. (2009) demonstrated increased numbers of leucocytes (both uNK cells and macrophages) within 25 ␮m of the vessel lumen in human decidual spiral arteries showing partial remodelling and an absence of EVT, compared with non-remodelled vessels and those with greater levels of remodelling (including the presence of EVT). In in vitro models using either chorionic plate arteries from human term placenta (Lash et al., 2010a) or non-pregnant myometrial arteries (Harris et al., 2010) we have shown that uNK cell supernatants from 8–10 weeks gestation can initiate vascular smooth muscle cell separation, while uNK cell supernatants from 12–14 weeks gestation have a greater effect on de-differentiation of the vascular smooth muscle cells (Robson et al., 2010; Lash, 2010; Harris et al., 2010) (Fig. 2). The uNK cell stimulation of EVT invasion at 12–14 weeks gestational age may play a role in attracting EVT cells towards the spiral arteries for completion of the remodelling process. The uNK cell derived factors responsible for mediating these effects are still being determined but are likely to include Ang-2 and VEGFC (Robson et al., 2009; Lash et al., 2010a). In addition, uNK cells have been shown to stimulate endothelial cell angiogenesis in both in vitro and in vivo models (Hanna et al., 2006), leading to the suggestion that uNK cells may contribute to placental angiogenesis during early pregnancy. A possible role for uNK cells in arteriogenesis, which involves the development of vascular smooth muscle cell coat in newly formed vessels, within the endometrium may be relevant for the role of uNK cells in reproductive disorders. 5. Uterine NK cells in reproductive disorders Altered numbers of uNK cells have been detected in the endometrium and in early pregnancy decidua from women with various reproductive disorders including recurrent miscarriage, recurrent implantation failure, uterine leiomyomata (fibroids), sporadic miscarriage, fetal growth restriction and preeclampsia. 5.1. Recurrent miscarriage and recurrent implantation failure Several studies from different groups have reported increased uNK cell numbers as determined by immunohistochemistry in mid-secretory phase endometrium of women with a history of recurrent miscarriage or recurrent implantation failure (Quenby et al., 1999; Clifford et al., 1999; Laird et al., 2005; Tuckerman et al., 2007, 2010). In contrast, other groups have reported no alteration in

159

endometrial uNK cell numbers in women with a history of recurrent miscarriage as determined by flow cytometry (Lachapelle et al., 1996; Shimada et al., 2004), although Lachapelle et al. (1996) did report altered proportions of the CD56bright CD16− (reduced) and CD56dim CD16+ (increased) uNK cell subsets. Using an immunohistochemical approach Michimata et al. (2002) also failed to detect any differences in endometrial uNK cell numbers in women with a history of recurrent miscarriage compared to controls as determined by immunohistochemistry but in contrast to other studies these authors defined recurrent miscarriage as two or more as opposed to three or more consecutive miscarriages. Therefore differences in the reported studies are likely to arise from the method of quantification of uNK cells (flow cytometry versus immunohistochemistry) and the definition of recurrent miscarriage (two versus three consecutive miscarriages). It is worth noting that immunohistochemical studies from different groups have consistently shown increased uNK cell numbers in the mid secretory phase of the menstrual cycle in women with a well defined history of recurrent miscarriage. Furthermore, it has been suggested that high uNK cell numbers in mid-secretory phase endometrium from women with recurrent miscarriage may predict miscarriage in subsequent pregnancy (Quenby et al., 1999), although this has been disputed (Tuckerman et al., 2007). Initial hypotheses suggested that the functional link between increased numbers of uNK cells and miscarriage would be due to increased uNK cell cytotoxic activity against the implanting conceptus. However, there is no evidence that uNK cells exhibit cytotoxic activity against EVT cells in the absence of stimulation by interleukin 2 or that this is altered in women with a history of recurrent miscarriage (Laird et al., 2003). Embryo implantation and early placental development occur in a relatively hypoxic environment (2–3% O2 ) (Yedwab et al., 1976; Rodesch et al., 1992), with oxygen levels in the intervillous space rising between 10 and 12 weeks gestation (Jauniaux et al., 2000). This is associated with a minor degree of oxidative stress damage to the placental villous syncytiotrophoblast, which is then able to adapt to the higher oxygen levels (Hung et al., 2001). Histological studies have reported disruption of the trophoblast shell and reduced plugging of spiral arteries by endovascular trophoblast in miscarriage (Hustin et al., 1990). Inappropriate maternal blood flow to the intervillous space may be underpinned by several different pathologies and has been proposed to be a final common pathway in miscarriage, both sporadic and recurrent (Jauniaux et al., 2000). Endometrial blood vessel development appears to be advanced in women with recurrent miscarriage and has been correlated with the percentage of uNK cells within endometrial stromal in the mid-secretory (LH+7) phase and lowered resistance to blood flow as determined by Doppler (Quenby et al., 2009). It has been hypothesised that this increased blood flow in the implantation site could compromise the implanting conceptus which prefers a hypoxic environment for early development (Quenby et al., 2009). Prednisolone treatment during non-pregnancy reduces uNK cell numbers (Quenby et al., 2005) as well as blood vessel development (Lash et al.,

