PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of capmcAbout manuscripts / A propos des manuscritsSubmit manuscript / soumettre un manuscrit
 
J Leukoc Biol. Author manuscript; available in PMC 2012 June 20.
Published in final edited form as:
PMCID: PMC3289395
CAMSID: CAMS2039

Unusual Timing of CD127 Expression by Mouse Uterine Natural Killer Cells

Abstract

Decidualization, a progesterone-dependent process that alters endometrial stromal cells at implantation sites in humans and rodents, is accompanied by a highly regulated, NK cell-dominated, leukocyte influx into decidua basalis (DB). Whether uterine NK cells differentiate from uterine progenitor cells is unknown as are the mechanisms restricting leukocytes to DB. We asked if cells expressing the early NK lineage marker CD127 (IL-7Rα) occurred in mouse decidua. CD127 was absent from gestation day (gd)6.5 decidual lymphoid cells but became expressed by a mature uNK cell subset in gd10.5 DB. DB and transient myometrial structures (MLAp) that ring maternal blood vessels supplying placentae both expressed IL-7 and thymic stromal lymphopoietin (TSLP), the CD127 ligands, but with differing temporal and spatial patterns. Uterine NK cells expressed TSLPR and study of gd10.5 implantation sites from mice deleted for IL-7, CD127 or TSLPR suggested that IL-7 and its receptor have physiological roles in limiting expansion of immature uNK cells within MLAp while the TSLP signalling pathway is used in DB to sustain IFN-γ production from a subset of mature uNK cells. Regionalized, dynamic expression of the additional lymphoid organ stromal markers gp38/Podoplanin and ER-TR7 but not CD157 was seen by immunohistochemistry in implantation sites and DB and MLAp contained transcripts for Aire, a tolerance54 promoting factor. These observations suggest that CD127+ NK lineage progenitors are not present in the early postimplantation period of mouse uterus and that decidualized endometrial stroma has key immunoregulatory properties.

Keywords: Decidua, interleukin 7, stromal factor, thymic stromal lymphopoietin

INTRODUCTION

Uterine (u)NK cells are abundant, transient lymphocytes found in the decidualizing endometrium of early pregnancy in species with hemochorial placentation, such as human, rat and mouse [1, 2]. Relationships between uNK and other NK cells are incompletely defined. It remains unclear whether the large uNK cell population arises from circulating or uterine progenitor/precursor cells. In mice, transplantable uNK progenitor cells have been identified in primary and secondary lymphoid tissues, including thymus and LN but not in uterine segments [1, 3]. UNK cells play key roles in the endometrial remodeling associated with pregnancy including roles in promotion of maternal angiogenesis and transformation of the major maternal arterial branches supplying the placenta, called spiral arteries [2, 4]. UNK cells are thought to interact with and share these functions with conceptus-derived placental trophoblast cells [4] and to have paracrine effects that alter gene expression in uterine stromal fibroblasts [5]. In contrast to human uNK cells that appear late in each menstrual cycle and are identified as CD56bright [6], mouse uNK cells appear after blastocyst implantation (gestation day (gd)4) [1] and are identified as Dolichos biflorus agglutinin (DBA) lectin-reactive [7].

Flow cytometry has resolved human and mouse NK cells into subsets that reflect progressive lineage differentiation and maturation [8, 9]. Application of this paradigm to human uNK cells suggested that only later, non-multipotent NK cell stages were present in cycling endometrium and in first trimester decidua [10]. CD34CD117+CD94 NK cells were the earliest stage identified in human uterus, a phenotype similar to “Stage 3” NK cells in secondary lymphoid organs. Many CD34CD117+CD94 human uterine cells strongly expressed CD127 (IL-7Rα) [10], a marker proposed as a thymic emigrant tag [11, 12]. CD127 is also expressed by mouse NK lineage cells early during differentiation [9]. When CD27 and CD11b are used to describe the progression of mouse NK cell differentiation, the earliest doubly negative cells are 50% CD127+. At the next stage of differentiation (CD11blow CD27high), characterized transcriptionally as highly proliferative, 32% of immature NK cells are CD127+. At later stages of NK cell maturation when the transcriptome is biased towards effector functions, CD127 expression is rare (1–2%). Thus, if mouse uNK precursors reside in the uterus, some of them should be identifiable as CD127+, particularly during their proliferative expansion that occurs initially within early gd5.5-8.5 DB and later within the MLAp [7].

Recently, unique CD127+ NK progenitor cells were indentified in mouse LN that differentiate by thymic-dependent and thymic-independent pathways [13]. These murine CD127+ NK cells, like human CD56highCD16 NK cells, produce significant amounts of cytokines but only become cytotoxic after long term activation [14, 15]. Since human LN is a known precursor source for CD56bright NK cells [16] and mouse uNK cells differentiate from LN [17], we asked whether CD127 expression was present on uNK cells in mouse implantation sites. UNK cells were not CD127 reactive at gd6.5 but, in a time course study, were shown to acquire CD127 with terminal differentiation. The CD127 ligands IL-7 and Thymic stromal lymphopoietin (TSLP) were locally expressed by decidual cells and genetic blockade of these signaling pathways differentially altered uNK cells and their synthesis of IFN-γ.

MATERIALS AND METHODS

Mice

C57BL/6J (B6), BALB/c, CD-1 (Jackson Laboratory, Bar Harbor, ME) and locally-bred alymphoid Rag2−/−/Il2rg−/− mice on a BALB/c background were housed under barrier conditions at Queen’s University. For timed matings, estrous females were selected, paired to syngeneic males and checked for copulation plugs the following morning. The morning of plug detection was named gd0.5. Gd10.5 uteri from syngeneic matings of B6 background Tslpr−/− (NIH), Il7−/− (University of Toronto, Canada) and Il7rα−/− (CD127 null; University of British Columbia, Canada) were shipped to Queen’s University. All procedures were conducted under Animal Utilization Protocols approved by the appropriate institutional Animal Care Committees.

