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Originally published online as doi:10.1189/jlb.0204090 on June 3, 2004

Published online before print June 3, 2004
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(Journal of Leukocyte Biology. 2004;76:667-675.)
© 2004 by Society for Leukocyte Biology

Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-ß

Mikael Eriksson*, Sarah K. Meadows*, Charles R. Wira{dagger} and Charles L. Sentman*,1

* Departments of Microbiology & Immunology and
{dagger} Physiology, Dartmouth Medical School, Lebanon, New Hampshire

1 Correspondence: Department of Microbiology & Immunology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756. E-mail: charles.l.sentman{at}dartmouth.edu


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ABSTRACT
 
Natural killer (NK) cells are a major population of lymphocytes in the human endometrium (EM), and NK cells can be a significant source of cytokines that alter local immune responses. The aim of this study was to determine the expression of NK cell receptors in situ and to test whether uterine NK (uNK) cells produce cytokines and how this activity may be regulated by transforming growth factor-ß (TGF-ß). We observed that human uNK cells were CD56+, CD3–, CD57–, CD9+, CD94+, killer inhibitory receptor+, and CD16+/– in situ by confocal microscopy. We examined cytokine production by uNK cells and uNK cell clones derived from human EM. Stimulation of uNK cells with interleukin (IL)-12 and IL-15, both of which are expressed in the human EM, induced interferon-{gamma} (IFN-{gamma}) and IL-10 production. IFN-{gamma} production by uNK cell clones was completely inhibited by TGF-ß1 in a dose-dependent manner with an inhibitory concentration 50% value of 20 pg/ml. IL-10 secretion by uNK cell clones was also inhibited by TGF-ß1 at similar concentrations. Furthermore, blocking endogenous TGF-ß in fresh human endometrial cell cultures increased the production of IFN-{gamma} by uNK cells. These data indicate that uNK cells have a unique phenotype that is distinct from blood NK cells. Further, data demonstrate that uNK cells can produce immunoregulatory cytokines and that inhibition of uNK cells by locally produced TGF-ß1 is a likely mechanism to regulate NK cell function in the human EM.

Key Words: cytokines • cellular activation • reproductive immunology


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INTRODUCTION
 
Natural killer (NK) cells play an important part in the innate immune system and were first defined functionally by their ability to kill certain tumors and virally infected cells without a requirement for major histocompatibility complex restriction or previous immunization [1 ]. NK cells produce immunoregulatory cytokines that contribute to the early host defense against several types of viruses, bacteria, and parasites. In humans, ~10% of peripheral blood lymphocytes are NK cells [2 ], which can be defined phenotypically by the expression of CD56 and the absence of CD3, and NK cells fall into two distinct subsets according to their surface density of CD56. The majority of NK cells in human blood has low CD56 expression (CD56dim) and expresses high levels of Fc receptor for immunoglobulin G (IgG; Fc{gamma}R)III (CD16) and CD57 [3 ]. A small subset of blood NK cells (~10%) expresses high levels of CD56 (CD56bright), low or no CD16, and lack CD57 expression. Uterine NK (uNK) cells account for a large percentage of leukocytes in the human endometrium (EM) and have similar expression of CD56, CD16, and CD57 as the CD56bright blood NK cell subset [3 , 4 ].

The human uterine EM is a complex mucosal tissue that has a unique immune cell component that is regulated by sex hormones throughout the menstrual cycle [5 , 6 ]. The EM must be prepared to respond to potential pathogen challenges yet be able to control immune cell responses to allow the development of a semi-allogeneic fetus. In the nonpregnant state, there is a tightly controlled influx, spatial compartmentalization, and regulation of immune cells [7 , 8 ]. The EM contains macrophages, NK cells, T cells, B cells, and neutrophils in contact with a variety of stromal and epithelial cells. The interplay among these different cell types and their roles in defense against pathogen invasion in this specialized tissue are poorly understood. Unlike the murine uterus, uNK cells in the human uterus are found in large numbers spread throughout the EM with increasing numbers as the menstrual cycle progresses [9 10 11 ].