160

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

8-10 weeks gestational age (GA)

uNK cell Lumen VSMC

Non-remodelled spiral artery associated with uNK cells uNK cells are observed in the vessel wall and adventia

8-10 weeks GA Partially remodelled spiral artery showing signs of VSMC separation, rounding and misalignment uNK cells are observed in adventia and are lost from within the wall

12-14 weeks GA Infiltration of the spiral artery wall and lumen by EVT Possibly stimulated by uNK cells? EVT cell

12-14 weeks GA VSMC migration, dedifferentiation and eventual complete loss Greater involvement of EVT cells than uNK cells

Fig. 2. Schematic of the role of uterine NK cells in spiral artery remodelling as gestational age increases.

2008) and may lead to a live birth (Quenby et al., 2003). However, the full efficacy of this treatment option is still to be determined, although a pilot study for a randomised clinical trial is currently underway (Tang et al., 2009).

The collected evidence as well as the known angiogenic growth factor expression pattern of uNK cells in non-pregnant endometrium (Li et al., 2001) suggests a causative role for uNK cells in advanced blood vessel devel-

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

opment in a subset of women with recurrent miscarriage. However, while altered numbers of endometrial uNK cells have been reported it is not yet known whether they are functionally altered in these women. Indeed, one study has suggested that endometrial uNK cells are non-functional (Manaster et al., 2008), while another suggests that several mRNA are upregulated in non-pregnant endometrial uNK cells compared to decidual uNK cells in early pregnancy (Kopcow et al., 2010). Therefore, a crucial area of future uNK cell research will be to determine their function in non-pregnant endometrium, as this may well be different from the function in early pregnancy decidua. Studies of uNK cell numbers in the decidua of women with a history of recurrent miscarriage undergoing a further miscarriage have provided differing results; this may reflect differences in methodology (immunohistochemistry versus flow cytometry) and any alteration in cell numbers may represent an effect of the miscarriage rather than cause (Chao et al., 1995; Yamamoto et al., 1999a; Kwak et al., 1999; Quack et al., 2001; Emmer et al., 2002). 5.2. Sporadic miscarriage Increased numbers of uNK cells, as determined by immunohistochemistry, have been reported in decidua of women with sporadic miscarriage compared with controls (Zenclussen et al., 2001; Plaisier et al., 2009), although we were not able to confirm this finding in an immunohistochemical study of placental bed biopsies from aneuploid and euploid miscarriages from 8 to 20 weeks gestation, compared with normal pregnancy (Scaife et al., 2004). In addition, using flow cytometry Yamamoto et al. (1999b) reported reduced numbers of CD56+ uNK cells in decidua of women with sporadic miscarriage. It is still therefore uncertain whether uNK cell numbers are altered in the decidua of women with sporadic miscarriage. How uNK cells may contribute to the aetiology of sporadic miscarriage is not clear and the increased numbers observed may be a consequence of the miscarriage rather than being causative. Indeed, altered uNK cell function is likely to be more important than altered uNK cell numbers. Vassiliadou and Bulmer (1998) reported decreased uNK cell cytotoxic activity against a K562 target when the uNK cells were isolated from decidua of women with sporadic miscarriage compared to normal controls. In contrast, Yamada et al. (2005) reported an increased proportion of uNK cells expressing perforin in the decidua of women with sporadic miscarriage compared to controls. Nakashima et al. (2008) reported no difference in the proportion of uNK cells expressing of perforin and granzyme B in decidua from sporadic miscarriage compared to normal controls, although they later reported an increase in the proportion of granulysin-positive uNK cells in decidua of women with sporadic miscarriage and related this functionally to an increase in EVT apoptosis (Nakashima et al., 2008). Taken together it is still not clear whether uNK cell numbers and function are altered in the decidua of women undergoing sporadic miscarriage. Due to the nature of sampling which inevitably follow miscarriage any alterations observed may be a result rather than the cause of the miscarriage. Further studies on uNK cell function and how this

