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Original Studies |
Reproductive Molecular Research Group (X.F.L., D.S.C.-J., E.Z., S.M., K.D., D.L., S.K.S.), Research Group in Human Reproductive Immunobiology (S.H., J.M.B., L.G., A.K., Y.W.L.), and Department of Obstetrics and Gynecology, Rosie Hospital (X.F.L., D.S.C.-J., E.Z., S.M., K.D., D.L., S.K.S.), Cambridge, United Kingdom CB2 2SW; and Department of Pathology, University of Cambridge (S.H., J.M.B., L.G., A.K., Y.W.L.), Cambridge, United Kingdom CB2 1QJ
Address all correspondence and requests for reprints to: Prof. S. K. Smith, Department of Obstetrics and Gynecology, The Rosie Hospital, Robinson Way, Cambridge, United Kingdom CB2 2SW. E-mail: sks1000{at}cam.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endometrium undergoes cyclical growth and regression, and inhibition of angiogenesis prevents proliferation (7). It is a rich source of angiogenic growth factors, including vascular endothelial growth factor (VEGF). The vascular endothelial growth factor family are important mediators of angiogenesis and consist of six members to date. In addition to VEGF, renamed VEGF-A, five additional proteins, VEGF-B (8), VEGF-C (9, 10), VEGF-D (11), VEGF-E (12), and placenta growth factor (PlGF) (13) have been identified. VEGF-A is the best characterized of this family and induces endothelial cell proliferation, migration, differentiation, tube formation with increased vascular permeability, and maintenance of vascular integrity (14, 15). The angiogenic responses induced by VEGF-A are mediated by two structurally related tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) (16). Recently, VEGF-C was characterized as a selective growth factor for lymphatic vessels and was found to affect the migration and proliferation of endothelial cells (8, 17, 18). VEGF-C is a ligand for two receptors, KDR and VEGFR-3, but not to Flt-1. PlGF is predominantly expressed in the placenta and binds with high affinity to Flt-1, but not to Flt-4, and KDR (19, 20). Endometrium expresses VEGF-A messenger ribonucleic acid (mRNA) and protein, and levels change during the menstrual cycle (21, 22). VEGF-A mRNA expression is stimulated by estradiol (21, 23) and progesterone (22). Although these studies suggest that expression of the VEGF-A gene is under ovarian steroid control, information on the exact roles of VEGF-A and its overall regulation with its receptors is lacking, and regulation of other VEGF family members in the endometrium has not been systemically investigated.
The angiopoietins have recently been shown to act in concert with the VEGFs in regulating vascular growth and integrity (24, 25, 26). Angiopoietin 1 (Ang1) induces autophosphorylation of Tie2 and is chemotactic for endothelial cells, whereas Ang2 competitively inhibits this effect (27). Mice lacking the Tie2 receptors die in utero at an older age than do those lacking VEGF or VEGFRs, indicating that angiopoietins exert their effects in the later stages of embryonic blood vessel formation (28, 29). Ang1 promotes angiogenic remodeling (25) as well as vessel maturation and stabilization (26, 29). Recently identified Ang3 in mouse and Ang4 in human have very different distributions in their respective species, and Ang3 appears to act as an antagonist, whereas Ang4 appears to function as an agonist (30). These factors may play a role in the mechanism of menstruation. However, the mRNA expression of this group of angiogenic growth factors has not been studied in human endometrium.
The uterine natural killer (uNK) cells are present as small granular cells in proliferative endometrium, but undergo vigorous proliferation during the secretory phase of the cycle (31). The factors regulating their proliferation and differentiation in vivo are unknown, but in vitro, IL-2, IL-15, and contact with endometrial stromal cells enhance their cytotoxicity, proliferation, and cytokine production (32, 33). uNK cells (CD56brightCD16-) are phenotypically distinguishable from the main population of circulating NK cells (CD56dimCD16bright). They also differ functionally from classical NK cells, having lower cytolytic activity against K562 cells and a different cytokine repertoire. The function of uNK cells is unclear. However, as they are found maximally in the decidua basalis in close proximity to infiltrating extravillous trophoblast at implantation, most studies have sought to elucidate their role in trophoblast invasion (34). Indeed, they do have specific receptors for human leukocyte antigen class I molecules expressed by extravillous trophoblast, establishing a molecular mechanism for maternal NK cell recognition of fetal trophoblast.
However, their cyclical presence in the nonpregnant uterus suggests that they have a role in endometrial differentiation and breakdown (35). Furthermore, uNK cells undergo apoptosis before other morphological features of menstruation become apparent, suggesting that they have a pivotal role in the triggering of menstruation.
