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BOR - Papers in Press, published online ahead of print May 4, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.033951
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BIOLOGY OF REPRODUCTION 73, 510–518 (2005)
DOI: 10.1095/biolreprod.104.033951
© 2005 by the Society for the Study of Reproduction, Inc.

Pathways Participating in Activation of Mouse Uterine Natural Killer Cells During Pregnancy1

Xuemei Xie 3, 4, Hong He 4, Marco Colonna 5, Tsukasa Seya 6, Toshiyuki Takai 7, and B. Anne Croy 2, 4

Department of Biomedical Sciences,4 University of Guelph, Guelph, Ontario, Canada N1G 2W1 Department of Pathology and Immunology,5 Washington University School of Medicine, St. Louis, Missouri 63110 Department of Microbiology and Immunology,6 Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan Department of Experimental Immunology,7 Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activated natural killer (NK) cells proliferate in large numbers in murine mesometrial endometrium from Day 6 to Day 12 of gestation (term = 19 gestation days) to become the most abundant uterine lymphocytes. Early human decidua contains analogous CD56+/CD16– cells. Murine uterine (u)NK cells localize to decidua basalis and mesometrial lymphoid aggregate of pregnancy (MLAp). Decidua and MLAp are transient, pregnancy-associated tissues traversed by maternal arteries to the placentas. Uterine NK cells sensitize these arteries, facilitating their structural changes into high-volume conduits by Gestation Day 10 through release of interleukin (IL)-18, interferon (IFN)-{gamma}, vascular endothelial growth factor (VEGF), and other molecules. Little information exists concerning where, when, or how murine or human uNK cells become activated. In murine lymphoid tissue, three NK cell adaptor-mediated activation pathways are known: FcR{gamma}/CD3{zeta}, DNAX-activating protein (DAP) 10, and DAP12 (genes Fcgr3/Cd3z, Hcst, and Tyrobp, respectively). Expression of ligands for these receptors was demonstrated in implantation sites of normal C57BL/6J mice. Then, histological and morphometric analyses of implantation sites in mice with genetic inactivation of each pathway were undertaken. Implantation sites in DAP10–/– (Hcst deleted) mice appeared normal, spiral artery modification occurred, and concentrations of IFN-{gamma} in MLAp and decidua basalis were similar to those in time-matched C57BL/6J. Implantation sites of FcR{gamma}–/–/CD3{zeta}–/– (Fcgr3/Cd3z double knockout), DAP12 (Tyrobp)-loss-of-function-mutant, and FcR{gamma}–/–/DAP12–/– (Fcgr3/Tyrobp double knockout) mice differentiated abundant but functionally impaired uNK cells that could not modify spiral arteries. These data reveal key importance of FcR{gamma}–/–/CD3{zeta}–/– and thus maternal IgG during activation of mouse uNK cells and assign DAP12 but not DAP10 signaling contributions.

decidua, DAP10, DAP12, FcR{gamma}/CD3{zeta}, immunology, lymphocyte activation, mouse pregnancy, pregnancy, signal transduction, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mice, uterine natural killer (uNK) cells are the dominant lymphocytes in the mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) to midgestation [1, 2]. Uterine NK cells contribute to normal implantation site development by promoting structural changes to spiral arteries, mediated by release of interferon (IFN)-{gamma} [3]. Murine uNK cells are also the major midgestational uterine source of inducible nitric oxide synthase (NOS2), the enzyme releasing the potent vascular relaxant NO [4] and they produce vascular endothelial growth factor (VEGF) [5].

NK cells have important cytokine production and target cell lysis functions in innate and adaptive immune responses. NK cells are tightly regulated by opposing receptors that induce inhibition or activation of function. Murine Ly49 (formally Klra) and human killer immunoglobulin-like receptors (KIR) are large gene families for NK cell receptors that recognize major histocompatibility complex (MHC) class I antigens. Their inhibitory receptors use immunoreceptor tyrosine-based inhibitory motifs (ITIM) and are more abundant and functionally dominant than activation receptors [6]. NK cell activating receptors recognize minor histocompatibility genes, including products of retinoic acid early inducible-1 (Rae1) gene family members and the heat shock 60 (Hspd1) gene in mice or of MICA, -B and UL16 binding proteins (ULBP) 1, 2, and 3 (also known as RAET1 gene family members) in humans [7, 8]. Activating receptors associate noncovalently with membrane-bound signaling adaptors. The adaptors CD3{zeta}, FcR{gamma} (Fc{epsilon}RI-{gamma}) and DNAX-activating protein (DAP) 12 (also called KARAP, formally TYROBP) contain immunoreceptor tyrosine-based activation motifs (ITAM) while DAP10 (formally HCST) uses an YxxM motif [9]. Receptor signaling is regulated by adaptor associations.

