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Contributions from Self-Renewal and Trafficking to the Uterine NK Cell Population of Early Pregnancy¹

Sirirak Chantakru^2,*, Craig Miller, Lindsay E. Roach^*, William A. Kuziel, Nobuyo Maeda, Wan-Chao Wang^¶, Sharon S. Evans^¶ and B. Anne Croy^*

^* Departments of Biomedical Sciences and Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada; Institute of Cellular and Molecular Biology, University of Texas, Austin, TX 78712; Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599; and ^¶ Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263

	Abstract

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Uterine NK (uNK) cells are abundant in human and murine uteri during decidualization. It is unclear whether precursors of uNK (pre-uNK) cells self-renew or are recruited from other sites. To assess self-renewal of pre-uNK cells, uterine segments from NK cell-competent mice were grafted orthotopically into NK/uNK cell-deficient or wild-type mice. Only in wild-type recipients did decidualized grafts contain uNK cells, indicating that pre-uNK cells do not self-renew in uterus. To identify pre-uNK cell sources, thymus, bone marrow, lymph node, or spleen cells were grafted from virgin or pregnant NK cell-competent donors into mated NK/uNK cell-deficient recipients. Cells from secondary lymphoid tissues of pregnant donors gave high level uNK cell reconstitution, which was independent of chemokine receptors CCR2 or CCR5. Pregnancy-induced changes to lymphocyte-endothelial cell interactions were documented using adhesion of human lymphocytes to frozen mouse tissue sections under shear. A dynamic increase was observed in L-selectin- and ₄ integrin-dependent adhesion of CD56^bright NK cells to decidualizing uterus and in human PBL adhesion to lymph node endothelium. These data support a model that attributes the dramatic increases in human and murine uNK cells during decidualization to precursor cell recruitment.

	Introduction

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Natural killer cells are the transiently dominant lymphocyte population in decidualizing human and murine uteri (1, 2). Decidualization is a process that transforms uterine stromal cells into large, endocrinologically competent, decidual cells. In mice, primary decidualization is triggered by the implantation of hatched blastocysts, and is followed by secondary decidualization that envelops each invading conceptus. A decidualization response (deciduoma) can also be artificially induced in hormone-primed uterus by introduction of inert objects (such as thread) into the lumen (3). Activation and terminal differentiation of uterine NK (uNK)³ cells happen only in a deciduoma or on the mesometrial side of the naturally pregnant uterus, in which decidua basalis (DB) develops between the placenta and the myometrial wall. Differentiating uNK cells divide, enlarge dramatically (45 µm diameter), acquire cytoplasmic granules containing lytic proteins, and initiate IFN- synthesis. In mouse pregnancy, uNK cells are found in two specific microdomains, DB and mesometrial lymphoid aggregate of pregnancy (MLAp, also termed metrial gland), a zone found in the uterine wall at implantation sites from gestation day 8 (gd8) (1, 2). The MLAp and DB are traversed by branches of the uterine artery that will supply the placenta beginning at gd9–10. Data from pregnancies in NK/uNK cell-deficient mice (tg26 and RAG-2^-/-/common -chain (_c)^-/-) indicate that DB and its spiral arteries are major targets of uNK cell actions, mediated via release of IFN- (4, 5, 6, 7). An unresolved question is whether adult uterus self-renews uNK cells or recruits precursors of uNK cells (pre-uNK) from other tissues.

Generation and maintenance of specialized lymphocyte subsets occur in some adult, nonlymphoid abdominal tissues, but have not been studied in uterus (8, 9). To establish the site of self-renewal for intestinal intraepithelial T lymphocytes, intestinal segment grafting into T cell-deficient hosts was used (10). Availability of tg26 and RAG-2^-/-/_c^-/- mice which have phenotypically similar anomalous implantation sites suggests that a uterine segment transplant approach would be possible. However, poor outcomes have been reported following grafting of uterine and oviductal tissues in rats, guinea pigs, rabbits, and primates (11, 12, 13, 14). Inflammation, necrosis, and fibrosis limited the ability of these grafts to support implantation.

NK cell progenitors (pro-NK) occur in fetal liver (15, 16) and fetal/neonatal thymus (17). In adults, bone marrow (BM) is the major source of pro-NK cells (18). Transplantable pro-NK cells are also found in spleen (19). Progenitors of uNK cells (pro-uNK) are in BM as transgene-marked adult BM injected into fetuses generates uNK cells following birth, adulthood, and pregnancy in the recipients (20). The more differentiated pre-uNK cell also occurs in BM, as pseudopregnant, irradiated mice given male rat BM differentiated some uNK cells with rat morphology (21). However, the origins of pre-uNK cells have not been studied during normal pregnancies.

