Published online before print October 4, 2005
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* Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Canada;
Biological Sciences, Efoa/Ceufe, Alfenas, MG, Brazil;
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts; and
Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada
1 Correspondence: Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1. E-mail: ctayade{at}uoguelph.ca
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(IFN-)-mediated, pregnancy-associated, structural changes in maternal placental arteries. In CD8+ T cells and NK cells, the transcription factors T-bet and eomesodermin (Eomes) regulate maturation and effector functions, including IFN- production. No studies are reported for uNK cells. Implantation sites in T-bet null mice, which have a defect in NK cell maturation, had uNK cells normal in morphology and number and normally modified spiral arteries. As Eomes null mice are not viable, real-time polymerase chain reaction comparisons between C57Bl/6J (B6) and alymphoid (Rag20/0c0/0) mice were used to assess uNK cell expression of T-bet, Eomes, and the target genes IFN-, granzyme A, and perforin. Gestation dated (gd) uterine tissues (mixed cell composition) and 200 morphologically homogeneous, laser-capture, microdissected uNK cells of different maturation stages were used. In uterus, Eomes transcripts greatly outnumbered those of T-bet, whether donors were nonpregnant or pregnant, and increased to gd10. In uNK cells, transcripts for T-bet, Eomes, and IFN- were most abundant in mature stage cells, and transcripts for granzyme A and perforin were lower at this stage than in immature or senescent cells. Thus, Eomes dominance to T-bet discriminates regulation of the uNK cell subset from that observed for peripheral NK cells.
Key Words: cell differentiation • granzyme • interferon- • perforin • reproductive immunology • transcription factors
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14 invariant NKT cells from a common stem cell [1
]. A related factor, Eomes, promotes interferon- (IFN-) production by T-bet0/0 lymphocytes [2
, 3
]. A specialized NK cell subset [uterine (u)NK cells, rodent; decidual CD56bright cells, human], with major IFN--mediated functions [4
], appears in rodent and human uteri during the progesterone-dependent transformation of uterine stromal cells into decidua [4
, 5
]. Although T-bet and Eomes are postulated to be the "Master" switches, which impart the defining attributes of T cell lineages, neither has been examined in uNK cells.
Murine uNK cells proliferate to become abundant during early pregnancy but stop division and degenerate from mid-gestation {gestation day (gd)11; [6
, 7
]}. Between gd6 and gd10, uNK cells differentiate in a restricted region of implantation sites known as decidua basalis (DB). They progress from small, agranualur (AG) lymphocytes into heavily granulated cells producing IFN- and vascular endothelial growth factor [4
, 8
, 9
]. The earliest event, which histologically distinguishes AG murine uNK cells as a unique subset, is a plasma membrane carbohydrate modification that results in molecules expressing terminal N-acetyl galactosamine. This sugar is recognized specifically by Dolichos biflorus lectin (DBA) and is absent from splenic NK cells. The membranes enclosing the cytoplasmic granules, which subsequently develop and accumulate in uNK cells, are also DBA+ [10
]. Although the cytoplasmic granules of uNK cells contain mucin 1 [11
], perforin [12
], granzymes [13
], phosphatases, and other enzymes [7
], the relationships between uNK and the NK cells found in lymphoid tissues are not fully defined. It is known that uNK cells are interleukin (IL)-15-dependent cells [14
], which bind prolactin-like molecules [15
] and develop in uterine microdomains rich in IL-12, -18, -23, and -27 [16
], independent of influences from lymphotoxin (LT) or LTß [17
].
Many murine uNK cells are localized to the spiral arteries [18
, 19
], where their activation and production of IFN- are essential for normal implantation site development. IFN- release allows pregnancy-associated structural changes to the maternal spiral arteries that feed into each developing fetal-placenta unit [4
]. In humans, limited spiral arterial modification is associated with hypertension in the common gestational complication of pre-eclampsia [20
]. Human spiral arterial change has been attributed largely to a fetally derived, invasive, extra-villous trophoblast [20
, 21
]. However, reduced uNK cell function, in women having killer cell immunoglobulin-like receptor (KIR) haplotypes with predominantly inhibitory alleles when combined with a specific group of trophoblast-expressed, paternal human leukocyte antigen-C alleles that engage these KIR, is postulated as an underlying cause of pre-eclampsia [22
, 23
].
