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Biology of Reproduction 60, 871-878 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Phenotypic Analysis and Proliferative Responses of Human Endometrial Granulated Lymphocytes during the Menstrual Cycle1

Roger F. Searle2,a, Rebecca K. Jonesa,b, and Judith N. Bulmerb

a Departments of Immunology and b Pathology, University of Newcastle upon Tyne, The Medical School, Newcastle upon Tyne, NE2 4HH, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The in vivo function of the unusual population of CD56+ CD16- endometrial granulated lymphocytes (eGLs) in human endometrium is unknown; their increased numbers in the secretory phase of the menstrual cycle suggests that they may play a role in the immunobiology of nonpregnant endometrium. In the present study, the phenotype and proliferative responses of eGLs at various phases of the menstrual cycle were compared with those in early pregnancy. Endometrial GLs were highly purified (> 98% CD56+) using immunomagnetic separation, and the expression of cell surface antigens was examined in smears using a double immunohistochemical labeling technique. Proliferative responses to mitogens and interleukin 2 (IL-2) were assessed in hanging drops in 60-well Terasaki plates. There was low to no expression of CD3, CD8, CD16, HML-1, L-selectin, and CD25 (IL-2 receptor {alpha}) on CD56+ cells isolated from nonpregnant and pregnant endometrium. The expression of CD2, CD49a, and CD122 (IL-2 receptor ß, IL-2Rß), however, increased from the proliferative to the late secretory phase of the menstrual cycle. In contrast, CD11a, CD69, and CD49d expression was high and did not vary with menstrual cycle phase; CD49d levels were significantly reduced in early pregnancy. Unlike early-pregnancy eGLs, none of the CD56+ eGL cultures throughout the menstrual cycle displayed phytohaemagglutinin (PHA)-induced lymphoproliferation. In contrast, eGLs from nonpregnant endometrium in the presence of 5 or 100 U/ml IL-2 after 48- and 120-h incubation showed significant proliferative responses, as did eGL cultures from early pregnancy. A significantly reduced number of proliferative phase eGL cultures proliferated in response to IL-2 compared to secretory phase and early-pregnancy eGL cultures. The IL-2-induced proliferative responses of CD56+ eGLs were associated with increased IL-2Rß (CD122) expression. These findings demonstrate 1) differential eGL expression of CD2, CD49a, and CD122 during the menstrual cycle; 2) differential IL-2-induced eGL proliferative responses during the menstrual cycle; and 3) differences between eGLs from nonpregnant and pregnant endometrium in CD49d expression and their ability to respond to PHA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human endometrium contains several leukocyte populations that vary with menstrual cycle phase [1–4]. Leukocytes account for approximately 5% of the total stromal cell population in proliferative endometrium but increase in number to comprise approximately 25% of stromal cells in the late secretory phase [3]; this altered leukocyte profile is due to the presence of phenotypically unusual CD56+ CD16- CD3- large granular lymphocytes, termed endometrial granulated lymphocytes (eGLs) [3–5]. During the menstrual cycle, eGL numbers increase dramatically from the proliferative to the late secretory phase and account for over 70% of endometrial leukocytes in early pregnancy [3,5]. By contrast, peripheral blood lymphocytes with a CD56+ CD16- phenotype represent fewer than 2% of the total lymphocyte population. Recently it has been reported that eGLs in premenstrual endometrium show high bcl-2 and Ki67 expression with no evidence of apoptosis, suggesting active proliferation in situ [6].

The antigenic phenotype and function of eGLs in nonpregnant endometrium are largely unknown, but numerous studies have addressed eGL immunobiology during early pregnancy. Endometrial GLs in first-trimester decidua display natural killer (NK) cell- and lymphokine-activated cytotoxicity [7–12], recognize trophoblast human leukocyte antigen (HLA)-G [13–15], produce various cytokines [16–18], and display proliferative responses [8]. Analysis of activation markers and adhesion molecules has provided additional information on early-pregnancy eGL cell activation and in vivo function. In first-trimester decidua, eGLs express a range of adhesion molecules, including certain ß1 integrins ({alpha}4ß1, {alpha}5ß1, {alpha}4ß7), ß2 integrins ({alpha}Lß2, {alpha}Mß2, {alpha}xß2), {alpha}Eß7, intercellular adhesion molecule, neural cell adhesion molecule (CD56), and CD2 (lymphocyte function-associated antigen-2) [11, 19–21]. Most decidual eGLs express interleukin 2 receptor ß (IL-2Rß; CD122) [10, 22–24], although they have low or no interleukin 2 receptor {alpha} expression (IL-2R{alpha}; CD25) [22–25]. Approximately 30–40% of eGLs from first-trimester decidua express CD69, an early activation marker as detected by flow cytometry [25–27].