161

may be altered are required before a causative link to sporadic miscarriage can be concluded. 5.3. Uterine leiomyomata (fibroids) Uterine leiomyomata (fibroids) impair optimal growth and development of the uterine vasculature and women with this condition are often sub-fertile. A recent study investigated leucocyte numbers in the endometrium of women with fibroids, comparing ‘near nodule’ endometrium and ‘non-near nodule’ endometrium of women with uterine fibroids with endometrium from controls (Kitaya and Yasuo, 2010). In proliferative and mid-late secretory phase endometrium uNK cell numbers were highest in control samples compared with non-near nodule endometrium from women with uterine fibroids, which in turn had higher uNK cell numbers than endometrium in those samples taken near nodules. In contrast, the number of CD45+ leucocytes showed the opposite correlation, with higher number of leucocytes being detected closer to the nodule (Kitaya and Yasuo, 2010). In recurrent miscarriage and recurrent implantation failure increased uNK cell numbers have been associated with advanced blood vessel maturation (Quenby et al., 2009). In endometrium of women with leiomyomata the composition of the total endometrial leucocyte population appears to differ from normal; it is possible that the altered numbers of uNK cells may contribute to the aberrant blood vessel development and sub-fertility observed with uterine leiomyomata. 5.4. Pre-eclampsia and fetal growth restriction Pre-eclampsia and fetal growth restriction are both characterised by inadequate spiral artery remodelling, particularly of the myometrial portions of the vessels. Some reports suggest inadequate or shallow invasion of interstitial EVT is also a feature of these conditions, although we and others have noted adequate invasion of interstitial EVT in these conditions (Pijnenborg et al., 1992, 1998). Interstitial EVT are also often prominent in myometrium surrounding non-remodelled vessels in pregnancies that were complicated with preeclampsia (Fig. 3). Some studies have reported increased numbers of uNK cells in decidua from women with preeclampsia compared with age matched controls as determined by immunohistochemistry (Stallmach et al., 1999; Bachmayer et al., 2006) or flow cytometry (Wilczynski et al., 2003). In contrast, in our laboratory using immunohistochemistry we have demonstrated a reduction in uNK cells in preeclampsia and fetal growth restriction in placental bed biopsies (Williams et al., 2009a). Eide et al. (2006) also reported a decrease in decidual uNK cell numbers in women with severe fetal growth restriction in the presence or absence of preeclampsia as determined by immunohistochemistry, although there were no differences in numbers of decidual uNK cells in women with preeclampsia alone without growth restriction. In a recent flow cytometry study of decidua from currettings from women with preeclampsia or controls there was no difference in the proportion of CD45+ cells that were CD56+ /CD16− in women with preeclampsia com-

162

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

Fig. 3. Photomicrographs of immunostained non-transformed spiral arteries from women with second trimester miscarriage (A–F) and preeclampsia (G and H). (A, D, G and H) Cytokeratin 7 immuno-reactivity in trophoblasts (brown) with PAS (pink) counterstain showing fibrinoid deposition in the vessel wall. Note interstitial extravillous trophoblast cells surrounding the wall of the spiral artery and some invasion of the vessel wall (D). (B and E) Desmin immunoreactivity in vascular smooth muscle cells (brown). (C and F) Factor VIII immunoreactivity in endothelial cell lining of the spiral artery. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

pared with controls, although there was a decrease in the proportion of CD45+ cells that were CD56+ /CD16+ (Rieger et al., 2009). Taking all the diverse studies together, it is still not clear whether uNK cell numbers are altered in preeclampsia and fetal growth restriction; differences in reported results may reflect differences in sampling, analysis and severity of disease. In addition, all of these studies are flawed by examining uNK cell numbers after delivery; it is not clear whether altered levels of uNK cells at term are reflective of those earlier in gestation. However, given the potential role of uNK cells in initiating spiral artery remodelling and

essentially priming the vessel wall for invasion by EVT it is tempting to speculate that reduced uNK cell numbers in the first trimester may be a significant contributor to the aetiology of preeclampsia and fetal growth restriction. 6. Conclusions Uterine natural killer cells are the most abundant leucocyte in mid and late secretory phase non-pregnant endometrium and early pregnancy decidua. Diverse roles have been proposed ranging from immune function, cytokine and growth factor secretion, regulation of tro-