Although T-bet transcription has been studied widely during lymphocyte activation and migration [1 , 2 , 24 ], Eomes transcription has been more thoroughly investigated during embryonic development. Eomes is essential for differentiation of early embryonic mesoderm in Xenopus [25 ] and mice [3 ]. Gene mapping studies have shown Eomes also contributes to development of placenta [3 , 21 , 26 ] and specific regions of the forebrain [27 ] and heart [28 ]. Conceptus-based expression of Eomes is essential for development to proceed beyond the primitive streak stage. We have asked if and when T-bet and Eomes are expressed during murine uNK cell differentiation.
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c0/0 mice on a B6 background were bred under barrier husbandry (OMAFRA Isolation Unit, University of Guelph) from foundation stock kindly provided by Dr. James P. Di Santo (INSERM, Paris, France). T-bet0/0 mice on a B6 background were mated homozygously under barrier husbandry at Harvard School of Medicine (Boston, MA). All animal procedures were conducted in accordance with approved animal use protocols. For timed matings, 7- to 10-week females selected for estrus were paired with strain-matched males and subsequently examined for copulation plugs. gd0 was the morning of plug detection. Mice were killed using CO2 and uteri dissected.
Histological procedures
For histology, gd10 uteri from four each B6 and T-bet null females were fixed in 0.4% paraformaldehyde, transected into implantation sites, routinely embedded in paraffin, serially sectioned at 7 µm, mounted on glass slides, and analyzed as previously reported [4
, 14
]. For survey histology and vascular morphometry, haematoxylin and eosin (H&E) staining was used. For uNK cell enumeration, sections were stained with periodic acid Schiff’s (PAS) reagent, which reacts with glyoproteins in the granules of uNK cells. Eleven mid-saggital sections were scored for each implantation site studied (three implants/pregnancy; three pregnancies/time-point). uNK cells were counted in subregions, the mesometrial lymphoid aggregate of pregnancy (MLAp) and DB, using a 1-mm2 ocular grid at 400x magnification. Every seventh serial section was scored to avoid duplicate counting of large (>40 µm) uNK cells. Myometrial circular smooth muscle was used as the boundry separating MLAp and DB. Total vessel area and vascular lumen area were measured on 11 comparable serial sections using Optimas image analysis software, Version 6.2 (Optimas Corp., Bothwell, MA). Total vessels to lumen area ratios are presented as means ± SD.
RNA isolation from C57Bl/6J and Rag20/0c0/0 mixed cell uterine tissues
Total RNA was extracted from nonpregnant uteri and dissected DB of all viable implantation sites at gd6, -8, -10, -12, -14, -16, and -18 from each three or more B6 and Rag20/0c0/0 females per gd time-point using RNeasy mini kits (Qiagen, Mississauga, ON, Canada). Briefly, 30 mg tissue was disrupted by Kontes pestles (Fisher Scientific, Ottawa, ON, Canada) in 600 µl RLT buffer supplied with the kit containing 1% 2-mercaptoethanol. The tissue lysate was centrifuged (15,000 g, 3 min), and the cleared lysate was mixed with 700 µl 70% ethanol. The reaction mixture was loaded on an RNeasy mini column and centrifuged (8000 g, 30 s). Columns were washed (500 µl each of wash buffer), centrifuged (8000 g, 2 min), and eluted using 50 µl nuclease-free water and centrifugation (8000 g, 1 min). cDNA was prepared from these RNA samples as described below.
