Molecular Human Reproduction, Vol. 8, No. 10, 930-933,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Expression of macrophage inflammatory protein-3
in an endometrial epithelial cell line, HHUA, and cultured human endometrial stromal cells
Department of Obstetrics and Gynecology, Oita Medical University, Hasama-machi, Oita 879-5593, Japan
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Abstract |
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It has been demonstrated that human endometrial epithelial cells (EEC) and stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of macrophage inflammatory protein (MIP)-3
in endometrial cells, the production of MIP-3 by an EEC line, HHUA, and cultured ESC stimulated with various inflammatory mediators was evaluated by ELISA. Unstimulated HHUA and ESC constitutively secreted MIP-3. Tumour necrosis factor- and interleukin-1ß significantly stimulated the secretion of MIP-3 by HHUA and ESC. Lipopolysaccharide also stimulated the secretion of MIP-3 by ESC, but not by HHUA. These results show that the concentration of MIP-3 in the endometrium is modulated by these inflammatory mediators. MIP-3 may contribute to the normal and pathological processes of human reproduction by regulating the trafficking of immature dendritic cells and memory T lymphocytes into the endometrium.
endometrium/interleukin-1ß/lipopolysaccharide/macrophage inflammatory protein-3/tumour necrosis factor-
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Introduction |
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Chemokines are a large superfamily of structurally and functionally related molecules demonstrating chemotactic activity targeted at specific leukocyte populations. They are 70–90 aa in length and are divided into four subfamilies based on the relative position of their cysteine residues (CC, CXC, C, CXC3) (Miller and Krangel, 1992
; Baggiolini et al., 1994; Luster, 1998). The CC chemokine subfamily includes macrophage inflammatory protein (MIP)-1, MIP-1ß, MIP-3, monocyte chemoattractant protein (MCP)-1, regulated upon activation, normal T cell expressed and secreted (RANTES), eotaxin, I-309 and HC14, all of which mainly chemoattract and activate mononuclear cells (Oppenheim et al., 1991; Baggiolini et al., 1994).
Human endometrial epithelial cells (EEC) and stromal cells (ESC) have been reported to produce and secrete various chemokines, including interleukin (IL)-8 (Arici et al., 1998; Nasu et al., 1998a,b, 1999, 2001c), epithelial neutrophil activating peptide (ENA)-78 (Nasu et al., 2001a), growth-regulated oncogene (GRO) (Nasuet al., 2001b), MCP-1 (Arici et al., 1995; Jolicoeur et al., 1998; Nasu et al., 1998a,b, 1999, 2001c), MIP-1 (Akiyama et al., 1999; Nasu et al., 1999), RANTES (Hornung et al., 1997; Arima et al., 2000) and eotaxin (Hornung et al., 2000; Zhang et al., 2000). The expression of these chemokines has been suggested to be important in menstruation, bacterial infection, implantation and the maintenance of early pregnancy (Chard, 1995; Garcia-Velasco and Arici, 1999).
MIP-3 (Rossi et al., 1997), also designated as liver- and activation-regulated chemokine (LARC) (Hieshima et al., 1997), exodus-1 (Hromas et al., 1997) or CCL20 (Zlotnik and Yoshie, 2000), is a 70 aa, 8 kDa protein that belongs to the CC chemokine family and has lymphocyte-activating and -chemoattracting properties (Hieshima et al., 1997). CCR6, which is expressed mainly in dendritic cells and lymphocytes, has been identified as the specific receptor for MIP-3 (Baba et al., 1997; Liao et al., 1997). MIP-3 expression has been observed in human liver, lung, small intestine, appendix, tonsillar crypts, Langerhans islets of pancreas, thymus, lymph nodes and peripheral blood lymphocytes (Hieshima et al., 1997; Hromas et al., 1997; Power et al., 1997; Rossi et al., 1997; Dieu et al., 1998; Dieu-Nosjean et al., 1999), and its expression has been implicated in the pathogenesis of atopic dermatitis (Nakayama et al., 2001 ) and psoriasis (Homey et al., 2000). It is now well known that its expression in vitro is inducible by a variety of inflammatory mediators, including lipopolysaccharide (LPS), IL-1 and tumour necrosis factor (TNF)-, in keratinocytes (Homey et al., 2000; Nakayama et al., 2001), intestinal epithelial cells (Izadpanah et al., 2001), dendritic cells, monocytes, granulocytes, lymphocytes (Hieshima et al., 1997; Hromas et al., 1997; Rossi et al., 1997), endothelial cells (Hromas et al., 1997; Homey et al., 2000) and skin fibroblasts (Homey et al., 2000; Schutyser et al., 2000). The regulation of MIP-3 expression is cell-specific (Schutyser et al., 2000) and its expression in human endometrium has not yet been elucidated.