NK cells express receptors for monocyte-derived cytokines (monokines) and can produce several cytokines in response to monokine stimulation. NK cells from peripheral blood have been shown to produce interferon-{gamma} (IFN-{gamma}), granulocyte macrophage-colony stimulating factor, interleukin (IL)-10, IL-13, and tumor necrosis factor-ß, and there is evidence that CD56bright NK cells are the major producers of cytokines by NK cells in response to monokines [12 ]. In contrast, CD56dim NK cells are more cytotoxic against tumor cells and produce smaller amounts of cytokines upon monokine stimulation than CD56bright NK cells [12 13 14 ].

Members of the transforming growth factor-ß (TGF-ß) family are powerful, immunoregulatory agents that act on a range of different target cells [15 16 17 18 ]. TGF-ß proteins are produced as inactive precursors that can bind to extracellular matrix and cell-surface proteins, where they are activated [19 ]. Low pH conditions can activate latent TGF-ß, but the mechanisms that regulate TGF-ß activation remain unclear. TGF-ß has been demonstrated to have activating and inhibitory effects on many parts of the human immune system, including cellular cytotoxicity, proliferation, cytokine production, and differentiation [20 ]. TGF-ß1 can inhibit proliferation of T cells [15 , 16 ], decrease antigen presentation [17 ], inhibit macrophage activity [18 ], and suppress cytotoxic activity, cytokine production, and proliferation in peripheral blood NK cells [21 ]. However, stimulatory effects from TGF-ß, such as murine T cell proliferation [22 ] and IL-2 release from human effector cells [23 , 24 ], have been reported.

In this study, we examined phenotype and function of uNK cells in the EM of nonpregnant women. In situ and in vitro analyses show that uNK cells have a unique phenotype compared with blood NK cells. We show that isolated human uNK cells produce cytokines and that these can be suppressed in a dose-dependent manner by TGF-ß1. In addition, we demonstrate that neutralizing endogenous TGF-ß promotes IFN-{gamma} production by uNK cells. Taken together, these results indicate that local TGF-ß-mediated inhibition is a mechanism that regulates NK cell-derived cytokine production in the human EM.


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MATERIALS AND METHODS
 
Isolation of human endometrial cells
Endometrial tissue specimens were obtained from women undergoing hysterectomy for various gynecological disorders. We used samples from 34 patients with an average age of 46 (±11) years. Initial patient diagnosis included fibroids, pelvic pain, menorrhagia, and prolapse. The tissue samples that we used were distal to any pathological changes. We found no differences in experimental outcomes between those patients that had been given hormonal therapy presurgery and those that had not been given hormonal therapy. We used a cell dispersion method that used an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase, followed by a mesh screen to facilitate cell dispersion [25 ]. Any red blood cells present were eliminated from the endometrial cells by treatment with lysis buffer (NH4Cl/Tris HCl) for 5–10 min at room temperature. Blood cell contamination of these endometrial tissue cells was less than 2% [25 ]. The isolated cells were cultured or used for experimental treatments directly. All human studies were done with approval of the Dartmouth Institutional Review Board (Hanover, NH).

Isolation of peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated from healthy donors and from patients undergoing hysterectomy who had consented to donate blood. Cells were separated on NycoprepTM or LymphoprepTM gradients according to protocols provided by the manufacturer (Axis-Shield, Oslo, Norway).

Generation of uNK cell clones
The enzymatically isolated EM cells were cultured in 500 U IL-2/ml for 2–3 days to allow for recovery of CD56 surface expression in complete media [RPMI 1640 supplemented with 2-mercaptoethanol (50 µM), penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), and 5% human serum]. Cells were harvested and stained for CD45, CD56, and CD3 surface antigens. uNK cells were then sorted using a FACStarTM cellsorter (Becton Dickinson, San Jose, CA) by gating on the CD45+CD56+CD3– cells. Sorted uNK cells were cloned using standard NK cell cloning procedures [26 ]. Briefly, sorted uNK cells were plated at one to three cells/well in U-bottom 96-well plates together with irradiated (100 Gy) feeder cells (105 allogeneic PBMCs together with 104 RPMI 8866 cells or 104 DAUDI cells), supplemented with 1 µg/ml phytohemagglutinin. After 10 days, the wells were examined for growth of clones. Cells from positive wells were expanded further and maintained in 500 U IL-2/ml for the remainder of their culture.