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

phoblast invasion and regulation of the initial stages of spiral artery remodelling. Altered numbers of uNK cells have been associated with several different reproductive disorders and given their known functions a causative role in some of these disorders may exist. In addition, adverse pregnancy outcomes (preeclampsia, fetal growth restriction and recurrent miscarriage) have been associated with mismatched KIR haplotype (KIR AA) expression by the decidual uNK cells and group 2 HLA-C (C2) expression by extravillous trophoblast cells (Hiby et al., 2010). The functional significance of these interactions on either the uNK or EVT cells is as yet unknown. It also remains uncertain, however, whether the function of uNK cells is altered in reproductive disorders and this may be a rewarding area for future research. Acknowledgements Research in the laboratory of JNB and GEL has been funded by BBSRC, Wellbeing of Women and The Royal Society. References Bachmayer, N., Rafik Hamad, R., Liszka, L., Bremme, K., SverremarkEkstrom, E., 2006. Aberrant uterine natural killer (NK)-cell expression and altered placental and serum levels of the NK-cell promoting cytokine interleukin-12 in preeclampsia. Am. J. Reprod. Immunol. 56, 292–301. Bulmer, J.N., Lash, G.E., 2005. Uterine natural killer cells: a reappraisal. Mol. Immunol. 42, 511–521. Bulmer, J.N., Morrison, L., Longfellow, M., Ritson, A., Pace, D., 1991. Granulated lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum. Reprod. 6, 791–798. Bulmer, J.N., Williams, P.J., Lash, G.E., 2010. Immune cells in the placental bed. Int. J. Dev. Biol. 54, 281–294. Champion, H., Innes, B.A., Bulmer, J.N., Robson, S.C., Lash, G.E., 2007. Interleukin 6 and its receptors in the placental bed: no role in trophoblast invasion. Placenta 28, A56. Chao, K.H., Yang, Y.S., Ho, H.N., Chen, S.U., Chen, H.F., Dai, H.J., Huang, S.C., Gill, T.J.3rd., 1995. Decidual natural killer cytotoxicity decreased in normal pregnancy but not in anembryonic pregnancy and recurrent spontaneous abortion. Am. J. Reprod. Immunol. 34, 274–280. Chen, L.J., Han, Z.Q., Zhou, H., Zou, L., Zou, P., 2010. Inhibition of HLA-G expression via RNAi abolishes resistance of extravillous trophoblast cell line TEV-1 to NK lysis. Placenta 31, 519–527. Chumbly, G., King, A., Robertson, K., Holmes, N., Loke, Y.W., 1994. Resistance of HLA-G and HLA-A2 transfectants to lysis by decidual NK cells. Cell. Immunol. 155, 312–322. Clifford, K., Flanagan, A.M., Regan, L., 1999. Endometrial CD56+ natural killer cells in women with recurrent miscarriage: a histomorphometric study. Hum. Reprod. 14, 2727–2730. Craven, C.M., Morgan, T., Ward, K., 1998. Decidual spiral artery remodelling begins before cellular interaction with cytotrophoblasts. Placenta 19, 241–252. Dallenbach-Hellweg, G., Nette, G., 1964. Morphological and histochemical observations on trophoblast and decidua of the basal plate of the human placenta at term. J. Anat. 115, 309–326. De Oliveira, L.G., Lash, G.E., Murray-Dunning, C., Bulmer, J.N., Innes, B.A., Searle, R.F., Sass, N., Robson, S.C., 2010. Role of interleukin 8 in regulation of extravillous trophoblast cell invasion. Placenta 31, 595–601. Eide, I.P., Rolfseng, T., Isaksen, C.V., Mecsei, R., Roald, B., Lydersen, S., Salvesen, K.A., Harsem, N.K., Austgulen, R., 2006. Serious foetal growth restriction is associated with reduced proportions of natural killer cells in decidua basalis. Virchows Arch. 448, 269–276. Emmer, P.M., Steegers, E.A., Kerstens, H.M., Bulten, J., Nelen, W.L., Boer, K., Joosten, I., 2002. Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies. Hum. Reprod. 17, 1072–1080. Hamperl, H., Hellweg, G., 1958. Granular endometrial stroma cells. Obstet. Gynecol. 11, 379–387.

