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  • 1. Journal of Reproductive Immunology 59 (2003) 175 Á/191 Review Update on pathways regulating the activation of uterine Natural Killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus B. Anne Croy a,*, Souad Esadeg a, Sirirak Chantakru a,b, Marianne van den Heuvel a, Valdemar A. Paffaro, Jr a,c, Hong He a, Gordon P. Black a, Ali A. Ashkar d, Yasuo Kiso e, Jianhong Zhang a,f a Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ont., Canada N1G 2W1 b Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand c Department of Histology and Embryology, Institute for Biology, UNICAMP, Campinas, Sao Paulo 13083-970, Brazil d Department of Pathology and Molecular Medicine, McMaster University, 1200 Main St. W., Hamilton, Ont., Canada L8N 4J5 e Department of Veterinary Anatomy, Yamaguchi University, Yamaguchi City 753-8515, Japan f Laboratory Animal Center, Shanxi Medial University, Taiyuan, Shanxi 030001, China Received 25 October 2002; received in revised form 12 December 2002; accepted 12 December 2002 Abstract Virgin adult C57Bl/6J mouse uterus contains a population of small, non-granulated Natural Killer (NK) cells with balanced expression of NK cell activating and inhibiting LY49 receptors. Coincident with blastocyst implantation and decidualization, uterine (u)NK cells become activated. The surface glycoslyation of uNK changes, the cells proliferate and they induce production of interferon (IFN)g, perforin, serine esterases and other molecules, * Corresponding author. Tel.: '/1-519-824-4120x54915; fax: '/1-519-767-1450. E-mail address: (B.A. Croy). 0165-0378/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0165-0378(03)00046-9
  • 2. 176 B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 including angiogenic factors. Mouse strains genetically ablated in uNK cells fail to undergo modification of spiral artery segments that branch from the uterine artery and feed into the placenta and these mice do not sustain a robust decidualization response. IFN-g is thought, from bone marrow transplantation and therapeutic studies, to be the key uNK-cell derived mediator regulating gene expression in vascular and decidual tissues. Here, we review recent studies showing that IL-15 is the critical cytokine controlling uNK cell differentiation and that uNK cells are activated by either IL-12 or IL-18 and by other factors when both IL-12 and IL- 18 are genetically absent from implantation sites. We address possible roles of the IFN-g regulated gene a2-macroglobulin (a2-M) in regulation of the position of fetal trophoblast within the walls of the spiral arteries, and we discuss approaches that have been successful in evaluating mechanisms involved in homing of mouse uNK cell precursors to the uterus. These approaches maybe applicable to studies in women. Our studies show that complex immuno- physiological events contribute to spiral artery modification by mid-gestation in mice. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Interleukin 15; Interleukin 12; Interleukin 18; Alpha 2-macroglobulin gene family; Lymphocyte Á/endothelial cell interactions; Mouse pregnancy 1. Introduction of uNK cell life history in mouse Cells expressing the surface phenotype of NK cells are found in mouse uterus only postnatally, beginning from week 2 of infancy (Kiso et al., 1992). They remain as small, agranular lymphocytes until blastocyst implantation and uterine decidualization (gestation day (gd) 4; Sharma et al., 1986; Peel, 1989; Parr et al., 1991; Kiso et al., 1992). Differentiation of uNK cells is regulated indirectly by estrogen (E) and progesterone (P4); there is no requirement for conceptus-derived tissue or antigens to trigger terminal uNK cell differentiation (Sharma et al., 1986; Peel, 1989). uNK cells change rapidly between gd 5 and 7. This subset of lymphocytes becomes recognized by the lectin Dolichos biflorus agglutinin (DBA), indicating a gain in cell surface expression of N-acetylgalactosamine as a terminal sugar (Stewart and Webster, 1997; Paffaro et al., 2003a). The cells begin to proliferate rapidly (Peel, 1989), acquire cytoplasmic granules that contain perforin, serine proteases, phosphatases, mucin-1 and other mole- cules (Peel, 1989; Parr et al., 1991; Croy et al., 1997) and they produce IFN-g (Platt and Hunt, 1998; Ashkar and Croy, 1999). uNK cells are found only on the mesometrial side of implantation sites. This is the side where the broad ligament (mesometrium) attaches to the uterus and serves as a conduit for the uterine vasculature and innervation. uNK cells initially appear in the decidua basalis (DB), a region characterized by a unique vascular addressin involving only V-CAM (Kruse et al., 2002). By gd 8, uNK cells have also established a transient lymphocyte-rich thickening in the myometrial wall called the mesometrial lymphoid aggregate of pregnancy (MLAp). This region is
