granulated lymphocytes of pregnancy

Placenta (1996), 17, 533-543

CURRENT TOPIC

Granulated Lymphocytes of Pregnancy

P. F. W h i t e l a w a and B. A. Croy

Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Paper accepted 10 April 1996

In pregnant mammals, the antigenically distinctive conceptus implants and grows in a uterine environment governed by maternal immune system. The uterus per se is not immunologically privileged and large numbers of lymphocytes accumula implantation sites. In mice and humans, many of these lymphocytes have been identified as uterine natural killer (uNK) cells exhibit a characteristic granulated morphology. In this review we focus on uNK cells and discuss their origin, differentiation possible roles in the maintenance of healthy pregnancies. In species with less invasive placentation (ruminants, pigs), lympho~ with similar granular morphology also appear during gestation and their identity and possible functions are examined.

© 1996 W. B. Saunders Company Placenta (1996), 17, 533-543

I N T R O D U C T I O N

The pregnant uterus is an immunologically regulated site where the maternal immune system permits the antigenically distinctive conceptus to implant and grow. Several hypotheses have been proposed as to the function of immunocompetent cells in the pregnant uterus. In the rodent, a population of morphologically distinctive granulated cells is found meso- metrially to the placenta at implantation sites. Previously known as granulated metrial gland (GMG) cells, these were recognized as bone marrow derived lymphocytes (Peel and Stewart, 1984) and have subsequently been identified as a subset of natural killer (NK) cells characteristic of the pregnant uterus (Mukhtar, Stewart and Croy, 1989; Parr et al., 1990; Linnemeyer and Pollack 1991). Mounting evidence from cell-surface phenotyping and in vitro lytic activity against known NK target cells indicates that they can be classed as NK cells.in their own right, and in this review they will be referred to as uterine NK (uNK) cells.

In cell suspensions prepared from human decidua during the first trimester pregnancy, 10-15 per cent of all cells are lymphocytes of which many are NK cells (Loke and King, 1995). In other species (ruminant, pig, horse) NK and/or 78 T cells have close association with the maternal-fetal interface and resemble the large granulated lymphocytes (LGL) of humans and rodents.

Little is known of the functional roles of uterine lymphocytes or their possible roles in the initiation of abortion. This review will focus on some recent findings related to LGL

"To whom correspondence'should-be addressed .

0143-4004/96/080533 + 11 $12.00/0

in the uterus of different species and discuss postu] mechanisms by which these cells may promote succe: pregnancy.

WHAT ARE NK CELLS?

NK cells are lymphocytes distinct from the other lymphc populations of B and T cells. Generally found in the bl lymphoid tissues and especially the spleen, NK cells bone marrow-derived and may appear as LGL due to I numerous cytoplasmic granules. NK cells do not rearr~ genes for the T-cell receptor (TCR) nor are they abl rearrange immunoglobulin genes. They can be identifiec the surface phenotype C D 3- , CDI6 ÷ and CD56 + in hur and CD3 NKI.1 + in mice (reviewed Trinchieri, 1989; L-" and Phillips, 1992). Endogenous lytic function against spe targets was originally used to characterize NK cells, and it found that upon activation with interleukin-2 (IL-2), NK became lymphokine activated killer (LAK) cells capabl killing a wide range of virally infected, malignant or noJ cells. Killing by NK and LAK cells is associated with release of cytolytic proteins, perforin and serine ester~ which induce apoptotic death in the target cell and probab the effecter cell. IL-2 stimulation also induces the expres of the cytokines including interferon (IFN)-% granule macrophage colony stimulating factor (GM-CSF) and tun necrosis factor (TNF)-ct. The lytic activity of NK was thought to be 'natural' because it did not involve recognition of antibodies or major histocompatability corn (MHC) Class II and was perceived as 'non-MHC restrict

© 1996 w. B. Saunders Compan 2

534 Placenta (1996), Vol. 17

Table 1. Terms currently related to NK cell reCeptors and activation molecules

Species Term Multiple genes Dominant signal Reference

Human CD16 No Stimulation Anegon et al. (1988) p58 Yes *Inhibition Moretta et al. (1993) NKBI Yes *inhibition Litwin et al. (1994) NKG2 Yes *Inhibition Houchins et al. (1991) CD69 No Activation Schnittger et al. (1993) CD94 (Kp43) No Ambivalent Perez-Villar et al. (1995) NKR-P1A No *Inhibition Lanier, Chang and Phillips, (1994) NKAT Yes *Inhibition Colonna et al. (1995) NKLA/Vl No *Inhibition Kornbluth, Kozlowski and Portis, (1995) p40 No *Inhibition Poggi et al. (I995)

NKR-PI Yes Activation Yokoyama et al. (1993) Ly-49 Yes InhibitiOn Smith, Karllaofer and Yokoyama, (1994)

Rodent

Structures possessing C-type lectin domains are shown in italics, others belong to the immunoglobulin superfamily. *Killer cell inhibitory receptor (KIR); a term applied to groups of receptors associated with inhibition of lyric activity on NK cells: It appears that signals are required to stimulate KIR suggesting that lysis is the default status for NK cells. Interestingly the murine C-lectin families are located on chromosome 6 while an NK complex is located on human chromosome 12 (Lanier, 1995).