163

Hanna, J., Goldman-Wohl, D., Hamani, Y., Avraham, I., Greenfield, C., Natanson-Yaron, S., Prus, D., Cohen-Daniel, L., Arnon, T.I., Manaster, I., Gazit, R., Yutkin, V., Benharroch, D., Porgador, A., Keshet, E., Yagel, S., Mandelboim, O., 2006. Decidual NK cell regulate key developmental processes at the human fetal–maternal interface. Nat. Med. 12, 1065–1074. Harris, L.K., Robson, A., Lash, G.E., Aplin, J.D., Baker, P.N., Bulmer, J.N., 2010. Physiological remodelling of the uterine spiral arteries during human pregnancy: uterine natural killer cells mediate smooth muscle cell disruption. Proc. Physiol. Soc. 19, C42. Hiby, S.E., Apps, R., Sharkey, A.M., Farrell, L.E., Gardner, L., Mulder, A., Claas, F.H., Walker, J.J., Redman, C.C., Morgan, L., Tower, C., Regan, L., Moore, G.E., Carrington, M., Moffett, A., 2010. Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2. J. Clin. Invest. 120, 4102–4110. Hu, Y., Dutz, J.P., Maccalman, C.D., Yong, P., Tan, R., Von Dadelszen, P., 2006. Decidual NK cells alter in vitro first trimester extravillous cytotrophoblast migration: a role for IFNgamma. J. Immunol. 177, 8522–8530. Hung, T.H., Skepper, J.N., Burton, G.J., 2001. In vitro ischemia–reperfusion injury in term human placenta as a model for oxidative stress in pathological pregnancies. Am. J. Pathol. 159, 1031–1043. Hustin, J., Jauniaux, E., Schaaps, J.P., 1990. Histological study of the materno–embryonic interface in spontaneous abortion. Placenta 11, 477–486. Jauniaux, E., Watson, A.L., Hempstock, J., Bao, Y.P., Skepper, J.N., Burton, G.J., 2000. Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure. Am. J. Pathol. 157, 2111–2122. Jokhi, P.P., King, A., Sharkey, A.M., Smith, S.K., Loke, Y.W., 1994. Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction. J. Immunol. 153, 4427–4435. Jones, R.K., Bulmer, J.N., Searle, R.F., 1997. Cytotoxic activity of endometrial granulated lymphocytes during the menstrual cycle in humans. Biol. Reprod. 57, 1217–1222. Kalkunte, S.S., Mselle, T.F., Norris, W.E., Wira, C.R., Sentman, C.L., Sharma, S., 2009. Vascular endothelial growth factor C facilitates immune tolerance and endovascular activity of human uterine NK cells at the maternal–fetal interface. J. Immunol. 182, 4085–4092. Kam, E.P., Gardner, L., Loke, Y.W., King, A., 1999. The role of trophoblast in the physiological change in decidual spiral arteries. Hum. Reprod. 14, 2131–2138. King, A., Wellings, V., Gardner, L., Loke, Y.W., 1989. Immunocytochemical characterization of the unusual large granular lymphocytes in human endometrium throughout the menstrual cycle. Hum. Immunol. 24, 195–205. Kitaya, K., Yasuo, T., 2010. Leukocyte density and composition in human cycling endometrium with uterine fibroids. Hum. Immunol. 71, 158–163. Kopcow, H.D., Allan, D.S., Chen, X., Rybalov, B., Andzelm, M.M., Ge, B., Strominger, J.L., 2005. Human decidual NK cells form immature activating synapses and are not cytotoxic. Proc. Natl. Acad. Sci. U. S. A. 102, 15563–15568. Kopcow, H.D., Eriksson, M., Mselle, T.F., Damrauer, S.M., Wira, C.R., Sentman, C.L., Strominger, J.L., 2010. Human decidual uNK cells from gravid uteri and NK cells from cycling endometrium are distinct NK cell subsets. Placenta 31, 334–338. Kwak, J.Y., Beer, A.E., Kim, S.H., Mantouvalos, H.P., 1999. Immunopathology of the implantation site utilizing monoclonal antibodies to natural killer cells in women with recurrent pregnancy losses. Am. J. Reprod. Immunol. 41, 91–98. Lachapelle, M.H., Miron, P., Hemmings, R., Roy, D.C., 1996. Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome. J. Immunol. 156, 4027–4034. Laird, S.M., Tuckerman, E.M., Cork, B.A., Linjawi, S., Blakemore, A.I.F., LI, T.C., 2003. A review of immune cells and molecules in women with recurrent miscarriage. Hum. Reprod. Update 9, 163–174. Laird, S.M., Tuckerman, E., Prakash, A., Ledger, W., LI, T.C., 2005. Endometrial CD56+ cells and implantation failure after IVF. Placenta 26, A26. Lash, G.E., 2010. Functional role of uterine natural killer cells in early human pregnancy. J. Reprod. Immunol. 86, 14. Lash, G.E., Otun, H.A., Innes, B.A., Kirkley, M., De Oliveira, L., Searle, R.F., Robson, S.C., Bulmer, J.N., 2006a. Interferon-gamma inhibits extravillous trophoblast cell invasion by a mechanism that involves both changes in apoptosis and protease levels. FASEB J. 20, 2512–2518. Lash, G.E., Schiessl, B., Kirkley, M., Innes, B.A., Cooper, A., Searle, R.F., Robson, S.C., Bulmer, J.N., 2006b. Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy. J. Leukoc. Biol. 80, 572–580.