In vitro and in vivo studies of activated NK cell adhesion to endothelial cells show that VEGF promotes adhesion, whereas basic fibroblast growth factor inhibits adhesion through the regulation of intracellular and vascular cellular adhesion molecules on tumor vasculature (36). Whether NK cells express angiogenic growth factors or their receptors has not been reported. We now show that in addition to VEGF-A, human endometrium expresses VEGF-C, Ang1, Ang2, and the Ang receptor, Tie2. Surprisingly, uNK cells, present in the secretory phase of the cycle, express high levels of VEGF-C, PlGF, and Ang 2. IL-2 and IL-15 up-regulate the level of VEGF-C mRNA expression. The results from our studies demonstrate for the first time that NK cells express angiogenic growth factors and suggest that uterine NK cells may play an important role in endometrial angiogenesis and regeneration.
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Materials and Methods |
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The present study was carried out with the approval of the ethical committee of the Addenbrooke’s Hospital National Health Service Trust. Normal endometrium was obtained from fertile subjects undergoing sterilization or from couples complaining of subfertility with solely tubal damage or male factor infertility. They all had regular menstrual bleeding with a menstrual cycle of 30 ± 5 days for the previous 3 months. Women who had received any form of exogenous hormones, had used an intrauterine device, or had exhibited demonstrable uterine disease in the previous 3 months were excluded from the study. Endometrial biopsy samples were collected by routine dilation and curettage. All specimens were rapidly rinsed in sterile saline and divided into two parts. For histological evaluation, immunocytochemistry, and in situ hybridization studies, tissue was immediately immersed in 10% formaldehyde, fixed for 6–8 h, and routinely embedded in paraffin wax. For RNA extraction, samples were snap-frozen in liquid N2 and stored at -70 C. Endometrial specimens were histologically dated from first day of the last menstrual period and were only used in the study if two independent observers agreed on their dating (37, 38).
Immunohistochemistry
To identify endometrial NK cells immunohistochemical staining was carried out as described by Clark et al. (39). Tissue sections serial in sequence to those hybridized with riboprobes for in situ hybridization were subjected to immunohistochemistry using a mouse monoclonal antibody to CD56 (antismall cell lung cancer) as the primary antibody, followed by a rabbit antimouse IgG as secondary antibody. Both antibodies were purchased from Zymed Laboratories, Inc. (South San Francisco, CA). To demonstrate the specificity of the immunohistochemical staining, the primary antibody was substituted an irrelevant mouse IgG at the same concentration, and no staining was observed. For each experiment known positive and negative endometrial sections were included as controls.
In situ hybridization
Riboprobes were designed to avoid regions of homology among the different growth factors, and the specificity of the probes was confirmed in practice by differences in the hybridization patterns observed in both placenta (40) and endometrium (see Results). Selected regions of VEGF-C, PlGF, Ang1, Ang2, Tie1, and Tie2 were amplified by RT-PCR from RNA extracted from term placenta, using the conditions and primers described in probes and RT-PCR sections. The probes were generated from PCR products amplified from complementary DNA (cDNA) obtained from term placenta and were cloned into the pCR-Script SK+ (Stratagene, Cambridge, UK). The orientation and identity of the clones were confirmed using an ABI 373A fluorescent sequencer (PE Applied Biosystems, Foster City, CA). Riboprobes were labeled with [33P]UTP, and in situ hybridization was performed as previously described in detail (41).
Probes
Primers to amplify a 183-bp fragment of VEGF-C were 5'-TGTACAAGTGTCAGCTAAGG-3' beginning at bp 590 and ending with 5'-CCACATCTATACACACCTCC-3' at bp 772 as previously described (9) (EMBL accession no. X94216). Primers for the amplification of PlGF were 5'-GARAARATGCCNGTNATG-3' (where R = AG and n = AGCT) beginning at bp 316 as previously described (13) and ending with 5'-CTCCAAGGGGTGGGTTA-3', with an expected product of 533 bp. The primers for Ang1 were 5'-AACCTTCAAGGCTTGGTT-3' and 5'-TACTGCCTCTGACTGGT-3' between bp 961 and 1421 (EMBL accession no. U83508), with an expected product of 461 bp. The primers for Ang2 were 5'-AAATAGTGACTGCCACGGTG-3' and 5'-ATCTCTTCTGTAGAATTAGGG-3' between bp 1047 and 1275 (EMBL accession no. AF004327), and the resulting product was 229 bp. The primers for Flt-4 were 5'-GCAGGGGCCTGCAAGAG-3' and 5'-AGGAACCACGGGTCTCAG-3'; the resulting product was 191 bp.
