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Original Studies |
Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi City, Osaka 570-8507, Japan
Address all correspondence and requests for reprints to: Hidetaka Okada, M.D., Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi City, Osaka 570-8507, Japan.
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Abstract |
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signal-transducing chains with the IL-2 receptor. It is suggested
that IL-15 is involved in regulating the proliferation and
differentiation of uterine natural killer cells. In the human
endometrium, we have recently reported that IL-15 messenger ribonucleic
acid (mRNA) levels significantly increased during the secretory phase
compared with those during the proliferative phase. In this study we
investigated whether the female sex steroids progesterone (P) and
estradiol (E2) regulate IL-15 messenger RNA (mRNA) and the
secretion in human endometrial stromal cells (ESC) in
vitro. Northern blot analyses revealed a significant increase
in IL-15 mRNA levels in ESC treated with P alone or E2 plus
P compared with vehicle. Furthermore, P is a potent inducer of IL-15
mRNA expression in ESC in a dose-dependent manner. On the other hand,
E2 alone did not increase IL-15 mRNA expression. By
enzyme-linked immunosorbent assay, IL-15 protein secretion was
stimulated by P and further enhanced by combined treatment with
E2 and P, whereas E2 alone was ineffective. It
is suggested that IL-15 is deeply involved in the hormonal control of
the human endometrium by P and E2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several molecular biology techniques of differential library
hybridization, suppression subtractive hybridization, and PCR-based
differential display have been used to isolate and characterize
differentially expressed genes (12, 13). In a recent
study, complementary DNA (cDNA) expression array system was used to
investigate the differential gene expression in human endometrium
between the proliferative and the secretory phases (14).
The level of interleukin-15 (IL-15) messenger ribonucleic acid (mRNA)
significantly increased during the secretory phase of the endometrium,
when serum P levels are known to be elevated (14). IL-15
is a novel cytokine that has recently been cloned and sequenced from
simian kidney epithelial cells CV-1/EBNA (15) and from the
human adult T cell leukemia cell line HuT-102 (16). It is
a member of the four 
-helix bundle cytokine family, which includes
IL-2. The effects of IL-15 are mediated by a trimeric membrane receptor
comprising the IL-2 receptor (IL-2R) ß- and -chains and a recently
identified specific -chain (17, 18). As signal
transduction is mediated by the ß- and -chains, many effects of
IL-2 and IL-15 are very similar. For example, like IL-2, IL-15 induces
T cell proliferation and chemotaxis, stimulates natural killer (NK)
cell growth and interferon- production, generates cytokine effector
cells, and costimulates B cell growth and Ig production
(19, 20, 21). Although IL-2 is selectively expressed in
activated T cells, IL-15 mRNA is constitutively expressed by a large
variety of cell types and tissues (15). The broad
expression of mRNA encoding IL-15 compared with the expression of IL-2
suggests that IL-15 has activities beyond the immune system. For
example, IL-15 stimulates muscle protein accretion in cultured skeletal
muscle fibers (22) and the differentiation of osteoclast
progenitors into preosteoclasts (23). However, although
Northern blot analysis indicated widespread constitutive expression of
IL-15 mRNA in a variety of tissues, such as skeletal muscle, placenta,
heart, kidney, lung, liver, and the dermal layers of skin
(15), it has been difficult to demonstrate IL-15 protein
in supernatants of many cells that express such mRNA. Indeed, although
activated monocytes expressed high levels of IL-15 mRNA, the culture of
these cells contained little or no IL-15 protein as assessed by
enzyme-linked immunosorbent assay (ELISA) (24). It has
also been shown that small cell lung carcinomas and adult T cell
leukemia cell lines express IL-15 mRNA, but do not appear to secrete
the cytokine (25, 26). In this study we investigated
whether the female sex steroids P and 17ß-estradiol
(E2) regulate IL-15 mRNA and protein production
in ESC in vitro.