Antibodies and reagents
The following fluorescein isothiocyanate (FITC)-conjugated antibodies were used: anti-CD16 (3G8), anti-CD57 (TB01), and anti-CD45 (HI30; Caltag, South San Francisco, CA) and anti-NKB1 (DX9), anti-CD158b (CH-L), and anti-CD94 (HP-3D9; PharMingen, San Diego, CA). Biotin-conjugated anti-CD3 and allophycocyanin (APC)-conjugated anti-CD3 were obtained from Caltag. R-Phycoerythrin-conjugated anti-CD56 (B159), APC-conjugated anti-IFN-{gamma} (B27), mouse IgG1 (MOPC-21) control, and streptavidin peridinin chlorophyll protein were purchased from Becton Dickinson. Streptavidin RED670TM was obtained from Invitrogen (Carlsbad, CA). Anti-TGF-ß1, -ß2, and -ß3 (1D11) monoclonal antibodies (mAb) and human recombinant (hr)TGF-ß1 were purchased from R&D Systems (Minneapolis, MN). hrIL-12 and IL-15 were obtained from PeproTech (Rocky Hill, NJ). Human FcR blocking reagent (Cohn’s fraction) was obtained from Sigma Chemical Co. (St. Louis, MO). For immunohistochemistry (IHC), FITC-conjugated anti-CD57, anti-CD158b, anti-CD94, and anti-CD14 were from PharMingen; IgG1, from Caltag; Cy3-goat anti-mouse IgG, Jackson Immunoresearch Laboratories (West Grove, PA); Cy5-anti-CD8 and Cy5-anti-CD3, Exalpha (Watertown, MA); and unconjugated anti-CD56, PharMingen.

IHC and confocal analysis
Live tissue sections of human EM were stained with fluorescently conjugated antibodies at 4°C, fixed, and imaged by confocal microscopy as described [8 ]. Briefly, thin (~80 µm), live endometrial tissue pieces were stained with unlabeled CD56 overnight at 4°C. All staining was done in the dark at 4°C in staining buffer (phosphate-buffered saline, 1% fetal calf serum, 10% Cohn’s fraction). After three washes, cells were labeled for 2 h at 4°C with Cy3-conjugated goat anti-mouse sera. After three washes, cells were stained for 4 h to overnight with FITC-conjugated antibodies and Cy5-conjugated antibodies against cell-surface markers. After extensive washing, samples were fixed in 2% paraformaldehyde and stored at 4°C in the dark. Sections were mounted onto microscope slides in antifade-mounting media (Molecular Probes, Eugene, OR) under coverslips. Cells were analyzed using a BioRad MRC 1024 laser scanning confocal system (BioRad, Hercules, CA) or a Zeiss LSM 510 meta laser scanning confocal system (Carl Zeiss Inc., Thornwood, NY). All samples were stained and analyzed as duplicate samples. Data were collected as a z-series stack of images with 2.5 µm steps over a 25- to 30-µm range. Five to seven image stacks from each pair of stained tissue sections were collected. The advantage of using z-series analysis is that we obtained two or three images from each cell to ensure identification. Isotype controls were used to ensure specific staining for each procedure. Projections of the entire image stack were prepared, and individual cells were counted using Image J software. The percent of NK cells (CD56+CD3– cells or CD56+CD8– cells) that expressed a given receptor was calculated from at least 200 NK cells/sample. The number of CD56+ cells expressing CD3 or CD8 was less than 0.1% of CD56+ cells.

Flow cytometry
A FACScaliburTM (Becton Dickinson) was used for flow cytometric analysis of cell-surface staining of all samples. For intracellular IFN-{gamma} analysis, freshly prepared endometrial cells were cultured at 3 x 105 cells/well in 96-well U-bottom microtiter plates for 18 h. Brefeldin A (10 µg/ml) was added to each well 5 h prior to harvesting to allow for accumulation of intracellular proteins. Cells were harvested and stained for CD3, CD45, and CD56, fixed, and permeabilized with saponin (0.1%). The cells were then stained intracellularly with anti-IFN-{gamma} mAb or IgG control, washed, and analyzed by flow cytometry.