RT-PCR
Total RNA was isolated from snap-frozen endometrium and decidual NK cells and analyzed for mRNA encoding vascular endothelial growth mediators by RT-PCR as previously described (21). cDNA was synthesized from 2 µg (2 µg from endometrial samples or total amount from decidual NK cell cultures) of the isolated RNA using avian myoblastosis virus reverse transcriptase enzyme (HT Biotechnology Ltd., Cambridge, UK) and primed with an oligo(deoxythymidine) (Pharmacia Biotech, St. Albans, UK). PCR, using a pair of forward and reverse primers, was performed with different PCR protocols. Primers used for RT-PCR were the same as those used for making probes in in situ hybridization unless otherwise indicated. cDNAs encoding VEGF-C were amplified for 35 cycles (30 s at 95 C, 1 min at 70 C, and 1 min at 57 C, then 1 min at 72 C for 15 cycles; 30 s at 95 C, 45 s at 57 C, and 1 min at 72 C for 20 cycles). PCR product for PlGF was amplified for 30 cycles (30 s at 94 C, 30 s at 52 C, and 30 s at 72 C). Flt-4 cDNA was amplified for 35 cycles (30 s at 94 C, 30 s at 56 C, and 30 s at 72 C). Ang1 and Ang2 cDNAs were amplified for 30 cycles (30 s at 94 C, 30 s at 50 C, and 30 s at 72 C for Ang1; 30 s at 94 C, 30 s at 56 C, and 30 s at 72 C). Tie1 cDNA was amplified for 30 cycles (30 s at 94 C, 30 s at 52 C, and 30 s at 72 C) with a pair of primers, 5'-CTGCACACGTGCTTCTC-3' and 5'-CCGTGGCCTCAACGCCA-3'; the resulting in cDNA fragment was 223 bp. Tie2 cDNA was amplified for 30 cycles between bp 2047 and 2289 (EMBL L06139). The primers were 5'-CCTTAGTGACATTCTTCC-3' and 5'-GCAAAAATGTCCACCTGG-3' with an expected product of 243 bp (30 s at 94 C, 30 s at 60 C, and 30 s at 72 C). cDNAs encoding ß-actin were amplified for 20 cycles (30 s at 94 C, 30 s at 55 C, and 30 s at 72 C). The primers used for ß-actin were 5'-CTACAATGAGCTGCGTGTGG-3' and 5'-AAGGAAGGCTGGAAGAGTGC-3', and the resulting cDNA fragment was 528 bp.
To exclude the possibility of amplification of contaminating genomic DNA, PCR procedures were carried out directly in RNA samples (i.e. without RT) using each primer set, and the results showed no positive PCR products. A negative control reaction in which no RNA or cDNA template was added was included in each experiment. For semiquantitative PCR, a trace amount of [32P]deoxy-CTP (0.001 µCi/mL) was included to label the PCR product. The PCR products were electrophoresed on 1% agarose gels containing ethidium bromide and visualized by UV illumination. Preliminary experiments were performed to determine the optimal numbers of PCR amplification cycles for different primer pairs to ensure that the amplification was in the exponential phase and had not reached the plateau. After photodocumentation, the agarose gels were fixed in 10% trichloroacetic acid, washed, and dried under vacuum. The dried gels were exposed to autoradiographic films and subjected to image analysis using NIH Image.
Isolation and culture of decidual NK cells
First trimester decidual tissue was obtained from termination of
pregnancy specimens collected at 6–12 weeks gestational age. Decidual
leukocytes were isolated as previously described (32). In
brief, pieces of decidua compacta were identified macroscopically and
washed in RPMI 1640 medium (Flow Laboratories, Rockville, MD). The
decidual fragments were finely minced between two scalpel blades and
incubated on a roller for 1 h at 30 C in 2 mg/mL collagenase type
IV (Sigma, St. Louis, MO) in RPMI/10% FCS. The resultant
cell suspension was filtered, washed with RPMI 1640, layered over an
equivalent volume of Lymphoprep (Flow Laboratories) at room
temperature, and spun at 800 x g for 20 min. The cells
at the interface were aspirated and washed in RPMI 1640. After
incubation with anti-CD56 microbeads (Miltenyi Biotec Ltd., Bisley,
Surrey, UK) together with human 
-globulin at 6 C for 20 min, the
cells were washed and then loaded onto a VS column in a VarioMACS
magnet (Miltenyi Biotec Ltd.). The column was washed, and the
CD56+ cells were eluted with buffer, washed, and
resuspended in RPMI/10% FCS. The purity of the decidual NK cells
isolated by this method (>95%) was confirmed by immunofluorescent
labeling for the antigens CD56, CD16, and CD3 and subsequent flow
cytometry (33).
Decidual NK cells and NKL cells, an NK cell line (42), were suspended in RPMI with 10% FCS at a concentration of 1 x 106 and cultured in six-well plastic culture plates (Nunc, Paisley, UK) for 24, 48, and 72 h with or without the addition of different cytokines at different concentrations. Recombinant human IL-2 was purchased from Roche Molecular Biochemicals (Lewes, UK), and IL-15 was obtained from R&D Systems (Abingdon, UK). At different times in culture, the cell culture supernatants were collected and spun down at 350 x g to pellet the cells for total RNA extraction, and the supernatants were frozen at -20 C until further use. The experiments were repeated between four and five times for each treatment.
Human umbilical vein endothelial cell (HUVEC) apoptosis assessment
HUVECs were plated onto 0.5% gelatin-coated flasks, grown in HUVEC medium, and maintained at 37 C in 5% CO2. Cells were used in passages 3–5 to avoid age-dependent variations in levels of apoptosis.