Tissue processing for laser-capture microdisssection (LCM)
Multiple implantation sites from B6 mice were collected on each gd at gd6, -8, -10, and -12, embedded in optimal cutting temperature compound (Thermo Shandon, Pittsburgh, PA) in a plastic biopsy mold, and snap-frozen in isopentane on dry ice. Cryostat sections were cut at 7 µm using an RNase-free blade, placed on glass slides (Fisher Scientific), and stored at –80°C for subsequent use.
Standard immunohistochemical staining protocols require relatively long incubations, which allow RNA degradation. A rapid DBA lectin-staining protocol was developed for uNK cell subset identification. All solutions, including haematoxylin, were supplemented with 0.5 unit/µl RNase inhibitor (Promega, Madison, WI). Frozen sections were thawed at room temperature (30 s) without drying, immediately fixed (30 s) in 70% ethanol in nuclease-free water (Gibco, Burlington, ON, Canada), and rehydrated in nuclease-free water (45 s). Sections were covered for 5 min with 1:10 hydrogen peroxide and Tris-Borate solution (TBS; 0.1 M, pH 7.5). Nonspecific binding was blocked using 4% bovine serum albumin in TBS (5 min), and slides were then incubated on an orbital shaker (37°C 10 min) with DBA lectin, which binds terminal N-acetyl galactosamine (Sigma Chemical Co., St. Louis, MO; 1:25 dilution in TBS). Sections were washed with TBS, and streptavidin (Sigma Chemical Co.) 1:50 in TBS was applied to each section (10 min). After rinsing with TBS, 3-3'-diaminobenzene (Sigma Chemical Co.) was applied (5 min). After rinsing in nuclease-free water, sections were counterstained for 15 s with haematoxylin (Fisher Scientific), dehydrated for 15 s in each of 75%, 95%, and 100% ethanol, and moved into histological xylene (Fisher Scientific) 5 min. Slides were air-dried (5 min), placed into a slide box over dessicator sand, and moved to the LCM station.
LCM was performed using a Pix Cell IIe (Arcturus Engineering, Mountain View, CA) and high-sensitivity-LCM caps (Arcturus Engineering). Laser pulse settings were optimized at 55 mW for 0.7 ms for uNK cell capture at 7.5 µm. Distinctly stained, brown uNK cells were captured by focal laser melting of the thermoplastic film from the cap onto individual desired cells from multiple implantation sites. On each gd studied, 200–300 uNK cells of the subtype dominant for that stage of gestation were collected as a morphologically homogeneous population. uNK cells were classified as AG precurors (DBA+, <9 µm diameter, gd6); immature (IM; DBA+, 13 µm, 
5 cytoplasmic granules, gd8); granular (G; DBA+, 20–30 µm, heavily granulated, 
10, round shape, gd10); or senescent (S; DBA+, >30 µm, apoptotic nuclei, vacuolated cytoplasm, gd12). These subtypes were defined ultrastructurally in previous studies [10
]. After capture, the cap was cleaned with a Capsure Clean Up Pad (Arcturus Engineering). Images documenting the success of the dissections were collected using the Pix Cell IIe image archiving workstation.
RNA extraction from LCM-isolated uNK cells, its amplification, and reverse transcription (RT)
RNA extraction was done using the Picopure RNA isolation kit as per the manufacturer’s instructions (Arcturus Engineering). The cap containing the dissected cells was assembled into an Extracsure assembly in an alignment tray (Arcturus Engineering), filled with 10 µl extraction buffer, covered by a microfuge tube, and incubated (42°C, 30 min). The microcentrifuge tube with Capsure-Extracsure assembly was then centrifuged (800 g, 2 min) to collect the cell extract. To this, 10 µl 70% ethanol was added, and this mixture was loaded onto a preconditioned RNA purification column. In facilitate RNA binding, samples were centrifuged (100 g, 2 min, then 16,000 g, 30 s). The RNA column was then washed (8000 g, 1 min) with 100 µl each provided wash buffers A and B. After additional wash with buffer B, RNA was eluted in 11 µl nuclease-free water and stored at –80°C.