Histological Procedures

Females mated at Queen’s University were deeply anaesthetized then perfused with 30 ml of freshly prepared 4% paraformaldehyde (PFA) in buffered saline. Pregnant uteri were dissected, transected between implantation sites and post fixed in PFA (15 min for gd6.5-10.5 sites, 60 min for gd12.5 sites). Standard procedures were used for automated processing into paraffin, embedding and cutting of 7 μm serial sections. Two or more viable implantation sites from each of multiple (2–5) females were studied for each gd reported. Fetal liver was collected as a positive control tissue and processed similarly.

Blood vessel morphometry and uNK cell enumeration were conducted as previously described [1, 18, 19] using 11 slides/implantation site and 3 implantation sites/pregnancy. Slides for vascular measurements were stained with hematoxylin and eosin; slides for uNK cell enumeration were stained with biotinylated DBA lectin (1μg/ml; Sigma) followed by streptavidin peroxidase and 3, 3-diaminobenzidine (DAB) to identify the granules and plasma membranes of uNK cells. Specificity control incubations incorporated N-acetylgalactosamine, the sugar that blocks DBA lectin binding. For some slides, FITC- or TRITC-conjugated DBA lectin (1μg/ml; Sigma) was used with epifluorescence for uNK cell visualization. Additional slides were stained with Periodic Acid Schiff’s (PAS) reagent to recognize uNK cell glycoproteins. Mathematic arterial wall thickness was calculated from: thickness = [square root (vessel area / π) - square root (lumen area / π)].

Immunohistochemistry for CD127 and TSLP was undertaken using pre-placental gd6.5, 7.5 or 8.5 implantation sites or placental gd10.5 or 12.5 implantation sites. After deparaffinization and rehydration, peroxidases were inactivated (3% H2O2 in 0.1M PBS, 6 min), a blocking reagent was applied (1% BSA in PBS, 20 min), then replaced by a primary rabbit antibody against CD127 (Santa Cruz Biotech; 1:200) or TSLP (Sigma; 1:400) overnight (4°C). After rinsing, slides were incubated with biotinylated goat anti-rabbit IgG (DAKO, 30 min), then visualized with DAB (Sigma) or with Alexa Fluor® 488/594 goat anti-rabbit IgG (Invitrogen; 1:200) when dual staining with FITC- or TRITC-conjugated DBA lectin was being conducted (Sigma, 1:200). Coverslips were mounted using Permount (Fisher Scientific) for immunohistochemistry or ProLong® Gold anti-fade reagent (Invitrogen) for immunofluorescence. Negative controls were serial sections processed identically except the primary antibody was replaced by non immune rabbit serum. For stromal cell study, gd7.5 and 10.5 implantation sites were stained with anti-mouse CD157, ER-TR7 (Erasmus University Rotterdam-Thymic Reticulum; BMA Biomedicals, Switzerland) or Hamster anti-mouse gp38 (RELIATech, Germany). All images were collected and analyzed using an AxioImager M.1 microscope and Axiovision software (Carl Zeiss, Oberkochen, Germany).

BM Transplantation

Three 6-wk-old BALB/c-Rag2−/−/Il2rg−/− females were pretreated with 150 mg/kg of 5-fluorouracil (5-FU) i.p. 48h before i.v. infusion of 2×107 BALB/c leukocytes flushed from bone marrow. Three-four wk later, these recipients were mated for study at gd6.5 (n=2) or 12.5 (n=3).

Flow Cytometry

Viable gd10.5 BALB/c implantation sites were dissected to isolate DB and MLAp. Each tissue was then mechanically dissociated to provide cell suspensions for flow cytometry as described elsewhere [20]. After incubation (30 min) in normal rat serum, leukocytes were stained (30 min, 4°C) with FITC-conjugated DBA (1μg/ml; Sigma), and antibodies purchased from eBioscience: PE-conjugated anti-mouse CD122 (5H4; 1μg/ml), PE-Cy5-conjugated anti-mouse CD3 (145-2C11; 1μg/ml) and APC-Alexa Fluor-conjugated anti-mouse CD127 (A7R34; 1.2μg/ml) or tagged isotype control IgG. 50,000 events were collected using a Cytomics FC500 Flow Cytometry (Beckman Coulter) and data were processed with FlowJo software (Tree Star Inc., Ashland, OR). Cell suspensions were prepared from mechanically dissociated, gd10.5 BALB/c female spleen, thymus and bone marrow and similarly labeled.

cDNA Synthesis and PCR Protocols

To evaluate Tslp and Tslpr expression in uNK cells, FACS sorted CD3CD122+DBA+ uNK cells were studied. In brief, decidual lymphocytes of gd10.5 CD-1 mice were isolated and stained with FITC-conjugated DBA, PE-conjugated CD122 and PE-Cy5-conjugated CD3. CD3CD122+DBA+ uNK cells were collected by EPICS Altra Flow HyPerSort Cytometer (Beckman Coulter). Then RNA was isolated, reverse transcribed and amplified using the Ovation Pico WTA System (NuGEN, San Carlos, CA) to obtain cDNA, which was used as the PCR template. PCR amplification used the Qiagen PCR kit with the following conditions: 94°C for 3 min (1 cycle); 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec (40 cycles); and 72°C for 10 min (1 cycle). PCR products were separated on 1.0% agarose gel and visualized by ethidium bromide staining. RNA was also prepared from the MLAp, DB and thymus of gd10.5 B6 mice to examine expression of autoimmune regulator (Aire). Primer sequences were given below. All PCR products were confirmed by sequencing.