In both human and murine NK cells, CD3{zeta} and FcR{gamma} are disulphide-bonded homodimers or heterodimers [10– 12] that associate with Fc{gamma}RIII (CD16 or FCGR3), the low-affinity Fc receptor for IgG. Fc{gamma}RIII mediates all antibody-dependent responses of NK cells, including cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC) [11]. Deletion of Fcgr3 results in loss of Fc{gamma}RIII surface expression and reduction of IFN-{gamma} production and of ADCC [13]. The role of CD3{zeta} in NK cell function is less clear because NK cell numbers and functions were thought to be normal in Cd3z-gene-deleted mice (CD3{zeta}–/–) [14, 15]. However, upon Fc{gamma}RIII-cross-linking of NK cells from CD3{zeta}–/– mice, IFN-{gamma} production and ADCC are upregulated, indicating a negative regulatory role for CD3{zeta} in Fc{gamma}RIII-mediated NK cell functions [16]. CD3{zeta} and FcR{gamma} also pair with NK cell-activating receptors other than CD16 in mice and humans [17–19]. Ligands for these additional receptors have not been identified but antibody cross-linking of these receptor complexes initiates cytokine production and cytotoxicity.

The activating receptor NKG2D (formally KLRK1) is constitutively expressed by all mouse and human NK cells. NKG2D provides activation through DAP12 and costimulation through DAP10 that results in cytokine production and cytotoxicity [17, 20, 21]. DAP12 is a type I transmembrane disulphide-linked homodimer with an ITAM in its cytoplasmic tail [22–24]. It associates with multiple additional NK cell-activating receptors, including LY49D (formally KLRA4) and H (formally KLRA8), CD94/NKG2C (formally KLRD1/KLRC2), and CD94/NKG2E (formally KLRD1/KLRC3) in mice and KIR2DS2, CD94/NKG2C, and NKp44 in humans [22–26]. Association of homodimeric DAP12 with these activating receptors is essential for phosphorylation of DAP12 following receptor triggering and leads to intracellular calcium mobilization, cell-mediated cytotoxicity, and cytokine production [26–29].

DAP10 has only 20% overall amino acid homology with DAP12 and is expressed as a disufide-bonded homodimer on NK cells [30]. Unlike DAP12, DAP10 does not contain an ITAM but has a consensus p85 PI3K-binding motif Tyr-Ile-Asn-Met (YINM) in its cytoplasmic tail. When this is phosphorylated, NK cell effector functions are promoted [30–32]. Because the same motif is found in CD28 and inducible costimulator (ICOS), engagement of NKG2D-DAP10 is thought to mediate costimulation rather than full NK cell activation [33, 34]. Ligation of NKG2D-DAP10 followed by DAP10 phosphorylation triggers NK cell cytotoxicity [32] and augments cytokine production initiated by signaling via DAP12-associated receptors [35].

Reproductively competent mice are available in which each of the documented NK cell activation signaling pathways has been blocked [13, 20, 23, 36]. To determine whether murine uNK cells use a predominant pathway during their activation, histological studies of implantation sites were undertaken in these strains to quantify uNK cell numbers within defined microdomains and to assess their functional ability to modify spiral arteries. Roles for FcR{gamma}/ CD3{zeta} and DAP12 signaling were revealed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

Breeding pairs of C57BL/6J (B6) background-related FcR{gamma}–/–/CD3{zeta}–/– (Fcgr3/Cd3z deleted) [14], DAP10–/– (Hcst deleted) [21], KARAP/DAP12 loss of function mutant (Tyrobp mutant) [23], and FcR{gamma}–/–/DAP12–/– (Fcgr3/Tyrobp deleted) [36] were shipped from their source colonies to barrier husbandry facilities at the University of Guelph. C57BL/6J (B6) mice, purchased from the Jackson Laboratory, Bar Harbor, ME, were used as controls and housed under conventional husbandry. Females were used between 7 and 12 wk of age. All procedures were conducted under Animal Utilization Protocols approved by the University of Guelph Animal Care Committee.

Estrous females were paired with genetically matched males and the day of copulation plug detection was called Day 0 of gestation. Mice were killed at specific gestation days, then dissected. Uteri were examined grossly for implantation site number and viability (based on size and color). For samples being dissected for RNA isolation or IFN-{gamma} quantification, killing was by CO2. For histological specimens, slow (15 min) whole-body perfusion of anesthetized (Avertin, 0.5 ml i.p. [37]) mice was undertaken using 30 ml freshly prepared 4% paraformaldehyde (PFA) in 0.1 M PBS with 0.1 M sucrose. Dissected tissue was then fixed in 4% PFA at 4°C (for 1 h if Days 6–9 of gestation or 6 h if >Day 10 of gestation) [38]. After fixation, the uteri were immersed in 0.1 M PBS for 15 min, then transferred into 70% ethanol (EtOH), and held at 4°C until subsequently processed. Only viable implantation sites were used for analyses.