Stimulation of cells with IL-12 and IL-15
uNK clones
NK cell clones were prepared as described previously [26 ] and plated at 5 x 104 cells per well in microtiter plates in triplicate wells. After stimulation by IL-12 (10 ng/ml)/IL-15 (100 ng/ml) in culture for 48 h or 72 h, cell-free supernatants were harvested, and the concentration of IFN-{gamma} or IL-10 was determined using specific Duoset enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems.

Fresh EM cells
Cells from enzymatic digestion of human endometrial tissue were seeded at 2 x 105 cells/well in microtiter plates. After 72 h of IL-12/IL-15 stimulation, supernatants were harvested and analyzed for the production of IFN-{gamma} using a hIFN-{gamma} Duoset ELISA kit from R&D Systems.

Statistics
Statistical comparisons were done using a two-sided, paired t-test as indicated. P < 0.05 was considered statistically significant.


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RESULTS
 
NK cells in the human uterine EM have a unique phenotype
To determine the phenotype and receptor expression of NK cells within the EM, we used in situ confocal microscopy. Whole human endometrial tissue was dissected into thin sections (~80 µm), stained with fluorescently labeled antibodies against cell-surface receptors, and fixed. Analysis by confocal microscopy demonstrated that uNK cells were CD56+, CD3–, CD8– cells. CD56+ cells, which expressed CD3 or CD8, were less than 0.1%. NK cell receptors were detected on uNK cells, and CD57 was absent on uNK cells and present on T cells (Fig. 1 and data not shown). A comparison of cell-surface molecule expression of uNK cells in situ (IHC), uNK cells after short-term culture (uNK), dim CD56, CD3– blood NK cells (CD56dim), and bright CD56, CD3– blood NK cells (CD56bright) is shown in Figure 2 . uNK cells express CD56 at high levels, and 78% on average expressed CD94 (Fig. 2A) . About 10–15% of CD56bright blood NK cells express CD16, whereas on average, 17% of uNK cells express CD16 with a range from 6% to 24% (Fig. 2C) . Thus, uNK cells in EM have a phenotype that is more similar to CD56bright blood NK cells than CD56dim blood NK cells. However, uNK cells also expressed the killer inhibitory receptors (KIRs) CD158b (45%) and NKB1 (34%), and their expression of these receptors was more similar to CD56dim blood NK cells (Fig. 2B and 2D) . Approximately 6% of CD56bright NK cells express KIR molecules. The high percentage of KIR-expressing uNK cells may reflect selective recruitment of KIR+CD56bright NK cells, expansion of KIR+ NK cells within the EM, or induction of KIR molecules on NK cells after they have entered the EM.



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  		Figure 1. uNK cells express NK cell receptors in situ. Live tissue sections of human EM were stained with fluorescently conjugated antibodies at 4°C, fixed, and imaged by confocal microscopy. Green color indicates positive cells in endometrial tissue samples that were stained with FITC-conjugated anti-CD158b (A), anti-CD94 (B), anti-CD14 (C), and anti-CD57 (D). Tissue sections were stained with Cy5-anti-CD3 (blue; A and B), or Cy5-anti CD8 (blue; C and D). (A–D) Staining with an unlabeled anti-CD56 mAb followed by a Cy3-goat anti-mouse IgG (red). (E) Control staining with FITC–IgG1 (green), Cy5-IgG (blue), and unlabeled IgG followed by Cy3-goat anti-mouse IgG (red).



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  		Figure 2. NK cell receptors are expressed on uNK cells. Panels show the proportion of NK cells expressing CD94 (A), NKB1 (B), CD16 (C), and CD158b (D). CD56bright and CD56dim cells from fresh PBMCs (A, B, and D) and cultured uNK cells isolated from endometrial tissue (A–D) were analyzed by flow cytometry and EM tissue sections (A–D), by IHC. Histograms for each NK cell receptor are from three to eight samples analyzed.