Although it has been demonstrated that N K cells lyse target cells that lack MHC Class I (reviewed Lanier and Phillips, 1992; Moretta et al., 1992; Yokoyama, 1993; Gumperz and Parham, 1995) reports over the last few years indicate that NK cell killing can have a Class I component. In rodents, two families of receptors responsible for NK cell activation have been identified which resemble calcium-dependent (C-type) lectins (re~iewed Gumperz and Parham, 1995). Receptors involved in directing N K mediated lysis include NKR-P1 in the rat and NKI.1 in the mouse and the ligands for these receptors have been shown to be multiple-cell surface oligo- saccharides (Bezouska et al., 1994). In the mouse the family of receptors LY-49 inhibits NK killing by recognition of I~LI-IC Class I (Yokoyama, 1993). Interestingly the human NK receptors which have been identified, represent a family of immunoglobulin superfamily genes but they do not appear to undergo any form of somatic rearrangement (Colonna and Samaridis, 1995). It is of great interest that rodents and humans express such different types of N K receptors. This could be viewed as convergent evolution, but is unlikely and it is more probable that the human lectin-like and mouse immunoglobulin-like receptors await discovery (reviewed Gumperz and Parham, 1995).

The definition of N K target recognition structures is currently an area of vigorous investigation. There is a consen- sus that N K cells do not recognize a single target but rather multiple target molecules that are independently recognized by the effector cell. The density of the multiple recognition structures may play a major role in determining whether a signal for activation or inhibition of lysis is sent to the effecter cell. The identification of these receptors on uNK will be an important step forward, and promises to demonstrate a further tier of cellular regulation with significant implications for the maternal-fetal interface. The definition of the multiple human NK cell receptor molecules has been highly dependent upon

the availability of long-term cloned human N K cell lines, none of which are uNK cells, and there are no widely recognized murine NK cell lines from any tissue. Because u N K cell cloning is required to identify these receptors, it is likely to be especially difficult in the mouse. However, a start has already been made with the identification of NK receptors on uNK cells; L G L - 1 and NKI.1 in the mouse (Parr et al., 1991; Croy and Kiso, 1993), NKR-P1 in the rat (Head et al., 1994) and CD94 in the human (Loke and King, 1995). Table 1 outlines some of the receptor families found on human and rodent N K cells.

LIFE H ISTORY OF R O D E N T uNK CELLS

The migratxon of uNK cells from bone marrow to the uterus is likely to be regulated by surface antigens expressed by immature precursor cells possibly stimulated by oestrogen, however this has not been elucidated and studies are needed to examine development and the trafficking Of uNK cells. The lineage from which these cells are derived has been clarified by cell-surface phenotyping. Consistent with their lymphocytic origin~ murine uNK cells express CD45 (Redline and Lu, 1989; Parr et al., 1990), Thy-1 (Bernard et al., I978;, Mukhtar, Stewart and Croy, 1989) and asialo-GM1 (Mukhtar, Stewart and Croy, 1989; Parr et al., 1990) and they have no expression of the B-cell marker IgM nor surface expression of the T-cell markers CD4 or CD8.

In virgin animals, immature uNK cells are distributed throughout the uterus (Kiso et al., 1992) and react with the N K marker LGL-1 (Parr et al., 1990, 1991). However, in pregnant animals the distribution and phenotype of these cells appear to change; initially the cells become localized at the sites of implantation and disappear from other parts of the decidualizing uterus. As gestation proceeds, proliferation of

Whitelaw and Croy: Granulated Lymphocytes of Pregnancy

the uNK cells is only observed in their preferred location, a developing area known as the metrial gland. Concurrently, the cells differentiate, increase dramatically in size (as much.as 50 ~tm) and gain a distinctive granular appearance due to the synthesis of numerous lytic protein granules in the cytoplasm which are detected histologically by periodic acid schiff (PAS) reagent. [Figure 1 (A) and (B)]. It has been known for sometime that the appearance of uNK cells is hormone dependent (reviewed Peel, 1989), however whether this was a direct effect on the cells or through an intermediary was not known. Therefore, the expression of the oestrogen receptor (ER) and progesterone receptor (PR) was determined with reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry (Van den Heuval et al., 1996) and in situ hybridization [Figure I(C)-(F)]. In situ hybridization was carried out as described previously (Whitelaw et al., 1995a) however, using digoxigenin probes (Boehringer Mannheim) (Whitelaw et al., 1995b). With immunohistochemistry ER was more strongly expressed (>90 per cent of cells expressing) than PR (63 per cent). PR expression while low early in gestation, appeared to increase during pregnancy. The expression of these transcriptional regulators opens new areas of investigation which will be important in understanding the biology of uNK cells and may distinguish the uNK subset as distinct from NK in lymphoid tissues.