Acquisition of Samples for Reverse Transcription-Polymerase Chain Reaction Analyses

Spleen, MLAp, decidua basalis, and placentas were dissected from B6 mice at Gestation Day 10 for RNA isolation. To obtain uNK cells for RNA isolation, a rapid, Dolichos biflorus agglutinin (DBA)-lectin-based magnetic bead separation was employed that enriches uNK cells to 98% [39]. RNA was isolated using the RNAeasy Mini kit (Qiagen, Mississauga, ON, Canada), followed by reverse transcription (First-strand cDNA Synthesis Kit; Amersham Pharmacia Biotechnology, Nutley, NJ) according the manufacturer's instructions. The following primers were used for reverse transcription-polymerase chain reaction (RT-PCR):

  1. Fcr{gamma} (formally Fcgr3), CCA GGA TGA TCT CAG CCG (forward) and ACA GTA GAG TAG GGT AAG (reverse);
  2. Nkg2d (formally Klrk1), GGC TTG CCA TTT TCA AAG AG (forward) and TGA GCC ATA GAC AGC ACA GG (reverse);
  3. Dap10 (formally Hcst), AGT CAG ACA TCG GCA GGT TC (forward) and GCC AGG CAT GTT GAT GTA GA (reverse);
  4. Dap12 (formally Tyrobp), CTG GTG TAC TGG CTG GGA TT (forward) and TGC CTC TGT GTG TTG AGG TC (reverse);
  5. Rae-1{alpha} (formally Raet1a), GGA CCC ACA GAC CAA ATG AC (forward) and CCC GTT GGT GTA TCC ATA GC (reverse);
  6. ß-actin (formally Actb), TTC TTT GCA GCT CCT TCG TT (forward) and CTG GGT CAT CTT TTC ACG GT (reverse).

Sizes for the correct products are 137 base pairs (bp) for Fcr{gamma} 374 bp for Nkg2d, 180 bp for Dap10, 208 bp for Dap12, 355 bp for Rae-1{alpha} and 411 bp for ß-actin. PCR conditions were a hot start at 94°C for 6 min, 35 cycles of 94°C for 45 sec, and 72°C for 45 sec, and then 72°C for 7 min before cooling to 4°C.

Histological Procedures

Fixed implantation sites were embedded in paraffin and blocks were serially sectioned transversely at 7 µm and mounted. For identification and enumeration of uNK cells, sections were stained with Periodic Acid– Schiff (PAS) reagent or DBA lectin [38]. Uterine NK cells in the central regions of MLAp and decidua basalis on 11 selected middle sections of each implantation site were enumerated using a 1 mm2 ocular grid at 400x magnification. Lumen areas of blood vessels were excluded. To avoid duplicate counting of individual uNK cells, every seventh section in sequence was scored. For general histological examination and morphometric analyses of tissues, sections were stained with hematoxylin-eosin (hematoxylin-eosin). Ratio measurements of external spiral artery vessel area to lumen area and surface area measurements of the MLAp, decidua basalis, and placentas were performed on the same 11 selected sections using Optimas image analysis software, version 6.2 (Optimas Corporation, Bothwell, MA). At least six viable implantation sites, collected from two or more females, were analyzed for each genotype at each gestational day (gd) studied.

For in situ hybridization, sections (7 µm) of 4% PFA-fixed implantation sites dissected from Day 6–10 pregnant B6 mice were prepared under RNase-free conditions. Hybridization was performed as described previously [40]. A PCR product (355 bp) from mouse Rae-1{alpha} (GenBank NM-009016) was subcloned into the Topo TA Cloning Kit (Invitrogen) as the probe, using T3 and T7 RNA polymerases to obtain sense RNA (negative control probe used in every assay) and antisense RNA (experimental probe).

For immunohistochemistry, sections (7 µm) of 4% PFA fixed implantation sites dissected from Day 5–10 pregnant B6 mice were immunostained using a rabbit anti-mouse-RAE1 polyclonal antibody [41] at a dilution of 1:1600 followed by incubation with 1:10 000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, Richmond, CA), as previously described [41]. Sections were counterstained with hematoxylin. Control sections without primary antibody showed no staining.

Tissue Acquisition for IFN-{gamma} Quantification by ELISA

To quantify IFN-{gamma}, the mesometrial triangle (MT) was dissected at Days 6 and 8 of gestation while mesometrial tissue at Days 10 and 12 of gestation was dissected into two components, decidua basalis and MLAp. Samples from a litter were pooled, minced in 100 µl of RPMI 1640 medium with 10% fetal calf serum and immediately homogenized using a Kontes micropestle (Fisher Scientific, Nepean, ON). Samples were centrifuged (800 x g, 4°C, 5 min) and supernatants were harvested and stored at –70°C until analysis. Supernatants were assayed for IFN-{gamma} by ELISA using paired capture and biotinylated detection antibodies (R4–6A2 rat anti-mouse IFN-{gamma} antibody; XMG1.2 rat anti-mouse IFN-{gamma} antibody; Pharmingen, Mississauga, ON, Canada) as previously described [3]. Concentrations of IFN-{gamma} were determined using standard curves obtained by serially diluting recombinant mouse IFN-{gamma} (1 mg/ml = 4 x 106 IU/ml; Sigma-Aldrich, Oakville, ON) over the range 4 ng/ml (16 IU/ml) to 0.0625 ng/ml (0.25 IU/ml). To calculate IU/implantation site, the units of IFN-{gamma} found in each sample were divided by the number of implantation sites pooled to produce the sample.