Endothelial cells were subcultured onto tissue culture plates at
approximately 50% confluence or onto four-well fibronectin-coated
microscope slides at a density of 1 x 105
cells/well and allowed to attach overnight. The medium was changed the
following day, and the cells were examined under phase contrast
microscopy. When the cells were at 70% confluence, the cultures were
divided into different treatment groups and analyzed for apoptosis
after treatment for 6, 12, 18, 24, and 30 h. The treatment groups
were: 100% endothelial culture medium as a negative control, 50%
endothelial medium plus 50% decidual NK cell conditioned medium, 50%
endothelial medium plus 50% decidual NK cell conditioned medium with
IL-2 (200 U/mL), 50% endothelial medium plus 50% decidual NK cell
conditioned medium with IL-15 (10 ng/mL), 50% endothelial medium plus
50% conditioned medium from decidual NK cells cultured IL-2 (200
U/mL), 50% endothelial medium plus 50 conditioned medium from decidual
NK cell cultured with IL-15 (10 ng/mL), and endothelial medium with
tumor necrosis factor-
(TNF; 30 ng/mL; R & D Systems, Inc.) plus chloroxine 20 µg/mL (Sigma) as a
positive control.
Morphological evaluation of apoptosis
Morphological evaluation of apoptosis was monitored by cell labeling with monoclonal anticytochrome C antibody (PharMingen, San Diego, CA) and Hoechst (Sigma) and then visualized by fluorescence microscopy. Nuclear morphology was assessed using Hoechst 33258 (Sigma) staining (43). Briefly, cells grown on four-well microscope slides were washed with phosphate-buffered saline (PBS) and fixed at room temperature with 3% paraformaldehyde in PBS for 15 min. The fixed HUVECs were incubated with anticytochrome C antibody (1:500) at room temperature for 60 min. After washing, the cells were incubated with antimouse IgG antibody conjugated with Cy3 (1:150; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 60 min. The cells were stained with Hoechst solution (2.5 mg/mL) before mounting. Apoptotic cells were distinguished by their characteristic patterns of nuclear condensation, cytoplasmic rounding, and membrane blebbing. The percentage of apoptotic cells in 10 fields (x20 magnification)/data point was determined.
Annexin V-fluorescein isothiocyanate (FITC) fluorescence-activated cell sorting (FACS) analysis
Annexin V, a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine (PS) was used to detect early stage apoptosis (44). Floating endothelial cells were collected, and adhesive cells were trypsinized [0.05% (wt/vol) trypsin in 0.02% (wt/vol) ethylenediamine tetraacetate], incubated for 5 min at 37 C, and harvested from 6-well plates. After washing twice in PBS, the pellet was resuspended in binding buffer [10 mmol/L HEPES/NaOH (pH 7.4), 140 mmol/L NaCl, and 2.5 mmol/L CaCl2]. Annexin V-FITC was added to a final concentration of 100 ng/mL, and the cells were incubated in the dark for 10 min, then washed and resuspended in 300 µL binding buffer. Five microliters of propidium iodide (PI) was added to each sample before flow cytometric analysis. The cells were analyzed using a Becton Dickinson and Co. FACStar Plus flow cytometer (Mountain View, CA). Electronic compensation was used to eliminate bleed-through fluorescence. In each sample, a minimum of 10,000 cells was counted, and analysis was performed with standard CellQuest software (Becton Dickinson and Co.).
Statistical analysis
All experiments were repeated a minimum of three time. Results were expressed as the mean ± SEM or the median and range. Normally distributed data were analyzed by ANOVA, with Scheffé’s F test correction for multiple comparisons. Nonparametric data were analyzed by the Kruskal-Wallis statistic procedure. P < 0.05 was interpreted to denote statistical significance.
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Results |
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In situ hybridization was performed to identify the site of expression of VEGF-B, VEGF-C, PlGF, Ang1, Ang2, Tie-1, and Tie-2 mRNA in 19 human endometrial samples from subjects with normal menstrual cycles.
uNK cells expressed VEGF-C mRNA
There was no hybridization signal detected in either endometrial
glands or stroma when proliferative endometrium (n = 10) was
incubated with 33P-labeled VEGF-C probe (Fig. 1
, A and D). In the secretory phase,
seven of nine sections showed hybridization signals in the endometrial
stroma. There was no hybridization with the sense
33P-labeled probe. The VEGF-C mRNA hybridization
signal was only localized to a group of stromal cells. They were
identified on serial sections as likely to be uNK cells, as the number
and distribution of the cells were virtually identical. In the early
secretory phase, a small number of cells that were positive for VEGF-C
hybridization were sparsely distributed in the stroma. The number of
VEGF-C mRNA-positive cells significantly increased in all of the
midsecretory phase endometrial samples (n = 3; Fig. 1, B and E).
They were particularly concentrated around glands and vessels (Fig. 1, C and F), and cells with a similar distribution stained positively with
CD56+ antibody on serial sections (Fig. 1I). In
late secretory phase (n = 3), the intensity of the VEGF-C
hybridization signal and the number of VEGF-C mRNA-positive cells
decreased and was more diffuse over the endometrial stroma. Glandular
epithelium, stromal, and endothelial cells did not show hybridization
signals in any endometrial sample at any time in the cycle.