164

G.E. Lash, J.N. Bulmer / Journal of Reproductive Immunology 88 (2011) 156–164

Lash, G.E., Innes, B.A., Bulmer, J.N., Drury, J.A., Robson, S.C., Quenby, S., 2008. Prednisolone treatment reduces endometrial angiogenic growth factor expression and vessel muscularisation in women with recurrent miscarriage. Placenta 29, 83A. Lash, G.E., Robson, S.C., Bulmer, J.N., 2010a. Functional role of uterine natural killer (uNK) cells in human early pregnancy decidua. Placenta 31, S87–92. Lash, G.E., Otun, H.A., Innes, B.A., Percival, K., Searle, R.F., Robson, S.C., Bulmer, J.N., 2010b. Regulation of extravillous trophoblast invasion by uterine natural killer cells is dependent on gestational age. Hum. Reprod. 25, 1137–1145. Li, X.F., Charnock-Jones, D.S., Zhang, E., Hiby, S., Malik, S., Day, K., Licence, D., Bowen, J.M., Gardner, L., King, A., Loke, Y.W., Smith, S.K., 2001. Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells. J. Clin. Endocrinol. Metab. 86, 1823–1834. Manaster, I., Mandelboim, O., 2008. The unique properties of human NK cells in the uterine mucosa. Placenta 29, S60–66. Manaster, I., Mizrahi, S., Goldman-Wohl, D., Sela, H.Y., Stern-Ginossar, N., Lankry, D., Gruda, R., Hurwitz, A., Bdolah, Y., Haimov-Kochman, R., Yagel, S., Mandelboim, O., 2008. Endometrial NK cells are special immature cells that await pregnancy. J. Immunol. 181, 1869–1876. Michimata, T., Ogasawara, M.S., Tsuda, H., Suzumori, K., Aoki, K., Sakai, M., Fujimura, M., Nagata, K., Nakamura, M., Saito, S., 2002. Distributions of endometrial NK cells, B cells, T cells, and Th2/Tc2 cells fail to predict pregnancy outcome following recurrent abortion. Am. J. Reprod. Immunol. 47, 196–202. Nakashima, A., Shiozaki, A., Myojo, S., Ito, M., Tatematsu, M., Sakai, M., Takamori, Y., Ogawa, K., Nagata, K., Saito, S., 2008. Granulysin produced by uterine natural killer cells induces apoptosis of extravillous trophoblasts in spontaneous abortion. Am. J. Pathol. 173, 653–664. Naruse, K., Lash, G.E., Bulmer, J.N., Innes, B.A., Otun, H.A., Searle, R.F., Robson, S.C., 2009a. The urokinase plasminogen activator (uPA) system in uterine natural killer cells in the placental bed during early pregnancy. Placenta 30, 398–404. Naruse, K., Lash, G.E., Innes, B.A., Otun, H.A., Searle, R.F., Robson, S.C., Bulmer, J.N., 2009b. Localization of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors for MMPs (TIMPs) in uterine natural killer cells in early human pregnancy. Hum. Reprod. 