A recent report has described expression of CD9 on decidual NK (dNK) cells but not on blood NK cells [27 ]. We have analyzed uNK cells and found that they express high levels of CD9 (Fig. 3A ), whereas CD56bright and CD56dim blood NK cells expressed little or no CD9 (Fig. 3B) , even after in vitro activation (Fig. 3C) . These data suggest that expression of CD9 is a marker that distinguishes uNK cells and dNK cells from blood NK cells and that the increase in CD9 expression is a result of factors within the unique endometrial environment and not linked to the uterus at the time of decidualization and early pregnancy.



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  		Figure 3. CD9 is expressed on uNK cells. Endometrial cells were cultured for 4 days and were stained for CD9, CD56, and CD3. (A) The CD9 expression profile for uNK cells gated on CD56+CD3– cells. Bottom two panels show CD9 expression on fresh (B) and IL-2-activated (C) CD56brightCD3– and CD56dimCD3– blood NK cells. Data are representative of three individuals for endometrial NK cells (A) and four individuals for blood cells (B and C).

uNK cell clones produce IFN-{gamma} and IL-10 after stimulation with IL-15 and IL-12
To obtain uNK cell clones from human EM, single CD56+CD3– cells were isolated from endometrial tissue samples and cloned using standard NK cell cloning procedures [26 ]. uNK cells clones were CD56+CD3–CD57–. Many uNK cell clones expressed CD94 (80%), NKB1 (50%), and CD158b (15%), and at least one of these receptors was expressed on every clone (data not shown). These findings are consistent with our data, which show that many NK cells in EM also express CD94 and KIRs in situ (Figs. 1 and 2) . In addition, most uNK cell clones expressed CD9 (88%) in a similar manner as isolated uNK cells. Taken together, these data indicate that uNK cell clones have similar cell-surface marker expression as uNK cells in situ. In addition, we tested the cytotoxic activity of these uNK cell clones and found that they lyse K562 cells but not RPMI 8866 cells (data not shown).

To test whether uNK cell clones could produce cytokines upon stimulation, uNK cell clones were stimulated with IL-12 (10 ng/ml) and IL-15 (100 ng/ml), as these cytokines have been shown to be important for regulating NK cell responses [12 , 28 , 29 ]. As shown in Figure 4 , all uNK cell clones produce significant amounts of IFN-{gamma} and IL-10 after stimulation with IL-15 and IL-12. These studies suggest that uNK cells have the potential to be a significant source of cytokines.



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  		Figure 4. TGF-ß1-mediated inhibition of cytokine production by uNK cell clones (5x104 cells/well), which were cultured in media only (solid bars), together with IL-12/IL-15 only (open bars), or IL-12/IL-15 together with 2000 pg/ml TGF-ß1 (hatched bars). Supernatants were harvested and assayed by ELISA for (A) IFN-{gamma} (48 h) or (B) IL-10 (72 h). Phorbol 12-myristate 13-acetate+ ionomycin treatment was included (B; shaded bars). Representative of data from >20 clones from three donors. (C) uNK cell clones (5x104 cells/well) were cultured with IL-12/IL-15 in the presence of variable amounts of TGF-ß1 for 48 h at 37°C. Supernatants were harvested and assayed for IFN-{gamma} production. Representative of two experiments from two different donors. M and A1 are two uNK cell clones.

TGF-ß1 suppresses cytokine production by uNK cell clones
As TGF-ß1 is present in human EM [30 ], we tested the effect of TGF-ß1 on cytokine-stimulated uNK cell clones and found that incubation in 2000 pg/ml TGF-ß1 completely inhibits production of IFN-{gamma} by all uNK cell clones stimulated with IL-12/IL-15 (Fig. 4A) . The production of IL-10 was also significantly lowered in the presence of TGF-ß1 (Fig. 4B) . Viability analysis of uNK cells indicated that the reduction in cytokine production was not a result of a toxic effect of TGF-ß1 on uNK cells (data not shown). The data in Figure 4C illustrate the dose-dependent inhibition of IFN-{gamma} production by TGF-ß1. When uNK cell clones were stimulated with IL-12/IL-15 for 48 h in the presence of increasing amounts of TGF-ß1 (0–2000 pg/ml), all clones tested from several patients were inhibited in a concentration-dependent manner with an inhibitory concentration 50% (IC50) value of ~20 pg/ml. In parallel studies using blood NK cell clones as a control, the IC50 value for inhibition of IFN-{gamma} production was similar to uNK clones (data not shown). These data indicate that TGF-ß1 has the potential to prevent cytokine production by uNK cells at low doses.