The ability to lyse NK target cells is relatively high in murine decidual cell suspensions early in gestation (day 6.5) but lytic activity has decreased in similar preparations from day I0 or 12 of pregnancy (Gambel et al., 1985). The placenta and metrial gland have fully formed by day 10, and as gestation progresses, the uNK cells become larger, more granular and often (>10 per cent) binucleate (Peel, 1989; Delgado et al., 1996). Studies to examine DNA fragmentation in these cells in ussue sections indicate that from at least day 12 onwards many are dying. Interestingly, in a binucleate cell it was not uncom- mon for one nucleus to be undergoing DNA fragmentation while the other appeared to have intact DNA (Delgado et al., 1996). By parturition at day 19 the majority of uNK cells had disappeared from the metrial triangle area or had aligned along the basal aspect of the placenta and were shed from the uterus with the placenta. It has been proposed that the composition and increased granularity of the mature uNK accompanied by cell death may be part of a mechanism to facilitate separation of the placenta from the uterine wall at parturition (Larkin, 1972; Straatsburg and Gossrau, 1993).

The classification of all uNK cells includes in part, evaluation of the ability to lyse traditional NK target cells in vitro. As uNK cells differentiate they appear histologically to resemble highly activated cells (Parr et al., 1990) and it has been thought for many years that uNK cells in the decidua are lyric cells present to actively limit the invasion of trophoblast (Mukhtar, Stewart and Croy, 1989; Parr et al., 1990, 1991). However, there is little evidence for this in vivo (Stewart, 1991, 1994). Even in vitro freshly isolated uNK are refractory to the lysis of traditional N K target cells (YAC-1) or trophoblast (Gambel et al., 1985; Drake and Head, 1989; Linnemeyer

535

and Pollack, 1991[). Indeed it is only after incubation with IL-2, when uNK cells are transformed into LAK cells that they become more efficient at lysis (Croy et al., 1991). However, some laboratories have found the requirement for IL-2 in vitro necessary for uNK migration and short-term viability (Linnemeyer and Pollack, 1991) while other laboratories con- sider IL-2 unable to support or prolong the growth of uNK in vitro (Croy et al., 1991). The confusion regarding tl~e necessity for IL-2 in culture is most probably an indication that the mouse culture system has not been successfully optimised rather than a situation that would be found in vivo (Croy and Kiso, 1993). This will be discussed later.

LGL IN THE H U M A N UTERUS

Leucocytes in the pregnant human uterus have also been extensively studied. In decidual cell suspensions NK cells are the predominant decidual leucocyte (70 per cent) in the first trimester of pregnancy (Ferry et al., 1990; King et al., 1991) with macrophages (20 per cent) and T cells (10 per cent) accounting for the remainder. There are virtually no B cells. Very little is known about uNK cell and distribution during the second trimester because of the paucity of normal placental tissue but by term the uNK cells have largely disappeared (Bulmer and Sunderland, 1984).

In 'the pregnant human, LGL are distributed throughout the decidua and do not form a distinct area such as the metrial gland. LGL are also present in the uterus of non-pregnant women where they proliferate and are greatest in number during the secretory phase of the menstrual cycle, whereas T cells and macrophages remain constant (King et al., 1989; Starkey, Clover and Rees, 1991). If pregnancy does not occur the cells become pyknotic and die. However, pregnancy sustains the continued existence of human LGLs, thus demonstrating the progesterone dependence of these cells (Starkey, Clover and Rees, 1991; King and Loke, 1991).

As in the rodent, human decidual LGLs have been idendfied as a subset of N K cells because of their cell- surface markers which are CD56 bright+, C D 1 6 - , CD2 ÷, C D 3 - (Saito et al., 1993; King et al., 1993). This subset of NK cells is present in a very small proportion (> 1 per cent) of peripheral blood where the majority of N K cells are CD16 +, CD56 aim÷ and lack cytoplasmic granules (Lanier et al., 1986). King and her colleagues (1991) have made a very detailed study of decidual C D 3 - LGL using flow cytometry and immunohistochemistry. They concluded that uterine LGL form a distinct subset o f N K cells. Mincheva-Nilsson and colleagues (1994) examined decidual lymphocytes with immuno-electron microscopy and classified ~he leucocytes in terms of the 78 TCR and CD56 reactivity. They found that approximately 50 per cent of TCR75 + Were also CD56 + thus questioning the assertion that decidual LGL are exclusively NK cells. They also determined that in addition to having the morphology of activated cells, the LGL expressed the activated T-cell marker CD45RO (Mincheva-Nilsson et al.,