  • 3. B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 177 enriched in the more immature (i.e. smaller and less granulated) proliferating stages of uNK cells (Peel, 1989; Paffaro et al., 2003a). Gd 8 is also the first day evidence for senescence and cell death is found amongst uNK cells (Peel, 1989; Paffaro et al., 2003a), suggesting that the lifespan of terminally differentiated uNK cells may be between 3 and 4 days. This is somewhat longer than the 28 h half life estimated for peripheral NK cells found in lymphoid organs (Miller, 1982). From histological time course studies in pregnant mice having no uNK cells (Guimond et al., 1998; Greenwood et al., 2000), two key actions of uNK cells and their timing were defined. Although these actions appeared to compromise fetal survival in the first uNK cell stain available (tg  26), quantifiable fetal compromise was not found in the / subsequent NK-/uNK-stains that have been developed (Greenwood et al., 2000). The first action of uNK cells was a role in sustaining decidual integrity. By gd 6, decidua showed compromise (Greenwood et al., 2000). Second was the absence of spiral artery modification at gd 9Á 10 (Guimond et/ al., 1998). uNK cells reach peak numbers in normal mouse pregnancy about gd 10 and decline in number from gd 12 (Delgado et al., 1996; Kather et al., 2003). A few can be found at term but the postpartum uterus lacks the heavily granulated mature uNK cell form (Peel, 1989). The transient development of uNK cells in the mouse uterus is illustrated in Fig. 1. It is not yet fully known what mechanisms trigger terminal differentiation and activation of uNK cells. Indeed, it can be asked whether uNK cells are cells with inhibited or activated function. In general, NK cells in other tissues express a mixture of receptors (up to four per cell) that are either activation receptors or inhibitory receptors. In mice, only lectin-like receptors have been described (Anderson et al., 2001). In humans, lectin-like and Ig-like receptors are both known (Blery et al., 2000). Under normal homeostatic conditions, inhibitory receptors are functionally dominant over activation receptors (Ortaldo et al., 1999; Anderson et al., 2001; Campbell and Colonna, 2001; Colucci et al., 2002). In mice and humans, uNK cells have limited cytotoxic potential (consistent with inhibitory receptor engagement) but produce numerous cytokines (consistent with activation) (Gambel et al., 1985; Ashkar and Croy, 1999; Moffett-King, 2002). 2. Activation of uNK cells To address the question of mechanisms that might control differentiation and activation of mouse uNK cells, we have used approaches productive for the study of NK cells in other tissues. NK cell differentiation occurs in a stepwise manner and it has been found that IL-15 is needed for survival of
  • 4. 178 B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 Fig. 1. Low power photomicrographs of cross-sections of the C57Bl/6J mouse uterus using DBA lectin to localize uNK cells. The broad ligament is at the top of each image. (A) Illustrates the virgin uterus ()/50) which lacks DBA lectin'/ cells, although it contains small lymphocytes expressing LY49G2 by immunohistology (Kiso et al., 1992) and a partial balanced repertoire of LY49 activation and inhibitory receptors by RT-PCR (Paffaro et al., 2003b). (B) Shows the gd 3.5 uterus ( )/50) just prior to blastocyst implantation. uNK cells remain as small (DBA lectin(/) lymphocytes. Decidualization has not commenced although uterine enlargement is seen. (C) Illustrates gd 6.5 ()/50). Most of the image is decidua. DBA lectin'/ uNK cells are present and localized in the DB and myometrium (arrowheads). Insert shows DBA'/ small agranular uNK cell ()/400). (D) illustrates a mid-sagittal section of a DBA lectin stained implantation site at gd 10 ()/25). High numbers of uNK cells (dark stain) are present, in the DB and more densely within the MLAp. Inset shows a mature granulated DBA'/ uNK cell, typical of mid-pregnancy ()/400). M, mesometrial; AM, anti-mesometrial; L, uterine lumen; DB, decidua basalis; MLAp, the transient mesometrial lymphoid aggregate of pregnancy; PL, placenta; EC, embryonic crypt containing primitive streak stage embryo; F, fetus. immature uNK cells (Waldmann and Tagay, 1999; Rosmaraki et al., 2001; Briard et al., 2002). IL-15 is reported as a stromal cell product in both mouse and human uteri undergoing decidualization (Ye et al., 1996; Kitaya et al., 2000; Okada et al., 2000; Dunn et al., 2002). In IL-15 ablated mice, uNK cells
