Molecular Human Reproduction, Vol. 6, No. 5, 469-473,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Cell proliferation effect of lactoferrin in human endometrial stroma cells
Department of Obstetrics and Gynecology, Showa University School of Medicine, Tokyo, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo, 142-8666, Japan
Abstract
The aim of this study was to evaluate the effect of lactoferrin (LF) on the proliferation of human endometrial stroma cells. In addition, we compared the effect of LF, oestradiol and epidermal growth factor (EGF) on the proliferation of human endometrial stroma cells. Human endometrial tissue was obtained from patients with a normal menstrual cycle in the proliferative phase and the stroma cells were isolated and cultured in vitro. When LF was added to the culture medium, the rate of cell proliferation increased significantly in comparison to controls (P < 0.01). The enhanced rate of proliferation induced by LF was neutralized by the addition of anti-LF monoclonal antibody. The effect of LF on cell proliferation at a concentration of 100 ng/ml was similar to that of 10 nmol/l oestradiol, but less than that of 10 mg/ml EGF. When LF was added in combination with either oestradiol or EGF, no additive effects on cell proliferation were observed. Based on the present results, it is suggested that LF has a potential biological effect in the proliferation of human endometrium.
cell proliferation/epidermal growth factor/endometrium/lactoferrin/oestrogen
Introduction
Lactoferrin (LF) is an iron binding glycoprotein with a molecular weight of 80 kDa, originally found in and isolated from human milk (Metz et al., 1984; Anderson et al., 1987
). This protein belongs to the transferrin family of proteins, and was found to exist in a wide variety of secretions and tissues, including human uterine endometrium (Cohen et al., 1987). A variety of functions has been proposed for LF, including resistance to infection, antibacterial activity, regulation of release of interleukins and cell-growth-promotion activity (Morrison and Allen, 1966). LF promotes growth in some human lymphocyte cell lines (Montreuil and Tonnelat, 1960; Morrison and Allen, 1966) and increases thymidine incorporation into the DNA of rat crypt enterocytes (Montreuil et al., 1960; Ballow et al., 1987) and mouse Balb/c 3T3 fibroblasts (Montreuil et al., 1960; Biserte et al., 1963). The DNA-synthesis-stimulating activity in enterocytes may explain the physiological role of LF in milk. In mouse mammary gland, it is reported that the prolactin response protein seems to be LF (Green and Pastewka, 1978). EGF also induces the expression of LF in the mammary tissues of mice (Nelson et al., 1991). Furthermore, the human LF gene was found to have a functionally imperfect oestrogen response element in the 5'-flanking promoter region (Rey et al., 1990; Teng et al., 1992). These studies have suggested that expression of LF is mediated by oestrogen. In addition, Mellor and Thomas (1995) suggested that the regulation of endometrial stroma cell proliferation involved not only an interaction between oestradiol and EGF, but also other factors.
In the present study, the effect of LF on cell proliferation using cultured human endometrial stroma cells was examined to elucidate the physiological role of LF.
Materials and methods
Materials
LF, oestradiol and EGF were obtained from Sigma Chemical Co. (St Louis, MO, USA). Hanks' balanced salt solution (HBSS), penicillin-streptomycin and Roswell Park Memorial Institute (RPMI) 1640 were obtained from Gibco BRL (Life Technologies, Inc., Rockville, MD, USA). Collagenase, EDTA, phosphate-buffered saline (PBS), bovine serum albumin (BSA), fetal bovine serum (FBS) and diaminobenzidine (DAB) were obtained from Wako Pure Chemical Industries Ltd (Tokyo, Japan). DNase was purchased from Boehringer Mannheim (Mannheim, Germany). Vimentin and cytokeratin were obtained from Novocastra Laboratories Ltd (Newcastle, UK).
Tissue samples
Informed consent was obtained from all patients who participated in this study. Human endometrial tissue was obtained from patients who were undergoing hysterectomy for benign gynaecological disorders and who had not received any hormonal treatment. The menstrual cycle phase during which the specimens were obtained was determined both by the date of the last menstrual period and by the serum concentrations of oestradiol and progesterone. Normal endometrial samples were dated by established criteria (Noyes et al., 1950).
Isolation and culture of endometrial stroma cells
The endometrial tissue obtained from patients in proliferative phase was washed with HBSS and minced to small fragments. The tissue fragments were then centrifuged at 1000 r.p.m. for 5 min and the supernatant was removed. Stroma cells were dissociated from glands by digesting the tissue fragments with HBSS containing 0.1% collagenase and 0.01% DNase in a humidified 5% CO2/95% air atmosphere at 37°C for 2 h. After digestion, the samples were filtered through 45 µm nylon meshes. The filtrates were centrifuged at 1000 r.p.m. for 5 min to collect stroma cells. Cell pellets were washed with culture medium containing 10 ml of RPMI 1640, 2% FBS, penicillin 100 IU/ml and streptomycin 100 µg/ml. After being washed twice with the medium, the cells were plated out on 10 cm plates. Stroma cells were allowed to adhere overnight in a humidified 5% CO2/95% air atmosphere at 37°C. The medium was changed every 48 h. After the cells became confluent, they were cultured for 24 h without FBS before the experiments.
