Molecular Human Reproduction, Vol. 7, No. 5, 453-458,
May 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Expression of epithelial neutrophil-activating peptide 78 in cultured human endometrial stromal cells
Department of Obstetrics and Gynecology, Oita Medical University, Hasama-machi, Oita 879-5593, Japan
Abstract
It has been demonstrated that human endometrial stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of epithelial neutrophil-activating peptide 78 (ENA-78) in the endometrium, concentrations of ENA-78 in cyclic endometrial tissues were measured using enzyme-linked immunosorbent assay. The expression of ENA-78 was also detected in cyclic endometrium by immunohistochemistry. Endometrial tissues in the secretory phase contained higher amounts of ENA-78 protein than did those in the proliferative phase. Immunofluorescence staining revealed that ENA-78 protein was localized mainly in the stroma of endometrium. In addition, to evaluate the involvement of inflammatory mediators and ovarian steroid hormones in the production of ENA-78 by ESC was evaluated by in-vitro studies. Unstimulated ESC constitutively secreted ENA-78. Progesterone, lipopolysaccharide, tumour necrosis factor-
, and interleukin-1ß significantly stimulated the expression of ENA-78 by ESC. It is suggested that the production of ENA-78 by ESC is regulated by progesterone as well as by the inflammatory mediators. The modulation of ENA-78 concentration in the local environment by these mediators may contribute to the normal and pathological processes of human reproduction through regulation of leukocyte trafficking into the endometrium.
cytokines/chemokines/ENA-78/endometrial stromal cell/progesterone
Introduction
Chemokines are a large superfamily of structurally and functionally related molecules with chemotactic activity targeted at specific leukocyte populations. They are 70–90 amino acids in length and are divided into two major subfamilies based on the relative position of their cysteine residues (CC, CXC) (Schall, 1991
; Miller and Krangel, 1992; Baggiolini et al., 1994). The CC chemokine subfamily includes RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1, MIP-1ß, monocyte chemoattractant protein-1 (MCP-1), I-309, and HC14, all of which mainly chemoattract and activate mononuclear cells (Oppenheim et al., 1991; Schall, 1991; Baggiolini et al., 1994). In contrast, the CXC chemokine subfamily includes interleukin (IL)-8, neutrophil-activating protein-2 (NAP-2), platelet factor 4, ß-thromboglobulin, growth-regulated oncogene (GRO), GROß, GRO
, interferon--inducible protein 10 and epithelial neutrophil-activating peptide 78 (ENA-78), many of which have been shown to chemoattract and activate neutrophils (Miller and Krangel, 1992; Baggiolini et al., 1994; Taub and Oppenheim, 1994). A role for these chemokines has been implied in a variety of human diseases, characterized histologically by the presence of neutrophils (Kunkel et al., 1995). Chemokines are produced by various cell types including leukocytes, haematopoietic cells, endothelial cells, fibroblasts, and tumour cells in response to viruses, bacteria, lipopolysaccharides (LPS), and pro-inflammatory cytokines such as tumour necrosis factor (TNF)- and IL-1 (Taub and Oppenheim, 1994). The responding cell can undergo morphological changes, intracellular calcium mobilization, release of intracellular stored enzymes, respiratory burst, and increased adhesion to extracellular matrix proteins (Oppenheim et al., 1991; Baggiolini et al., 1994; Taub and Oppenheim, 1994).
ENA-78 is a 78 amino acid, 8 kDa protein that belongs to the CXC chemokine family and has neutrophil-activating and -chemoattracting properties similar to those of IL-8 (Walz et al., 1991). The ENA-78 cDNA encodes a 114 amino acid precursor protein with a putative 31 amino acid leader sequence (Walz et al., 1991). An essential structural element for neutrophil activation is a Glu-Leu-Arg (ELR) motif in the 5'-structure of the protein (Clark-Lewis et al., 1991; Hebert et al., 1991). The CXC chemokines with neutrophil-activating properties exhibit very similar activities on neutrophils, though they differ markedly in their cellular origin and/or kinetics of induction, suggesting a different pathophysiological role. ENA-78 is as equally potent as IL-8 in inducing neutrophil chemotaxis, though it is consistently less active in inducing the release of granules from neutrophils. ENA-78 was originally isolated from the human type II-like alveolar epithelial cell line A549 (Walz et al., 1991), and was initially thought to be exclusively a product of epithelium. It is now well known that its expression is inducible by a variety of inflammatory mediators, including LPS, IL-1, and TNF, in monocytes (Strieter et al., 1992), neutrophils (Strieter et al., 1992), platelets (Power et al., 1994), alveolar epithelial cells (Walz et al., 1991, 1997; Strieter et al., 1992), intestinal epithelial cells (Keates et al., 1997), renal cortical epithelial cells (Strieter et al., 1992; Schmouder et al., 1995), fibroblasts (Strieter et al., 1992; Walz et al., 1997), arterial smooth muscle cells (Lukacs et al., 1995), and endothelial cells (Strieter et al., 1992; Lukacs et al., 1995). In research focused on inflammatory diseases, ENA-78 expression has been implicated in the pathogenesis of rheumatoid arthritis (Koch et al., 1994), adult respiratory distress syndrome (Goodman et al., 1996), renal allograft rejection (Schmouder et al., 1995), inflammatory bowel diseases (Keates et al., 1997; Z'Graggen et al., 1997), and chronic pancreatitis (Walz et al., 1997).