536 Placenta (1996), Vol. 17

. [~ ' . •

Figure 1. (A) Micrograph showing cross section of day 14 routine implantation site. mt: inertial triangle, d: decidua, p: placenta, e: embryo. Bar=500gm. (B) Micrograph showing typical uNK cells from routine inertial triangle. Characteristic PAS ÷ granules are highlighted with arrowheads. Bar=25gm. (C) and (D) Micrographs showing the expression of sense (12) and antisense (D) oestrogen receptor mRNA detected by in situ hybridization within the metrial triangle area. Bar=50gm. (E) and (F) Micrographs demonstrating the expression of sense (E) and antisense (F) progesterone receptor mRNA detected by in situ hybridization within the metrial triangle area. Day 16 of gestation. Bar=50gm.

1994). However, this study is disputed (Loke and King, 1995) and it is generally accepted that the predominant leucocytes in the human uterus are N K derived.

The cytolytic capacity of human uNK cells has been characterized both in the presence and absence of IL-2 (King et al., 1989, King and Loke, 1990). Freshly isolated human decidual uNK cells were unable to kill first trimester human

trophoblast, despite being able to lyse the human NK-sensitive cell line K562. However, incubation of uNK cells with IL-2 proved to be mitogenic and promoted their differentiation into LAK cells that were then able to kill trophoblast model JEG choriocarcinoma cells and, after an extended period of incubation (4-6 days), normal trophoblast (King and Loke, 1990). King and Loke (1990) point out that the preference for killing


malignant cells rather than trophoblast after IL-2 activation may indicate an in vivo role for uNK in the containment of exceptionally invasive or malignant trophoblast.

UTERINE NK CELL R E S P O N S I V E N E S S TO IL-2

The IL-2 receptor consists of three chains ct, ~ and y, each with different affinities for, and responses to, the cytokine. The high-affinity receptor found on activated T cells consists of all three subunits. When activated it induces the expression of immediate early genes involved in proliferation such as c-myc and c-fos (Naume et al., 1993). The ~t chain of the IL-2R has a molecular weight (MW) of 55kDa (also referred to as p55 or CD25) and binds IL-2 with low affinity and little biological response alone. The [3 chain has a MW of 75kDa and intermediate affinity for IL-2 (reviewed Smith, 1987) while the 64kDa y chain has 70 per cent homology between mice and humans and has been demonstrated to be important for cell signalling (Nakamura, 1994). NK cells in PBL express IL-2R[37 and can be stimulated by high levels of IL-2. Activated NK cells express all three chains. Interestingly, the IL-2R7 chain is common to several cytokine receptors; IL-4, IL-7, and IL-15, an NK cell growth factor (Giri et al., 1994).

Clearly uNK cells from both rodents and humans are responsive to IL-2 in vitro, however it is important to assess the levels of both cytokine and receptor present in the decidua. Starkey (1991) and King et al. (1992) used immunohistology and flow cytometry to examine first trimester human decidua and found expression of the p75 [3 chain on CD56 bright+ cells but no expression of the p55 ct chain. Incubation with IL-2 in vitro did not induce expression of p55 but did stimulate proliferation and down regulated the p75 IL-2R[3. A monoclonal antibody to the IL-2R[3 blocked IL-2 induced proliferation indicating that the activation of decidual NK cells occurs through the IL-2R[3 alone (King et al., 1992). Van den Heuval et al. (1996), employing PCR and immunohistochemistry techniques found that at day 8 of gestation all three IL-2 R chains were detectable on mouse uNK cells, but by day 12 only the y chain was expressed and by day 16 this had also disappeared. These results support the observations (Gambel et al., 1985) that murine uNK cells have greater capacity to lyse NK target cells early in gestation but have loss of viability later in gestation. It also adds credence to the hypothesis that uNK cells are important in early gestation for limiting trophoblast invasion because it is only at this time that the uNK are capable of responding to IL-2.

M E C H A N I S M S OF I M M U N E S U P P R E S S I O N IN THE P R E G N A N T UTERUS

Despite all the evidence that demonstrates uNK as lytic cells in vitro there is virtually no evidence of trophoblast destruction in vivo (Stewart, 1994). Several mechanisms are probably present in the uterus to protect against the activation of uNK

537

cells. The CD 16 antigen also known as the FcvRIII is present on peripheral NK ceils and is known to be important for theu- activation and for antibody-dependent cell cyotoxicity (ADCC). Human uNK cells are CD16 - , arid therefore, would be unable to perform ADCC. However, incubation with IL-2 does increase the number of CD16 + cells in decidual LGL (King et a1.,1989).