Lymphocyte mobilization is induced by pregnancy; primary lymphoid organs (BM and thymus) involute (22, 23, 24), while secondary lymphoid organs, such as spleen and some lymph node (LN), hypertrophy (25, 26, 27). Chemokine signaling directs cell movement toward tissues, while egress of circulating cells from vessels requires lymphocyte/endothelial cell interactions. Receptors for CC chemokines are found on NK cells (CCR2 for monocyte chemoattractant protein-1, CCR5 for macrophage-inflammatory protein-1 and RANTES) (28, 29), and these chemokines are up-regulated in pregnant uteri (30, 31). L-selectin and ₄ integrin are critical molecules involved in slowing circulating leukocytes and facilitating rolling, tethering, and docking for vessel egress in secondary lymphoid tissues and at extralymphoid sites of inflammation (32). In humans, functionally active L-selectin is expressed at extremely high levels by the numerically minor, circulating CD56^bright NK cell subset, compared with the more prevalent CD16⁺, CD56^dim subset (33). The ₄ integrin is expressed equivalently by CD56^bright and CD56^dim NK cells (33). Human uNK cells are CD56^bright (2), suggesting a potential for preferential recruitment of L-selectin^bright, ₄ integtrin⁺, CD56^bright cells into the uterus. Goals of this study were 1) to determine whether uterus self-renews uNK cells; 2) to examine lymphoid tissues as sources for pre-uNK cells; and 3) to explore underlying chemokine- and adhesion-related mechanisms that contribute to mobilization of cells into decidualizing uterus.

	Materials and Methods

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Animals

Immunocompetent mice (random bred CD1; Charles River Laboratories, St. Constant, Quebec, Canada), C57BL/6J (B6), and C57BL/6 x 129/J F₁ (F₁; The Jackson Laboratory, Bar Harbor, ME)) were housed under standard husbandry in the Central Animal Facility, University of Guelph. Immunodeficient mice (random bred ICR-scid/scid (SCID; NK⁺, T^-, B^-; Taconic Farms, Germantown, NY), tg26 (H-2^k/b; NK^-, T^-, B⁺), and RAG-2^-/-/_c^-/- (H-2^b; NK^-, T^-, B^-) were housed in the University of Guelph’s barrier-husbandry facility. Both tg26 and RAG-2^-/-/_c^-/- lack uNK cells and are referred to as NK/uNK cell deficient (5). Mice ablated for CCR2 (H-2^k/b) and CCR5 (H-2^k/b) (34, 35) were bred at University of Texas (Austin, TX) and shipped to Guelph. Virgin females were used at 8 wk of age, unless different ages are stated and, if bred, were mated to males of the same strain. gd0 was the morning of copulation plug detection. Euthanasia was by CO₂, followed by cervical dislocation.

Uterine segment transplantation

Uterine segments (10 or 5 mm), trimmed of mesentery and vessels, were grafted from virgin donors to virgin recipients in an orthotopic manner meant to preserve both cranial-caudal and mesometrial-antimesometrial orientations. For autotransplantation (n = 2), CD1 females were anesthetized (0.35 ml xylaxine (20 mg/ml) and ketamine (100 mg/ml)), and the donor horn was reanastomosed with simple interrupted 8-0 Vicryl (Polysorb, Norwalk, CT) sutures. For all other grafting, donors (CD1 or SCID) were euthanized, while recipients (tg26 or CD1) were anesthetized as above. Recipient horns were cut at their midpoint, and donor tissue was inserted and anastomosed. No vascular anastomosis was attempted. The abdomen was closed surgically. After 7 days, recipients were paired for breeding and euthanized at gd10.

Thymic engraftment

Thymuses were dissected from nonpregnant or pregnant (gd3 or 5) adult, or neonatal (48 h) B6 mice and grafted under the renal capsule of anesthetized gd0 RAG-2^-/-/_c^-/-.