  • 5. B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 179 fail to develop, spiral arteries are not modified and decidua is hypocellular and edematous. This phenotype is identical to that seen in alymphoid, RAG- 2 null/common cytokine chain g (gc) null mice and in NK and T cell deficient tg  26 mice (Fig. 2; Guimond et al., 1998; Greenwood et al., 2000). / Transplantation of bone marrow from T and B cell deficient or normal B6 mice to either RAG-2 null/gc null or tg  26 establishes the uNK cell / precursor pool and normal numbers of uNK cells differentiate and localize properly when recipients are mated (Guimond et al., 1998; Ashkar et al., 2000). In contrast, transplantation of B6 marrow to IL-15 null females restores no uterine lymphocyte population (Croy et al., 2002a; Ashkar et al., manuscript in preparation), indicating that the deficit in the single stromal molecule IL-15 blocks terminal uNK cell differentiation. Fig. 2. The images illustrate the spiral arteries (long arrows) of uNK cell deficient (tg  /26) (A, C) and normal (B, D) mice at gd 12 by scanning electron microscopy of vascular casts (A and B) and by histopathology using Periodic Acid Schiff’s stain (C and D). uNK cells (arrowheads) are only present in the decidua of the normal mice (D). Wall diameter to lumen diameter measurements made on the histological sections indicated a failure of spiral artery modification (arrows). The smooth muscle coat of the spiral artery is retained in the uNK cell deficient mouse. By comparing the casts, deficits in spiral artery width, elongation and coiling are obvious. The footprints left by endothelial cells in each cast differ. The footprints in the uNK cell deficient animal were deeper, indicating a more swollen endothelium. This is confirmed in the histopathology where raised endothelium is seen projecting into the lumen of the tg  /26 artery (C).
  • 6. 180 B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 While IL-15 is essential to maintain the viability of uNK cells and their precursors, it is unlikely to be the molecule that activates them to produce IFN-g. In extrauterine sites, IL-12, a heterodimer of p35 and the p40 signaling chain, is the key cytokine regulating IFN-g production (Trinchieri, 1998; Walker et al., 1999). It is derived from macrophages and dendritic cells (Gerosa et al., 2002). IL-18 is also a major enhancer of IL-12-promoted IFN- g production (Trinchieri, 1998; Nakanishi et al., 2001). Implantation sites have been examined in mice ablated for IL-12p40 and/or IL-18 and their congenic controls (Takeda et al., 1998). Spiral artery modification was deficient in the absence of either or both cytokines with a 30Á 44% difference/ in Wall:Lumen ratio measurements compared with ratios in normal cogenic mice at the same day of pregnancy (Zhang et al., 2003). Some change in spiral artery Wall:Lumen ratios was found compared with gd matched alymphoid mice, suggesting additional factors are involved in inducing IFN-g in early pregnancy. Recently, murine and human IL-27 were reported (Pflanz et al., 2002). IL-27 is a heterodimer in which each chain is highly homologous to IL-12’s p35 and p40. The chains of IL-27 are known as p28 and EBI3, respectively. EBI3 has been localized as a virally-derived protein in human syncytiotrophoblast and extravillous trophoblast in all trimesters and it is strongly expressed in intramural trophoblast of the spiral arteries (Devergne et al., 2001). The IL-27 receptor is TCCR (Chen et al., 2000). IL-27 synergizes with IL-12 to drive IFN-g production in naive CD4' T cells. / Availability of these reagents should permit rapid future assessment of IL-27 production in the mesometrial decidua, of IL-27 receptor expression by uNK cells and of IL-27’s role in induction of IFN-g in the vicinity of the spiral arteries. 