Statistical Analyses

Data are presented as means ± SD. Statistical analyses were done by ANOVA using SAS software (SAS 8.2; SAS Institute, Cary, NC). Significance was set at P < 0.05. Graphs were produced using excel (MS Office) software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of C57BL/6J Implantation Sites for Expression of Fcr{gamma} (Fcgr3), Dap10 (Hcst), Dap12 (Tyrobp), Nkg2d (Klrk1), and Rae-1{alpha} (Raet1a)

To evaluate whether the adaptors of interest (Fcr{gamma}, Dap10, and Dap12) were expressed in normal, B6 implantation sites, RT-PCR was employed to analyze mesometrial tissues and uNK cells collected at Day 10 of gestation. Spleen was used as the positive control. Gene expression was detected in all samples (Fig. 1A). RNA for Nkg2d was also detected at Day 10 (Fig. 1A) and Rae-1{alpha} from Days 6 to 10 of gestation (not shown). Cells transcribing and translating Rae-1{alpha} between Gestation Days 6–10 were identified using in situ hybridization and immunohistochemistry. Expression occurred in trophoblast, vascular endothelium, and stromal cells (Fig. 1B). Raet1a expression in fetal brain was also confirmed [42]. Transcription of Raet1d and Raet1e was detected by RT-PCR from Gestation Days 6–10 in the same tissues that transcribed Raet1a and in Day 10 and 13 fetal brain (not shown).



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  		FIG. 1. A) Analysis of mRNA expression of Fcr{gamma} (formally Fcgr3), Dap10 (Hcst), Dap12 (Tyrobp), and Nkg2d (Klrk1) in Gestation Day 10 B6 mice by RT-PCR. Lane 1: 100-bp ladder; lane 2: uNK cells; lane 3: decidua basalis; lane 4: MLAp; lane 5: placenta; lane 6: spleen; lane 7: genomic DNA; lane 8: negative control. B) In situ hybridization of B6 implantation sites (Gestation Day 10) for Rae-1{alpha} (Raet1a) message using antisense (B-a, x50) and sense probes (B-b, x100). Immunohistochemistry for RAE1 on Gestation Day 8 B6 (B-c, x50) and negative control (B-d, x50). Transcripts and protein were strongly expressed by decidual vascular endothelium, placental trophoblasts (PL), and the transition regions between myometrium and decidua basalis (DB) that included the mesometrial lymphoid aggregate of pregnancy (MLAp) (B-a, B-c). Em labels embryonic tissue. Inserts show strongly stained decidual spiral arteries (SA, B-a, x200, B-c, x400) and trophoblasts (B-c; arrow, x400). In B-c, the regions shown in the higher power images are outlined by dashed squares

Reproductive Performance of FcR{gamma}–/–/CD3{zeta}–/–, DAP10–/–, DAP12 Loss of Function and FcR{gamma}–/–/DAP12–/– Mice

Adult FcR{gamma}–/–/CD3{zeta}–/–, DAP10–/–, DAP12 mutant, and FcR{gamma}–/–/DAP12–/– male and female mice resembled control mice in weight and size. They mated and produced offspring. For most of these strains, success in establishing pregnancy following mating (i.e., detection of a copulation plug) was lower than for B6 (Table 1, right-hand column). In particular, uteri from 75% to 85% of the mated DAP12 mutant females showed no evidence that conception had occurred (Table 1). No blastocysts could be flushed from the uteri of four mated mice and postmortem examination of two of our three stud males showed bilateral pathology of the epididymis that was histologically diagnosed (Animal Health Laboratories, Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, ON) as congenital adenosis of the epididymis [43]. Obstructed ducts result in an aspermic ejaculate that retains its ability to form a copulation plug. The source colony had previously noted sterile males but had not reported their findings nor conducted postmortem examinations of the male reproductive tract. This problem is a recognized congenital pathology of mice [43] and does not appear related to the functional absence of DAP12 because sterile matings were not present in FcR{gamma}–/–/DAP12–/–, a newer strain. This pathology more likely reflects a background mutation in the single male we received from Europe to establish our breeding program and it limited our investigations of this strain.


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  		TABLE 1. Pregnancy and fetal survival in FcR{gamma}–/–/CD3{zeta}–/–, DAP10–/–, DAP12 mutant, FcR{gamma}–/–/DAP12–/– mice, and their congenic controls

In pregnant females of all genotypes, fetal loss was seen. Early postimplantation fetal loss (Days 6 and 8 of gestation) was elevated in FcR{gamma}–/–/CD3{zeta}–/– and DAP10–/– females compared with B6 or FcR{gamma}–/–/DAP12–/– mice. The absence of implantation sites in mated DAP12 mutant mice prevented collection of comparable data. Midgestation losses occurred in all mutant strains and were at rates higher than in B6 on Gestation Day 10 in DAP10–/– and on Gestation Day 12 in FcR{gamma}–/–/DAP12–/–. This suggests that none of the genetic mutations cause major impairment of fetal development and that many more litters would be needed to establish whether these mutations have minor developmental effects.

Assessment of Implantation Site Morphology in Mice with Disrupted NK Cell Activation

The general histological appearance of viable implantation sites from the four genetically impaired strains and B6 controls at Day 12 of gestation is depicted in Figure 2. In comparison with B6, the FcR{gamma}–/–/CD3{zeta}–/– sites appeared hypercellular, with a very large MLAp (Fig. 2A). Sites from DAP10–/– appeared hypocellular (Fig. 2B). Decidua basalis seemed relatively reduced in DAP12 mutants (Fig. 2C). Thick-walled arteries were unusually prominent in FcR{gamma}–/–/ CD3{zeta}–/–, DAP12 mutant, and FcR{gamma}–/–/DAP12–/–. These general impressions were extended by morphometry.