In contrast, investigations of eGLs in nonpregnant endometrium are sparse, although it has recently been shown that these eGLs exhibit differential NK cell lytic activity during the menstrual cycle; eGLs from early proliferative phase endometrium lack lytic activity, unlike eGLs from other phases of the menstrual cycle [12]. Their increased numbers in the secretory phase suggest that eGLs may have an important, but as yet undetermined, in vivo role in nonpregnant endometrium. No studies to date have addressed either the expression of adhesion molecules and activation antigens or the proliferative responses of eGLs at different phases of the menstrual cycle. Investigation of their antigenic phenotype and proliferative properties may further understanding of the function of eGLs during the menstrual cycle, particularly in the period approaching implantation in a fertile cycle. In the present study the lymphoproliferative responses of eGLs freshly purified from nonpregnant endometrium to interleukin 2 (IL-2) and mitogens, stimuli routinely used to generate early-pregnancy eGL clones in vitro, and their expression of activation antigens and adhesion molecules were studied throughout the menstrual cycle and compared with that of eGLs from early pregnancy.


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

Samples of nonpregnant endometrium (n = 29) were obtained from specimens from hysterectomy performed for non-endometrial pathology (e.g., benign leiomyomata, cervical or ovarian abnormalities) at various phases of the menstrual cycle (11 proliferative, Days 5–13; 7 early secretory, Days 14–22; 11 late secretory, Days 23–28). The separation of specimens into early and late secretory phases was based on previous studies that have shown maximal changes in the endometrial leukocyte populations in the late secretory phase [1, 3]. Menstrual cycle phase was established using standard histological dating criteria [28]. All endometrial samples had been examined histologically prior to inclusion in the study. Samples were excluded if the patients were subsequently found to be receiving hormonal therapy, if the endometrium showed histological changes inconsistent with the menstrual dates provided, or if the endometrium showed inflammation or was abnormal in any way.

Early-pregnancy endometrium (n = 12) was obtained from elective terminations of normally progressing first-trimester pregnancy between 6 and 12 wk of gestation. Heparinized venous blood was obtained from normal healthy female volunteers; peripheral blood mononuclear cells (PBMCs) were prepared as previously described [12].

Ethical approval was granted by the Newcastle Joint Ethics Committee, and samples were obtained with informed consent.

Purification of eGLs

CD56+ eGLs were purified from nonpregnant and early-pregnancy endometrium using immunomagnetic separation as previously described [12]. Briefly, endometrium was finely minced and digested using 0.1% collagenase type II (Sigma Chemical Co., Poole, UK) and 0.01% DNase type IV (Sigma) for two 40-min periods at room temperature, sieved (40 µm), treated with 0.84% ammonium chloride solution to lyse erythrocytes, and then incubated sequentially for 30 min and overnight at 37°C to remove macrophages and stromal cells, respectively. CD56+ eGLs were positively selected from the unseparated endometrial cell preparation using the Mini MACS magnetic separation technique (Miltenyi Biotec Ltd., Camberley, UK); endometrial cells were treated with 5 µl anti-CD56 antibody (NKH-1; Coulter Immunology, Luton, UK) per 1 x 106 CD56+ cells for 30 min, washed, incubated with 20 µl MACS indirect IgG1 and IgG2 mixed microbeads (Miltenyi Biotec Ltd.) per 10 x 106 total cells for 15 min, and then positively selected using a Mini MACS Separation Column (Miltenyi Biotec Ltd.). The purity of the purified CD56+ eGLs was > 98% as assessed using flow cytometric and immunohistochemical methods [12].