Tslp (266bp), 5′-CCAGGCTACCCTGAAACTGA-3′ (forward),

5′-TCTGGAGATTGCATGAAGGA-3′ (reverse);

Tslpr (267bp), 5′-CCGGCTTCCCGTTTTCGGCT-3′ (forward),

5′-AAGTTGGCGCCGTGGTGGTC-3′ (reverse),

Aire [21], 5′-TGTGCCACGACGGAGGTGAG-3′ (forward),

5′-GGTTCTGTTGGACTCTGCCCTG-3′ (reverse).

For quantitative RT-PCR, total RNA was extracted from gd10.5 B6 DB or MLAp using Qiagen RNeasy Mini Kit. cDNA was synthesized from 1.5 μg total RNA using Invitrogen SuperScript® III First-Strand Synthesis System. Then 20 ng cDNA were subjected to realtime PCR in 96-well plates, as triplicates according to the manufacturer’s protocol (10 min at 95°C, 40 cycles of 5 sec at 95°C for denaturing and 33 sec at 60°C for annealing and extension using iTaq Fast SYBR Green Supermix with ROX (Bio-Rad Laboratories, Hercules, CA; ABI Prism 7500 (Applied Biosystems, Foster City, CA)). Primer sequences were given below and products were confirmed by sequencing:

Il7 [22], 5′-TGGAATTCCTCCACTGATCC-3′ (forward),

5′-ACCAGTGTTTGTGTGCCTTG-3′ (reverse);

Cd127 [23], 5′-TCTGACCTGAAAGTCGTTTATCGC-3′(forward),

5′-CATCCTCCTTGATTCTTGGGTTC-3′(reverse);

Tslp [24], 5′-CCAGGCTACCCTGAAACTGA-3′ (forward),

5′-TCTGGAGATTGCATGAAGGA-3′ (reverse);

hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1),

5′-GCTGACCTGCTGGATTACAT-3′ (forward),

5′-TTGGGGCTGTACTGCTTAAC-3′ (reverse).

Relative expression levels of target transcripts were normalized to Hprt1 transcripts.

Statistical Analysis

Data are expressed as mean±SD. Student’s t-test was applied for statistical analysis, p-values of < 0.05 were considered significant.

RESULTS

CD127 expression in B6 implantation sites between gd6.5-12.5

Serial sections from gd6.5-12.5 B6 implantation sites were stained with CD127 or/and DBA lectin (Fig. 1A&B). At gd6.5, occasional CD127 signal (arrow heads) was found on decidual stromal cells but not DBA+ uNK cells. At gd8.5, some DBA+ uNK cells were very weakly CD127-reactive. At gd10.5 (midgestation), CD127+ uNK cells were present. These were more frequent in DB than in MLAp. Both endothelium and smooth muscle cells of the spiral arterial walls were CD127. Gd10.5 placentas were also CD127 reactive over trophoblast cells and some nucleated fetal blood cells. By gd12.5 when uNK cell numbers are in decline, CD127 reactivity appeared to be weaker over uNK cells DB in and barely detectable on uNK cells in the MLAp. Gd 12.5 fetal liver, used as a positive control tissue, contained CD127 reactive cells.

Figure 1
CD127 expression in midsagittal serial sections from gd6.5-12.5 B6 implantation sites. A, immunohistochemistry for CD127 (top) or DBA-lectin (bottom). B, immunofluorescence staining of gd10.5 B6 implantation site. Detection was nuclear DNA, DAPI (blue); ...

To quantify CD127 expression by DBA+ uNK cells, flow cytometry was undertaken using gd10.5 BALB/c mice (Fig. 2A). About half of the leukocytes from DB or MLAp were CD3CD122+ cells (putative NK cells), which were 4–15 times more than thymus, spleen or BM. Analyses of CD3CD122+ cells for DBA lectin expression revealed significantly more DBA+CD3CD122+ uNK cells from DB (88%) than in uNK cells from the MLAp (66%; p<0.05). Many fewer surface DBA+CD3CD122+ cells were present in lymphoid tissues. The parallel analysis of frequency of CD3CD122+ cells for CD127 expression revealed the highest frequency of this subset in thymus (90%, as reported by others [15]) with gd10.5 DB having the 2nd highest frequency (37%), followed by spleen. The MLAp and bone marrow (BM) had similar frequencies of CD127+CD3CD122+ cells. Analyse of median fluorescence intensity (MFI) revealed that expression of glycosylation sites binding DBA lectin was significantly higher on CD3CD122+ NK cells in DB than on uNK cells in the MLAp or in any other tissue (p<0.05). UNK cells from DB again showed greater MFI than uNK cells from MLAp, BM or spleen (p<0.05). MFI for CD127 on NK cells from the thymus was similar to that for NK cells from DB (Fig. 2B). These data suggest that midpregnancy DB but not MLAp is a specialized site that briefly supports CD127+ NK cells. The CD127+ cells appear from their size, side scatter profile indicative of granularity and positional location to be mature, activated NK cells rather than progenitors or early lineage cells.

Figure 2
CD127 expression in different mouse tissues. A, isolated uterine lymphocytes were stained with FITC-conjugated DBA lectin, PE-conjugated CD122 (5H4), PE-Cy5-conjugated CD3 (145-2C11) and APC-Alexa Fluor conjugated CD127 (A7R34). CD3 CD122+ NK ...