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  		FIG. 2. Midsagittal sections, spiral arteries and uNK cells in Gestation Day 12 implantation sites of FcR{gamma}–/–/CD3{zeta}–/– (A), DAP10–/– (B), DAP12 mutant (C), FcR{gamma}–/–/DAP12–/– (D) mice, and their congenic B6 (E) controls. Sections were stained with hematoxylin and eosin. Bar = 100 µm. MLAp, Mesometrial lymphoid aggregate of pregnancy; DB, decidua basalis; and PL, placental trophoblast region. Inserts show hematoxylin and eosin-stained decidual spiral arteries (x400) in the lower left corner of each panel and PAS-stained uNK cells (arrow, x1000) in the MLAp at lower right of each panel. Compared with B6 controls, the spiral arteries of FcR{gamma}–/–/CD3{zeta}–/– (A), DAP12 mutant (B), and FcR{gamma}–/–/DAP12–/– (D) mice showed restricted modification, whereas those of DAP10–/– (C) mice were well modified

Area Morphometry of MLAp, Decidua Basalis, and Placentas

Morphometric analyses of MLAp, decidua basalis, and placentas were performed for the four mutant strains and control B6 mice (Fig. 3). Placental enlargement (growth) occurred in all strains between Days 10 and 12 of gestation. In comparison with B6 at Day 10 of gestation, the MLAp, which is the site preferentially enriched in immature, dividing uNK cells, was larger in FcR{gamma}–/–/CD3{zeta}–/– and DAP12 mutants, similar in DAP10–/–, and smaller in FcR{gamma}–/–/DAP12–/–. The decidua basalis and placenta of all mutants except FcR{gamma}–/–/DAP12–/– were larger than B6. Both of these regions were statistically smaller in FcR{gamma}–/–/ DAP12–/–. At Day 12 of gestation, the MLAp of FcR{gamma}–/–/ CD3{zeta}–/– remained significantly larger than control and all other strains while the decidua basalis and placenta of FcR{gamma}–/–/DAP12–/– remained much smaller than the others. The surface areas of the placenta and decidua basalis were more variable between the other mutant strains at Day 12 than Day 10 of gestation. Decidua basalis was relatively stable in surface area in B6, DAP10, and FcR{gamma}–/–/DAP12–/– but had begun to regress in FcR{gamma}–/–/CD3{zeta}–/– and in the DAP12 mutants.



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  		FIG. 3. Comparison of the cross-sectional areas (mean ± SD) of mesometrial lymphoid aggregate of pregnancy (MLAp), decidua basalis (DB), and placenta (PL) from FcR{gamma}–/–/CD3{zeta}–/–, DAP10–/–, DAP12 mutant, FcR{gamma}–/–/DAP12–/– mice with congenic B6 controls at Days 10 and 12 of gestation (gd). Area enlargement of MLAp was observed in FcR{gamma}–/–/ CD3{zeta}–/– mice at both gd 10 (# P < 0.01;) and 12 (* P < 0.05), and in DAP10–/– mice at gd 10 (P < 0.01). No difference in MLAp was found between DAP12 mutant mice and B6 controls at each gestation day examined, while FcR{gamma}–/–/DAP12–/– mice had smaller MLAp at gd 10 (P < 0.01). In comparison with B6, DB was enlarged in FcR{gamma}–/–/CD3{zeta}–/– mice at Day 10 (P < 0.05) and in DAP10–/– mice at both Days 10 and 12 of gestation (P < 0.05). DAP12 mutant mice had larger DB at gd 10 (P < 0.05) and smaller DB at Day 12 (P < 0.01), while smaller DB areas were found in FcR{gamma}–/–/DAP12–/– mice at both Days 10 (P < 0.01) and 12 (¶ P < 0.0001) of gestation. In comparison with B6, placental areas were enlarged in FcR{gamma}–/–/CD3{zeta}–/– and DAP10–/– mice at gd 10 (P < 0.01) and in DAP12 mutant mice at both Days 10 (P < 0.05) and 12 (P < 0.0001), while placental area was reduced in FcR{gamma}–/–/DAP12–/– mice at both Days 10 (P < 0.01) and 12 (P < 0.0001)