Assessment of Activation Marker and AdhesionMolecule Expression

Expression of cell surface antigens by CD56+ eGLs was assessed in air-dried acetone-fixed cell smears using a modified double immunohistochemical staining technique as described previously [6]. Briefly, anti-CD56 antibody (used for positive selection) was detected using biotinylated horse anti-mouse antibody and avidin-biotin-peroxidase complex (Vectastain Elite; Vector Laboratories, Peterborough, UK) and developed with 3'-amino-9-ethyl carbazole substrate (Vector) to produce a red reaction product. After washing, the smears were incubated with the primary monoclonal antibody (Table 1) followed by biotinylated secondary antibody and avidin-biotin-peroxidase complex before the reaction was developed with nickel-modified 3,3'-diaminobenzidine (Sigma) to produce a black reaction product. Peripheral blood lymphocyte smears were used as positive controls for all antibodies except HML-1. Smears of endometrial cells before separation were prepared as a positive control for HML-1. Omission of one of the primary antibodies allowed assessment of nonspecific binding of the secondary antibodies, or spurious double labeling and negative controls were performed for each sample and for each stage of the technique.


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  		TABLE 1. Primary monoclonal antibodies.

The percentage of CD56+ cells coexpressing activation markers or adhesion molecules was assessed by counting the number of single (red) and double-labeled (red and black) cells to give a total of at least 200 cells. Statistical analysis was performed using the Mann-Whitney test with the conventional p < 0.05 significance level.

Proliferation Assays

The proliferative responses of CD56+ eGLs to recombinant human interleukin 2 (rhIL-2) and phytohaemagglutinin (PHA) were assessed in 60-well Terasaki plates [29]. In parallel assays, the response to Concanavalin A (ConA), an accessory cell-dependent T cell mitogen, was included to check eGL contamination by other leukocyte populations. Unseparated endometrial cells and positively selected CD56+ cells were adjusted to 4 x 105 cells/ml in RPMI-1640 medium (HyClone, Cramlington, UK) supplemented with 2 mM glutamine (Gibco, Paisley, Scotland), 50 U/ml penicillin (Gibco), 50 µg/ml streptomycin (Gibco), and 10% fetal calf serum (Advanced Protein Products, Brierly Hill, UK) (RF10) and added at 10 µl/well in triplicate into 60-well plates (Nunc, Life Technologies Ltd., Paisley, Scotland). To every well, 10 µl of RF10, PHA (2 µg/ml in RF10; Sigma), ConA (10 µg/ml in RF10; Sigma), or rhIL-2 (5–100 U/ml in RF10; First Link, Brierly Hill, UK) was added to achieve a final volume of 20 µl/well. Optimal levels of mitogens and rhIL-2 were evaluated in preliminary experiments (data not shown). Controls included cells and RF10 alone, RF10 alone, and mitogen or rhIL-2 alone. In each assay, PBMCs (4 x 105 cells/ml) in RF10 served as positive controls.

The Terasaki plates were inverted and incubated at 37°C in 5% CO2 in a humidified atmosphere for the duration of the assay. On the basis of our previous optimization of experimental protocols, Terasaki plates were incubated for 72 h in the presence of mitogens and for either 48 or 120 h in the presence of rhIL-2. After this period the cultures were pulsed with 0.16 µCi/well [3H]thymidine diluted in PBS (Amersham Life Sciences, Little Chalfont, UK), incubated for a further 6 h, and then harvested. Tritiated thymidine incorporation, as an indicator of cellular proliferation, was measured by liquid scintillation counting. The filter discs were placed in 60 wells of a polystyrene 96-well plate (Opitplate; Packard Canberra, Pangbourne, UK) with 20 µl of scintillant (Microscint-O; Packard Canberra) and counted on a microplate scintillation counter (Top Count; Packard Canberra). In each experiment, the cell viability in replicate wells was assessed with 50% nigrosin; in all experiments > 98% of cells were viable.

Results are expressed as mean counts per minute (CPM) ± SEM for each triplicate culture. The stimulation index (SI) was then determined as follows: SI = (mean eGL CPM in presence of mitogen / IL-2) / mean eGL CPM in RF10 alone.

Statistical analysis was performed in two stages. First, results were analyzed to determine whether the SI produced by the addition of mitogen or rhIL-2 to the eGLs was significant using a standard t-test comparing the CPMs from test wells with those from control wells. Secondly, the significance of individual between-group differences was determined using the chi-square test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypic Analysis

The percentage of CD56+ eGLs expressing adhesion molecules and activation markers throughout the menstrual cycle is summarized in Figure 1. Expression of CD16 by CD56+ eGLs was low to absent and did not vary with menstrual cycle phase; this finding showed the high degree of eGL purity.