24, 553–561. Pijnenborg, R., Vercruysse, L., Ballegeer, V., Anthony, J., Davey, D.A., Hanssens, M., Spitz, B., Tiltman, A., Van Assche, A., 1992. The distribution of fibronectin in the placental bed in normotensive and hypertensive human pregnancies. Troph. Res. 6, 343–350. Pijnenborg, R., Vercruysse, L., Verbist, L., Van Assche, F.A., 1998. Interaction of interstitial trophoblast with placental bed capillaries and venules of normotensive and pre-eclamptic pregnancies. Placenta 19, 569–575. Pijnenborg, R., Vercruysse, L., Hanssens, M., 2006. The uterine spiral arteries in human pregnancy: facts and controversies. Placenta 27, 939–958. Plaisier, M., Dennert, I., Rost, E., Koolwijk, P., van Hinsberg, V.W.M., Helmerhorst, F.M., 2009. Decidual vascularization and the expression of angiogenic growth factors and proteases in first trimester spontaneous abortions. Hum. Reprod. 24, 185–197. Quack, K.C., Vassiliadou, N., Pudney, J., Anderson, D.J., Hill, J.A., 2001. Leukocyte activation in the decidua of chromosomally normal and abnormal fetuses from women with recurrent abortion. Hum. Reprod. 16, 949–955. Quenby, S., Bates, M., Doig, T., Brewster, J., Lewis-Jones, D.I., Johnson, P.M., Vince, G., 1999. Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Hum. Reprod. 14, 2386–2391. Quenby, S., Farquharson, R., Young, M., Vince, G., 2003. Successful pregnancy outcome following 19 consecutive miscarriages: case report. Hum. Reprod. 18, 2562–2564. Quenby, S., Kalumbi, C., Bates, M., Farquharson, R., Vince, G., 2005. Prednisolone reduces preconceptual endometrial natural killer cells in women with recurrent miscarriage. Fertil. Steril. 84, 980–984. Quenby, S., Nik, H., Innes, B., Lash, G., Turner, M., Drury, J., Bulmer, J., 2009. Uterine natural killer cells and angiogenesis in recurrent reproductive failure. Hum. Reprod. 24, 45–54. Rieger, L., Segerer, S., Bernar, T., Kapp, M., Majic, M., Morr, A.K., Dietl, J., Kammerer, U., 2009. Specific subsets of immune cells in human decidua differ between normal pregnancy and preeclampsia—a prospective observational study. Reprod. Biol. Endocrinol. 7, 132. Ritson, A., Bulmer, J.N., 1989. Isolation and functional studies of granulated lymphocytes in first trimester human decidua. Clin. Exp. Immunol. 77, 263–268. Robson, A., Lash, G.E., Innes, B.A., Robson, S.C., Bulmer, J.N., 2009. Potential role of VEGF-C, Ang2 and IFN␥ in spiral artery remodelling. Reprod. Sci. 16, 110A.