Uterine NK Cell Density in Implantation Sites

Uterine NK cells were numerous and appeared in appropriate microdomains within implantation sites in all four mutant strains. Morphology of the uNK cells was not distinct from B6 at any time point for which samples were available (Fig. 2). Day 6 samples were not collected for histology from DAP12 mutants or FcR{gamma}–/–/DAP12–/– due to limited availability of these animals. For the other strains, there were no significant differences in uNK cell numbers/mm2 of mesometrial triangle (MT) compared with the control (Fig. 4). At Day 10 of gestation, there were 46% more uNK cells in the MLAp of FcR{gamma}–/–/CD3{zeta}–/– than B6 but fewer than B6 in the other strains. The deficits were statistically significant in DAP10–/– and DAP12 mutants; the deficit in FcR{gamma}–/–/DAP12–/– was not. In Gestation Day 10 decidua basalis, uNK cell numbers were lower than control for FcR{gamma}–/–/DAP12–/– and higher than control for FcR{gamma}–/–/CD3{zeta}–/– and the DAP12 mutant. Uterine NK cells in DAP10–/– were equivalent to B6. At Gestation Day 12, uNK cell numbers in the MLAp were lowest in FcR{gamma}–/–/ DAP12–/– and DAP12 mutant mice and statistically different from those in control B6. Uterine NK cells in the MLAp of DAP10–/– mice were also lower than control, but uNK cells in the MLAp of FcR{gamma}–/–/CD3{zeta}–/– were equivalent. In Day 12 decidua basalis, numbers of uNK cells for only FcR{gamma}–/–/CD3{zeta}–/– and FcR{gamma}–/–/DAP12–/– differed from the control. Both were elevated. Uterine NK cell numbers in FcR{gamma}–/–/DAP12–/– did not show the decline typical of Gestation Day 12 in normal mice.



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  		FIG. 4. Comparison of uNK cell density (mean ± SD) in implantation sites of FcR{gamma}–/–/CD3{zeta}–/–, DAP10–/–, DAP12 mutant, and FcR{gamma}–/–/DAP12–/– mice with B6 controls at Gestation Days (gd) 6, 10, and 12. At Day 6, no significant differences were found in uNK cell numbers in the mesometrial triangle (MT) between mutant and control mice. Compared with B6, more uNK cells were present in the mesometrial lymphoid aggregate (MLAp) and decidua basalis (DB) at Day 10 and in the DB at Day 12 in FcR{gamma}–/–/CD3{zeta}–/– mice (¶ P < 0.0001), while DAP10–/– mice had fewer uNK cells at Days 10 and 12 in the MLAp (P < 0.0001) but not in the DB. In DAP12 mutant mice, fewer uNK cells were observed in the MLAp at Days 10 and 12 gestation and more in the DB at Day 10 (* P < 0.05). FcR{gamma}–/–/DAP12–/– mice had fewer uNK cells in the DB at gd 10 (P < 0.0001) and in the MLAp at Day 12 (P < 0.0001)

Ratios of Vessel to Lumen Areas of Spiral Arteries

One of the major functions of uNK cells is promotion of spiral artery remodeling during pregnancy. The functional effects of the gene modifications were assessed by measurements of decidual spiral artery vessel to lumen area ratios (Fig. 5). Compared with gestation length-matched controls, DAP10–/– mice showed no differences at Days 10 or 12 of gestation. In contrast, vessel-to-lumen-area ratios were greater than in the control for FcR{gamma}–/–/CD3{zeta}–/– and FcR{gamma}–/–/DAP12–/– mice at both Days 10 and 12 of gestation (Fig. 5, P < 0.01). In DAP12 mutant mice, statistically significant restriction to vascular changes (P < 0.01) was demonstrated only on Day 12. In B6 controls, vessel-to-lumen-area ratios of the decidual spiral arteries were significantly reduced at Day 12 compared with Day 10 (Fig. 5, P < 0.05). However, no similar changes were found in any genetically altered mice, indicating further impairment of spiral artery modification. Indeed, a significant increase in ratio was observed at Day 12 compared with Day 10 in DAP12 mutant mice (Fig. 5, P < 0.01). These results suggest that lack of FcR{gamma}/CD3{zeta} and/or DAP12 but not DAP10 induces functional impairment in uNK cells.



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  		FIG. 5. Comparison of spiral arterial vessel-to-lumen-area ratios (mean ± SD) in FcR{gamma}–/–/CD3{zeta}–/–, DAP10–/–, DAP12 mutant, FcR{gamma}–/–/DAP12–/– mice and their congenic B6 controls at Gestation Days 10 and 12. Compared with B6 controls, the vessel-to-lumen-area ratios were much greater in FcR{gamma}–/–/CD3{zeta}–/– (# P < 0.01) and FcR{gamma}–/–/DAP12–/– mice at both Day 10 (¶ P < 0.001) and Day 12 (P < 0.01), while in DAP12 mutant mice, the ratio was greater only at Day 12 (P < 0.01). No significant differences were found between DAP10–/– mice and B6 controls at each time point. In B6 controls, a significant reduction in ratio was found at Day 12 of gestation compared with Day 10 (* P < 0.05). No similar changes were observed in the gene deleted mice. In contrast, a significant increase in ratio was found in DAP12 mutant mice at Day 12 of gestation compared with Day 10 (P < 0.01)

IFN-{gamma} Assessment in Pregnant Uteri of DAP10–/– and FcR{gamma}–/–/DAP12–/– Mice