To confirm that homed uNK cell progenitors indeed lack CD127 when the lineage is established but acquire the capacity to express CD127 at midgestation, CD127 immunohistochemistry was undertaken on implantation sites from Rag2−/−/Il2rg−/− recipients of BALB/c bone marrow. In the absence of transplantation, implantation sites in this strain lack uNK cells (Fig. 2Ci). CD127+ uNK cells were undetectable at gd6.5 in non transplanted and in transplanted Rag2−/−/Il2rg−/− recipients although DBA+ NK cells were numerous in the transplant recipients. CD127+ uNK cells were detectable in transplant recipients at gd12.5 (Fig. 2C). For the gd12.5 transplant-derived CD127+ uNK cells, it was again observed that their frequency and staining intensity were greater in DB than in MLAp.

CD127 ligands in mouse implantation sites

CD127 is a component of two cytokine receptors, that for IL-7 which is paired with IL-2Rγ and that for TSLP which is paired with TSLPR [25]. UNK cells do not differentiate in mice genetically deleted for Il15 or its receptor Il2rγ [26] and this has been attributed to the absence of IL-15 signalling [27, 28] since previous work indicated that Il7 mRNA was absent from mouse decidua between gd3.5-18 [29]. Using our primers, Il7 mRNA was detected in gd7.5 B6 DB and in gd10.5 DB and MLAp (Fig. 3A). Transcripts were relatively more abundant at gd7.5 than at 10.5 and, at gd10.5, Il7 transcripts were more abundant in the MLAp than in DB. Immunohistochemistry confirmed IL-7 production at both times (Fig. 3Bi–iii). At gd10.5 but not gd7.5, a few DBA+ uNK cells were amongst the IL-7+ cell population. IL-7+ uNK cells were more frequent in DB than in MLAp. CD127 transcripts were relatively more abundant at gd10.5 than at gd7.5 and, at gd10.5, transcript abundance was greater in the DB than in the MLAp (Fig. 3C). Dual immunohistochemistry for IL-7 and CD127 at gd7.5 showed non overlapping patterns of reactive cells with IL-7+ cells in the central DB and CD127+ cells in the lateral sinusoid regions. At gd10.5, CD127+ cells co-mingled with IL-7+ cells in central DB but were not detectable in MLAp (Fig. 3Biv–vi).

Figure 3
IL-7 and CD127 in B6 implantation sites. A, realtime PCR analysis of relative Il7 transcript abundance in gd7.5 DB and in gd10.5 DB and MLAp. B (i–vi), immunofluorescence co-localization of IL-7 (red) with DBA+ uNK cells or CD127+ cells (green). ...

Tslp transcripts were detected in gd7.5 DB and in gd10.5 DB and MLAp (Fig. 4A). Transcript abundance was greater in both gd10.5 tissues than in gd7.5 DB and statistically higher in gd10.5 MLAp than DB. TSLP was localized in gd7.5 and 10.5 implantation sites by immunohistochemistry. At gd7.5, TSLP+ decidual cells and DBA+ uNK cells did not appear to interact (Fig. 4B, higher power 4C and supplementary Fig. 1). At gd10.5, TSLP+ cells were more frequent in DB than MLAp, an outcome inverse to that predicted from RNA analysis. Gd10.5 DB was the only region where an occasional DBA+ uNK cell co-stained with anti-TSLP. Using mRNA extracted from FACS isolated CD3CD122+DBA+ uNK cells, Tslp and Tslpr expression by uNK cells was confirmed (Fig. 4F).

Figure 4
TSLP expression in B6 implantation sites. A, relative Tslp expression analyzed by realtime PCR. B and C, immunohistochemical detection of TSLP at gd7.5 in DB at lower and higher power revealed that TSLP+ decidual cells (green) were not DBA+ uNK cells ...

To address the functional significance of Il-7 and Tslp signaling pathways in implantation sites, histological and morphometric studies were conducted on gd10.5 uteri from Il7−/−, Il7ra−/− and Tslpr−/− mice, then compared to normal B6 implantation sites. UNK cell numbers in DB were not altered from control in any gene deleted strain (Fig. 5A). However, uNK cell numbers in the MLAp were elevated in Il7−/− and Il7ra−/− but not in Tslpr−/− implantation sites. Additionally, decidual spiral arterial modification, a feature linked with uNK cell-derived IFN-γ, was not impaired (arterial wall thickness to vessel lumen ratios are given in supplementary Fig. 2B). This outcome was surprising because immunohistochemistry for IFN-γ, which reaches its peak in normal implantation sites at gd10.5 and is largely uNK cell derived, was undetectable in gd10.5 Tslpr−/− uNK cells (Fig. 5E&I; supplementary Fig. 2A). In contrast, IFN-γ-producing DBA+ uNK cells were abundant in DB of gd10.5 implantation sites from B6, Il7−/− and Il7ra−/− mice (Fig. 5B–D, F–H).

Figure 5
Distribution and IFN-γ reactivity of uNK cells in gd10.5 B6, Il7−/−, Il7rα−/− and Tslpr−/− mice. A, uNK cell enumeration in MLAp and DB. More uNK cells (DBA+) were present in the MLAp of ...