Human endometrial stromal cells (ESC) have been reported to produce and secrete various chemokines, including IL-8 (Arici et al., 1993; Nasu et al., 1998a,b, 1999), GRO (Oral et al., 1996), MCP-1 (Nasu et al., 1998a,b, 1999), MIP-1 (Nasu et al., 1999), and RANTES (Arima et al., 2000). The expression of these cytokines has been suggested to be important in menstruation, bacterial infection, and in embryo implantation and the maintenance of early pregnancy (Garcia-Velasco and Arici, 1999). However, the expression of ENA-78 in human endometrium has not yet been elucidated.
In this report, we demonstrate the expression of ENA-78 in endometrial tissue and the effects of known modulators of endometrial function on the expression of ENA-78 protein by cultured ESC which have been shown to produce several kinds of CC and CXC chemokines. We also discuss the regulation of ENA-78 expression in the cytokine network in the cyclic endometrium.
Materials and methods
Tissue preparation, protein isolation and measurement of ENA-78
Normal endometrial specimens were obtained from 15 pre-menopausal patients who had undergone hysterectomies for intramural leiomyomas. Six specimens were diagnosed as being from the proliferative phase (days 4–14 of the menstrual cycle) and nine as being from the secretory phase (days 16–27 of the menstrual cycle) on the basis of standard histological criteria. To isolate protein, each tissue sample (100 mg) was soaked in 1 ml of TRIzol reagent (Gibco BRL, Gaithersburg, MD, USA), homogenized by use of a Polytron (Type PT 10/35; Kinmatica GmbH, Luzern, Switzerland), stored at room temperature for 5 min, and shaken vigorously for 15 s after the addition of 0.2 ml of chloroform. The homogenates were centrifuged at 12 000 g at 4°C for 15 min, then 0.3 ml of 100% ethanol was added to the interphase and the organic phase. The samples were stored at room temperature for 3 min, shaken vigorously, and centrifuged at 2000 g at 4°C for 5 min, then 1.5 ml of isopropyl alcohol was added to the supernatant. The samples were stored at room temperature for 10 min and centrifuged at 12 000 g at 4°C for 10 min. The protein precipitate was washed three times with 0.3 mol/l guanidine hydrochloride (Wako, Osaka, Japan) in 95% ethanol and once with 100% ethanol. The protein precipitate was then dried briefly and dissolved in 0.3 ml of PBS containing 10 mmol/l PMSF (Sigma, St Louis, MO, USA) and 1 mmol/l leupeptin (Sigma), followed by a 20 min centrifugation at 12 000 g and 4°C. The supernatant was collected and total protein was measured using a Coomassie protein assay reagent kit (Pierce, Rockford, IL, USA). The amount of ENA-78 was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). Sensitivity of the assay for ENA-78 was 15 pg/ml.