Triggering of spiral arterial modification is attributed to uNK cell-derived IFN-{gamma}. The morphometric results suggested that midgestation mesometrial IFN-{gamma} would be comparable in DAP10–/– and B6 but relatively deficient in the other strains. Inadequate numbers of FcR{gamma}–/–/CD3{zeta}–/– and DAP12 mutant pregnancies were available for cytokine quantification. However, pregnancies in FcR{gamma}–/–/DAP12–/– mice, mutant in both receptors of interest, were available. IFN-{gamma} was undetectable in homogenates of the MT, MLAp, or decidua basalis of FcR{gamma}–/–/DAP12–/– from Day 6 through Day 14 of gestation. Concentrations of IFN-{gamma} for DAP10–/– and B6 were similar in Gestation Day 8 MT (3.19 ± 0.2 IU vs. 3.43 ± 0.52 IU, P = 0.49), Day 10 decidua basalis (6.39 ± 1.4 IU vs. 6.56 ± 1.06 IU, P = 0.87), Day 12 MLAp (5.29 ± 0.66 IU vs. 6.54 ± 0.93 IU, P = 0.13) and Day 12 decidua basalis (3.80 ± 0.78 IU vs. 4.12 ± 0.23 IU, P = 0.53). The only statisticially significant difference in IFN-{gamma} between DAP10–/– and B6 was in Gestation Day 10 MLAp (4.95 ± 0.28 vs. 6.34 ± 0.67, P < 0.05). This may reflect a mild functional difference in DAP10–/– mice because there was no deficiency in the surface area of the Gestation Day 10 DAP10–/– MLAp or in the number of cells it contained.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is not yet fully known when, during uNK differentiation, the lineage diverges from NK cells found in lymphoid tissue and blood. Transplantable NK and uNK cell progenitors are widely codistributed in lymphohematopoetic tissues [44], and no distinctive uNK precursor cell has been identified, although human gene profiling studies suggest its existence [45]. Onset of terminal murine uNK cell differentiation involves IL-15, estrogen priming, and progesterone [46–48], but other activation steps are not reported. Trophoblast is not required for initiation of uNK cell differentiation because uNK cells develop in artificial decidua [48]. However, growth factors from trophoblasts are required to sustain decidua for its normal life span and must, either indirectly or directly, also enhance viability of uNK cells [48].

Outside of the uterus, involvement of FcR{gamma}/CD3{zeta}, DAP10, and DAP12 in NK cell activation and functions is well documented. We established that uNK cells express Fcr{gamma}, Dap10, and Dap12. We also showed uNK cell transcription of the activation receptor Nkg2d and transcription and translation of its ligand RAE1, in implantation sites. Uterine NK cell activation must occur early during decidualization because murine uNK cells are sources of IL-18, IFN-{gamma}, perforin, and serine esterases by Day 6 of gestation [39, 49, 50]. Embryonic tumor (F9) cells and developing fetal brain (Day 13 of gestation) are reported to express Rae1 gene family members [41, 42]. We found gene and protein expression much earlier and localized them to trophoblast cells between Days 6 and 14 of gestation. Thus, RAE1 expression by fetal trophoblast could be of major importance in maintenance of maternal uNK cell activation through the period of spiral arterial modification to midgestation. RAE1 was also induced in endometrium, especially in endothelium lining the spiral arteries. Uterine NK cells are unusual cells because they appear to leave the circulation via arterioles rather than veins [48]. Our data therefore suggest that important steps in uNK cell precursor maturation/activation may occur via interactions with uterine endothelium during egress from the circulation. Rae1 expression was confirmed in 129 strain-derived F9 cells and in endometrium, placenta, and fetal brain of 129/P3J (genes Rae1tb and Raeltc) by RT-PCR at Day 10 of gestation (not shown).

Mice, genetically modified to lack FcR{gamma}, CD3{zeta}, DAP10, and/or DAP12 signaling, were used to address the relative importance of each pathway during uNK cell activation. Each strain differentiated abundant numbers of morphologically normal uNK cells, indicating that none of the normal NK cell activation mechanisms contributes exclusively to differentiation of the lineage, its production of cytoplasmic granules, or the timing of uNK cell senescence (not shown). The elevated numbers of uNK cells found in the MLAp and decidua basalis in FcR{gamma}–/–/CD3{zeta}–/– may reflect compensation for impaired uNK cell function or unregulated overgrowth of immature stages. Because no differences in the numbers of NK cells or their distribution are found in FcR{gamma}–/– mice [12] and high numbers were not found in the MLAp of FcR{gamma}–/–/DAP12–/–, our results suggest that absence of CD3{zeta} promoted the uNK cell increase. This supports the proposed negative regulatory role of CD3{zeta} in Fc{gamma}RIII-mediated functions of murine NK cells [16].

Morphometric study revealed that midgestation implantation sites in FcR{gamma}–/–/DAP12–/– were smaller than in the other strains studied, even though this strain had about one less offspring per litter (Table 1). For the other mutants, growth of implantation site tissues appeared slightly accelerated compared with the B6 control. The numerical data may be somewhat misleading because hypercellularity enlarged tissue regions in FcR{gamma}–/–/CD3{zeta}–/–, while edema enlarged regions in DAP10–/– (Fig. 2). Deletion of Dap10 had the least structural effect on implantation sites. DAP10–/– mice produced IFN-{gamma}, indicative of successful uNK cell activation, at levels similar to those in B6. This affected normal spiral artery modification, indicating that DAP10 does not contribute significantly to uNK cell activation or function. This conclusion is consistent with the finding that NKG2D-DAP10 is sufficient to trigger cytotoxicity but not cytokine production in activated mouse NK cells [51]. Given normal vascular changes, it is difficult to understand the mechanism for elevated fetal loss seen in DAP10–/– between Days 6 and 10 of gestation.