3. LY49 gene expression in virgin and pregnant uteri For many lymphohematopoietic cell lineages, changes in expression of surface receptors separate major steps in cell differentiation. For NK lineage cells in B6 bone marrow, precursors express CD122 (IL-2Rb) but are negative for NK1.1, DX5 and LY49 family members. Immature NK cells gain NK1.1 and at maturity the cells gain DX5, LY49 and lytic ability. The mature pattern observed in marrow is also seen on NK cells in spleen (Rosmaraki et al., 2001). The LY49 gene family is the major NK cell receptor family in mice and any one NK cell will express up to four different LY 49 genes. The LY49 genes have been well characterized in B6 and 129 strains and exhibit significant differences between the strains (Yokoyama and Seaman, 1993; Anderson et al., 2001). In B6 mice, 14 genes and five
  • 7. B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 181 pseudogenes are known. Only two of these, LY49D and LY49H are thought to be activation receptors, signaling through immunoreceptor tyrosine-based activation motif-containing adaptor proteins (ITAMs)s, (usually DAP-12 and DAP-10) and promoting cytolytic action by the NK cell. The remaining LY49 molecules are classified as killer inhibitory receptors (KIRs) and signal through immunoreceptor tyrosine-containing inhibitory motifs (ITIMs), blocking cytolytic function (Blery et al., 2000; Lopez-Botet et al., 2000; Colucci et al., 2002; Kabat et al., 2002). Most NK cells have balanced expression of activation and inhibitory receptors, and display dominance of the inhibitory signals. We have investigated the expression of LY49 genes in mesometrial tissue of virgin B6 mice and B6 females at gd 6 and 10 using RT-PCR. Parallel studies were also conducted in three strains of immune-deficient mice (Paffaro et al., 2003b). In all strains, part of the LY49 repertoire was expressed in virgin uterus. The activation receptors LY49D and LY49H were expressed in all strains and balanced by expression of two or more inhibitory receptors. At gd 6 in B6 and in T cell deficient B6-nu/nu mice, the entire repertoire was induced. The full repertoire was sustained in both strains at gd 10. Thus, the induction of LY49 receptor expression in uNK cells does not correlate to any morphological steps in terminal differentiation of the cells but to the initial activation of small agranular uNK cells. It is not clear that the gain in KIR expression occurs on NK cells within the uterus. It has not been excluded that recruitment of new cells to the uterus could account for the detected gain in inhibitory receptor expression. These data showing that pregnancy induces a gain in inhibitory receptors by uNK cells appear to contradict directly the morphological and biochem- ical evidence suggesting that uNK cells are activated cells. In other models studying uNK cell function in non-lymphoid tissue, it has been suggested that the LY49 activation receptors will override the signals from KIR when the cell is in a pro-inflammatory environment (Ortaldo et al., 2001). Time-course studies localizing IL-18 in the pregnant mouse uterus indicate that the entire decidua on the day of implantation (gd 4) is producing IL-18 (Chaouat et al., 2002; Zhang et al., 2003). Other studies (Peel, 1989; Paffaro et al., 2003a,b) indicate that the earliest DBA lectin-positive uNK cells appear in the uterus at gd 4Á 5 of normal pregnancy or following transplantation into alymphoid / mice (Chantakru, 1998). Thus, the IL-18, IL-12 (Chaouat et al., 2002) (and putatively IL-27)-rich environment would bias the NK cells towards dominance of their activation receptors and induction of cell division and synthesis of IFN-g, perforin and other mediators, which is observed. Interestingly, decidual stromal cells appear, by immunohistochemistry, to rapidly downregulate IL-18 production and, by gd 7Á 8, uNK cells are /