Detection of ENA-78 protein in endometrium by double indirect immunohistochemistry
Seven endometrial specimens in proliferative phase and eight in secretory phase were processed for double indirect immunohistochemistry as previously described (Nasu et al., 2000). Briefly, tissues were fixed in 3% paraformaldehyde for 30 min, washed three times in PBS, infiltrated with 5–15% sucrose followed by OCT (Miles Scientific, Naperville, IL, USA), and frozen in liquid nitrogen. Sections (6 µm) were prepared using a cryostat (Slee International Inc., Tiverton, RI, USA) and collected on poly-L-lysine-coated microscope slides (Dako, Copenhagen, Denmark). Fixed sections were permeabilized in cold methanol for 5 min, washed three times in PBS for 5 min, and incubated for 30 min with 1% bovine serum albumin (Sigma) in PBS. Sections were then incubated for 1 h with two primary antibodies [mouse anti-ENA-78 monoclonal antibody (R&D Systems) and rabbit anti-cytokeratin polyclonal antibody (Dako)], followed by rinsing three times in PBS for 5 min. The sections were then incubated with secondary antibodies conjugated to fluorescein or rhodamine (fluorescein-labelled donkey anti-goat IgG and rhodamine-labelled donkey anti-rabbit IgG; Jackson Immunoresearch Laboratories, West Grove, PA, USA), washed three times in PBS for 5 min, and mounted with Vectorshield (Vector Laboratories, Burlingame, CA, USA). All incubations and washes were performed at room temperature. Samples were viewed with a Zeiss Axiophot Epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) equipped with filters to selectively view the fluorescein and rhodamine fluorescence.
ESC isolation procedure
Normal endometrial specimens were obtained from eight additional pre-menopausal patients. All of the specimens were diagnosed as being from the late proliferative phase (11th to 13th day of the menstrual cycle). Normal ESC were separated from epithelial glands by digesting the tissue fragments with collagenase as previously described (Arici et al., 1993; Nasu et al., 1998a,b, 1999). Briefly, the tissue was minced into 2–3 mm pieces and incubated with collagenase (200 U/ml) (Gibco BRL) in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco BRL) with stirring for 2 h at 37°C. The suspension was then filtered through a 150 µm wire sieve to remove mucus and undigested tissue. The filtrate was then passed through an 80 µm wire sieve, which allowed the stromal cells to pass through while the intact glands were retained. After washing three times with serum-free RPMI 1640, cells were transferred to culture flasks (Corning, New York, NY, USA) at a density of 106 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco BRL), streptomycin (100 U/ml) (Gibco BRL), and penicillin (100 U/ml) (Gibco BRL). Culture medium was replaced every 4 days. After 3 passages (15–20 days after isolation) using standard methods of trypsinization, the cells, which were >98% pure as analysed by immunocytochemical staining with antibodies to vimentin (V9; Dako), keratin (Dako), factor VIII (Dako), and leukocyte common antigen (2B11+PD7/26; Dako), were used for the experiments. The cultures were incubated at 37°C in an atmosphere of 5% CO2 in air at 100% humidity.
Detection of ENA-78 in ESC culture media by ELISA
To study the production of ENA-78 by ESC, 1x106 cells were plated on 6-well culture plates (Corning) in 1 ml of culture medium with 10% heat-inactivated FBS and cultured until they were fully confluent. The supernatant was then replaced with fresh culture medium containing various amounts of LPS (0.001–1 µg/ml) (Sigma), recombinant human IL-1ß (0.001–1 ng/ml) (R&D systems), and recombinant human TNF- (0.1–100 ng/ml) (R&D systems). The effects of ethinyl oestradiol-17 (10 nmol/l) (Sigma), medroxyprogesterone acetate (MPA) (100 nmol/l) (Sigma), and the combination of these two steroid hormones on ENA-78 production were also examined. Under these conditions, the supernatant was collected 24 h after stimulation and stored at –70°C until assayed. Isolated cells from each individual patient were used per experiment, and each experiment, performed in triplicate, was repeated four times. All experiments were performed in the presence of 10% heat-inactivated FBS. The concentration of ENA-78 was determined in each supernatant with a commercially available ELISA (R&D Systems). The number of cultured cells was quantified by the Methylene Blue method (Nasu et al., 1995) to evaluate the effects of the above reagents on the cell growth. Briefly, after collecting the supernatant and washing once with PBS, the cells were fixed with 3% paraformaldehyde. The cells were then rinsed with 0.1 mol/l borate buffer (pH 8.5), followed by staining with 1% Methylene Blue. After washing four times with 0.1 mol/l borate buffer, the stained cells were lysed with 1 N HCl, and subjected to photocytometry analysis at an absorbance at 595 nm.
Statistical analysis
Data are presented as mean ± SD and were appropriately analysed by Mann-Whitney U-test and Bonferroni/Dunn test with StatView 4.5 (Abacus Concepts, Berkeley, CA, USA). P < 0.05 was accepted as statistically significant.