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  		FIG. 1. Variation in the expression of different antigens on CD56+ cells positively selected from endometrium at various phases of the menstrual cycle (proliferative, n = 11; early secretory, n = 7; late secretory, n = 11) and in early pregnancy (n = 12) (bars represent mean ± SEM).

Adhesion Molecules

CD2 expression on CD56+ cells was high, and it increased from the proliferative to the late secretory phase (p = 0.015); but in early pregnancy, the level of CD2 expression by decidual CD56+ eGLs was comparable to levels in the proliferative phase. Similarly, levels of CD49a expression on CD56+ cells increased from the proliferative to the early (p = 0.062) and late (p = 0.005) secretory phases, and then decreased in decidua compared with the late secretory phase (p = 0.019). Although CD49d expression on CD56+ cells also increased with advancing menstrual cycle phase, these differences were not significant; CD49d expression, however, was significantly lower in early-pregnancy CD56+ cells (proliferative phase, p = 0.011; early secretory phase, p = 0.002). Expression of CD11a was high and did not vary throughout the menstrual cycle. In contrast, expression of HML-1 and L-selectin by CD56+ cells was low and did not vary with menstrual cycle phase or in early pregnancy.

Activation Markers

The percentage of CD69+ CD56+ eGLs increased from the proliferative to the late secretory phase and then decreased in first-trimester decidua, but the differences were not significant. Although CD25 coexpression was low in nonpregnant and pregnant endometrium, there was a significant reduction between the proliferative and late secretory phase (p = 0.018) and between the early and late secretory phase (p = 0.008). CD25 expression by CD56+ eGLs from decidua was comparable to that in late secretory phase samples but was significantly lower than on CD56+ cells purified from proliferative (p = 0.029) and early secretory (p = 0.028) phase endometrium. In contrast, CD122 coexpression significantly increased in late secretory phase endometrium as compared with the proliferative (p = 0.002) and early secretory phases (p = 0.042). Similarly, CD122 expression on decidual CD56+ cells was significantly higher than in proliferative phase endometrium (p = 0.010).

Proliferation Assays

As no significant differences were detected in the proliferative responses of eGLs from early and late secretory phase endometrium, these data were combined and treated as one group (these eGLs are termed secretory phase eGLs). In all assays, eGLs from both nonpregnant and early-pregnancy endometrium displayed a low level of basal proliferative activity in the absence of stimulation by PHA or IL-2, and this did not differ with menstrual cycle phase.

Response to Mitogens

The proliferative responses of CD56+ eGLs to PHA are summarized in Figure 2. None of the CD56+ eGL cultures from nonpregnant endometrium (n = 9) produced significant PHA-induced proliferation throughout the menstrual cycle, in contrast to all the unseparated endometrial cell cultures. On the other hand, 3 of 5 decidual CD56+ eGL cultures showed significant proliferative responses to PHA, as did all the unseparated decidual cultures. The proliferation of early-pregnancy eGLs differed significantly from that of eGLs during the menstrual cycle regardless of menstrual cycle phase (p < 0.01 > 0.001, X 6.87). In parallel cultures, PBMCs had a mean PHA-induced SI of 130.6 ± 22.0 (n = 16).



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  		FIG. 2. The effect of PHA and ConA on the proliferation of unseparated and CD56+ endometrial and decidual cells after 72-h incubation. Solid circles represent nonsignificant SI; open circles represent significant SI.

None of the CD56+ cultures from either nonpregnant (n = 8) or pregnant (n = 5) endometrium produced a significant proliferative response to ConA (Fig. 1). The lack of ConA-induced lymphoproliferation shown by eGL cultures confirmed the absence of contaminating endometrial T cells and macrophages in these eGL cultures. In contrast, 10 of 13 unseparated cultures produced significant proliferation in response to ConA. ConA-induced PBMC proliferation showed a mean SI of 68.1 ± 10.6 (n = 16).