Studies using cells isolated from both rodent (Ci'oy et al., 1988; Scodras et al., 1990; J. Head, personal communication) and human (Lala, Kennedy and Parhar, 1988, Parhar, Yagel and Lala, 1989) decidual tissues have shown that decidual cells were able to prevent uNK cells cultured in IL-2 from becoming activated LAK cells capable of killing trophoblast. The addition of the cyclo-oxygenase inhibitor indomethacin restored LAK activity indicating that prostaglandins, a major metabolite of pregnancy, were responsible for the suppression (Parhar, Yagel and Lala, 1989; J. Head, personal communication). Parhar, Yagel and Lala (1989) showed that prostaglandin E 2 (PGE2) down regulated IL-2R expression. Linnemeyer and Pollack (1993) investigated the effect ofPGE 2 on murine spleen LAK cells and demonstrated a conversion to a large granular phenotype resembling uNK cells via a cAMP- dependent mechanism. These spleen derived cells also gained expression of the uNK cell specific antigen 4H12 (Linnemeyer and Hamilton, 1990) and had lower lytic activity compared with controls.

TGF-[3 is known to be an immunosuppressive factor (Rook et al., 1986; Graham et al., 1992; Cheng et al., 1993) and has been found in granulated cells in the pregnant uterus of human and rodent. Indeed Northern analysis of decidual and placental tissue has shown the presence of two TGF-[32 mRNA bands which was confirmed by immunohistochemistry demonstrating expression of TGF-[32 between day 8.5 and 12.5 in murine decidua (Lea et al., 1992). The TGF-[3 producing cells, are however, small and relatively infrequent lymphocytes and there is no data linking them to uNK cells.

The cytokines IL-4 and IL-10 have also been implicated in the down regulation of IL-2 induced activity (Starkey, 1991) and have been demonstrated in murine decidua (Wegmann et al., 1993). However, there is contradictory evidence that IL-4 may activate NK cells (Tunru, Suzuki and Kobayashi, 1993). Perhaps IL .-4- triggers the differentiation of uNK pro- genitors into their mature phenotype without the acquisition of cytolytic activity characteristic of LAK cells.

In addition to the suppression of putative IL-2 activation, it is apparent that uNK cells in both rodents and humans are considerably less cytolyw;ic than NK cells from peripheral blood lymphocytes (PBL). The identification of adhesion molecules on the surface of cells is useful in predicting cytolytic activity. The adhesion molecule CDllb also known as Mac-1 and found on NK cells and granulocytes has been extensively studied in human PBL and decidua (Sluvkin et al., 1994). CDIIb is important in lymphocyte-target binding, and was found to be decreased in decidual cells when compared with PBL perhaps accounting in part for their decreased lytic activity toward trophoblast.

538

The discovery that human extravillous trophoblast expressed a novel, nonclassi.cal, non-polymorphic, MHC class I antigen, human lymphocyte antigen-G (HLA-G) (Ellis, Palmer and McMichael, 1990; Kovats et al., 1990) has added another clue to the puzzle of decidual-trophoblast interactions (reviewed Loke and King, 1991; Schmidt and Oral, 1993). An isoform (37kDa) of the HLA-G may be secreted and expression appears highest in the first trimester of pregnancy suggesting an involvement in early placental development. Interestingly this correlates with the presence of LGL in the decidua. King et al. (1989) reported that lysis of trophoblast by CD56 + did not occur because of lack of a target structure. With the identification of HLA-G, it is now thought that the presence of an MHC class I inhibitor molecule could prevent lysis of extravillous trophoblast. To address this idea, Chumbley et al. (1994) transfected either HLA-G or a classical Class I MHC molecule HLA-A2 into HLA-null cells. They found that the antigens were able to protect the target cells from lysis by NK cells from decidua or PBL although HLA-G did not confer as much protection as HLA-A2. The results however demonstrated that the nonclassical HLA-G correlated with NK resistance in the same way as the classical MHC molecule.

STUDIES ON I M M U N O D E F I C I E N T M U T A N T MICE

Previous work from this laboratory has used immunodeficient mutant mice to answer questions relating to reproductive immunology (reviewed Croy and Kiso 1993; Croy 1993). With mice it is possible to examine the biology of pregnancy in much more depth than is possible with humans. Despite having fewer cells, mice have an advantage over other animals in that they reproduce quickly and their genetics and immunology have been extensively mapped and manil~ulated. In addition mice and rats have a haemichorial placenta which, with the exception of most primates, is more similar to the human than are other experimental animals.