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After in situ hybridization, PlGF mRNA signals were
detected in the secretory endometrial stroma and again were apparently
localized in the NK cells by examining CD56-stained serial sections
(Fig. 3). The pattern of the PlGF mRNA localization was similar to that
of VEGF-C mRNA in the endometrium throughout the menstrual cycle,
although the overall levels of the hybridization were lower than
VEGF-C. There was no detectable hybridization signal in any of the
proliferative endometrial samples (n = 10). Weak PlGF mRNA
expression was noted in a small number of stromal cells in two of three
early secretory endometrial samples. Stronger hybridizations of PlGF
mRNA were detected in midsecretory endometrial NK cells (n = 3).
As with VEGF-C, the number of positive cells and the level of
hybridization decreased in the late secretory endometrium (n = 3).
The hybridization signal was only seen in cells that were likely to be
NK cells as judged from staining of serial sections with
CD56+ antibody, but not in any other compartments
of the endometrium. There was no hybridization observed during
experiments when the sense 33P probe was
labeled.
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During the proliferative phase (n = 10), low levels of Ang2
mRNA hybridization were observed in endometrial stroma. Weak
hybridization was diffuse in the stroma and was not limited to a
particular cell type (Fig. 2, A and B).
In the secretory phase, six of eight sections showed very strong
hybridization signals in the endometrial stroma. Similar to VEGF-C
mRNA, hybridization signals were strictly localized to uNK cells, which
were identified by immunocytochemistry as before. In the early
secretory phase, a small number of NK cells expressed low levels of
Ang2 (n = 2). The level of hybridization increased in the
midsecretory phase (n = 3), but, unlike VEGF-C and PlGF, reached a
maximum in late secretory endometrium (n = 3; Fig. 2, E–G). The
late secretory phase endometrial NK cells demonstrated the strongest
hybridization on Ang2 mRNA compared with those in the early and
midsecretory phases of the cycle. Glandular epithelium, stromal, and
endothelial cells did not show any hybridization signals. When the
related serial tissue sections were incubated with sense
33P-labeled probe no specific hybridization was
observed (Fig. 2, C and D).
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Hybridization to mRNAs encoding Ang1, KDR, Flt-4, Tie-1, and Tie-2 mRNAs did not show signal above the background despite signal being encoded in positive control samples.
Cyclic changes in levels of angiogenic growth factors and their receptor mRNA expression in human endometrium
To investigate cyclical changes in the endometrial levels of mRNAs
encoding VEGF-C, PlGF, Ang1, Ang2, Tie1, and Tie2 mRNA (n = 25;
Fig. 4), a semiquantitative RT-PCR
method was used. To control for variation in mRNA loading, ß-actin
mRNA levels were determined concurrently, and data were analyzed as
ratios of the autoradiographic density of angiogenic growth factors to
ß-actin.
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The relative level of mRNA encoding VEGF-C in human endometrium
from various phases of the menstrual cycle was shown in Fig. 4A
. Low levels were detected in all
proliferative phase endometrium. The median level was 0.52 (range,
0.49–0.64) in early proliferative endometrium (n = 4) and 0.28
(range, 0.18–0.54) in midproliferative phase endometrium (n = 3).
Levels increased in early secretory endometrium (median, 0.85; range,
0.77–1.22) and midsecretory endometrium (median, 0.91; range,
0.90–0.95). Levels declined significantly in the late secretory phase
(median, 0.49; range, 0–0.68).
Cyclic changes in levels of PlGF mRNA expression
Low levels of PlGF mRNA relative to ß-actin were detected in early and midproliferative endometrium (early: median, 1.25; range, 1.06–1.78; mid: median, 0.54; range, 0.53–1.63; n = 3 each). The PlGF mRNA level was higher in the late proliferative (median, 2.24; range, 2.02–2.46; n = 3), early secretory (median, 2.07; range, 1.79–2.56; n = 3), and midsecretory (median, 2.08; range, 1.88–2.14; n = 2) phase. mRNA expression then decreased in the late secretory endometrium (n = 5; median, 1.35). The median levels in the late proliferative and early and midsecretory endometrium were significantly higher than those in the midproliferative endometrium (P < 0.05).
Levels of Ang1 and Ang2 mRNA expression in endometrium
The levels of mRNA for both Ang1 (Fig. 4C) and Ang2 relative to
ß-actin (Fig. 4D) in human endometrium were relatively steady
throughout the menstrual cycles. There were considerable individual
variations in the levels of Ang1 mRNA within the same phase of the
menstrual cycle. The median relative levels of Ang1 mRNA varied from
9.98 in the early proliferative endometrium to 2.02 in the late
secretory endometrium. The median relative level of Ang2 mRNA was 10.90
in the early proliferative endometrium and slightly decreased to 9.64,
8.36, 10.16, and 9.82 in mid- and late proliferative, and early and
midsecretory endometrium. The median level for Ang2 in the late
secretory endometrium was higher (median, 13.29) than that in all other
groups; however, there was no statistically significant difference in
the relative levels of either Ang1 or Ang2 mRNA across the menstrual
cycle.