Inhibition of TGF-ß increases IFN-{gamma} production by fresh endometrial cells stimulated by IL-12/IL-15
To test whether TGF-ß inhibition in human EM is a mechanism that suppresses local uNK cell production of IFN-{gamma}, we cultured fresh human endometrial cells with IL-12/IL-15 for 72 h in the presence of blocking anti-TGF-ß mAb, control IgG1, or media only. This anti-TGF-ß mAb blocks the three TGF-ß isoforms: TGF-ß1, -ß2, and -ß3. We found in every patient sample tested (seven samples) that anti-TGF-ß mAb increased the amount of IFN-{gamma} produced by endometrial cells, with an average increase of 61% (range: 15–120%; Fig. 5A and 5B ). No IFN-{gamma} was detected from endometrial cells cultured in media alone (Fig. 5A) . These data indicate that the presence of endogenous TGF-ß in the EM can inhibit production of IFN-{gamma} by endometrial cells.



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  		Figure 5. Increased cytokine production by "fresh" EM cells in the presence of blocking TGF-ß mAb. (A) Fresh human endometrial cells (3x105 cells/well) were cultured with IL-12/IL-15 for 72 h in the presence or absence of blocking anti-TGF-ß mAb. Cell-free supernatants were then assayed for IFN-{gamma} by ELISA. **, P < 0.01; paired t-test compared with control (IL-12/IL-15 only). (B) Relative increase in IFN-{gamma} release by IL-12/IL-15 activated EM cells in the presence of anti-TGF-ß mAb (P<0.05; t-test) from seven different endometrial cell samples.

Fresh uNK cells produce IFN-{gamma} within the EM in response to IL-12 and IL-15 stimulation
To determine whether freshly prepared uNK cells produce IFN-{gamma}, we analyzed the production of IFN-{gamma} by uNK cells in response to cytokine stimulation. Fresh, human endometrial cells were cultured in the presence of IL-12 and IL-15 prior to the analysis of intracellular IFN-{gamma} production. As shown in Figure 6 , IFN-{gamma} production was observed in 5.9% of CD56+CD3– NK cells from endometrial cells cultured in the presence of IL-12/IL-15 (Fig. 6A) compared with 0.7% of uNK cells from endometrial cultures grown in media without cytokine stimulation (Fig. 6D) . In the presence of anti-TGF-ß mAb, the proportion of IFN-{gamma}-producing uNK cells increased about two-fold (from 5.9% to 9.6%; Fig. 6A and 6B ). This was observed in all five patients tested (Fig. 6E) . Analysis of IFN-{gamma} production by IL-12/IL-15-stimulated blood NK cells (within PBMCs or enriched blood NK cells) showed on average (n=4) that 7% of blood CD56bright NK cells produced IFN-{gamma} after stimulation (data not shown). The increase in the number of IFN-{gamma}-producing NK cells among IL-12/IL-15-stimulated EM cells in response to anti-TGF-ß mAb was evident from all patients examined (Fig. 6E) and varied between 1.6- and 3.1-fold (mean: 2.02, n=5, P<0.0001). The presence of IgG1 control mAb did not increase the IFN-{gamma} production by the NK cells in response to IL-12/IL-15 (Fig. 6C) . These data demonstrate that uNK cells are a source of IFN-{gamma} in the EM upon stimulation and that the presence of endogenous TGF-ß within EM can affect the ability of uNK cells to respond to monokine stimulation.