RNA amplification was carried out using MessageAmp II antisense RNA (aRNA) kit (Ambion, Austin, TX) as per the manufacturer’s instructions. The procedure consisted of RT with oligo(dT) primer bearing a T7 promoter and in vitro transcription of DNA with T7 RNA polymerase to produce copies of aRNA for each mRNA in the sample. Briefly, to 11 µl total RNA, isolated using the picopure extraction kit, 1 µl T7 oligo(dT) primer was added, and the mixture was incubated (10 min, 70°C) in a thermal cycler and then placed on ice. RT (8 µl) master mix [2 µl 10x first-strand buffer, 1 µl RNase inhibitor, 4 µl deoxy-unspecified nucleoside 5'-triphosphate (dNTP) mix, 1 µl RT] was added to each sample, and incubation was continued (2 h, 42°C). Second-strand cDNA synthesis followed immediately using a master mix comprised of 20 µl cDNA sample just obtained, 63 µl nuclease-free water, 10 µl 10x second-strand buffer, 4 µl dNTP mix, 2 µl DNA polymerase, and 1 µl RNAase H and incubation (2 h, 16°C). The resulting cDNA was purified using a cDNA filter cartridge as per kit instructions, eluted in 20 µl nuclease-free water, and used for in vitro transcription to synthesize aRNA (16 µl-purified cDNA, 4 µl each T7 adenosine-, cytidine-, guanosine-, and uridine 5'-triphosphate 75 mM solutions, T7 10x reaction buffer, and T7 enzyme mix, 6 h, 37°C). Then, 60 µl elution solution was added, and the aRNA was purified using aRNA filter cartridge as per kit instructions. The aRNA was eluted in 100 µl nuclease-free water and stored at –80°C until further use.
aRNA obtained after RNA amplification from the LCM-isolated uNK cells as well as total RNA extracted from implantation sites were reverse-transcribed using the first-strand cDNA synthesis kit (Amersham, Bioscience, Piscataway, NJ). Total RNA (1 µg) from uterine tissue samples or 200 ng aRNA from LCM samples was brought to 20 µl using nuclease-free water, incubated in a thermal cycler (10 min, 65°C), and then mixed with 1 µl dithiothreitol, 1 µl Not-1(dT) primer, and 11 µl bulk first-strand cDNA mix. This reaction mixture was incubated (37°C, 60 min, then 90°C, 5 min) and then placed on ice. The resulting cDNA was stored at –20°C.
Cloning and sequencing of partial coding sequences of T-bet and Eomes
Polymerase chain reaction (PCR)-amplified products of T-bet and Eomes from uNK samples were cloned using the TOPO-TA cloning kit (Invitrogen, Burlington, ON, Canada) as per the manufacturer’s instructions. Plasmid DNA was purified by the Genelute plasmid DNA purification kit (Sigma Chemical Co.). Sequencing was done at the Molecular Biology SuperCentre, University of Guelph. Sequences were analyzed by BLASTN program of National Center for Biotechnology Information portal and deposited to GenBank.
Quantitative real-time PCR
Realtime PCR (LightCycler, Roche Diagnostics, Laval, QU, Canada) was used to quantify expression of target genes relative to ß-actin in uterine samples and in uNK cells. Primers were designed using the Primer 3 software program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and are given in Table 1
. Quantitect SYBR Green I PCR mix kit (Qiagen) was used for the quantification of gene expression. LightCycler reactions were performed in 20 µl total reaction volume as per the manufacturer’s instructions. PCR products were gel-purified using the Wizard DNA purification system (Promega) and/or plasmid DNA, and specific inserts were quantified and diluted serially to generate standard curves for each gene. Each reaction mixture contained 2 µl cDNAs, 10 µl SYBR Green I master mix, 1 µl sense and antisense primers (0.5 uM each), and 6 µl PCR-grade water supplied with the kit. The LightCycler program for each gene was denaturation (94°C, 15 min): PCR amplification and quantification (95°C, 10 s; 58°C, 5 s; 72°C, 20 s), and the fluorescence measurement was at specific acquisition temperatures for 5 s, repeated for 45 cycles. The melting program was 70–95ºC at the rate of 0.1°C/s with continuous fluorescence measurement, and the final cooling step was at 40°C. Data were quantified using RelQuant LightCycler analysis software. For each real-time PCR run, three replicates were used, and the runs were repeated three time to determine PCR efficiency and variance between samples. Second-derivative maximum analysis, arithmetic baseline adjustment, and polynomial calculation methods were used for the quantification. Baseline curve, melting curve, melting point, crossing point, slope error (0.1– 0.5), and correlation (r–1) were critically monitored for each round of analysis.