Adoptive transfer of BM, LN, or splenocytes

BM and spleen cell (SC) donors were nonpregnant or pregnant SCID mice, while LN donors were B6 (gd3, 5, or 7). As pregnancy changes cellularity of these organs, one donor tissue equivalent dose was used for each recipient, pooling donors if several mated recipients were available on the same day. Uteri from all gd3 donors were flushed to confirm pregnancy by detection of preimplantation blastocysts. BM was flushed from femurs and tibias of each donor. Twelve s.c. LN (PLN), three iliosacral LN, and the mesenteric LN chain were harvested from each donor. The mesenteric and iliosacral LN were pooled (MLN). LN and spleens were dissociated mechanically. PBS (400 µl with/without cells) was infused via tail veins into gd0 tg26 or RAG-2^-/-/_c^-/- recipients, who were sacrificed on the recipients’ gd10.

Morphometric analyses

Abdominal contents were examined grossly, then uteri were dissected, fixed in Bouin’s solution, processed routinely for paraffin embedding, serially sectioned at 7 µm (transversely for normal uteri and longitudinally for surgically manipulated uteri), and stained with H&E for routine histopathology or periodic acid Schiff’s (PAS) for uNK cell enumeration. Eleven central tissue sections from each implantation site were scored as previously described (3). One square millimeter per section was analyzed in each mesometrial microdomain, DB, and MLAp. Circular smooth muscle was used as the boundary between these. When the MLAp was absent or rudimentary, mesometrial triangle (MT) is the term used to describe the scored region, rather than MLAp. For longitudinally sectioned uteri, those containing a conceptus were scored as above. Uteri containing a deciduoma were scored in two independent 1-mm² areas of 10 of the serial sections separated by 42 µm to avoid duplicate counting of uNK cells, which may reach 45 µm by gd10. Diameter and granularity were estimated from a total of 30 cells from 10 sections at 1000x. Means and SDs of uNK cells/mm (2) diameters, granularity, p values, and Student-Newman-Keul test for ANOVA were conducted using PC-SAS 6.12 for Windows (SAS Institute, Cary, NC).

Lymphocyte adhesion to frozen mouse tissues under shear

Human PBL were isolated from buffy coat leukocyte concentrates of anonymous donors (American Red Cross, Buffalo, NY) by Ficoll/Hypaque centrifugation and adherent cell depletion (36). Information regarding donor sex and hormonal status was not available; thus, equal numbers of nonpregnant female and male donors are probable. Uteri and PLN from nonpregnant and pregnant (gd3, 6, or 10) B6 mice were cryopreserved immediately upon collection. PBL were either unstained or stained with anti-CD56 PE mAb (1/100; Coulter Immunology, Hialeah, FL) and goat anti-mouse IgG-rhodamine isothiocyanate (RITC) Ab to permit analysis of NK cell subset adhesion to high endothelial venules (HEV) by fluorescence microscopy, as described previously (36). Before assay, PBL were incubated with or without blocking mAb (10 µg/ml) specific for L-selectin (DREG-56; American Type Culture Collection, Manassas, VA) or ₄ integrin (HP2/1; Coulter). Treated PBL (5 x 10⁶) were overlaid on 12-µm cryosections of mouse tissues and rotated at 112 rpm (Labline Instruments, Melrose Park, IL) for 30 min at 4°C. After removal of nonadherent cells, specimens were fixed vertically in 3% formaldehyde in PBS for 1 h. For unlabeled PBL, glutaraldehyde-fixed specimens were permeabilized in 70% ethanol and stained with 0.5% toluidine blue in absolute ethanol. Adhesion of PBL was scored in 300–500 HEV per specimen. All enumeration was done in triplicate. If CD56-prelabeled cells were used, specimens were washed in PBS and fluorescent cells were quantified in double-blind analysis of 10 high powered fields (equivalent to 5 mm²) at x200 magnification, while total cells were quantified in the same section by bright field using an Olympus BH2-RFL fluorescence microscope (Olympus Optical, Tokyo, Japan). As reported previously (33), the accuracy of using fluorescence microscopy to visually discriminate CD56^bright and CD56^dim cells in human PBL suspensions and adherent cell populations was verified by flow cytometry and confocal fluorescence microscopy, respectively, to quantify the fluorescence intensity of individual cells. Fluorescent-labeled cells were not detected in specimens labeled with isotype-matched negative control murine IgG1 Ab (Coulter).