Levels of Tie-1 and Tie-2 mRNA in endometrium
Unlike the levels of Ang1 and Ang2 mRNA in endometrium, the
relative level of Tie-2 mRNA showed cyclic changes during the menstrual
cycle (Fig. 4E). Low levels of Tie-2 mRNA were detected in all early
proliferative endometrial samples (median, 0.75; range, 0.63–0.84).
Expression levels increased slightly in the midproliferative phase
(median, 0.90; range, 0.32–1.15), then further to much higher levels
in the late proliferative (median, 1.90; range, 1.6–2.2) and early
secretory (median, 1.96; range, 1.0–2.2) phases. The mRNA levels were
low in mid- and late secretory phases. The levels of Tie2 mRNA
expression in late proliferative and early secretory phases were
significantly higher than those in early and midproliferative as well
as late secretory phases (P < 0.05). There was
considerable individual variation in the levels of Tie1 mRNA expression
in all phases of the menstrual cycle (Fig. 4F), but there was no
significant difference between different stages of the menstrual
cycle.
Regulation of mRNAs of angiogenic growth factors and their receptors by IL-2 and IL-15 in isolated uterine NK cells
To investigate whether cytokines such as IL-2 and IL-15 regulate VEGF-C, PlGF, Ang2, Flt-4, and KDR mRNA levels in uNK cells, purified CD56+ NK cells isolated from first trimester decidua were cultured in different concentrations of IL-2 and IL-15. Total RNAs were extracted from decidual NK cells, and semiquantitative RT-PCR was performed.
IL-2 and IL-15 stimulated VEGF-C mRNA expression
NK cells immediately separated from the first trimester decidua
contained relatively high levels of VEGF-C mRNA. This declined after
24, 48, and 72 h in culture. In some of the cell samples cultured
for 72 h VEGF-C mRNA was undetectable. Figure 5A shows a densitometric evaluation of
the VEGF-C mRNA levels induced by different concentrations of IL-2 from
four separate experiments. A dose-dependent increase in VEGF-C mRNA
expression was observed, which peaked at 200 U/mL for IL-2. NK cells
exposed to different concentrations of IL-15 (2.5, 5, 10, and 20
ng/mL), demonstrated a maximum induction at 10 ng/mL (not shown).
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Neither IL-2 nor IL-15 altered Ang 2 or PlGF mRNA levels (Fig. 5B).
Effect of IL-2 and IL-15 on Flt-4 and KDR mRNA level
To understand the regulation of receptor mRNAs for angiogenic
growth factors, Flt-4 and KDR mRNA levels in decidual NK cells was
investigated. A relatively high level of Flt-4 mRNA was detected in NK
cells that were immediately separated from the first trimester decidua.
However, unlike VEGF-C and Ang2 mRNA, after culture with or without
IL-2 for 3 days, they did not express Flt-4 mRNA (Fig. 5C). Similarly,
treatment with IL-15 did not prevent the disappearance of Flt-4 mRNA
from decidual NK cells in culture.
Peripheral blood NK cells and an NK cell line (NKL) were also used to study the regulation of angiogenic growth factor mRNA by cytokines, IL-2, and IL-15. Although all of the above-mentioned angiogenic growth factor mRNAs were detected in peripheral blood NK cells and the NKL cell line, the level of their expression was very low, and they did not respond to IL-2 or IL-15 treatment.
Effect of NK cell-conditioned medium on HUVEC apoptosis
HUVEC apoptosis was evaluated by two independent methods,
microscopic detection of characteristic morphology of apoptosis and
quantification of apoptosis cells by FACS. HUVEC were treated with
NK-conditioned cell medium with or without IL-2 and IL-15 for
26 h before analysis for apoptosis. TNF treatment served as a
positive control, and HUVEC medium was the negative control.
Characteristic morphology of apoptosis in HUVEC exposed to conditioned NK medium
To investigate whether the conditioned NK cell medium causes
endothelial cell death, quantification of the total number of cells was
evaluated by direct cell counting. There were no differences in the
total number of cells counted between control and conditioned NK cell
medium or among the conditioned NK cell medium with or without IL-2 and
IL-15 (Fig. 6). Under a light microscope,
HUVEC showed normal intact morphology attached to plates and chamber
slides in both negative control and various conditioned NK cell medium
culture condition. In contrast, the cells appeared rounded, and some of
them detached from the plates and chamber slides in TNF-positive
control treatment (Fig. 6).
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Mitochondrial membrane breakage with release of cytochrome C,
condensation of chromatin (seen as intensified fluorescence),
contraction of the cytoplasm, and formation of projections or blebs are
all evident after exposure to TNF (Fig. 6). Quantification of the
relative number of apoptotic cells was achieved by direct cell
counting. The result showed that more than 20% of cells were apoptotic
after TNF treatment. However, less than 5% of cells showed these
characteristic morphological apoptotic changes in control cells and NK
cell-conditioned medium-treated cells (Fig. 6).