  • 8. 182 B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 recognized as the major producer cells (Chaouat et al., 2002; Zhang et al., 2003). As uNK cells do not reach peak IFN-g production until gd 10 (Ashkar and Croy, 1999), it appears that uNK cells are forced to use autocrine signaling to maintain their pro-inflammatory environment and sustain production of IFN-g. It is important to point out that all of the matings we studied were syngeneic. Thus, trophoblast would not express foreign MHC, a typical KIR ligand, although it is possible that conceptus-derived MHC could differ from maternal MHC by carbohydrate or lipid modifica- tions. Recent studies have shown that the developmental antigen RAE-1 complexes with the mouse NK cell activation receptor NKG2D (Li et al., 2002). The ligands for human NKG2D are MIC-A and MIC-B (Steinle et al., 2001). RAE-1 is induced by retinoic acid on embryonic stem cell and teratocarcinoma cell lines and its expression is reported on neural tube. There is no published information on expression of RAE-1 by trophoblast cells, the conceptus-derived population that interacts with uNK cells (Zou et al., 1996; Nomura et al., 1996). Possible engagement of a receptor on uNK cells by this and other developmental antigens merits investigation. 4. Members of the Alpha 2 macroglobulin gene family regulate spiral arteries and trophoblast positioning IFN-g is a regulatory cytokine that changes levels of transcription in targeted genes in many tissues. It has been estimated that transcription of 0.5Á 1% of the genes expressed in mice can be regulated via IFN-g (Boehm et / al., 1998). To identify some of the genes that might be IFN-g-regulated in mesometrial tissue of pregnant mice, a cDNA microarray was employed (Mouse GEM#, Incyte, St. Louis, MO). RNA was prepared from pregnant B6 mice using the mesometrial triangle at gd 6 and the MLAp at gd 10. Both dissections included uNK cells, some DB, some myometrial fibers and the major vessels crossing these regions. Among the genes with upregulated expression at gd 10 compared with gd 6 that might be influenced by IFN-g was mouse a2-M (MAM) and an EST called 777415, defined as ‘‘similar to human a2-M precursor’’. The receptor for MAM, called LRP, was moderately upregulated (Croy et al., 2002b). The differential expression of these three genes was confirmed using Northern analysis (Ashkar, 2000; and Esadeg et al., manuscript in preparation). To determine the biological significance of this information, implantation sites in mice genetically deficient in MAM were examined. The mice were also deleted for an a2-M gene family member MUG- 1 (Umans et al., 1999) but we subsequently established that MUG-1 was not expressed in normal implantation sites. Full
  • 9. B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 183 details of the abnormalities in the implantation sites in MAM null/MUG-1 null mice will be published elsewhere (Esadeg et al., in preparation). Briefly, however, trophoblast cells (cytokeratin') had invaded very deeply into the / walls of the spiral arteries by midgestation, a position they never achieve in normal mice and this was accompanied by spiral artery dilation (Esadeg et al., 2002). Ultrastructural analysis revealed that endothelial cells in the spiral arteries were damaged but endothelium remained overlying the mural trophoblast cells (Fig. 3). The labyrinth of the placenta was very small in the MAM null/MUG-1 null mice and the cells between the fetal and maternal capillaries were packed with unusual inclusion bodies, indicative of physiological stress. These data suggest that, in normal pregnancy, MAM must bind molecules which limit the depth of trophoblast invasion. MAM binds a large molecular array of proteases, hormones and cytokines (LaMarre et al., 1991). To establish which class of molecules might be best pursued as being key to unlocking the question of control of depth trophoblast invasion, human a2-M in its native or activated form was infused into pregnant RAG-2 null/gc null females. Both molecules were active in promoting spiral artery dilation at several dosages indicating that cytokines rather than proteases are the most probable regulators (Esadeg et al., manuscript in preparation). Fig. 3. Ultrastructural images from the spiral arteries of MAM/MUG females on gd 11. Endothelial cell (EC) desquamation is shown in (A) )/3000. The area of detachment is indicated by *; the vessel (BV) contains an erythrocyte (arrow). One of the endothelial cells (left) shows nuclear pyknosis. Trophoblast cells were always intramural never intraluminal, i.e. always covered by cytoplasmic processes from endothelial cells. In normal mice at gd 11, trophoblast cells are absent from the spiral arteries. In (B), a trophoblast cell (TC) with a large nucleus is shown in the wall of a spiral artery adjacent to an endothelial cell )/3000.