In this report, we investigate the effects of inflammatory mediators on the expression of MIP-3 protein by an EEC-derived cell line, HHUA (Ishiwata et al., 1984), and cultured ESC, which have been shown to produce several kinds of CC and CXC chemokines. We also discuss the regulation of MIP-3 expression in the cytokine network in the cyclic endometrium.
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Materials and methods |
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ESC isolation procedure and cell culture conditions
The study was approved by the Institutional Review Board of Oita Medical University and informed consent obtained from each patient. Normal endometrial specimens were obtained from eight premenopausal patients (aged 35–42 years) who had undergone hysterectomies for intramural leiomyomas. Their menstrual cycles were regular. All of the specimens were diagnosed as being from the late proliferative phase (day 11–13 of the menstrual cycle) on the basis of standard histologic criteria. Normal ESC were separated from epithelial glands by digesting the tissue fragments with collagenase as previously described (Nasu et al., 1998a
). Briefly, the tissue was minced into 2–3 mm pieces and incubated with collagenase (200 IU/ml; Gibco BRL, Gaithersburg, MD, USA) in RPMI 1640 (Gibco) while stirring for 40 min at 37°C. The suspension was then filtered through a 150 µm wire sieve to remove mucus and undigested tissue. The filtrate was 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, NY, USA) at a density of 106 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), streptomycin (100 IU/ml; Gibco) and penicillin (100 IU/ml; Gibco). Culture medium was replaced every 4 days. After three 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), cytokeratin (Dako), factor VIII (Dako) and leukocyte common antigen (2B11+PD7/26; Dako), were used for the experiments. An endometrial epithelial cell line, HHUA (Ishiwata et al., 1984), was cultured under the same conditions and used for the experiments. The cultures were incubated at 37°C in an atmosphere of 5% CO2 in air at 100% humidity.
Detection of MIP-3 in culture media of HHUA and ESC by ELISA
To study the production of MIP-3 by HHUA and ESC, 5x106 cells were plated on 6-well culture plates (Corning) in culture medium with 10% heat-inactivated FBS and cultured until they were fully confluent. The supernatant was then replaced with 1 ml of fresh culture medium containing various amounts of LPS (0.001–1 µg/ml; Sigma), recombinant human IL-1ß (0.01–10 ng/ml; R&D Systems) and recombinant human TNF- (0.1–100 ng/ml; R&D Systems). Under these conditions, the supernatant was collected 24 h after stimulation and stored at –70°C until assay. Isolated cells from each individual patient were used for each 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 MIP-3 was determined in each supernatant, after appropriate dilution, with a commercially available ELISA kit (R&D Systems). Sensitivity of the assay for MIP-3 was 0.47 pg/ml. Inter- and intra-assay coefficients of variation were 6.8 and 2.4% respectively.
Statistical analysis
Data are presented as mean ± SD and were analysed by means of the Bonferroni/Dunn test employing StatView 4.5 (Abacus Concepts, Berkeley, CA, USA). P < 0.05 was accepted as statistically significant.
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Results |
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Detection of MIP-3
in the culture media of HHUA and ESCThe concentration of MIP-3
in the culture medium without cells was below the level of detection (<0.47 pg/ml). Low levels of MIP-3 protein were detected in the culture medium of non-stimulated HHUA incubated for 24 h (Figure 1). The levels of MIP-3 increased significantly with increasing concentrations of TNF- [12.4-fold increase (908.0 ± 152.6 pg/ml) versus non-stimulated control (73.4 ± 12.1 pg/ml) at a concentration of 100 ng/ml; P < 0.0001] and IL-1ß [21.2-fold increase (1406.6 ± 147.0 pg/ml) versus non-stimulated control (66.5 ± 9.5 pg/ml) at a concentration of 10 ng/ml; P < 0.0001]. However, LPS did not affect the production of MIP-3 by HHUA (data not shown).