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Materials and Methods |
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ESC were purified from the proliferative phase endometrium and cultured as described previously (6, 27). Briefly, tissue samples were washed with DMEM/F-12 medium (Life Technologies, Inc., Grand Island, NY) and minced into small pieces of less than 1 mm3. The tissues were then incubated for 2 h at 37 C in DMEM/F-12 medium containing 1 mg/mL collagenase (Wako Pure Chemical Co. Ltd., Osaka, Japan) and 0.005% deoxyribonuclease (DNase) type I (Roche Molecular Biochemicals, Mannheim, Germany). After subsequent pipetting, the cell suspension was diluted with 2 vol DMEM/F-12 medium and placed in a centrifugation tube (Corning, Inc., Corning, NY), where it remained upright for 10 min at unit gravity. The supernatant, excluding the lowermost 2 mL, was transferred into a new tube to collect suspended single cells. After repeating this procedure several times, the cell suspension was washed three times and used as a source of ESC. The viability, determined by die exclusion, was at least 90%. Two million viable ESC were cultured in 75-cm2 flasks in DMEM/F-12 medium supplemented with 10% FCS (HyClone Laboratories, Inc., Logan, UT), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Life Technologies, Inc.) at 37 C in humidified atmosphere of 5% CO2 in air.
Immunohistochemical analyses were conducted using factor VIII as a marker of endothelial cells, cytokeratin as a marker of epithelial cells, leukocyte common antigen as a marker of leukocytes, CD68 as a marker of macrophages, and vimentin as a marker of ESC (DAKO Corp., Kyoto, Japan). Initially, the purified fraction contained about 1–2% endothelial cell, 2–3% epithelial cells, 1–2% leukocytes, 1–2% macrophages, and 95% ESC by immunohistochemistry. CD10 antigens are expressed on human ESC (6, 28, 29), and the ratio of CD10 antigen-positive cells in confluent ESC was more than 99% by immunohistochemical staining.
Steroid hormones treatment
After ESC from passages 1–2 were nearly confluent (cultures were maintained for 1–2 weeks), they were plated in 75-cm2 flasks for Northern blot analyses and six-well plates for ELISA. To remove endogenous steroid hormones, FCS was treated as follows. FCS (100 mL) mixed with 0.25 g activated charcoal (Sigma, St. Louis, MO) and 0.025 g dextran (clinical grade; Sigma) was stirred at 56 C for 30 min and centrifuged to separate the dextran-coated charcoal pellet. The supernatant was then subjected to the same treatment at 37 C, and the dextran-coated charcoal stripped (DCS)-FCS was filtered through a 45-µm sterilization unit (Corning, Inc.) and stored at -20 C. ESC were grown to confluence, and the media were then replaced with phenol red-free DMEM/F-12 supplemented with 10% DCS-FCS. After 48 h, ESC were washed and cultured in DCS-FCS-supplemented medium with P (Sigma), estrogen (E2; Wako), or ethanol as vehicle control. The culture media were changed every 3 days.
RNA extraction and Northern blotting
Total RNA was prepared from frozen tissues and cultured cells by the acid guanidinium-phenol-chloroform method using TRIzol reagent (Life Technologies, Inc.). Total RNA (20 µg) was separated in a 1.2% formaldehyde gel and transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The probe was labeled by the multiprime DNA labeling system (Amersham Pharmacia Biotech, Arlington Heights, IL). Human IL-15 probe (14) and human S26 probe (6), which is identified as the mRNA-binding human ribosomal protein RNA, were prepared as described previously. Hybridization was performed at 42 C for 18 h in 5 x standard saline-phosphate-ethylenediamine tetraacetate (SSPE)/5 x Denhardt’s solution/50% formamide/0.5% SDS/100 µg/mL salmon sperm DNA. The filters were washed at room temperature in 2 x SSC (standard saline citrate)/0.1% SDS, following by 0.1 x SSC/0.1% SDS at 50 C, and then autoradiographed. The membranes were deprobed and rehybridized with the human S26 probe as an internal control, because its expression level is virtually constant in many tissues (30). The mRNA levels were calculated after normalization to S26 mRNA expression on the basis of the hybridized signal as measured in a BAS 2000 Bioimage Analyzer (Fujix, Tokyo, Japan).
IL-15 assay by ELISA
The IL-15 protein in cell culture supernatants was assessed using the human IL-15 ELISA kit (Genzyme, Cambridge, MA) in which recombinant human IL-15 was used as a standard according to the manufacturer’s instructions. The kit has a lower detection limit of 3.9 pg/mL IL-15. Intra- and interassay variations were less than 10%.