Additional immune-like properties of decidual stroma

To determine whether decidua expresses molecules other than TSLP that are associated with lymphoid organ stroma, immunohistochemistry of gd7.5 and 10.5 B6 implantation sites was undertaken using antibodies against CD157 (a marker of B cell stroma), gp38 (Podoplanin, a marker of stroma in T cell regions) and ER-TR7 (an extracellular matrix protein produced by lymphoid tissue reticular fibroblasts). All antibodies reacted appropriately in spleen (not shown). No CD157+ decidual stroma was found (not shown), consistent with the known profound deficit in B cells in DB [30]. For gp38 and ER-TR7, intense mesometrial reactivity was observed although neither antibody stained cells of the central DB (Fig. 6A–D). At gd7.5, gp38 was expressed circumferentially by the myometrium and lateral DB and was not associated with uNK cells (insert). At gd10.5, gp38 reactivity remained present across the entire myometrium and was strong in the MLAp, where many uNK cells appeared to be surrounded by gp38+ stromal cells (Fig. 6B). When CD127 replaced DBA lectin as a cell marker in specimens co-stained with gp38, no CD127 expression was detected in the central DB at gd7.5. However, CD127+ cells were found in lateral sinusoid of gd 7.5 implantation site and closely associated with gp38+ stromal cells (Fig. 6E, F). At gd10.5, gp38+ cells were distributed mainly in B6 MLAp and the pattern was unaltered in Il7−/−, Il7ra−/− or Tslpr−/− mice (Fig. 6B, G and supplementary Fig. 3). The pattern of ER-TR7 localization overlapped with that of gp38 at both time points studied (Fig. 6C, D). Importantly however, ER-TR7+ cells were present in the central DB and had contact with gd10.5 DBA+ uNK cells (Fig. 6D inset).

Figure 6
Stromal networks and uNK cells in gd7.5 and 10.5 B6 implantation sites. Immunofluorescent reactivity using DBA lectin (red) and gp38/podoplanin (green; A, B) or ER-TR7 (green; C, D) identified decidual stromal regions expressing lymphoid stroma-associated ...

Lymphoid organ stromal cells have important roles in regulation of tolerance and immune responses [43]. We hypothesized that the distinct and dynamic stromal microdomains we observed within decidua might be components of a structural network promoting tolerance of the conceptus. We therefore asked if DB and MLAp expressed Aire, a gene associated with induction of self tolerance. As illustrated in Fig. 6I, Aire transcripts were detected in gd10.5 B6 DB and MLAp, providing support for further exploration of this hypothesis.

DISCUSSION

The goal of this study was to determine if CD127+ NK lineage progenitor cells were present in the mouse uterus during decidual differentiation. At gd6.5, DBA+ uNK cells, including small, early, non-granulated cells with an average diameter of 9 μm [7] were present but none were CD127+. This observation suggests that uNK cells do not differentiate directly from CD127+ progenitor cells migrating from LN or thymus. Alternatively, this observation could indicate that resident uterine progenitors of uNK cells do not share their steps in differentiation with NK cells found in LN, thymus, marrow, spleen, liver or blood because the earliest stages do not express CD127 [31]. The latter interpretation seems unlikely since genetic deletions that ablate peripheral NK cells, block uNK cell differentiation. A simpler hypothesis that supports conclusions drawn in human pregnancies [10], is that only relatively mature NK cells are able to leave the circulation, enter decidual tissue and become specialized uNK cells. Only 1–2% of later stage CD11bhigh CD27high/low NK cells are CD127+ in other mouse organs [9].If some of these cells enter decidua, their frequency may have been below detection by immunohistochemistry.

Unexpectedly, many uNK cells became CD127+ by gd10.5 and their frequency was unevenly distributed between the maternal mesometrial subregions. There were three fold more CD127+ uNK cells in DB than in MLAp. Morphometric and ultrastructural approaches have estimated that central DB has 10% early, 70% mature and 20% early senescent uNK cells while MLAp has 90% immature, 10% mature and no senescent uNK cells [7]. The finding that 37% of uNK cells in gd10.5 DB were CD127+, suggests not all mature uNK cells induce or sustain CD127 expression. Yadi et al, using a similar mechanical dissociation protocol and FACS analyses found no CD127+ uNK cells in gd9.5 uteri [32]. However, our immunohistochemistry and flow cytometric data clearly show developmentally regulated CD127 expression with the critical gain in expression between gd9.5 and gd10.5. Highly significant changes occur in conceptus development and in maternal physiology at gd9.5-10.5. At this time, fusion of the allantois to the chorion opens the placental circulation [33], maternal spiral arterial modification occurs [1] and declining maternal blood pressure reaches its nadir and begins increasing [34]. All of these features would alter perfusion and oxygenation of the uNK cell environment.