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  		Figure 6. Increased IFN-{gamma} production by fresh uNK cells in the presence of blocking anti-TGF-ß mAb. Total endometrial cells (3x105 cells/well) were isolated and cultured with IL-12/IL-15, and after 24 h, the cells were analyzed for intracellular IFN-{gamma} production. The numbers indicate the percentage of IFN-{gamma}-positive NK (CD45+CD56+CD3–) cells from EM cells cultured in the presence of IL-12/IL-15 alone (A) or together with anti-TGF-ß mAb (B) or control IgG1 (C) or from media only without IL-12/IL-15 (D). (E) Relative increase in the number of IFN-{gamma}-producing uNK cells within IL-12/IL-15-stimulated EM cells from five different donors in response to anti-TGF-ß mAb (P<0.0001; t-test).


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DISCUSSION
 
This study examines the phenotype and functional responses of human uNK cells. We described the unique phenotype of uNK cells within whole EM and demonstrated that uNK cells were able to produce cytokines upon stimulation and that cytokine production was inhibited by endogenous TGF-ß. These data indicate that TGF-ß is likely an important regulatory mechanism for controlling uNK cell function in human EM.

NK cells are found throughout the human EM and increase in number during the secretory phase of the menstrual cycle [9 10 11 ]. The term uNK cell generally refers to any NK cell found within the uterus without regard to pregnancy. We use the term uNK cell to refer to those NK cells in the EM and the term dNK cell to identify those NK cells found in decidua and throughout pregnancy. uNK cells are most probably the precursors of dNK cells. Pregnancy induces a set of hormonal and cellular events that results in unique immune cell phenotypes and functions, so the factors that regulate NK cell phenotype and function are likely different in EM and decidua. Thus, we believe these two subsets of NK cells should not be considered as identical. uNK cells can account for a significant number of leukocytes within the EM, and the uNK cells may be derived from the CD56bright subset of blood NK cells. Unlike murine uNK cells that only appear after decidualization and are localized near the metrial gland, the presence of human uNK cells in the uterus during the menstrual cycle is independent of decidual formation [9 10 11 ]. Thus, the role of uNK cells and the mechanisms that recruit and activate uNK cells may be quite different between humans and mice as well as between pregnant and nonpregnant women.

We examined the phenotype of uNK cells in situ and found that they expressed a unique set of cell-surface markers that distinguishes them from blood NK cells. These uNK cells were CD56+, CD57–, CD3–, CD94+, and CD9+. Further, CD9 was expressed on all uNK cells (our data) and on dNK cells [27 ] but was absent on blood NK cells. CD9 is a member of the tetraspanin family of proteins, has been suggested to be involved in various cellular and physiological functions [31 , 32 ], and is a useful marker for differentiating blood NK cells from uNK cells. In addition, many uNK cells expressed KIR molecules, and a low percent expresses CD16 in situ. The percentage of peripheral blood NK cells that express KIR molecules is donor-dependent, such that some individuals tend to express more KIR molecules on their blood NK cells than others [33 , 34 ]. Our data indicate that uNK cells are not identical to CD56dim or CD56bright blood NK cell subsets. This unique expression of cell-surface molecules may be a result of selective recruitment of rare blood NK cell subsets or expression of new molecules within the EM as a result of unique factors within the microenvironment.

Blood NK cells and uNK cells, as indicated in our study, have been shown to be a significant source of cytokines upon monokine stimulation [35 36 37 ]. Data indicate that several of the monokines that activate NK cells are present in the human EM [38 39 40 41 ]. However, inflammation must be tightly regulated in this tissue to allow reproduction to take place, so mechanisms must exist that control the activity of uNK cells and limit the production and/or action of potent, proinflammatory cytokines.

NK cell-derived IFN-{gamma} is a proinflammatory cytokine that is important as a defense mechanism against a variety of pathogens, and IFN-{gamma} production by NK cells has also been shown to be important for the restructuring of arteries in the murine placenta during pregnancy [42 ]. However, unregulated IFN-{gamma} production may be detrimental to successful pregnancy [43 ]. CD56+ NK cells in EM do not express IFN-{gamma} spontaneously, although cytokines that can induce IFN-{gamma} production by blood NK cells are found in the human EM. The IFN-{gamma} present in fresh human EM has been found to be localized to PMNs [44 ].