Statistical analysis
Data are expressed as the mean ± SD. Results were analyzed with a statistical software package (StatView II, version 4.0, Abacus Concepts, Inc., Berkeley, CA). Differences in the measured parameters across the different groups were statistically assessed using ANOVA with repeated measurements, followed by Fisher’s protected least significant difference, multiple range test. P < 0.05 was considered statistically significant.
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Results |
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Cultured human ESC, after E2 priming,
undergo proliferation and differentiation in response to P. We
evaluated whether E2 plus P regulates the
expression of IL-15 mRNA using the cultured ESC. The induction of PRL
mRNA, a typical marker for decidualization, was detected after 6 days
of culture (data not shown). Figure 1
shows patterns of regulation of IL-15 mRNA by Northern blot analysis
alongside control unregulated S26 mRNA. IL-15 transcripts of 1.5 kb
were detected in all of the total RNA extracts prepared from the
cultured ESC. The addition of vehicle to the culture medium had no
significant effect on the levels of the IL-15 mRNA. In contrast,
E2 and P caused a significant increase in IL-15
mRNA levels after 6 days of culture in the presence of this steroid.
The expression of IL-15 mRNA continued to increase until the end of
these studies at 12 days. Figure 2
presents Northern blots demonstrating the effects of
E2, P, or E2 and P on IL-15
mRNA. After 9 days of culture, the IL-15 mRNA level after treatment
with P, alone or in combination with E2, was 4-
to 5-fold higher than with vehicle. In contrast,
E2 did not affect the IL-15 mRNA level. There was
no apparent synergism between P and E2 in the
induction of IL-15 mRNA. These findings suggest that P, but not
E2, participate in the induction of IL-15 mRNA by
cultured ESC.
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To analyze the dose dependence of the effects of P on IL-15 mRNA
levels, ESC were incubated with various doses of P for 9 days. P
increased the IL-15 mRNA levels in a dose-dependent manner, with P
(10-8,
10-7, and
10-6 mol/L) resulting in
2.7 ± 0.7-, 3.8 ± 1.5-, and 4.2 ± 1.3-fold increases,
respectively, compared with the level without P (Fig. 3). E2 did not
affect IL-15 mRNA expression at any of the doses examined (data not
shown).
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To study the secretion of IL-15, we measured the IL-15
concentration in the cell culture supernatants using an ELISA kit. The
temporal release of IL-15 from ESC exposed to sex steroid hormone
treatment is shown in Fig. 4. The levels
of IL-15 protein were significantly increased after 6 days of treatment
with P, alone or in combination with E2. IL-15
secretion was induced progressively by E2 in the
presence of P after 12 days, but was very low in control cells or cells
treated with E2 alone. Simultaneous treatment
with P and E2 resulted in a marked synergistic
effect. However, treatment of ESC with E2 alone
failed to trigger IL-15 secretion. The number of ESC after 12 days with
E2, P, and E2 plus P
increased 1.35-, 1.2-, and 1.52-fold, respectively, compared with that
of control without steroid.
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To determine whether the ability of E2 to
potentiate the P-mediated increase in IL-15 secretion was dose
dependent, ESC were cultured in the presence of P plus varying doses of
E2. As shown in Fig. 5, IL-15 protein secreted from ESC was
dose dependently induced by P in the presence of
E2 after 12 days. Maximum IL-15 protein levels
were observed in ESC cultured in the presence of
10-8 mol/L
E2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-15 has biological activities including the induction of T cell
proliferation, the activation of cytotoxic effector cells, the
costimulation of Ig synthesis by B cells, and the activation monocytes
(19, 20, 21). In particular, previous reports suggest that
IL-15 is important in the differentiation, survival, and function of NK
cells. For example, mice made deficient in the ß-chain of IL-2R are
markedly deficient in NK cells (32). The IL-15R contains
the ß- and -chains of IL-2R and a recently identified specific
-chain (17, 18). IL-2Rß is required for the action of
IL-2 and IL-15, but is not used by other growth factors. In contrast,
mice deficient in IL-2 or IL-2R, the private receptor used by IL-2,
have a normal number of NK cells, suggesting that IL-15 may be required
for NK cell development (32). Indeed, NK cells are
virtually absent in mice deficient in the signaling molecules required
for IL-15 expression or in receptors or signaling molecules required
for IL-15 action (33, 34, 35). These finding suggest the
importance of IL-15 in NK cell differentiation and proliferation.