To begin to address whether CD127 acquisition is biologically important in uNK cell function, midgestation implantation sites in mice deleted for the receptor were studied. In comparison to control B6 implantation sites, uNK cell frequency in Il7rα−/− mice was increased in MLAp but normal in DB, suggesting that one or both of the cytokines using CD127 is important in limiting recruitment, expansion or differentiation of immature uNK cells. Because Il7−/− but not Tslpr−/− mice matched in phenotype, this action is attributed to IL-7. Dual antibody immunohistochemistry was used to address expression of IFN-γ by gd10.5 DBA+ uNK cells. B6, Il7−/− and Il7rα−/− decidua contained dually positive cells of similar appearance. Tslpr−/− decidua lacked detectable IFN-γ producing uNK cells despite abundant non-granulated and granulated uNK cells. In T cells, TSLP and IL-7 activate STAT5, the former via JAK1 and JAK2 and the latter via JAK1 and JAK3 [35]. STAT5 directly promotes Ifng transcription through binding to multiple conserved non-coding sequences identified in a region extending 60–70 kilobases upstream and downstream of the mouse Ifng locus, and binding to the Ifng promoter [36]. These epigenetic changes facilitate T-bet binding to the Ifng promoter. With STAT5 common to both cytokine signaling pathways, it is not obvious why uNK cell production of IFN-γ differs between the mutant strains studied. Eomes rather than T-bet appears to be the dominant transcription factor used by uNK cells to regulate Ifng gene expression [37]. Thus, the importance of specific regulatory elements in preventing IFN-γ production in the absence of Tslpr remains an open question, although RUNX3 and GATA3 are strong candidates [36, 38]. Failure to detect IFN-γ in uNK cells of Tslpr−/− mice was expected to result in failure of normal, mid gestational, spiral arterial modification as seen in Rag2−/−/Il2rg−/− females with uNK cells differentiated from Ifng−/− BM donors [39]. This was not observed; instead, fully modified spiral arteries were present. At present, there is no explanation for this observation except unmasking of other cytokine interactions and compensation at the fetal-maternal interface. Trophoblast cells contribute to decidual vascular remodeling but anti-cytokeratin staining of gd10.5 Tslpr−/− implantation sites to define the position of trophoblast did not reveal over invasion or trophoblast in the vicinity of the dilated arteries (BA Croy et al. unpublished). Further, our IFN-γ immunohistochemistry excluded other cells as providing compensatory IFN-γ in Tslpr−/− decidua. Thus, IFN-γ and intravascular trophoblast invasion are not the only mechanisms promoting gestational spiral arterial remodeling. A possible reason for late onset of CD127 expression in some uNK cells is suggested by the above observations. That is, that gain of CD127 by a subset of uNK cells is a “savior” signal that prolongs their viability as IFN-γ-producing cells at the time when widespread senescence is triggered in uNK cells [40]. This could be a mechanism to provide gradual withdrawal of IFN-γ from its peak levels in decidua.

TSLP was originally described as a thymic stromal cell line product; it is now recognized as prevalent in epithelial cells [41]. Previous reports on TSLP in mouse and human implantation sites have focused on trophoblast and dendritic cells [42]. To ascertain whether endometrial decidualization initially creates a trophoblast-free, mesometrial domain that not only recruits specific leukocyte subsets but may also regulate their responsiveness, we sought positional information on additional lymphoid tissue stromal markers. Expression of CD157, a molecule important in B cell and granulocyte extravasation in secondary lymphoid tissues [43, 44] was not detected. This is consistent with reports that B cells and granulocytes are very rare in gd7.5 decidua [45] especially in barrier-husbandry raised mice (BA Croy et al., unpublished). Gp38 and ER-TR7, markers of reticular fibroblasts found in T cell zones of secondary lymphoid tissue, were detected. In gd7.5 and 10.5 implantation sites of the normal and null strains investigated, both markers were strongly associated with the uterine wall (myometrium) but were not polarized mesometrially where leukocytes accumulate. Gd7.5 central DB lacked gp38 but it was strongly expressed in transient anti-mesometrial decidua and in the lateral decidua where large vascular sinuses occur. Scattered ER-TR7 reactive cells were present in gd7.5 central DB where unique endothelium expressing only VCAM-1 is reported and considered to be important for recruitment of uNK cells and intravascular trophoblasts [46]. In contrast, TSLP was localized to the central DB rather than myometrium at gd7.5. Thus, stromal markers that contribute to compartmentalization of lymphoid organs have unique distributions in gd7.5 mouse implantation sites.

At midpregnancy (gd10.5), relationships between the stroma and uNK cells had changed. Myometrium and the newly differentiated MLAp region were strongly gp38+ and ER-TR7+, suggesting regulation of uNK cell differentiation within the MLAp may share features with T cell differentiation within lymphoid tissues. Immature uNK cells of the MLAp were surrounded by gp38+ cells while more mature uNK cells in the DB, many now CD127+, had no association. CD127+ non-uNK cells, which may have been DCs [47], continued to associate with gp38+ stroma in the lateral decidua. TSLP continued to be detected at midgestation in the central DB, where ER-TR7+ cells appeared. TSLP was more abundant at gd7.5 than gd10.5 and in DB than MLAp, the inverse of relative transcript abundance suggesting post-transcriptional regulation of TSLP in implantation sites. Detection of Aire indicated that decidual stroma may have more similarities to thymic stroma than simply production of TSLP and perhaps participates in a number of ways to gestational tolerance of the genetically distinct conceptus [48]. Of note, LN endothelial cells have roles in Aire-independent tolerance induction [49]. Early decidua is an exceptionally vascular tissue and its endothelial cells may also have this capability, particularly when the conclusion suggested from our study is that the mouse uNK cell lineage is established by decidual homing of relatively mature CD127+ NK cells. In sum, this study has shown that neither IL-7 nor TSLP is essential for uNK development during normal pregnancy. However, each of these cytokines has a distinct functional impact on uNK cells in separate mesometrial regions of implantation sites.

Supplementary Material

Acknowledgments

We thank Mr. K. Hatta and Drs. G. N. Smith, C. Tayade (Queen’s University) for helpful discussions and Dr. C. Paige and Mr. S. Corfe (University of Toronto) for provision of pregnant Il7−/− uteri.

This study was supported by the Natural Sciences and Engineering Research Council, Canada and Canadian Institutes of Health Research (A. C., J. F. and J. G.), the Canada Research Chairs Program (A. C.), Province of Ontario/Queen’s Postdoctoral Fellowship (J.H. Z.), the Intramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health (Y.R. and W.J.L).