	Results

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Assessment of uNK cells in uterine segment transplants

Feasibility of orthotopic uterine grafting was assessed in autologously grafted CD1 mice using grafts of 10 mm (n = 3) and 5 mm (n = 4). The longer grafts showed gross and histological full-length necrosis, while the shorter grafts were viable. All recipients of the 5-mm grafts were pregnant, with three grafts containing implantation sites and the fourth a deciduoma (Table I). Typical uNK cells (16–45 µm diameter with 9–25, PAS-reactive cytoplasmic granules/cell) were found in these decidualized grafts (Fig. 1, A and B). Thus, orthotopically grafted mouse uterus supports uNK cell differentiation. Next, 5-mm uterine segments were grafted from uNK cell-competent CD1 or SCID donors into NK/uNK cell-deficient tg26 recipients (n = 7). Two females mated, but were not pregnant at euthanasia, despite grossly and histologically normal grafts. Five recipients were pregnant, and each grafted segment contained a large deciduoma indicative of viable, hormonally responsive graft tissue. By serial section analysis, neither the deciduomata in donor tissues nor implantation sites in host tissues contained uNK cells (Fig. 1, C and D). Thus, the donor segments did not contain self-renewing pro/pre-uNK cells that could differentiate in situ or migrate to adjacent implantation sites.

View larger version (176K): [in this window] [in a new window]   FIGURE 1. Photomicrographs of grafted uterine segments at gd10. Boxed regions in A, C, and E are enlarged in B, D, and F. A and B, Lower and higher power images of a CD1 graft site in a CD1 recipient with a normally developed implantation site including the MLAp, DB, and placenta (pl). B, Shows numerous, mature uNK cells (arrowheads). C and D, Lower and higher power images of a SCID graft segment in a tg26 recipient revealing development of a deciduoma (DC). Image D is representative of serial sections of deciduoma that contained no uNK cells. E, uNK (arrowheads) cells established within the DB by SC from gd5 SCID donors were mature granulated cells. F, SC from nonpregnant SCID donors did not generate uNK cells during the 10-day assay protocol. The blood vessel (asterisk) is an unmodified decidual spiral artery. pl, Placenta. A–F, Stained with PAS; bars in A and C, 400 µm; bars in B, 40 µm; and bars in D–F, 25 µm.

At gd10 in normal and SCID mice, the range in uNK cell frequency is 27–53 cells/mm² in DB and 72–129 cells/mm² in MLAp (Table II). Uterine segment transplantation suggested that migration of pre-uNK cells accounts for filling of these microdomains. Lymphoid tissues were assessed for pre-uNK cells by grafting to mated, NK/uNK cell-deficient mice. Thymic engraftment generated limited numbers of uNK cells at gd10 (Table II). There were no statistical differences in reconstitution of DB or MT by thymuses of different ages or from different donor pregnancy states (p > 0.05). BM from nonpregnant donors or donors at three early times of pregnancy also gave low uNK cell reconstitution in all recipients (Table II). No significant differences were found in uNK cells/mm² in DB or MT between the BM donor groups (p > 0.05). MLN gave low uNK cell restoration, while implantation sites in recipients of PLN showed MLAp development. Both MLAp and DB of PLN-grafted mice contained mature uNK cells. Implantation sites in NK/uNK cell-deficient mice receiving SC from pregnant donors also showed development of MLAp and high levels of uNK cells in both MLAp and DB. However, if the SC donors were not pregnant, uNK cell reconstitution was much lower (p < 0.001) in both areas. As shown in Fig. 1, E and F, levels of engraftment resulting from inoculation of SC from pregnant donors were sufficient to modify the decidual spiral arteries. In sharp contrast, host arterial vasculopathy persisted in recipients of SC from nonpregnant donors. For all SC donors, uNK cells were present at higher frequencies in MLAp than in DB (p < 0.01), the typical gd10 pattern in normal mice. Morphological assessment of graft-derived uNK cells showed that uNK cells derived from thymus, BM, LN, and SC were similar in diameter (means ± SD of 14.5 ± 4, 15.6 ± 4.5, 18.7 ± 7.5, and 13.9 ± 4.7 µm, respectively) and in numbers of granules/cell (12.8 ± 5.8, 15.6 ± 5.5, 20.5 ± 11.9, and 17.8 ± 7.7 granules/cell, respectively). These properties are identical with uNK cells in unmanipulated B6 (14.3 ± 2.9 µm diameter and 18 ± 8.2 granules/cell) and SCID (15.9 ± 4 µm diameter, 19.4 ± 9 granules/cell) mice, implying equivalent maturity.