The scid/scid mouse lacks both B and T cells but appears to be fertile and reproduces normally thus indicating that neither B nor T cells are essential for successful pregnancy (Croy and Chapeau, 1990). It was expected that double mutant mice of the genotype scid/scid.bg/bg which have neither B, T nor lytically active NK cells might have problems in fertility. However, scid/scid.bg/bg mice bred successfully for seven generations. When implantation sites from these mice were examined they appeared to have morphologically normal metrial glands. The beige mutation results in a defect in the lytic ability of NK cells without a reduction in NK cell numbers. Therefore, it appears that cytolytic ability is not an important factor of uNK cells in viva (Croy, 1993). This has been more recently supported in the perforin knockout mouse (Kagi e't al., 1994). Thus, while the lytic capability of NK cells is not required for successful pregnancy perhaps other functions such as cytokine production and the secretion of extra-

Placenta (1996), Vol. 17

cellular matrix (ECM) proteins are more important (Croy et al., 1991; Braga and Gendler, 1993).

A recent development in our lab has been the arrival of the immunodeficient transgenic mouse line TgE26. This transgenic mouse has a profound block to T and NK cell development in fetal life due to the insertion of high copy numbers (>30 copies) of the human CD3~ gene into the genome (Wang et al., 1994). It is not clear how the transgene affects the development of T and NK cells because disrup- tion of the murine CD3E chain in another transgenic stock resulted in a block to T cell development alone (Renard et al., 1995). Reproductively, TgE 26 females seem to ovulate, mate and progress through early pregnancy normally with an average litter size of nine. However between 8 and 10 days gestation, there is an onset in reabsorption of conceptuses and by term about half of each litter has died (Guimond et al., 1996). Morphological examination of the implantation sites shows that the placenta is 50 per cent smaller and contains only 3 per cent of uNK cells when compared with controls.

It is too early to say for certain that the lack of uNK cells causes the dramatic effects on the placenta and on the success of pregnancy. However, this mouse offers the best model system to date to understand the functional aspects of the uNK-lineage because it represents the only successful protocol for in vivo depletion of NK cells from a pregnant uterus. Antibody-mediated depletion of this compartment has previously only been achieved in non-pregnant animals (Guimond et al., 1996). The changes in the placenta of TgE26 mice may be an indirect effect because changes are also seen in the decidua and uterine vasculature. Continued characterization of reproductive events in these transgenic mice and those with a reconstituted NK cell lineage are in progress.

CYTOKINE PRODUCTION BY uNK CELLS

Using RT-PCR Croy et al., (1991).demonstrated the expression of IL-1, CSF-1, and leukaemia inhibitory factor (LIF) in uNK cells collected from explant cultures of murine metrial glands. However, IFN-y, IL-2, IL-3, IL-4 IL-6, IL-7 and TNF-a were not detectable. The addition of IL-2 to the medium was found to stimulate the production of IFN-7. Because murine uNK cells are habitually cultured in medium containing IL-2, Croy and Kiso (1993) observed that the conditions under which murine uNK cells were isolated may affect gene expression, and are therefore, a poor indication of what happens in vivo. The situation is complicated by the terminal differentiation of the majority of the isolated uNK.

Human decidual lymphocytes isolated by flow cytometry on the basis of their cell-surface phenotype can be expanded for a short-term in cultures (Christmas et al., 1993). These cells were assessed by RT-PCR for the expression of a panel of cytokine mRNAs (Saito et al., 1993; Jokhi et al., 1994). Both


Table 2. Proposed functions of cytnkines produced by decidual lymphocytes

539

Cytokine Potential effects on pregnancy

CSF I

GM-CSF

IFN- y

LIF TGF-~

TNF-ct

Trophoblast differentiation and production of hCG (Croy et al., 1991; Saito et al., 19.93) DNA replication leading to terminally differentiated trophoblast giant cells (Drake and Head, 1994; Jokhi, King and Loke, 1994) Increases HLA-A on trophoblast; protects against lysis by LAK cells (King and Loke, 1993; Grabowska et al., 1990) Crucial for implantation in mice (Stewart et al., 1992) Cell differentiation, suppression of trophoblast invasion, immunosuppression of NK and T cells (Rook et al., 1986; Graham et al., 1992; Cheng et al., 1993) Cell differentiation, possibly limits trophoblast invasion (Hunt et al., 1989)

bright+ CD56 , CD16 + NK cells and CD3 + T cells were found to express mRNA for CSF-1, TNF-Gt, IFN-% TGF-13, and LIF. GM-CSF mRNA was only detected on the CD56brigh'+NK cells. IL-2 was detectable in a couple of samples only after 55 cycles of PCR. In contrast, PBL with a similar surface phenotype expressed only T N F - a and TGF-I3. An examination of the actions of these cytokines in vivo suggests that their expression may regulate the differentiation and invasion of trophoblast cells within the decidua (Jokhi et al., 1994). Cytokines produced by uNK cells which have been implicated in immunosuppression are detailed in Table 2.