  • 10. 184 B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 5. Origins of mouse uNK cells It is well established in both rodents and women (Peel, 1989; King et al., 1996) that uNK cells proliferate within the uterus. Uterine segment transplantation studies in mice suggest that this follows mobilization and activation of precursors that are unable to self renew within the uterus (Chantakru et al., 2002). Subsequent studies, in which lymphoid tissues from virgin or pregnant donors were transplanted to mated alymphoid RAG-2 null/gc null females, showed that all tissues, except lymph nodes draining the pregnant uterus, contained progenitor cells that could differentiate into uNK cells (Fig. 4). Spleen cells from pregnant donors were identified as the richest source for transplantable pre-uNK cells. Peripheral lymph nodes were also efficient in reconstitution and this was independent of whether or not donors were pregnant. These data suggest that hormonal events, with or without chemokine signaling from the decidualizing uterus, mobilize some lymphoid cells in peripheral tissues to cross their organ endothelium, enter the blood stream, flow to the uterus, cross the uterine endothelium to exit the blood Fig. 4. Summary of lymphohematopoietic transplantation experiments. NK cell deficient mice (tg  /26 or RAG-2 null/gc null) were mated by syngeneic males and grafted the morning of copulation plug detection. Donors were usually randombred SCID females (T ( B () unless the organ being assessed was rudimentary, then C57Bl/6 donors were used. Donors who were virgin, gd 3, 5 and 7 were examined for all groups, except liver where gd 5 donors were not available. Also included were neonatal donors for thymus. All tissues contained progenitors differentiating into a uNK cell when implantation sites in the recipients were studied at recipient gd 10, except for the lymph node pool (MLN) draining the pregnant uterus. Absence of progenitors from that pool suggests that pregnancy retains uNK progenitor cells within the uterus. Secondary lymphoid tissue gave higher levels of reconstitution than primary lymphoid tissue. Donor pregnancy induced a dramatic increase in repopulation ability only for progenitors found in spleen. For further details, see Chantakru et al. (2002).
  • 11. B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 185 stream and localize within the DB and mesometrial triangle. A series of studies has been undertaken to explore mechanisms that may participate in recruitment of lymphoid cells to the pregnant uterus. 6. Interactions between human blood lymphocytes and mouse endothelium Established in vitro assays were used and modified to explore pregnancy effects on interactions between lymphocytes and endothelium in the uterus and in peripheral tissues (Chantakru et al., 2002, 2003). Test indicator cell suspensions were applied to thick cryostat sections of organs collected from virgin, time-mated or hormone- or vehicle-treated ovariectomized mice. The number of lymphocytes adhering per high endothelial venule or capillary was determined for lymphoid tissue or pancreatic tissue, respectively. Numbers of lymphocytes adhering per unit area of virgin endometrium or decidua were determined for uterus. Specificity of the adhesion was demonstrated using specific, function blocking monoclonal antibodies. PBL from random human donors, mouse splenocytes and mouse TK-1 lymphoma cells that are a4b7 integrinhigh, L-selectinlow, each detected pregnancy or steroid hormone (E2 and/or P4) induced gains of adhesive function in endothelium of lymphoid or uterine tissue. No gain of adhesive function was seen in pancreatic endothelium, indicating that endothelial cell alteration was organ specific rather than systemic. This assay approach permitted independent assessment of the effects of pregnancy and steroid hormones on the adhesiveness of lymphocytes themselves. When mouse splenocytes were used, pregnancy or hormone treatment of ovariectomized animals promoted adhesion to endothelium in organs from either virgin or pregnant mice, when compared with adhesion by splenocytes from virgin donors or placebo-treated ovariectomized donors. Thus, pregnancy promotes independent but coordi- nated increases in adhesive function of peripheral lymphocytes and of endothelium in lymphoid and uterine tissues that could be involved in mobilizing uNK progenitor cells to the mouse uterus. When normal human donors (non-pregnant and not practising hormone-based contraception) were serially bled over their menstrual cycles, a hormone-dependent gain in adhesion to gd 8 mouse uterus or peripheral lymph nodes was also observed. For these women, the gain in adhesion coincided with ovulation (van den Heuvel et al., manuscript submitted). The key molecules involved in the alterations to adhesion of both human and murine lymphocytes appear to be L-selectin, which binds to peripheral node addressin (PNAd) and a4 integrins which bind to mucosal addressin cell adhesion molecule (MadCAM). These data indicate that decidualized mouse uterine tissue sections maybe very