The ability of TGF-ß to alter NK cell cytotoxicity and cytokine production has been demonstrated for blood-derived NK cells. Previous studies demonstrated that addition of exogenous TGF-ß inhibits NK cell cytotoxicity and IFN-{gamma} production by blood-derived NK cells and human blood NK cell clones in culture [21 ]. In these studies, the inhibitory effects of TGF-ß were observed at concentrations of TGF-ß in excess of 1 ng/ml. It has been reported previously that TGF-ß2 reduced the cytotoxicity and proliferative ability of decidual CD16–CD56bright NK cells, although it did not suppress the IL-2 augmentation of effector responses [45 ]. Our data extend the role of TGF-ß to demonstrate that EM produces biologically active TGF-ß in culture and that this endogenous TGF-ß can inhibit NK cell cytokine production. These data suggest that sufficient amounts of TGF-ß are present to inhibit uNK cell cytokine production in EM.

Previous studies have shown that TGF-ß is widely produced and processed from an inactive proform to an active molecule [19 , 46 ]. The biology of the TGF-ß family is complicated, and the various roles for TGF-ß in the uterus remain unclear. As aberrant T helper cell type 1 responses are speculated to contribute to miscarriages [43 ], our finding in the present study that TGF-ß1 was able to regulate production of IFN-{gamma} by uNK cells suggests that TGF-ß1 may play a role in regulating fetal implantation and preventing miscarriages. The cells in the EM that can produce TGF-ß may include epithelial cells, fibroblasts, and leukocytes [30 ]. The local activation of TGF-ß within tissues is poorly understood, but our study demonstrates that isolated endometrial cells produce biologically active TGF-ß. As the blocking antibody used in our study is able to inhibit the activity of TGF-ß1, -ß2, and -ß3, we cannot determine from these experiments which of the three TGF-ß isoforms is responsible for the suppression of IFN-{gamma} production in fresh EM. Epithelial cells isolated from rat uteri have been shown to produce biologically active TGF-ß, and the production of TGF-ß was regulated by estradiol [17 ]. The recognition that latent TGF-ß attached to extracellular matrix can be activated locally suggests that production and/or activation of TGF-ß may be one of the key elements that regulates uNK cells and mucosal immunity in general within the EM.

NK cells and other elements of the innate immune system are present in the EM and are likely involved in successful pregnancy and in defense against attack by microorganisms. NK cells are able to activate immune defenses by the production of a range of cytokines. Our data demonstrate that uNK cells in EM express a unique set of cell-surface molecules and are capable of producing cytokines but that this activity is kept in check by the presence of TGF-ß. Our findings suggest that uNK cells can play a role in immune defense within the EM and that local alteration of TGF-ß function can promote or prevent uNK cell activity.


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ACKNOWLEDGEMENTS
 
This study was supported by Grant AI 51877 from the National Institutes of Health (Bethesda, MD). The authors thank Virginia L. Kelly and Kim M. Wood (Norris Cotton Cancer Center, Lebanon, NH) for assistance with blood donation, Gary Ward for cell sorting, Ken Orndorff and Alice Givan for help with confocal microscopy, and Barbara Schaeffer for assistance with hysterectomy specimens. The authors also thank Vincent A. Memoli, M.D., section chief of Anatomic Pathology, Department of Pathology, for facilitating procurement of tissues; Peter Seery, Maryalice Achbach, Judy Rook, Elizabeth Rizzo, John Vitale, medical technologists, Section of Anatomic Pathology, for inspecting and dissecting tissue specimens; Linda Hallock for interfacing with respect to patient surgical schedules; surgeons of the Department of Surgery: Barry Smith, Emily Baker, Joan Barthold, Jackson Beecham, Deb Birenbaum, John Currie, Leslie Demars, Paul Hanissian, Diane Harper, John Ketterer, Michele Lauria, Benjamin Mahlab, Paul Manganiello, Misty Porter, Karen George, Stanley Stys, William Young, Kris Strohbehn, Tina C. Foster, Judith McBean, and Roger C. Young; surgical nurses: Jeannette Sawyer, Tracy Stokes, Fran Reinfrank, and Jaclyn Logren; and clinical support: Joanne Lavin, Karen Carter, Chris Ramsey, Nancy Leonard, Laura Wolfe, and Tamara Krivit.

Received February 16, 2004; revised April 20, 2004; accepted April 25, 2004.


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