Response to IL-2

48-Hour assay The proliferative response of CD56+ eGLs to rhIL-2 at either 5 U/ml or 100 U/ml in 48-h or 120-h assays is summarized in Figure 3. After 48-h incubation with 5 U/ml rhIL-2, only 2 of 5 CD56+ cultures from proliferative phase endometrium showed significant rhIL-2-induced proliferation, unlike all eGL cultures from the secretory phase (n = 6). Similarly, the majority of unseparated endometrial cultures showed significant rhIL-2-induced proliferative responses throughout the menstrual cycle. In early pregnancy, all CD56+ eGL cultures from decidua showed significant proliferation in response to 5 U/ml rhIL-2 (n = 5), as did the majority of unseparated decidual cell cultures. Recombinant human IL-2-treated PBMCs showed a mean SI of 3.4 ± 0.6 (n = 16). The IL-2-induced proliferation of secretory phase and early-pregnancy eGL groups differed significantly from that for the proliferative phase group (p < 0.05 > 0.01, X 4.95).



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  		FIG. 3. The effect of 5 U/ml (a, c) and 100 U/ml (b, d) rhIL-2 on the proliferation of unseparated and CD56+ endometrial and decidual cells after 48-h (a, b) and 120-h (c, d) incubation. Solid circles represent nonsignificant SI; open circles represent significant SI.

Similar CD56+ eGL proliferative responses were obtained with 100 U/ml rhIL-2 after 48-h incubation. Four of seven CD56+ eGL cultures during the proliferative phase of the menstrual cycle showed significant proliferation, as did the majority (7 of 8) of eGL cultures from secretory endometrium. Eleven of fifteen unseparated endometrial samples also showed significant proliferative responses. In early pregnancy, all CD56+ eGL cultures showed significant proliferation (n = 5) with SIs ranging from 3.0 to 29.1; the majority of unseparated cultures showed similar rhIL-2-induced proliferation. PBMCs in the presence of 100 U/ml rhIL-2 showed a mean SI of 3.9 ± 0.6 (n = 19). With 100 U/ml there were no significant differences in eGL proliferation between the various phases of the menstrual cycle and early pregnancy.

120-Hour assay Increasing the period of incubation did not affect the pattern of rhIL-2-induced eGL proliferation, although overall, fewer eGL cultures proliferated. After 120-h incubation with 5 U/ml rhIL-2, only 3 of 6 proliferative phase and 3 of 5 secretory phase CD56+ eGLs showed significant proliferation. Similarly, only 6 of 11 unseparated endometrial cultures showed significant proliferation. In contrast, 4 of 5 early-pregnancy CD56+ cultures showed significant proliferative responses, as did the corresponding unseparated cultures. PBMCs in parallel assays showed a mean SI of 13.0 ± 2.6 (n = 16). With 5 U/ml rhIL-2 there were no significant differences between the various phases of the menstrual cycle and early pregnancy.

In the presence of 100 U/ml rhIL-2, only 4 of 9 CD56+ proliferative phase and 5 of 8 secretory phase eGL cultures showed significant rhIL-2-induced proliferation after 120-h incubation; similarly, 11 of 17 unseparated endometrial samples showed significant proliferation. In early pregnancy, 5 of 6 CD56+ cultures and all unseparated cell cultures showed significant proliferation. PBMCs showed a mean SI of 15.7 ± 2.0 (n = 20). With 100 U/ml rhIL-2, there were no significant differences between the various menstrual cycle phases and early pregnancy.

In each experiment cell viability was assessed and shown to be > 98%. Overall, for both IL-2 levels and incubation times, secretory phase and early-pregnancy eGLs differed from proliferative phase samples in the responsiveness to IL-2 (p < 0.01 > 0.001, X 7.67; p < 0.001, X 12.46, respectively). Table 2 shows that an IL-2-induced eGL proliferative response was generally associated with high levels of CD122 expression prior to IL-2 treatment in vitro.


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  		TABLE 2. Comparison of CD122 expression with IL-2 induced eGL proliferative responses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The majority of studies to date on eGL phenotypic and functional properties have focused on cells from early-pregnancy decidua, while the status of eGLs in nonpregnant endometrium has received scant attention. Application of the Terasaki plate hanging-drop approach and cell smear immunohistochemistry enabled the analysis of eGL phenotype and proliferation responses throughout the menstrual cycle, although the yield of purified eGLs from nonpregnant endometrium was typically 100-fold less than that from early-pregnancy decidua. The present study has shown that differences exist between eGLs from pregnant and nonpregnant endometrium, and at different stages of the menstrual cycle, in their proliferative responses and phenotype. Interestingly, eGLs show a similar pattern of differential NK cell lytic activity at different phases of the menstrual cycle [12]. Unfortunately, the changes in phenotype and proliferative responses across the menstrual cycle were assessed in different women, as it is not feasible in practice to obtain endometrial samples from the same woman at different phases of the cycle.