Conditioned NK cell medium did not cause translocation of PS from the inner face of the plasma membrane to the cell surface in the HUVEC
FACS of HUVEC was used to quantify apoptosis by measuring the
percentage of cells that stained annexin V but not PI. After initiation
of apoptosis, most cell types translocate the membrane PS from the
inner surface of the plasma membrane to the outside (44).
PS can be detected by staining with an FITC conjugate of annexin V that
binds naturally to PS. During programmed cell death, PS externalization
typically precedes membrane bleb formation and DNA fragmentation
(45). PI was used as the DNA stain. Figure 7 shows annexin V-FITC staining in
conjunction with PI staining in HUVEC treated for 26 h with a
different NK cell-conditioned medium compared with that used for TNF
treatment. Under basal conditions, 6.22 ± 0.22% (mean ±
SE) of HUVEC were apoptotic. The result showed no
significant changes in percentages of annexin and/or PI
fluorescence-positive cell numbers between the negative control and
treated samples. However, the percentages of annexin- and PI-positive
cells were significantly higher in the TNF-treated groups
(23.28 ± 0.58% and 40.54 ± 4.45%, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF-C stimulates predominantly (9), but not exclusively (46), lymphatic endothelial cell proliferation and migration via the VEGF-R2 and -R3 receptors (9). Both are present in endometrium. Conversely, PlGF acts only through the VEGF-R1 receptor (19), which is also present in endometrium. Expression of both of these angiogenic growth factors was restricted to uNK cells. The highest levels of expression were found in the midsecretory phase of the cycle, which is coincident with increased lymphocytes in the endometrium (31). Ang2 expression was also restricted to uNK cells, but here levels peaked in premenstrual endometrium. These differential effects are not the direct consequence of progesterone, as uNK cells do not express either of the progesterone receptors (47).
It is clear that factors produced within the endometrium can affect endothelial cells. For example, it has been reported previously that a slight, but not significant, increase in endothelial migration occurred when cells were treated with supernatants from cultured midsecretory phase endometrium. However, because of wide interperson variability it was not possible to identify the temporal regulation of the factor(s) responsible for endothelial cell proliferative activity (48). The data presented here suggest that there is a complex interaction between angiogenic factors and the cells that produce them. Uterine NK cells are characteristically situated just beneath the epithelial glandular layer and around spiral arterioles (49). The localization of uNK cells suggests a possible link between local VEGF-A production and the location of these cells. The highest levels of VEGF-A are found in glandular epithelium and vascular smooth muscle surrounding the spiral arterioles (21, 22). Although most VEGF-A is secreted from the luminal surface of epithelial cells (50) sufficient amounts may diffuse across the epithelial basement membrane to induce a gradient affecting the subepithelial complex of capillaries. VEGF-A promotes adhesion and migration of NK cells to tumor endothelial cells by induction of ICAM-1 and VCAM-1 (36), and this may lead to the focal migration of uNK cells.
Ang1 is expressed throughout the cycle at low levels, but it is only the uNK cells that express Ang2. This strongly implicates these cells in vascular remodeling. Ang1 is assumed to function by mediating the dialogue between pericytes and vascular smooth muscle and the endothelium, so as to promote the stability of blood vessels. Ang2 is highly expressed only at sites of vascular remodeling in the adult, notably in the ovary (27). Ang2 mRNA was either expressed together with VEGF at sites of vessel sprouting and ingrowth or in the absence of VEGF at sites of obvious vessel regression (e.g. atretic follicles). In the present study intense and specific hybridization for Ang2 was found in uNK cells situated near subepithelial capillaries and spiral arterioles. These expression patterns lead us to propose a model in which Ang2 plays a facilitative role at sites of vascular remodeling by blocking a constitutive stabilizing action of Ang1, allowing the vessels to revert to a more plastic and unstable state, leading to frank vessel regression before menstruation. As the sole source of Ang2 in human endometrium, this suggests that uNK cells play a critical part in the process of menstruation.
NK cell activity is regulated by cytokines, and several induce the
differentiation and activation of NK cells. IL-2 and IL-15 are
regulators of T and NK cell activation, and most activated T and NK
cells express cytolytic mediators (51). It was previously
reported that IL-15 is produced by cells in endometrium and decidua
and, like IL-2, is capable of inducing proliferation and augmenting
cytotoxic activity in decidual NK cells (33). Our studies
not only demonstrated that decidual NK cells expressed several
angiogenic growth factor mRNAs, but also showed that uNK cells
activated by IL-2 increased VEGF-C mRNA expression, but not Ang2 mRNA
expression. There was an increase in levels of VEGF-C mRNA expression
in decidual NK cells treated with IL-15, but the difference was less
than that after treatment with IL-2, and the response was more
variable. Cytokines regulate the expression of the lymphatic
endothelial mitogen VEGF-C (52). IL-1ß, also expressed
in endometrium, induced a concentration- and time-dependent increase in
VEGF-C. TNF and IL-1 also elevated VEGF-C mRNA steady state
levels, but Ang1 was down-regulated by IL-1ß (53).