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Low levels of MIP-3
protein were also detected in the culture medium of non-stimulated ESC incubated for 24 h (Figure 2). The levels of MIP-3 increased with increasing additions of TNF- [8.7-fold increase (38.1 ± 3.2 pg/ml) versus non-stimulated control (4.4 ± 0.2 pg/ml) at a concentration of 100 ng/ml; P < 0.0001], IL-1ß [8.8-fold increase (119.1 ± 7.6 pg/ml) versus non-stimulated control (13.5 ± 2.8 pg/ml) at a concentration of 10 ng/ml; P < 0.0001] and LPS [10.9-fold increase (139.5 ± 36.1 pg/ml) versus non-stimulated control (12.8 ± 3.1 pg/ml) at a concentration of 1 µg/ml; P < 0.0001].
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Discussion |
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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. In the present study, we demonstrated for the first time that MIP-3
is expressed in an EEC cell line, HHUA, and cultured ESC. It has been reported that a number of chemokines, including IL-8 (Arici et al., 1998), ENA-78 (Nasu et al., 2001a), GRO (Nasu et al., 2001b), MCP-1 (Arici et al., 1995; Jolicoeur et al., 1998), RANTES (Hornung et al., 1997), MIP-1 (Akiyama et al., 1999) and eotaxin (Hornung et al., 2000; Zhang et al., 2000) were detected in endometrial tissue. Although further studies with immunohistochemistry and in-situ hybridization are necessary to elucidate the localization of MIP-3 in vivo, MIP-3 may be an additional chemokine that is expressed in the endometrium. At present, MIP-3 is the only chemokine detected in the endometrial cells that affects immature dendritic cells and memory T lymphocytes.
We demonstrated that MIP-3 expression by ESC is up-regulated by TNF-, IL-1ß and LPS, whereas its expression in HHUA is up-regulated by TNF- and IL-1ß, but not by LPS. Although further experiments involving primary EEC cultures are necessary, this characteristic may be specific to this cell line. The regulation of MIP-3 expression in HHUA and ESC is similar to that reported in other tissues (Hieshima et al., 1997; Hromas et al., 1997; Rossi et al., 1997; Homey et al., 2000; Schutyser et al., 2000; Izadpanah et al., 2001; Nakayama et al., 2001). These results suggest that endometrial cells may respond to inflammatory signals and secrete MIP-3 to attract certain types of leukocytes when infection occurs in the endometrium.
MIP-3 is shown to function as a NF
B target gene in other cell types, and its expression is up-regulated by inflammatory stimulation in EEC and ESC. We have demonstrated that other CC chemokines expressed in the endometrium, such as RANTES and MCP-1, are also up-regulated by IL-1ß, TNF- or LPS in ESC (Nasu et al., 1998a; Arima et al., 2000). However, unlike RANTES and MCP-1, MIP-3 mainly signals immature dendritic cells and memory T lymphocytes which are important in host adaptive immune responses. Endometrial MIP-3 is suggested to have a specific implication in the recruitment of these immature dendritic cells and memory T lymphocytes into the endometrium during normal and pathological human reproductive processes. It is possible that constitutive low levels of epithelial expression of MIP-3 production may serve to maintain immature dendritic cells and memory T cells in endometrium (Saito et al., 1994; Olivares et al., 1997), the first site of host contact with bacterial antigen.
In summary, we here demonstrate that MIP-3 is expressed in HHUA and cultured ESC. TNF-, IL-1ß and LPS stimulate MIP-3 secretion by ESC, and TNF- and IL-1ß stimulate MIP-3 secretion by HHUA. Further investigations are necessary to connect these in-vitro data with in-vivo phenomenon; however, these observations may provide insights into the mechanisms involved in the recruitment of inflammatory cells during human reproduction.
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Acknowledgements |
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This work was supported in part by the Ministry of Education, Science and Culture of Japan Grant-in-Aid No. 13770927 (to K.N.) and No. 13671733 (to I.M.) for Scientific Research.
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1 To whom correspondence should be addressed. E-mail: nasu{at}oita-med.ac.jp
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References |
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Submitted on December 10, 2001; resubmitted on March 4, 2002; accepted on June 14, 2002.
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