It is considered unlikely that either enzymatic extraction or positive immunomagnetic selection would have resulted in the failure of eGLs from nonpregnant endometrium to mount PHA-induced proliferative responses, since these cells are capable of IL-2-induced proliferation. Additionally, early-pregnancy eGLs purified using an identical protocol displayed significant PHA-induced proliferation. The detection of PHA-induced proliferation by early-pregnancy CD56+ eGLs concurs with a previous report of decidual eGL proliferation in response to PHA and ionomycin/phorbol 12,13-dibutyrate [11]. The exact cellular mechanisms of mitogen-induced early-pregnancy eGL lymphoproliferation remain unclear. As PHA is a T cell mitogen, it would not be expected to induce eGL proliferation, since eGLs do not express the T cell receptor or the complete CD3 molecule—although decidual eGLs have recently been shown to express cytoplasmic CD3{epsilon} and CD3{zeta}, unlike peripheral blood NK cells [30–32]. It is highly likely that PHA-induced decidual eGL proliferation involves the CD3{epsilon} chain as in the case of T cell proliferation induced by anti-CD3 antibodies [33]. The CD3{epsilon} status of eGLs during the menstrual cycle is unknown. It is possible that either a change in eGL phenotype takes place during pregnancy or the different hormonal milieu of pregnancy activates the eGLs so that they are capable of responding to PHA. Other workers have recently suggested that CD56+ eGLs in early pregnancy are the immature progenitors for {gamma}{delta} T cells [34].

In contrast to the lack of PHA-induced lymphoproliferation, CD56+ eGLs from nonpregnant endometrium responded differentially to IL-2 with menstrual cycle phase. Secretory phase eGLs responded significantly to 5 U/ml IL-2 in 48-h cultures, unlike proliferative phase eGL cultures. Analysis of IL-2R expression demonstrated that IL-2-induced eGL proliferative activity was consistently associated with CD122 (IL-2Rß) expression in the secretory phase and in early pregnancy, although there was no apparent correlation between the level of CD122 expression and the magnitude of the SI; CD25 (IL-2R{alpha}) coexpression was virtually absent in both nonpregnant and pregnant endometrium. Others have also reported high IL-2Rß expression and no or low CD25 expression by decidual eGLs [10, 22–26]. Increased eGL expression of IL-2Rß during the menstrual cycle is the likely explanation for the present finding of differential IL-2-induced proliferative responses. The observation that proliferative responses of eGLs from nonpregnant endometrium were induced with low levels of IL-2 during the menstrual cycle concurs with previous studies of early-pregnancy eGLs [8–11, 22–24, 26]. The significance of the ability of eGLs to respond to IL-2 during the menstrual cycle is unclear, as IL-2 protein and mRNA are absent from endometrium during pregnancy [18]; the IL-2 status of nonpregnant endometrium is unknown. IL-2Rß expression, however, would allow eGLs with NK cell activity to respond to IL-2 production by endometrial T cells and/or IL-15 production by endometrial non-T cells. IL-15 closely mimics the biological properties of IL-2 and is expressed in both the nonpregnant (S. Verma, personal communication) and pregnant endometrium [35].

Although it has been suggested that eGLs may be important for successful pregnancy by controlling trophoblast invasion into the decidua, or acting as local immunosuppressor cells down-regulating the immune response to the fetal allograft [36–38], their role in nonpregnant endometrium is unclear. Their expression of adhesion molecules and activation antigens suggests that eGLs have a role throughout the menstrual cycle rather than solely during early pregnancy, possibly related to mucosal defense against infection. Up-regulated expression of adhesion molecules such as CD2, CD11a, CD49a, and CD49d during the menstrual cycle is likely to reflect eGL function and may be directly or indirectly regulated by steroid hormones [39].