Robson, A., Innes, B.A., Lash, G.E., Robson, S.C., Bulmer, J.N., 2010. Uterine natural killer cells promote vascular smooth muscle cell dedifferentiation in a vessel culture model. Reprod. Sci. 17, 324A. Rodesch, F., Simon, P., Donner, C., Jauniaux, E., 1992. Oxygen measurements in endometrial and trophoblastic tissues during early pregnancy. Obstet. Gynecol. 80, 283–285. Rouas-Freiss, N., Goncalves, R.M., Menier, C., Dausset, J., Carosella, E.D., 1997. Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc. Natl. Acad. Sci. U. S. A. 94, 11520–11525. Saito, S., Nishikawa, K., Morii, T., Enomoto, M., Narita, N., Motoyoshi, K., Ichijo, M., 1993. Cytokine production by CD16-CD56bright natural killer cells in the human early pregnancy decidua. Int. Immunol. 5, 559–563. Scaife, P.J., Searle, R.F., Robson, S.C., Innes, B.A., Bulmer, J.N., 2004. Comparison of decidual leucocyte populations in spontaneous early pregnancy loss. J. Soc. Gynecol. Investig. 11, 285A. Shimada, S., Kato, E.H., Morikawa, M., Iwabuchi, K., Nishida, R., Kishi, R., Onoé, K., Minakami, H., Yamada, H., 2004. No difference in natural killer or natural killer T-cell population, but aberrant T-helper cell population in the endometrium of women with repeated miscarriage. Hum. Reprod. 19, 1018–1024. Smith, S.D., Dunk, C.E., Aplin, J.D., Harris, L.K., Jones, R.L., 2009. Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy. Am. J. Pathol. 174, 1959–1971. Stallmach, T., Hebisch, G., Orban, P., LÜ, X., 1999. Aberrant positioning of trophoblast and lymphocytes in the feto–maternal interface with pre-eclampsia. Virchows Arch. 434, 207–211. Tang, A.W., Alfirevic, Z., Turner, M.A., Drury, J., Quenby, S., 2009. Prednisolone trial: study protocol for randomised controlled trial of prednisolone for women with idiopathic recurrent miscarriage and raised levels of uterine natural killer (uNK) cells in the endometrium. Trials 10, 102. Tuckerman, E., Laird, S.M., Prakash, A., Li, T.C., 2007. Prognostic value of the measurement of uterine natural killer cells in the endometrium of women with recurrent miscarriage. Hum. Reprod. 22, 2208– 2213. Tuckerman, E., Mariee, N., Prakash, A., Li, T.C., Laird, S., 2010. Uterine natural killer cells in peri-implantation endometrium from women with repeated implantation failure after IVF. J. Reprod. Immunol. 87, 60–66. Vassiliadou, N., Bulmer, J.N., 1998. Characterization of tubal and decidual leukocyte populations in ectopic pregnancy: evidence that endometrial granulated lymphocytes are absent from the tubal implantation site. Fertil. Steril. 69, 760–767. Wilczynski, J.R., Tchorzewski, H., Banasik, M., Glowacka, E., Wieczorek, A., Lewkowicz, P., Malinowski, A., Szpakowski, M., Wilczynski, J., 2003. Lymphocyte subset distribution and cytokine secretion in third trimester decidua in normal pregnancy and preeclampsia. Eur. J. Obstet. Gynecol. Reprod. Biol. 109, 8–15. Williams, P.J., Bulmer, J.N., Searle, R.F., Innes, B.A., Robson, S.C., 2009a. Altered decidual leucocyte populations in the placental bed in preeclampsia and foetal growth restriction: a comparison with late normal pregnancy. Reproduction 138, 177–184. Williams, P.J., Searle, R.F., Robson, S.C., Innes, B.A., Bulmer, J.N., 2009b. Decidual leucocyte populations in early to late gestation normal human pregnancy. J. Reprod. Immunol. 82, 24–31. Yagel, S., 2009. The developmental role of natural killer cells at the fetal–maternal interface. Am. J. Obstet. Gynecol. 201, 344–350. Yamada, H., Shimada, S., Morikawa, M., Iwabuchi, K., Kishi, R., Onoe, K., Minakami, H., 2005. Divergence of natural killer cell receptor and related molecule in the decidua from sporadic miscarriage with normal chromosome karyotype. Mol. Hum. Reprod. 11, 451–457. Yamamoto, T., Takahashi, Y., Kase, N., Mori, H., 1999a. Decidual natural killer cells in recurrent spontaneous abortion with normal chromosomal content. Am. J. Reprod. Immunol. 41, 337–342. Yamamoto, T., Takahashi, Y., Kase, N., Mori, H., 1999b. Role of decidual natural killer (NK) cells in patients with missed abortion: differences between cases with normal and abnormal chromosome. Clin. Exp. Immunol. 116, 449–452. Yedwab, G.A., Paz, G., Homonnai, T.Z., David, M.P., Kraicer, P.F., 1976. The temperature, pH, and partial pressure of oxygen in the cervix and uterus of women and uterus of rats during the cycle. Fertil. Steril. 27, 304–309. Zenclussen, A.C., Fest, S., Sehmsdorf, U.S., Hagen, E., Klapp, B.F., Arck, P.C., 2001. Upregulation of decidual P-selectin expression is associated with an increased number of Th1 cell populations in patients suffering from spontaneous abortions. Cell. Immunol. 213, 94–103.