We demonstrated that P enhances IL-15 production in ESC in
vitro. This result suggests the importance of IL-15 during the
secretory phase and early pregnancy, when P from corpus luteum affects
the uterus. What is the role of IL-15 in human endometrium? IL-15 may
not be involved in regulating the proliferation and differentiation of
ESC, because ESC was not observed to express IL-2R ß- and -chains
(Okada, H., et al. unpublished data). During the secretory
phase and the early pregnancy, there is a dense mucosal infiltration of
uterine NK (uNK) cells that are thought to play an important role in
the maintenance of pregnancy (36, 37). These cells are
characterized by the marked expression of the NK cell marker CD56
despite the absence of CD16, another NK cell marker. Such
CD16- CD56bright NK cells
differ from the CD16+
CD56dim NK cells that constitute a large
proportion of NK cells in peripheral blood (38, 39). In
nonpregnant women, few CD16-
CD56bright NK cells are apparent in the
endometrium during the proliferative phase, but these cells increase in
number during the secretory phase, and they account for 70–80% of all
lymphocytes in the early stages of pregnancy (38, 39). The
variation in NK cell number in the mucosa over the menstrual cycle and
in pregnancy suggests that the recruitment/maintenance of uNK cells is
likely to be under hormonal control, but the identities of the stimuli
are presently unknown. We reported that the percentage of
CD56+ cells in endometrial leukocyte-rich
fractions cultured with P was significantly higher than that in
fractions without P (40). P might be an important factor
for the proliferation and differentiation of
CD56+ cells in human endometrium. However, all
lymphocytes, including NK cells, in both nonpregnant endometrium and
decidua do not express E2 or P receptors
(41). Therefore, if these hormones were to influence uNK
cells, they would have to do so indirectly, perhaps through an effect
on ESC. IL-2 is the only substance identified to date that is capable
of stimulating uNK cell proliferation in vitro, and NK cells
have been observed to express IL-2R -, ß-, and -chains.
However, IL-2 is unlikely to be involved in vivo because
this cytokine cannot be detected at the placental-uterine interface
(42, 43). An alternative candidate is IL-15, whose
receptor shares with the IL-2R. Therefore, IL-15 production in ESC
implies an important role of this cytokine in the regulation of
CD16- CD56bright NK cells
in the endometrium. More recently, IL-15 has induced the proliferation
of uNK cells in vitro in a dose-dependent manner without
transforming them into potent cytolytic cells capable of destroying
trophoblast (44). In recent studies, large granulated
lymphocytes (LGLs) and metrial gland structure were absent from the
pregnant uteri in IL-2R-deficient mice, but implantation sites in
IL-2-deficient mice displayed normal placentation and decidualization
with metrial gland and LGL development (45, 46). LGLs
belong to the NK cell lineage and has been identified in humans as uNK
cells (38, 47). These findings suggest that IL-15 might
regulate the differentiation of LGLs at implantation sites. In fact, it
is suggested that IL-15 is involved in regulating the differentiation
of LGLs during mouse pregnancy (48, 49).
The specific functions of CD16- CD56bright NK cells in human endometrium are unclear. However, because these cells are present in such abundance in the uterus at the time of implantation and are in close contact with invading placental trophoblast cells, they might have a role to play in the implantation process and the subsequent orderly growth and development of the placenta (46, 50). Other previously proposed uNK cell functions include lysis of virus-infected cells present in the uterus and placenta, nutritive function, and cytokine production (47, 51).
In conclusion, we revealed that ESC could produce IL-15, which is thought to play important roles in uNK cell proliferation and differentiation, and suggested that IL-15 production in ESC was under the control of P in the process of decidualization. We believe that IL-15 production by ESC is a key event for the successful pregnancy establishment, and further studies on endometrial IL-15 may be important to understand implantation failure and early pregnancy loss.
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Acknowledgments |
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Footnotes |
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Received December 2, 1999.
Revised May 30, 2000.
Revised July 31, 2000.
Accepted August 25, 2000.
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