CD2 (lymphocyte function-associated antigen-2, LFA2) is an adhesion molecule whose ligand, LFA3, is expressed by endometrial epithelium during the menstrual cycle [36]. Binding of CD2 to LFA3 may account for the aggregation of eGLs around endometrial glands or eGL migration into intraepithelial locations [40]. CD49a ({alpha}1 integrin) expression by eGLs in nonpregnant endometrium has not hitherto been addressed, although up-regulation of CD49a expression in secretory phase endometrial stroma has been reported [39]. Using a flow cytometric approach, others have reported that most decidual CD56+ cells express CD49a [19, 21], but immunohistochemical studies of sections of early-pregnancy decidua have failed to detect widespread expression of CD49a by eGLs in situ (unpublished results). In contrast, CD49d ({alpha}4 integrin) expression did not significantly vary with menstrual cycle phase. CD49d is one of the principal surface proteins mediating lymphocyte homing, and it has been proposed that CD49d fibronectin interactions regulate eGL movement in decidua and mediate trophoblast interactions [21]. Fibronectin is also expressed by endometrial stromal cells [41, 42], and interactions with very late activation antigen 4 may play a role in controlling eGL movement and/or function in nonpregnant endometrium. Although L-selectin is primarily responsible for homing of lymphocytes to high endothelial venules in peripheral lymph nodes and HML-1 is proposed to have a role in leukocyte homing or retention within epithelium, low to no L-selectin and HML-1 expression by eGLs from nonpregnant and pregnant endometrium suggests that these adhesion molecules have no role in the homing of endometrial or decidual eGLs from the circulation and their subsequent local expansion by proliferation. CD11a expression was also high on all samples examined, with eGLs from first-trimester decidua showing the lowest levels. LFA1 has been proposed to have a role in mediating NK adhesion and target cell lysis [43, 44]. The lack of variation in LFA1 expression during the menstrual cycle, however, is concomitant with the lack of variation in cytotoxic activity displayed by the eGLs between the late proliferative and early and late secretory phases [12].

The present and previous studies have noted marked heterogeneity in the surface phenotype of the endometrial CD56+ leukocyte population. This may be due to differentiation or maturation of eGL subpopulations during the menstrual cycle and early pregnancy. Altered phenotype during the secretory phase could also reflect differing function in preparation for implantation. Alternatively it could represent a response to changes in mucosal defense against infection around the time of ovulation. Future studies should address whether these subpopulations of eGLs have differing functional properties that may relate to movement of eGLs into an intraepithelial location.

The NK cell lytic activity displayed by eGLs in the period approaching the time of implantation in a fertile cycle [12], in combination with their capacity to respond to IL-2, may be associated with the production of a favorable uterine environment for implantation, perhaps through elevated immunological defense systems that protect the developing pregnancy from infection. The differential expression of activation markers such as CD69 during the menstrual cycle suggests early eGL activation and concurs with previous flow cytometric studies of first-trimester decidua [25–27, 45], but there is the proviso that CD69 can be induced in vitro by cell isolation and purification [45]. Endometrial GLs may be important for defense against genital tract infection at ovulation, when sperm and pathogenic organisms are most likely to enter the reproductive tract. Although the genital tract is generally held to be protected from infection by the "hostile" vaginal and cervical environment, high levels of estrogen at ovulation mediate changes that facilitate sperm passage and may leave the reproductive tract vulnerable to infection. The increased proliferative capacity of eGLs in the secretory phase of the menstrual cycle therefore would be highly advantageous, especially as endometrial CD8+ T lymphocyte cytotoxicity is down-regulated during this period [46]. Clearly, a detailed study of endometrial cellular responses during immunological challenge and detailed analysis of endometrial T cells are urgently required before the role of eGLs in the nonpregnant endometrium can be fully understood.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the financial support provided by the Sir Jules Thorn Charitable Trust and The Wellcome Trust. We would also like to thank Professor S. Robson, Department of Obstetrics and Gynaecology, Royal Victoria Infirmary, and the pathologists of the Department of Pathology, Royal Victoria Infirmary, for their help in obtaining specimens.


   FOOTNOTES
 
1 This work was supported by grants from The Sir Jules Thorn Charitable Trust (9312A) and The Wellcome Trust (33166/Z/91). Back

2 Correspondence: Roger F. Searle, Department of Immunology, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. FAX: 191 222 8803; r.f.searle{at}newcastle.ac.uk Back

Accepted: November 12, 1998.

Received: August 6, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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