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Table 1. Primers Used for Real-Time Quantitative PCR
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production in uNK cells. As Eomes null mice die early in gestation, pregnancy could not be assessed in this strain, and a molecular approach was used to evaluate Eomes expression by uNK cells.
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Figure 1. Midsaggital sections of implantation site from B6 control (A) and T-bet null (B) mice at gd10 (H&E, original magnification, 50x). PL, Placenta; SA, loops of spiral arteries crossing the plane of section. (C) B6 and (D) T-bet null spiral arterial loops in MLAp (H&E, original magnification, 400x). (E and F) uNK cells in MLAp of B6 and T-bet null mice, respectively (PAS, original magnification, 1000x). (G and H) Images represent uNK cells in the DB. Here, uNK cells are much larger and more heavily granulated than in the MLAp (PAS, original magnification, 1000x). No major histological differences were found between implantation sites of T-bet null mice and their B6 congenic controls.
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Figure 2. (A) Comparison of means uNK cell/mm2 of tissue ± SD in the MLAp and in the DB between T-bet null and B6 control mice at gd10. No significant differences were found. (B) Comparison of ratios for mean total spiral arterial vessel area to mean lumen area ± SD between T-bet null and B6 control mice at gd10. There was no statistical difference.
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c0/0mice
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Figure 3. Expression of Eomes (A) and T-bet (B) in uterine tissue samples of C57Bl/6J and Rag20/0c0/0 mice during pregnancy. Level of gene expression was quantified by real-time RT-PCR (Roche Diagnostics) using ß-actin as a housekeeping gene (shown). In other studies, comparisons were made with reference to glyceraldehyde 3-phosphate dehydrogenase and to ß2 microglobulin as the housekeeping genes. The conclusions were consistent, independent of the housekeeping gene used (not shown). (B) Image represents quantification of T-bet in uterine tissues from B6 and Rag20/0c0/0. Methodological details are provided in Materials and Methods. *, Statistical significance compared with transcripts in genetically matched, nonpregnant (NP) uteri (P<0.05).
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Expression of Eomes and T-bet in DBA lectin-stained, laser-captured uNK B6 cells
Pools of 200 morphologically homogeneous uNK cells collected by LCM between gd6 and gd12 (Fig. 4
) gave good quality RNA. This cDNA prepared from uNK RNA was used to clone partial coding sequences for Eomes and T-bet. Amplified PCR products were cloned in TOPO-TA cloning vectors. BLAST search analysis revealed 100% sequence identity with the coding sequences for Eomes and T-bet. GenBank accession numbers for the deposited partial coding sequences are AY836552 (Eomes) and AY836551 (T-bet). No DBA lectin-reactive lymphocytes were present in virgin B6 uterus or in virgin or pregnant uteri of alymphoid mice (ref. [10
] and not shown).
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Figure 4. Rapidly stained DBA lectin+ NK cells in frozen gd10 B6 uterine sections used for LCM. (A) Mature-stage uNK cells, indicated by arrows. *, Test a laser shot before the capture of uNK cells. (B) Successful removal of uNK cells by melting of thermoplastic film over the cells. Arrowheads indicate empty spaces.