INTERACTIONS WITH ECM

Murine uNK cells do not proliferate in culture and have reduced viability compared with in vivo. However, culture on ECM prolongs their lifespan beyond what would be expected in vivo (Croy and Kiso, 1993). Both trophoblast cells and the stromal cells of the decidua produce ECM in large amounts which is important in directing the behaviour and shaping the environment of cells within the fetomaternal unit (reviewed Loke and King, 1995). NK and uNK cells in rodents and humans have been shown to express receptors for ECM although the distribution and specificity of the receptors is altered according to the degree of differentiation or activation (Somersalo et al., 1992; Burrows, King and Loke, 1993; Kiso et al., 1994; Sluvkin et al., 1994). For example in mice at early stages (day 3-6) of gestation LGL-1 + cells expressed the integrin chains 131 and ctl, ct2, ct4, ct5 and ct6 which are binding receptors for collagen, fibronectin and laminin. However, during mid-pregnancy (day 8-15) the uNK cells only expressed 131, Gt4, and ct5 demonstrating that major changes had occurred between days 6-8 (K_iso et al., 1994). The remaining integrin proteins are involved in binding to fibronectin and are important in target cell recognition and regulating the migration and extravasation of NK cells. In mutant mice overproducing collagen, uNK cells do not colonize the mesometrial t'l'iangle efficiently, further support-

ing the importance of ECM in establishing the architecture of the maternal-fetal interface. (Kiso et al., 1992).

Human decidual CD56 bright+ cells sorted by flow cytometry, also strongly expressed the fibronectin receptors ct4~l, ct5131 and ct4137 (Burrows, King and Loke, 1993). They expressed the antigen HML-1 which is known to be important for the rnucosal localization of lymphocytes. However, it was found that the integrin ct4 subunit, HML-1, and C D l l b were decreased after IL-2 stimulation of uNK cells in culture while CD18 increased. CD18 and CDl l a are known to be correlated with increased cytolytic activity. (Burrows, King and Loke, 1993). The identifying antigen for human uterine uNK is CD56, and this has been shown to correspond to an embryonic form of the neural cell adhesion molecule (NCAM). The function of this cell-surface molecule on uNK is unknown.

ONTOGENY OF DECIDUAL NK CELLS AND SIMILARITIES WITH 78 T CELLS

With the exception of the ability to rearrange the T-cell receptor genes, T and NK cells have many similarities and it has been proposed that they are derived from a common progenitor (Lanier, Spits and Phillips, 1992). Thymocytes that are triple negative (TN: CD3 - , C D 4 - , CD8 - ) can give rise to both NK and T cells under the appropriate conditions, and activated adult NK cells contain germline transcripts of TCR[3 and 8 but translate CD3e in the cytoplasm. (reviewed Lanier, Spits and Phillips, 1992). There are two populations o fT cells, both associated with the CD3: the well characterized cq3 T cells and ?8 T cells arose first in phylogeny and are important in the surveillance of epithelial and mucosal surfaces (Janeway, Jones and Hayday, 1988). They are relatively non-polymorphic and appear to be largely specific for MHC class I (Bluestone and Matis, 1989). y8 T cells are capable of lysing NK sensitive targets and of recognizing tumour cells. NK and 78 T cells appear to be important remnants of primitive immune systems which together with the more sophisticated Qtl3 T and B cells give protection in an additive manner (Loke and King, 1995).

540

LG.L IN OTHER SPECIES--RUMINANTS

Placentation in ruminants (reviewed Wooding, 1992) is described as synepitheliochorial because there is fusion between the fetal trophectoderm and maternal epithelium. The areas 6f trophoblast invasion are known as placentomes, where the fetal cotyleden is enmeshed in a maternal crypt (caruncle). Placentomes vary in number from six in deer to 150 in the goat. Fetal trophectoderm ceils modify the maternal epithelim by fusing with maternal epithelial cells to form hybrid bi- nucleated cells (BNC). These are present from just before implantation and persist until parturition. The function of BNC is to form a syncytium and to deliver protein and steroid hormones such as placental lactogens.