Human PBL adhesion under shear to murine tissue sections

Human anti-CD56 mAb-labeled cells were assessed for adhesion under shear to frozen uterine sections from nonpregnant or pregnant B6 mice (gd3, 6, 10). Consistent with previous reports (33, 47), the proportion of labeled cells in test PBL suspensions was 10–12% CD56⁺, with <1.5% being CD56^bright. Prelabeling with anti-CD56 (murine IgG1) did not affect the total number of cells bound to uterine tissues at any of the time points (data not shown). CD56⁺ cells adhered to nonpregnant endometrial stroma in a randomly dispersed, low frequency manner (Fig. 2). Numbers of CD56⁺ cells adhering to nondecidualized uterine stroma markedly increased in gd3 tissue, and the adhering cells remained randomly distributed. At gd6 and 10, cell adhesion was significantly elevated, compared with nonpregnant uterus (p < 0.001), and the cells were restricted to the mesometrial side of the uterus and localized exclusively to DB. The adhering CD56⁺ cells were predominantly small sized (6.8 ± 0.4 µm, measured from 12 adherent cells). A minor adhering population (1–10%) of larger, CD56⁺ cells (8.01–9.01 µm, measured from three adherent cells) was also detected (Fig. 2B). In comparison with the numbers of CD56^bright cells in the initial overlay cell suspension, adhesion to uterine tissue significantly enriched the CD56^bright cells (Fig. 2). Pregnancy-induced adhesion was dependent on both L-selectin and ₄ integrin homing receptors (Fig. 2) because it was inhibited by specific function-blocking Abs, i.e., DREG-56 and HP2/1, respectively, but not by an IgG1 isotype-matched control Ab. Pregnancy induced dramatic and unexpected gains in lymphocyte adhesion in HEV of PLN from gd6 in C56/BL6 mice (Fig. 2). As predicted from earlier reports, lymphocyte adhesion to LN HEV from nonpregnant and pregnant mice was L-selectin dependent and did not involve ₄ integrin (32). Only uniformly sized small lymphocytes bound to PLN HEV.

View larger version (53K): [in this window] [in a new window]   FIGURE 2. Human lymphocyte adhesion in uterine and peripheral LN tissues. A, Uterine or PLN tissues were isolated from nonpregnant (NP) and pregnant mice at various times of gestation, cryopreserved, and used in adhesion assays. Before assay, lymphocytes were incubated for 30 min with function-inhibiting mAb specific for L-selectin (DREG-56; ), 4 integrin (HP2/1; ), or an isotype-matched control Ab (mouse IgG1; ). To permit discrimination of NK cells bound to uterine tissues, PBL were initially stained with anti-CD56 mAb and goat anti-mouse IgG-RITC secondary Ab. The initial PBL sample was comprised of 11% CD56+ cells and <1.5% CD56bright cells (not shown). The relative proportion of CD56bright cells within the total lymphocyte adherent populations was determined by direct analysis of 10 high powered fields (5-mm2 area) under fluorescence and light microscopy. *, Denotes statistical differences calculated using an unpaired two-tailed Student’s t test for the increase in adhesion detected in response to pregnancy, p < 0.001. Data are the mean ± SD of triplicate specimens prepared using a single lymphocyte pool. Results are representative of three independent studies, each using different human donors and a different series of gestationally timed murine tissues. B, Photomicrographs showing adhesion of CD56bright cells to uterine tissues (left panels) or toluidine-stained PBL bound to PLN HEV (right panels). Fluorescently labeled lymphocytes were not detected in uterine tissues when PBL were stained with isotype-matched control Ab and goat anti-mouse IgG-RITC secondary Ab (not shown). Bar, 25 µm.

	Discussion

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To explain the dramatic rise in human uNK cells during decidualization, some authors suggest that uNK cells self-renew in the uterus (38), while others suggest direct trafficking from BM is the major supply mechanism (39). Our studies indicate that both ideas may be incorrect. In our adoptive transfers, BM made only a minor contribution to uNK cell numbers during early to mid gestation. Despite the known and observed pregnancy-induced involution of BM (Table II), there was no loss in BM-derived progenitors able to populate uterus with uNK cells, during the first trimester of pregnancy. Levels of uNK cell generation from thymus were also low and independent of donor age or pregnancy status. However, our results show for the first time that thymus retains its capacity for NK cell generation into adulthood.