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Figure 5. Expression of Eomes (A), T-bet (B), IFN- (C), granzyme A (D), and perforin (E) in homogenous populations of 200 LCM-isolated uNK cells, which were classified for dissection as AG (<9 µm diameter); IM ( 13 µm, few (5) cytoplasmic granules); G (20–30 µm, heavily granulated, round shape); and S (>30 µm, apoptotic nuclei, vacuolated cytoplasm). cDNA used for expression profile quantification was prepared from amplified aRNA, obtained from captured uNK cells. Expression of Eomes, T-bet, IFN-, granzyme A, and perforin is relative to ß-actin (*, P<0.05).
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, granzyme A, and perforin in uterine samples and DBA lectin+, laser-captured uNK cells, granzyme A, and perforin was assessed. In B6 uteri, transcripts for IFN- increased in early gestation to peak sharply at gd10 (P<0.05, compared with IM uNK at gd8). From gd14 to gd18, IFN- transcription was relatively less than in virgin uterus (Fig. 6
6A
). In B6 uNK cells, IFN- transcription was not significant until cytoplasmic granules were present. It peaked in the mature cells and fell rapidly with cell senescence (Fig. 5C)
. Peak relative transcription was 10 logs higher in uNK cells than in uterine mix cell samples, indicating uNK cells are a significant source of IFN- transcripts within the uterus. In uterine tissue sections from alymphoid mice, IFN- transcripts were low and relatively stable between virgin animals and those at any day of gestation (Fig. 6A)
.
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Figure 6. Quantification of IFN- (A), granzyme A (B), and perforin (C) in uterine tissues obtained from C57Bl/6J and Rag20/0c0/0 mice. Target gene expression is relative to ß-actin (*, P<0.05, compared with nonpregnant).
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Transcriptional elevation of Eomes occurred in fully mature uNK cells, and elevation in T-bet expression commenced in more IM cells just beginning to acquire cytoplasmic granules. T-bet expression continued to rise as the cells matured. Both transcription factors declined significantly when uNK cells became postmitotic (S). From gd8 to gd12, implantation sites contain all four subtypes of uNK cells in varying relative quantities. IM stages are located murally, and more mature stages predominate in the DB. Thus, levels of transcription found in the uterine mix tissue samples reflect differing mixtures of the uNK cells studied as homogenous cell populations and contributions from nonlymphoid cells, revealed by the studies of pregnancy in alymphoid mice.
For Eomes, analyses of B6 mixed cell uterine tissues gave a pattern of expression similar to uNK cells but at much lower relative transcript frequency. For T-bet, not only were transcripts in the uterus samples relatively lower than in uNK cells, but the pattern of T-bet expression in uterus differed to that in uNK cells with a continued increase from gd10 to gd12. After an abrupt decline at gd14, T-bet transcripts began to increase again gradually. This pattern may reflect increasing T-bet transcription in a second cell population that overlaps with uNK cell transcription. Summed transcripts from two or more sources may provide the later gd12 peak and continued rise in T-bet expression from gd14 to gd18. The postulate that this second cell type may be a T cell cannot be sustained, as increasing T-bet expression from mid-gestation to term was found in uterine samples from alymphoid mice (Fig. 3B) . In these mice, activated monocytes, macrophages, or dendritic cells are more probable sources of elevated T-bet mRNA [29 ]. Elevated T-bet transcription probably reflects activation of these cells, as only neutrophils are thought to increase in number in late gestation implantation sites [30 ].
Transcripts for granzyme A and perforin were elevated in IM uNK cells, suggesting that T-bet is a more probable initiator of transcription for these genes than Eomes. The sudden drop in levels of transcripts for these effector molecules in mature uNK cells was not anticipated, as T-bet is reported to be regulated by IFN-, which is maximally present in implantation sites on gd10 [4
]. However, the negative fluctuation occurred as high levels of IFN- were initiated. Spiral arterial modification is recognized histologically at gd9.5–10 [31
], suggesting a switch in effector molecule transcription and cell function specific to that time. The rebound of transcription for granzyme A and perforin in uNK cells is consistent with morphological studies showing that postmitotic uNK cells become exceedingly heavily granulated before disintegrating [7
, 18
]. The low levels of granzyme A and perforin expression in uteri from alymphoid mice indicate minor transcriptional contributions by activated, nonlymphoid cells.