Results
Detection of ENA-78 protein in the endometrial tissue
ENA-78 protein was detected in all protein extracts from endometrial tissues (87.8 ± 41.0 pg/mg protein in the proliferative phase and 204.0 ± 161.0 pg/mg protein in the secretory phase). As shown in Figure 1, ENA-78 protein levels in the secretory phase endometrium were significantly higher (2.3-fold) than those in the proliferative phase endometrium (P < 0.05, Mann-Whitney U-test).
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Using fluorescence immunohistochemistry, ENA-78 protein was localized in the stroma of the endometrium in all specimens (Figure 2
).
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Detection of ENA-78 in the culture media of ESC
The concentration of ENA-78 in the culture medium without cells was below the detection level (<15 pg/ml). Low levels of ENA-78 protein were detected in the culture medium of non-stimulated ESC incubated for 24 h (Figures 3–6
). As shown in Figure 3, the concentrations of ENA-78 increased with increasing concentrations of LPS. LPS showed the strongest effects at the concentration of 0.1 µg/ml (10-fold increase versus non-stimulated control). The concentrations of ENA-78 were also increased with increasing concentrations of IL-1ß (380-fold increase versus non-stimulated control at the concentration of 1 ng/ml) (Figure 4). TNF- also increased the ENA-78 secretion by ESC in a concentration-dependent manner (32-fold increase versus non-stimulated control at the concentration of 100 ng/ml) (Figure 5). As shown in Figure 6, ethinyl oestradiol-17 did not affect ENA-78 production by ESC, whereas MPA enhanced ENA-78 production by these cells (2.5-fold increase). Among these agents, IL-1ß showed the strongest effects on ENA-78 production by ESC. The cell number was not affected under these culture conditions.
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Discussion
Chemokines are key components in the process of leukocyte recruitment from the vasculature into tissues. The interaction of different chemokines with their receptors on leukocytes allows selective activation and chemotaxis of the neutrophils, eosinophils, lymphocytes, or monocytes appropriate for migration to sites of evolving inflammation. We have previously reported on the production of the chemokines IL-8 (Nasu et al., 1998a,b, 1999), MCP-1 (Nasu et al., 1998a,b, 1999), macrophage inflammatory protein-1 (Nasu et al., 1999), and RANTES (Arima et al., 2000) by human ESC, and our findings have suggested paracrine regulation of the cytokine network by these chemokines in the normal cyclic endometrium and in early pregnancy.
In the present study, we demonstrated that ENA-78 was expressed in the stroma of normal cyclic endometrium and that the concentration of ENA-78 in the tissue was higher in the secretory phase than in the proliferative phase. Progesterone, in the form of MPA, significantly enhanced the production of this protein by cultured ESC. These results show that ENA-78 is produced constitutively in the endometrium and may be regulated, at least in part, by ovarian steroid hormones. The expression of IL-8, another member of the CXC chemokine family, is also regulated by progesterone (Arici et al., 1996). Together these results suggest that ovarian steroid hormone-regulated expression of CXC chemokines might be important in the physiological changes of normal cyclic endometrium.
Because ENA-78 and IL-8 are similar in many respects and are constitutively co-expressed by ESC, we examined the effects of known modulators of endometrial function on ENA-78 production by ESC. In this study, we demonstrated that LPS, IL-1ß and TNF-, all of which have been shown to induce IL-8 production by ESC (Nasu et al., 1998a), also enhanced ENA-78 production by these cells. While IL-8 and ENA-78 are similar in this regard, CXC receptor 1 has a high affinity for IL-8 only, whereas other ELR+CXC chemokines, including ENA-78, bind to this receptor with low affinity. All of these ELR+CXC chemokines have a high affinity for CXC receptor 2 (Ahuja and Murphy, 1996; Bozic et al., 1996). The differential receptor usage of the structurally related ELR+CXC chemokines is indicative of a different role in inflammatory reactions.
In summary, we here demonstrate for the first time that ENA-78 is expressed in cyclic endometrium and in cultured ESC. MPA, LPS, TNF- and IL-1ß also stimulated ENA-78 secretion by cultured human ESC. These observations may provide insights into the mechanisms involved in the recruitment of inflammatory cells during human reproductive processes.
Acknowledgements
This work was supported in part by the Ministry of Education, Science and Culture of Japan Grant-in-Aid No. 11770945 (to K.Nasu) and No. 09671699 (to I.Miyakawa) for Scientific Research.
Notes
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Oita Medical University,Hasama-machi, Oita 879-5593, Japan. E-mail: nasu{at}oita-med.ac.jp 
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Submitted on December 19, 2000; accepted on February 14, 2001.
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