LN was shown to be a source of pre-uNK cells, but not all LN had transplantable pre-uNK. Hypertrophy was anticipated in the iliosacral LN draining the uterus (26, 27), but was not measurable when these smaller nodes were pooled with the much larger mesenteric LN chain. This pool, which includes the LN draining the pelvic region, hind limb, and uterus, had limited pre-uNK cells. Transplantable pre-uNK cells were present in PLN. However, further study would be necessary to precisely define which LN in the PLN pool contained transplantable pre-uNK. Development of the LN in the PLN that were pooled is heterogeneously regulated with some nodes (i.e., cervical) differentiating analogously to MLN (40). These nodes would perhaps lack transplantable pre-uNK cells.

Spleen contained pre-uNK cells that were mobilized to the uterus by pregnancy. In comparison with nonpregnant donors, SC numbers doubled in pregnant donors, while numbers of uNK cell progeny increased 4–47 times (Table II). This suggests that numerical alterations are not the sole pregnancy-induced changes in SC accounting for uNK cell reconstitution. The developmental stages of hemopoietic cells that move into the uterus are not yet known. Because uNK cells differentiating from thymus, BM, LN, and SC are identical morphologically and morphometrically and match those in gd10 unmanipulated, normal mice, the cells that moved into the uterus from these tissues were probably at relatively similar stages of differentiation. Alternatively, uNK cells may differentiate rapidly, and cells at various pro/pre-uNK stages may have had sufficient time to complete differentiation under our experimental conditions. The heterogeneity in size of human lymphocytes adhering to murine uterus suggests that circulating cells at more than one stage of differentiation/activation may have uterine homing potential. Despite the fact that splenic NK cells express the genes for chemokine receptors CCR2 and CCR5 (Refs. 28, 29 ; W. A. Kuziel, unpublished data) and that pregnant mouse and human uteri express high levels of the ligands for these receptors (30, 31), we found that the targeted absence of either CCR2 or CCR5 on pre-uNK cells did not reduce homing from spleen to uterus. These observations raise the possibility that the CCR2 and CCR5 chemokine receptors are functionally redundant, or that pre-uNK cells use alternative receptors for their recruitment.

Functional assays of CD56⁺ lymphocyte (male or female) adhesion to frozen tissue sections under shear showed that all uteri (nonpregnant and gestational) bound labeled cells, including a rare population of larger cells. Adherent cell localization was dramatically altered by development of decidua, changing from random to mesometrial indistribution. At gd6, adhering cells were absent over fetal trophoblasts and maternal MT/MLAp regions and localized exclusively over DB. In gd10 mice, 10% of the large, mature uNK cell population is intravascular in DB, while intravascular uNK cells are rare in MLAp, a domain containing uNK cells with less mature morphology (41). Thus, adherence of CD56⁺ cells in DB could indicate a specialized endothelium participating in pre-uNK cell recruitment and/or serving as a target of uNK cell functions within implantation sites.

Selective recruitment of NK cells to uterus most likely involves dual control of adhesion molecules on both trafficking NK cells and target uterine vascular endothelial cells. CD56⁺ cell adhesion to uterine tissues was dependent on both L-selectin and ₄ integrins. Coordinate expression of these molecules has been previously documented on circulating CD56^bright cells, with L-selectin expressed at very high density (33). Both L-selectin and ₄ integrins (i.e., ₄₇ and ₄₁) function as gatekeepers, controlling lymphocyte extravasation through their ability to bind to vascular addressins under shear (32, 43). Thus, it is tempting to speculate that pregnancy induces differential display of vascular adhesion molecules on uterine microvessels. The almost complete blocking of CD56^bright cell adhesion by mAb to both L-selectin and ₄ integrin suggested that these adhesion molecules are used by the same CD56^bright subset. The data are consistent with the coutilization of a common endothelial ligand for both L-selectin and ₄ integrin, analogous to the situation in Peyer’s patches (PP) in which trafficking of naive lymphocytes is mediated by cooperative interactions of L-selectin and ₄₇ integrins with spatially distinct domains within the mucosal addressin cell adhesion molecule (MAdCAM-1) (44, 45). Blockade of L-selectin and ₄ integrins under the same conditions as used in the present study results in complete inhibition of human PBL binding to PP HEV (46). Alternatively, if independent ligands are involved in mediating CD56⁺ NK cell adhesion to uterine tissues, partial inhibition by L-selectin and ₄ integrin-blocking mAb might be expected in frozen-section adhesion assays, as reported for mesenteric LN that dually express peripheral LN addressins (PNAd; an L-selectin ligand) and MAdCAM-1 (a ligand for ₄ integrins and L-selectin) (44, 45).