Abbreviations

DB
decidual basalis
DBA
Dolichos biflorus agglutinin
gd
gestation day
MLAp
mesometrial lymphoid aggregate of pregnancy
TSLP
thymic stromal lymphopoietin

Footnotes

Authorship:

J. Z. designed the study, performed experiments, analyzed data and wrote the manuscript, Z. C. performed experiments and analyzed results, J. H. F., Y. R., A. P. and N. A. prepared samples and performed experiments, J. H. F., W. J. L. and J. L. G. discussed the results and edited the manuscript, B. A. C. designed the study, discussed the results, and edited the manuscript.

References

1. Croy BA, van den Heuvel MJ, Borzychowski AM, Tayade C. Uterine natural killer cells: a specialized differentiation regulated by ovarian hormones. Immunol Rev. 2006;214:161–185. [PubMed]
2. Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol. 2002;2:656–663. [PubMed]
3. Chantakru S, Miller C, Roach LE, Kuziel WA, Maeda N, Wang WC, Evans SS, Croy BA. Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy. J Immunol. 2002;168:22–28. [PubMed]
4. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med. 2006;12:1065–1074. [PubMed]
5. Germeyer A, Sharkey AM, Prasadajudio M, Sherwin R, Moffett A, Bieback K, Clausmeyer S, Masters L, Popovici RM, Hess AP, Strowitzki T, von Wolff M. Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts. Mol Hum Reprod. 2009;15:39–48. [PubMed]
6. King A, Burrows T, Verma S, Hiby S, Loke YW. Human uterine lymphocytes. Hum Reprod Update. 1998;4:480–485. [PubMed]
7. Paffaro VA, Jr, Bizinotto MC, Joazeiro PP, Yamada AT. Subset classification of mouse uterine natural killer cells by DBA lectin reactivity. Placenta. 2003;24:479–488. [PubMed]
8. Aharon G, Freud MAC. Human natural killer cell development. Immunol Rev. 2006;214:56–72. [PubMed]
9. Chiossone L, Chaix J, Fuseri N, Roth C, Vivier E, Walzer T. Maturation of mouse NK cells is a 4-stage developmental program. Blood. 2009;113:5488–5496. [PubMed]
10. Male V, Hughes T, McClory S, Colucci F, Caligiuri MA, Moffett A. Immature NK cells, capable of producing IL-22, are present in human uterine mucosa. J Immunol. 2010;185:3913–3918. [PMC free article] [PubMed]
11. Di Santo JP, Vosshenrich CAJ. Bone marrow versus thymic pathways of natural killer cell development. Immunol Rev. 2006;214:35–46. [PubMed]
12. Di Santo JP. Natural killler cell developmental pathways: a question of balance. Annu Rev Immunol. 2006;24:257–286. [PubMed]
13. Luther C, Warner K, Takei F. Unique progenitors in mouse lymph node develop into CD127+ NK cells: thymus-dependent and thymus-independent pathways. Blood. 2011;117:4012–4021. [PubMed]
14. Shi FD, Ljunggren HG, La Cava A, Van Kaer L. Organ-specific features of natural killer cells. Nat Rev Immunol. 2011;11:658–671. [PMC free article] [PubMed]
15. Vosshenrich CAJ, Garcia-Ojeda ME, Samson-Villeger SI, Pasqualetto V, Enault L, Goff ORL, Corcuff E, Guy-Grand D, Rocha B, Cumano A, Rogge L, Ezine S, Di Santo JP. A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127. Nat Immunol. 2006;7:1217–1224. [PubMed]
16. Freud AG, Becknell B, Roychowdhury S, Mao HC, Ferketich AK, Nuovo GJ, Hughes TL, Marburger TB, Sung J, Baiocchi RA, Guimond M, Caligiuri MA. A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells. Immunity. 2005;22:295–304. [PubMed]
17. Chantakru S, Wang WC, van den Heuvel M, Bashar S, Simpson A, Chen Q, Croy BA, Evans SS. Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones. J Immunol. 2003;171:4011–4019. [PMC free article] [PubMed]
18. Zhang JH, Yamada AT, Croy BA. DBA-lectin reactivity defines natural killer cells that have homed to mouse decidua. Placenta. 2009;30:968–973. [PubMed]
19. Croy BA, Xie X. In vivo models for studying homing and function of murine uterine natural killer cells. Methods Mol Med. 2006;122:77–92. [PubMed]
20. Croy BA, Zhang J, Tayade C, Colucci F, Yadi H, Yamada AT. Natural Killer Cell Protocols of Methods in Molecular Biology. Humana Press; New York: 2010. Analysis of uterine natural killer cells in mice; pp. 465–503. [PubMed]
21. Fletcher AL, Seach N, Reiseger JJ, Lowen TE, Hammett MV, Scott HS, Boyd RL. Reduced thymic Aire expression and abnormal NF-κB2 signaling in a model of systemic autoimmunity. J Immunol. 2009;182:2690–2699. [PubMed]
22. Ueda Y, Kondo M, Kelsoe G. Inflammation and the reciprocal production of granulocytes and lymphocytes in bone marrow. J Exp Med. 2005;201:1771–1780. [PMC free article] [PubMed]
23. Laouar Y, Sutterwala FS, Gorelik L, Flavell RA. Transforming growth factor-[beta] controls T helper type 1 cell development through regulation of natural killer cell interferon-[gamma] Nat Immunol. 2005;6:600–607. [PubMed]
24. Demehri S, Liu Z, Lee J, Lin MH, Crosby SD, Roberts CJ, Grigsby PW, Miner JH, Farr AG, Kopan R. Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity. PLoS Biol. 2008;6:e123. [PMC free article] [PubMed]