We were unable to demonstrate a direct effect of uNK cell supernatants on HUVEC endothelial cell apoptosis. This could be explained in several ways. HUVECs are characteristic of large vessel, not microvascular, endothelial cells. The experimental design did not accurately reflect the close cellular proximity found in endometrium. NK cells may not need to use the VEGF/Ang system to play a part in vascular remodeling. IL-2 and IL-15 up-regulate the expression of mRNAs of various cytolytic mediators, such as granzyme D-G and TNF-related apoptosis-inducing ligand, and augment NK cell cytotoxicity (51, 54, 55). Once activated by IL-2, NK cells can adhere to and lyse human endothelial cells (56, 57, 58), which directly contribute to the antitumor effect of IL-2 by reducing tumor vascularization. The destruction of endothelial cells by IL-2-activated NK cells was proposed to contribute to the toxicity of high dose IL-2 treatment, particularly to the capillary leakage syndrome (58, 59).
The targeted localization of NK cells by VEGFs may have broader consequences for endometrial function. NK cells induce target cell apoptosis through exocytosis of perforin/granzymes and signaling via death receptors, the FasL/TNF-Fas/TNFR system (59). IL-2 and IL-15 induce granzyme D-G expression in primary cultures containing granulated metrial gland cells (NK cells) from murine uterus. Apoptosis in human endometrium may be mediated by perforin and granzyme B released from NK cells and cytotoxic T lymphocytes (60). However, the results of this study did not show a direct effect of NK cells on the induction of endothelial cell necrosis or apoptosis.
The presence of Flt-4 mRNA on freshly isolated NK cells suggests the possibility of an autocrine action of VEGF-C in NK cells. Its disappearance in cultured decidual NK cells implies that other cellular interactions may be necessary for its continued expression.
The findings of this study suggest that uNK cell activity is involved
in the vascular remodeling that occurs during human implantation. This
view is supported by the finding of vascular pathology at implantation
sites of mice deficient in NK cells. Two strains of mice, the tgE26 and
the IL-2Rß null x RAG-2 null hybrid, that lack both NK and T
cells demonstrate abnormal decidual vessels in the mesometrial segment
of the uterus on days 7 and 8 of gestation (61).
Endothelial cells become tall and columnar with evidence of cell death
and separation from the basement membrane. Arterioles demonstrated
increased thickness and had not undergone the thinning of the vascular
smooth muscle cells, characteristic of the vascular adaptation of these
vessels in pregnancy. Mice lacking T cells, but not NK cells,
demonstrated a normal phenotype (62). Bone marrow
transplantation to restore the NK cell lineage resulted in normal
arterioles (63). In part, this is mediated by
interferon- (64). These studies demonstrate that
uNK cells express angiogenic growth factors. The findings that abnormal
development of spiral arterioles underlies heavy regular uterine
bleeding (65) and that failure of the spiral arterioles to
adapt is a critical feature of preeclampsia indicate that further
studies are needed to define the role of uNK cells in reproductive
angiogenesis.
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Acknowledgments |
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Footnotes |
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2 Fellow in Medical Sciences at King’s College, Cambridge; research
supported by Action Research.
Received July 24, 2000.
Revised November 15, 2000.
Accepted December 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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26 mice. J Exp Med. 187:217–223. contributes
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T. A. Henderson, P. T. K. Saunders, A. Moffett-King, N. P. Groome, and H. O. D. Critchley Steroid Receptor Expression in Uterine Natural Killer Cells J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 440 - 449. [Abstract] [Full Text] [PDF]
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L. C. Kao, S. Tulac, S. Lobo, B. Imani, J. P. Yang, A. Germeyer, K. Osteen, R. N. Taylor, B. A. Lessey, and L. C. Giudice Global Gene Profiling in Human Endometrium during the Window of Implantation Endocrinology, June 1, 2002; 143(6): 2119 - 2138. [Abstract] [Full Text] [PDF]
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O. Gubbay, H. O. D. Critchley, J. M. Bowen, A. King, and H. N. Jabbour Prolactin Induces ERK Phosphorylation in Epithelial and CD56+ Natural Killer Cells of the Human Endometrium J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2329 - 2335. [Abstract] [Full Text] [PDF]
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L. S. Gambino, N. G. Wreford, J. F. Bertram, P. Dockery, F. Lederman, and P. A.W. Rogers Angiogenesis occurs by vessel elongation in proliferative phase human endometrium Hum. Reprod., May 1, 2002; 17(5): 1199 - 1206. [Abstract] [Full Text] [PDF]
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P. Hewett, S. Nijjar, M. Shams, S. Morgan, J. Gupta, and A. Ahmed Down-Regulation of Angiopoietin-1 Expression in Menorrhagia Am. J. Pathol., March 1, 2002; 160(3): 773 - 780. [Abstract] [Full Text] [PDF]
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