  • 12. 186 B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 useful surrogates for human uterine tissue in defining mechanisms potentially controlling lymphocyte movement to the human uterus and in exploring pathogenic alterations that could contribute to suboptimal human gesta- tional outcomes. 7. Model for uNK cell differentiation and function in normal mouse pregnancy Fig. 5 summarizes our current thinking, derived from studies in mice, regarding recruitment, activation and functions of the uNK cell lineage in normal pregnancy. Self-renewing progenitors and precursors of uNK cells are found predominantly in spleen as small, DBA lectin-negative, agranular lymphocytes. There is continual replacement of mature but nonactivated pre- uNK cells in the virgin adult uterus. With pregnancy, changes in uterine stroma associated with decidualization elevate IL-15, which promotes Fig. 5. Model for uNK cell differentiation, activation and function in the decidualizing uterus. Precursors of uNK cells move from spleen to the blood stream, then cross VCAM-1'/ endothelium into the vessels of the DB. Here, stromal/decidual cell produced IL-15 sustains viability of the precursors and triggers their terminal differentiation and gain of KIRs. Other decidual cell-derived factors, including IL-12, IL-18, and possibly IL-27, activate the uNK cells, overriding their KIR expression and inducing IFN-g production. To sustain IFN-g production, the uNK cells induce autocrine IL-18 production which supports their proinflammatory milieu. IFN-g targets different genes in the stromal, vascular smooth muscle (SM) and endothelial cells of the DB, including the spiral arteries and modifies levels of transcription. Among these genes are a2M-X, MAM and the MAM-receptor LRP. This family of molecules regulates bioavailability of cytokines, and perhaps chemokines, in implantation sites. The molecules bound via a2M-X and MAM include negative regulators of trophoblast invasion, pro-apoptoic molecules acting on uNK cells and factors regulating spiral artery modification.
  • 13. B.A. Croy et al. / Journal of Reproductive Immunology 59 (2003) 175 Á/191 187 survival of pre-NK cells present in and mobilized to the uterus. Decidualiza- tion also induces IL-18 and perhaps additional pro-inflammatory signals. This induces terminal differentiation of the lineage with full expression of KIR. The pro-inflammatory environment permits uNK cell activation and production/release of IFN-g. IFN-g induces gene transcriptional alterations in uterine stromal (decidual) cells, in vascular smooth muscle cells and in endothelial cells of the spiral arteries. One of the gene families altered by IFN-g and by at least one independent alternate pathway, is the a2-M gene family. By their ability to bind cytokines, a2-M gene family members regulate the functional bioavailability of these molecules. The key cytokines affected are those that negatively regulate the depth of trophoblast invasion. These or other a2M transported molecules alter uNK cell and decidual cell prolifera- tion and spiral artery modification. uNK cells have a life span of 3Á 4 days / and, from studies not discussed here (Ashkar and Croy, 1999), use IFN-g- induced pathways for autocrine regulation of their own senescence and death about midgestation (Delgado et al., 1996; Paffaro et al., 2003a). Many attributes of mouse uNK cells seem to be shared by human intravascular trophoblast cells. It is possible that both the mother and conceptus are endowed with mechanisms that will promote gestation time-specific dilation of the major vessels supplying the placenta, to ensure reproductive success. Acknowledgements We thank Barbara Mitchell, Tony Cenjiga and Dave Bridle of the OVC- OMAFRA isolation unit for their dedicated care of our immune deficient mouse colony and Dr C. Terhorst, Dr T. Mak, Dr J. Di Santo, J. Pleschon, Dr F. van Leuven, Dr S. Akira and Dr K. Takeda for providing breeding stocks of the various immune deficient mice used in our investigations. The input of Dr S. Evans, Dr P. Aplan and Dr A. Yamada to collaborative studies is gratefully acknowledged as is assistance from Kanwal Minhas in the ultrastructural studies and from Kim Best in manuscript preparation. This work was supported by the Natural Sciences and Engineering Council, Canada; Ontario Ministry of Agriculture, Food and Rural Affairs, the OVC Bull Travel Fellowship Program, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Shanxi Scholarship Council of China and a Royal Thai Government Scholarship.
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