25. Rochman Y, Spolski R, Leonard WJ. New insights into the regulation of T cells by [gamma]c family cytokines. Nat Rev Immunol. 2009;9:480–490. [PMC free article] [PubMed]
26. Croy BA, Guimond MJ, Luross J, Hahnel A, Wang B, van den Heuvel M. Uterine natural killer cells do not require interleukin-2 for their differentiation or maturation. Am J Reprod Immunol. 1997;37:463–470. [PubMed]
27. Ashkar AA, Black GP, Wei Q, He H, Liang L, Head JR, Croy BA. Assessment of requirements for IL-15 and IFN regulatory factors in uterine NK cell differentiation and function during pregnancy. J Immunol. 2003;171:2937–2944. [PubMed]
28. Ma A, Koka R, Burkett P. Diverse functions of IL-2, IL-15 and IL-17 in lymphoid homeostasis. Annu Rev Immunol. 2006;24:657. [PubMed]
29. Ye W, Zheng LM, Young J, Liu C. The involvement of interleukin (IL)-15 in regulating the differentiation of granulated metrial gland cells in mouse pregnant uterus. J Exp Med. 1996;184:2405–2410. [PMC free article] [PubMed]
30. Trundley A, Moffett A. Human uterine leukocytes and pregnancy. Tissue Antigens. 2004;63:1–12. [PubMed]
31. Crellin NK, Trifari S, Kaplan CD, Cupedo T, Spits H. Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells. J Exp Med. 2010;207:281–290. [PMC free article] [PubMed]
32. Yadi H, Burke S, Madeja Z, Hemberger M, Moffett A, Colucci F. Unique receptor repertoire in mouse uterine NK cells. J Immunol. 2008;181:6140–6147. [PubMed]
33. Anson-Cartwright L, Dawson K, Holmyard D, Fisher SJ, Lazzarini RA, Cross JC. The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta. Nat Genet. 2000;25:311–314. [PubMed]
34. Burke SD, Barrette VF, Bianco J, Thorne JG, Yamada AT, Pang SC, Adams MA, Croy BA. Spiral arterial remodeling is not essential for normal blood pressure regulation in pregnant mice. Hypertension. 2010;55:729–737. [PMC free article] [PubMed]
35. Rochman Y, Kashyap M, Robinson GW, Sakamoto K, Gomez-Rodriguez J, Wagner KU, Leonard WJ. Thymic stromal lymphopoietin-mediated STAT5 phosphorylation via kinases JAK1 and JAK2 reveals a key difference from IL-7-induced signaling. Proc Natl Acad Sci U S A. 2010;107:19455–19460. [PubMed]
36. Wilson CB, Rowell E, Sekimata M. Epigenetic control of T-helper-cell differentiation. Nat Rev Immunol. 2009;9:91–105. [PubMed]
37. Tayade C, Fang Y, Black GPVAP, Jr, Erlebacher A, Croy BA. Differential transcription of Eomes and T-bet during maturation of mouse uterine natural killer cells. J Leukoc Biol. 2005;78:1347–1355. [PubMed]
38. Yagi R, Junttila IS, Wei G, Urban JF, Jr, Zhao K, Paul WE, Zhu J. The transcription factor GATA3 actively represses RUNX3 protein-regulated production of Interferon-[gamma] Immunity. 2010;32:507–517. [PMC free article] [PubMed]
39. Ashkar AA, Di Santo JP, Croy BA. Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J Exp Med. 2000;192:259–270. [PMC free article] [PubMed]
40. Delgado SR, McBey BA, Yamashiro S, Fujita J, Kiso Y, Croy BA. Accounting for the peripartum loss of granulated metrial gland cells, a natural killer cell population, from the pregnant mouse uterus. J Leukoc Biol. 1996;59:262–269. [PubMed]
41. Ziegler SF, Artis D. Sensing the outside world: TSLP regulates barrier immunity. Nat Immunol. 2010;11:289–293. [PMC free article] [PubMed]
42. Ziegler SF, Liu Y-J. Thymic stromal lymphopoietin in normal and pathogenic T cell development and function. Nat Immunol. 2006;7:709–714. [PubMed]
43. Fritz JH, Gommerman JL. Cytokine/stromal cell networks and lymphoid tissue environments. J Interferon Cytokine Res. 2011;31:277–289. [PubMed]
44. Taylor RT, Patel SR, Lin E, Butler BR, Lake JG, Newberry RD, Williams IR. Lymphotoxin-independent expression of TNF-related activation-induced cytokine by stromal cells in cryptopatches, isolated lymphoid follicles, and Peyer’s patches. J Immunol. 2007;178:5659–5667. [PubMed]
45. Kruse A, Merchant MJ, Hallmann R, Butcher EC. Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface. Eur J Immunol. 1999;29:1116–1126. [PubMed]
46. Kruse A, Martens N, Fernekorn U, Hallmann R, Butcher EC. Alterations in the expression of homing-associated molecules at the maternal/fetal interface during the course of pregnancy. Biol Reprod. 2002;66:333–345. [PubMed]
47. Lin Y, Zhong Y, Shen W, Chen Y, Shi J, Di J, Zeng S, Saito S. TSLP-induced placental DC activation and IL-10+ NK cell expansion: Comparative study based on BALB/c x C57BL/6 and NOD/SCID x C57BL/6 pregnant models. Clin Immunol. 2008;126:104–117. [PubMed]
48. Kyewski B, Peterson P. Aire, Master of Many Trades. Cell. 2010;140:24–26. [PubMed]
49. Cohen JN, Guidi CJ, Tewalt EF, Qiao H, Rouhani SJ, Ruddell A, Farr AG, Tung KS, Engelhard VH. Lymph node–resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct antigen presentation. J Exp Med. 2010;207:681–688. [PMC free article] [PubMed]