The ligands for ₄ integrins and L-selectin in uterine stroma remain to be defined. Recent studies by Kruse et al. (42) showed VCAM-1 is restricted to endothelium of the central DB in gd9 mice. While this could account for the ₄ integrin-mediated binding observed in the present study, caution must be used in this interpretation because extracellular matrix is also exposed in the assayed tissue and ₄ integrins bind to fibronectin, a molecule prevalent in mesometrial decidua (47). High level MAdCAM-1 expression has also been reported on mesometrial endothelium in the vascular zone, lateral to the DB in gd9 mice (42). This counterreceptor for L-selectin and ₄₇ in PP (32) may have the same function in decidualized uterus. Message for glycosylation-dependent cell adhesion molecule-1, an L-selectin ligand (48), is up-regulated mesometrially between gd6 and 10 in B6 uterus, while P-selectin glycoprotein ligand-1, a P-selectin ligand (49) used by NK cells, is constitutively expressed at these times (S. Chantakru and B. A. Croy, unpublished data). Regardless of the mechanisms involved, exposure of indicator PBL to pregnant uterine tissue results in preferential adhesion and the enrichment of the CD56^bright NK cell subset. Further studies are required to determine whether pregnancy alters human lymphocyte function in the binding assay. This outcome is anticipated because pregnancy conferred increased potential for reconstitution of uNK cells in the splenocyte adoptive transfer experiments and from recent studies in our laboratories showing that pregnancy promotes mouse splenocyte adhesion to HEV in LN from virgin mice (S. Chantakru, W. C. Wang, B. A. Croy, and S. S. Evans, manuscript in preparation).

Pregnancy, unexpectedly, altered lymphocyte/endothelial interactions in nonuterine sites. Thus, elevated adhesion to LN HEV was observed by gd3, whereas maximal adhesion occurred by gd6 that was sustained throughout pregnancy (shown in this study, S. Chantakru, W. C. Wang, B. A. Croy, and S. S. Evans, manuscript in preparation). The physiological roles of these changes in endothelial cells in peripheral lymphoid tissues are undefined, but they may contribute to cell recruitment to the uterus or in immune protection by surveillance for fetal Ag-sensitized cells. This study has shown that pregnancy induces functional changes in lymphocytes in vivo and adhesion molecules on endothelial cells, and it provides novel transplantation and adhesion assays that will facilitate further studies on the mechanisms regulating pre-uNK cell recruitment to the uterus in both mice and women.

	Acknowledgments

 
We thank Drs. C. Terhorst and J. P. Di Santo for providing foundation breeding stocks of tg26 and RAG-2^-/-/_c^-/- and for discussions of unpublished transplantation protocols. We are indebted to the Ontario Ministry of Agriculture, Food, and Rural Affairs Isolation Unit staff for the outstanding level of husbandry provided to our immunodeficient mice.

	Footnotes

 
¹ These studies were supported by National Sciences and Engineering Research Council Ontario Ministry of Agriculture, Food, and Rural Affairs and Bull Travel Fellowship awards (to B.A.C.), a Thai Royal Government Scholarship (to S.C.), and National Institutes of Health Award CA79765 (to S.S.E.).

² Address correspondence and reprint requests to Dr. Sirirak Chantakru, Department of Biomedical Sciences, Building No. 40, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. E-mail address: schantak{at}uoguelph.ca

³ Abbreviations used in this paper: uNK, uterine NK; BM, bone marrow; DB, decidua basalis; gd, gestation day; HEV, high endothelial venule; LN, lymph node; MAdCAM-1, mucosal addressin cell adhesion molecule-1; MLAp, mesometrial lymphoid aggregate of pregnancy; MLN, pools of mesenteric and iliosacral LN; MT, mesometrial triangle; PAS, periodic acid Schiff’s; PLN, pools of s.c. LN; PP, Peyer’s patch; pre-NK, precursors of NK cell; pro-NK/uNK, progenitors of NK/uNK cell; RITC, rhodamine isothiocyanate; SC, spleen cell; _c, common -chain.

Received for publication July 9, 2001. Accepted for publication October 22, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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