Restricted spiral artery remodeling was seen in mice lacking FcR{gamma}/CD3{zeta}, DAP12, or FcR{gamma}/DAP12. This suggests that NK cell-activating receptor signaling transduced through FcR{gamma}/CD3{zeta} and DAP12 is important for murine uNK cell activation and IFN-{gamma} production. The male infertility in DAP12 loss of function mice that led to euthanasia of females mated by sterile males, reduced animal numbers below those necessary for a time-course analysis of IFN-{gamma} production. FcR{gamma}–/–/CD3{zeta}–/– mice were also unavailable, but sufficient numbers of the newly described FcR{gamma}–/–/ DAP12–/–, that combined these mutations, were accessible. No IFN-{gamma} was detected in homogenates of MT collected on Day 6 or 8 of gestation or in homogenates of MLAp or decidua basalis collected on Day 10 or 12 of gestation, showing that FcR{gamma} and DAP12 are required for induction of IFN-{gamma} synthesis in implantation sites. This outcome differs from our earlier findings in Tg{varepsilon}26 and alymphoid mice [50]. In these strains, low concentrations of IFN-{gamma}, representing about 10% of that in normal mouse implantation sites, were consistently detected. These low amounts were insufficient for modification of spiral arteries and were attributed to nonlymphoid cells such as macrophages and granulocytes. Absence of low amounts of IFN-{gamma} in FcR{gamma}–/–/ DAP12–/– suggests that one or both of these activation pathways is also used by the nonlymphoid uterine cells capable of pregnancy-induced IFN-{gamma} synthesis. The importance of DAP12 in spiral artery modification appeared at Day 12 of gestation. We have not previously found a strain impaired at Day 12 that was not also impaired at Day 10 of gestation. This result could imply sequential roles for FcR{gamma}/CD3{zeta} and DAP12 signaling, dominance of the FcR{gamma}/ CD3{zeta} signal, or may relate to the premature (gd12) regression of decidua in DAP12 loss of function mice.

The histological abnormalities in implantation sites FcR{gamma}–/–/CD3{zeta}–/– strongly suggest that murine uNK cell activation involves signals transduced by Fc{gamma}RIII and/or NKR-P1C (formally called KLRB1C) through FcR{gamma} and/ or CD3{zeta} pathways known to lead to IFN-{gamma} production [16, 17]. The physiological ligands for NKR-P1C are unknown. Our study design did not define whether only FcR{gamma} or CD3{zeta} or both regulate uNK cell activation. FcR{gamma} appears more relevant for murine NK cell activation and function because expression of Fc{gamma}RIII, Fc{gamma}RIII-mediated ADCC, and IFN-{gamma} production are markedly diminished in NK cells from FcR{gamma}–/– but not CD3{zeta}–/– mice [13]. In rodents, implantation is associated with vascular leakage that elevates all antibody classes in decidual tissue. IgG, however, is the only maternal antibody actively transported across the placenta via the IgG transporter, FcRn (formally FCGRT) [52]. Because IgG has been considered the only ligand for Fc{gamma}RIII on murine NK cells, our data suggest an important new role of IgG in activation of mouse uNK cells. Recently, IgE has been shown to activate NK cells through Fc{gamma}RIII and to induce cytokine and chemokine production and ADCC [53]. This implicates contributions by IgE antibodies to uNK cell activation and may be particularly relevant to pregnancies complicated by disease. In sum, results of the present study suggest that molecules able to induce murine NK cell activation by multiple known pathways are present in the uterus shortly after implantation. Uterine NK cell activation depends more on DAP12 adaptation than DAP10 cosignaling, but the most important activation stimuli for mouse uNK cells are antibody-mediated through FcR{gamma}/CD3{zeta}.


   ACKNOWLEDGMENTS
 
We thank the staff of the OMAFRA Isolation Unit for their outstanding animal care and Dr. Patricia Turner, Guelph, for pathology consultations. We are grateful to Dr. P.E. Love (Laboratory of Mammalian Genes and Development, NICHD, NIH, Bethesda, MD), Dr. E. Vivier, and Dr. E. Tomasello (Center d'Immunologie, INSERM-CNRS de Marseille-Luminy, 13288 Marseille, Cedex 09, France) for their generous gifts of immune-deficient mice and critical suggestions for the manuscript. We also thank Y. Nishizawa (Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Japan) for technical assistance.


   FOOTNOTES
 
1 Supported by the Natural Sciences and Engineering Research Council Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs. Back

2 Correspondence: Anne Croy, Department of Anatomy and Cell Biology, Room 924, Botterell Hall, Stuart Street, Queen's University, Kingston, Ontario, Canada K7L 3N6. FAX: 613 533 2566; croya{at}post.queensu.ca Back

3 Current address: Department of Environment Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Back

Received: 16 July 2004.

First decision: 19 August 2004.

Accepted: 3 May 2005.


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