The leucocyte distribution in PBL of ruminants is unusual in that up to 75 per cent of the cells are C D 4 - C D 8 - T I9 + T cells and a large population of these are 3'5 TCR +. 3'8 T cells predominantly patrol epithelial surfaces, and are thought to have evolved as an adaptation to the bacterial and protozoan load peculiar to ruminants (reviewed Hein and Mackay, 1991). In sheep, the uterine leucocyte population is restricted to the epithelial layer and falls into three subsets; a major (50 per cent) population of the phenotype CD8 +, C D 4 5 R - , y8 T C R - and two equal subsets of CD8 +, CD45R +, y8 T C R - and CD8 +, CD45R +, 3'8 TCR + (Meeusen et al., 1993; reviewed Hansen 1995). From mid- to late gestation in sheep CD45R + 3'8 TCR cells were found to disappear from the plaeentomes and to increase dramatically in the areas of uterine epithelium where no invasion occurs (Gogolin Ewens et al., 1989). These cells had virtually disappeared after parturition. In both the cycling and pregnant uterus 3'8 TCR + cells were found to contain large membrane bound granules (Lee, Gogolin Ewens and Brandon, 1988; Lee et al., 1992; Meecusen et al., 1993). The absence of these cells from the early part of pregnancy argues against these cells playing a role in the prevention of interplacentomal invasion of trophoblast but they may secrete cytokines to promote conceptus development in a manner somewhat analogous to human and murine uNK.

Electron microscope studies in deer (Lee et al., 1995) have identified increasing numbers of LGL throughout the placenta and especially in close proximity to trinucleate cells. Tri- nucleate cells are a transient hybrid between maternal and fetal cells,, do not form syncytia and, after releasing hormone granules, are reabsorbed by the trophectoderm. Lee et al. (1995) postulate that the LGL are necessary to prevent expansion of trinucleate cells into invasive syncytium. Classification of LGL in deer was not possible because there was no cross reactivity with available sheep or human antibodies (Lee, Wooding and Morgan, 1995). There are few other studies on the distribution and activity of ruminant uNK cells.

PIGS

The epitheliochorial class of placentation is the least invasive and exemplified in the pig. The uterine epithelium does not


form decidua and the chorioallantoic membrane attaches to the epithelium with no invasion of maternal tissue. There are no reports of uterine LGL in pigs but several studies have examined the distribution and lytic activity of N K cells in the pregnant uterus (Croy, Wood and .King, 1987; Croy et al., 1988; Yu et al., 1993; reviewed Croy, Yu and King, 1994; Engelhardt and King, 1995). During early stages of pregnancy there appear to be increasing numbers of NK recruited to the uterus as compared with cycling controls (Engelhardt and King, 1995). N K cell activity, determined by the ability to kill K562 targets, was not detected in suspensions of the uterine epithelium of cycling pigs but in uterine cells from pregnant pigs, NK activity was substan- tially elevated on days I0 and 20 of gestation. However, by day 30, the NK activity was almost undetectable (Croy, Wood and King, 1987; Croy et al., 1988; Yu et al., 1993). The NK activity in the pig appeared before attachment (day 12) but required the presence of a conceptus, because pseudopregnant pigs did not exhibit the same level of lyric NK activity. This is in contrast to the mouse where the uNK cells are dependent upon hormone stimulation alone and uNK cells are found in the deciduomata of pseudo- pregnancy (Peel, 1989). Pig uNK cells also killed trophoblast without prior IL-2 activation, and are therefore, more aggressive than u N K cells from human or mouse (Yu et al., 1993). Lytic activity may not have any biological significance in epitheliochorial placentation but the decline at day 30 of gestation correlates with the completion of placentation. It is conceivable that porcine NK cells are present in the uterus solely to destroy trophoblast should it become invasive and the lack of direct contact between maternal and fetal compartments may render NK cell suppression superfluous. However, this raises the question: are there NK-like cells present in the epithelium after day 30 which are no longer lytic and perhaps have a role in cytokine secretion?

CONCLUSIONS

Decidual LGL populations whether derived from NK or T lineages are more primitive than their PBL counterparts and appear to be arrested in a semi-activated state. The decidual milieu and the presence of non-polymorphic MHC Class I expression on trophoblast deliver multiple immunologically suppressive signals that prevent activation of the cytolytic machinery of the lymphocytes. It seems that placentation has evolved in synergy with the immune system with the primary function of u N K / L G L being to co-ordinate trophoblast invasion through ECM proteins and the production of cytokines. Cytolytic activity appears to be tightly regulated and important as a safety mechanism during placental development but as pregnancy progresses, the decrease in cytolytic activity, con- comitant death and disappearance of uNK suggests that there is less need for cytolytic cells after the terminal differentiation of trophoblast.

Whitelaw and Croy: Granulated Lymphocytes of Pregnancy 541

ACKNOWLEDGEMENTS

The progesterone and oestrogen probes were the gifts of Dr Ok Kyong Park-Sarge and Dr Malcolm Parker, respectively. Many thanks to Jeff Luross for his assistance with the figures. P.F.W. holds a Canada International Fellowship from the Natural Sciences and Engineering Research Council of Canada.

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