The pattern of IFN- transcription is consistent with enzyme-linked immunosorbent assay results previously reported. Rising protein concentration was found from gd6–10 followed by rapid decline. Low levels of IFN- protein are found in alymphoid mice and have been attributed to nonlymphoid cell types such as macrophages and neutrophils [4
, 32
]. The steep gain in transcription after gd8 suggests Eomes rather than T-bet as the more effective or dominant transcription factor used by uNK cells. Eomes dominance and T-bet transcription below that of ß-actin have been reported previously for activated, cultured CD8+ T cells studied by Northern analysis [2
]. Uterine samples from an alymphoid mouse showed low levels of Eomes transcription. These transcripts could arise from trophoblast cells found in microscopic islands within the mouse DB [26
]. Alternatively, Eomes is induced muscle during development. It is possible that the unusual muscle coat retained in the spiral arteries of alymphoid mice contributes to Eomes transcripts.
A major function attributed to Eomes is specification of the dorsal axis and promotion of meosdermal cell migration [26 ]. T-bet expression is also reported to confer migratory capability in human T cells through induction of CXC chemokine receptor 3 (CXCR3) [24 ]. uNK cells accumulate in the region of the uterus, which would be most dorsally specified. The uterus is suspended embryologically by the dorsal mesentery, and uNK cells are thought to migrate to the mesometrial uterus during decidua induction [33 ]. In humans, it is postulated that CXCR3 is a major receptor involved in localization of uNK cells within implantation sites [34 35 36 ]. Thus, it is possible that expression of Eomes and T-bet in uNK cells has supplementary roles to the activation of transcription of immune effector molecules. Use of Eomes knockout mice to examine the centrality of this transcription factor to uNK cell differentiation will be difficult. Eomes null mice die at approximately gd6–7 and are not available as adult females for study. This early, primitive streak stage loss also precludes lymphocyte complementation assays of using gd10 fetal liver or the aortic-gonadal-mesonephros primordium [37 , 38 ]. Production of somatic chimeras by aggregation of morulae from alymphoid mice with Eomes null embryonic stem cells would be an approach for evaluation of this question. The present study, demonstrating pregnancy-regulated expression of Eomes and T-bet in uNK cells, reinforces earlier findings that uNK cells share many features with NK cells in lymphoid organs, but it does not assist in defining where differences arise. Are uNK cells normal NK cells simply differentiating in an unusual environment (decidua), or are their precursors rare cells in blood and the only subset able to populate the decidualizing endometrium [19 ]? Our work provides, however, a unique protocol for recovery of high-quality RNA from rodent uNK cells using DBA lectin identification. This should advance studies of uNK cells, a population that has been refractory to successful culture. Our study also documents that caution will be necessary when trying to extract information concerning the functions of uNK cells from endometrial tissue sections, particularly human endometrial needle biopsies. Markedly different levels of gene transcription were found in uNK cells and in the mixed cell mixtures. In addition, the mixed stages of differentiation of uNK cells within a single biopsy will provide confounding data, as the transcription factors and their target genes are not stably expressed from day to day of pregnancy.
Received March 11, 2005; revised July 29, 2005; accepted August 16, 2005.
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14i NKT cells Immunity 20,477-494[CrossRef][Medline] contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy J. Exp. Med. 192,259-270 J. Reprod. Immunol. 49,33-47[CrossRef][Medline] in lymphoid and myeloid cells Proc. Natl. Acad. Sci. USA 98,15137-15142
26 mice J. Exp. Med. 187,217-223 contributes to the normalcy of murine pregnancy Biol. Reprod. 61,493-502This article has been cited by other articles:
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