Immunohistochemical staining
The characterization of cultured stroma cells was detected immunohistochemically using vimentin and cytokeratin as antibodies by the streptavidin–biotin method (ABC). The samples were stained first with a monoclonal mouse anti-human antibody against vimentin or cytokeratin. Then the samples were stained with antibody using Histifine SAB PO (M) Kit (Nichirei Corporation, Tokyo, Japan). Finally, ABC staining was performed using DAB. Negative controls for the immunostaining were carried out with PBS instead of primary antibody (Osborn et al., 1984; Osteen et al., 1989)
Cell proliferation
Cell proliferation was detected immunocytochemically using a cell proliferation enzyme-linked immunosorbent assay (ELISA), BrdU (colorimetric) kit (Boehringer Mannheim, Germany), which measures 5-bromo-2'-deoxyuridine incorporation. Stroma cells were removed from the plates using 0.05% trypsin + 0.2% EDTA. 2x103 cells/100 µl were plated in each of 96-well chambers and incubated in medium containing various combinations of 1 µg/ml LF, 10 nmol/l oestradiol and 10 ng/ml EGF for 24 h in 95% CO2/5% H2O atmosphere at 37°C. As controls, cells were cultured in the same medium in which 0.01% ethanol was added to avoid the influence of diluents of test substances. Ten µl of diluted BrdU (1:100) was added to each well and further incubation was performed for an additional 18 h. The cells were fixed for 60 min at room temperature. The cells were then incubated with anti-BrdU monoclonal antibody-POD for 90 min. The wells were washed three times using washing solution, tetramethylbenzidine was added, and the wells were incubated at room temperature for 10 min. The reaction was stopped using 1 mol/l H2SO4. Finally, the 450 nm absorbance of each well was measured using a contrast absorbance 690 nm (International Reagent Corporation ELISA Reader) and the rate of proliferation was calculated.
Statistical analysis
All the data were analysed statistically using one-way analysis of variance with multiple comparison test.
Results
Characterization of stroma cells
Cultured cells were immunohistochemically stained using vimentin and cytokeratin as antibodies to prove the stromal origin of the cultivated cells. Positive staining to vimentin and negative immunohistochemical reaction to cytokeratin was observed (Figure 1). These results indicate that the cultured cells possess the characteristics of stroma cells.
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The effect of LF on proliferation of endometrial stroma cells
When 10–1000 ng/ml LF were added into the medium without FBS, BrdU incorporation into endometrial stroma cells increased significantly compared to controls (Figure 2
). This result indicated the stimulatory effect of LF on stroma cell proliferation. When medium containing 2% FBS was used, LF was even more effective in promoting cell growth than in FBS-free medium (Figure 3). Again, LF stimulated cell growth in serum-containing medium. This stimulatory effect by the addition of 1000 ng/ml of LF was diminished by the addition of anti-LF monoclonal antibody (Figure 4).
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Comparison of the effects of LF, oestradiol and EGF on proliferation of endometrial stroma cells in culture
Oestradiol and EGF were added to the wells to obtain final concentrations of 10 nmol/l and 10 ng/ml, respectively. LF was added to obtain a final concentration of 100 and 1000 ng/ml, respectively. Addition of oestradiol and EGF stimulated cell growth to 140 and 330% of the controls, respectively. The results indicated that 1000 ng/ml LF was more effective than 10 nmol/l oestradiol but less effective than 10 ng/ml EGF in stimulating the proliferation of endometrial stroma cells in culture. When LF and EGF were added to the medium at the same time, a significant increase of cell proliferation was observed compared with the addition of LF alone. However, there was no significant difference between EGF alone and EGF plus LF (Figure 5
).
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Discussion
The present study clearly demonstrated the proliferation effect of LF on human stroma cells in culture.
It is well known that oestrogen and progesterone play an important role in the proliferation and differentiation of endometrium. Mouse and human LF genes contain a functionally imperfect oestrogen response element (ERE) in the 5'-flanking promoter regions (Rey et al., 1990; Teng et al., 1992). Furthermore, since LF expression was induced by 17ß-oestradiol in the immature mouse uterus, but not in the mammary gland (Teng et al., 1986, 1989; Walmer et al., 1992), regulation of LF expression by ovarian sex steroids has been found to be tissue-specific (Teng et al., 1989). In the mouse uterus, LF is an oestrogen-induced uterine secretory protein present throughout the epithelium, and it is expressed concomitantly with epithelial cell proliferation (Hagiwara et al., 1995). These studies suggest that sex steroids such as oestrogen regulate LF.
In humans (Kelver et al., 1996), serum concentrations and the expression of LF by endometrial tissue obtained from subjects in both proliferative and secretory phases have been investigated. Serum LF concentrations were found to be higher in the proliferative phase than in the secretory phase, and the expression of LF assessed by immunohistochemical staining was also greater during the proliferative phase than the secretory phase. The LF concentration in human vaginal mucus is found to be highest just after menses and lowest just before menses (Cohen et al., 1987). Immunohistochemical staining (Walmer et al., 1995) showed that LF was localized to a region of normal endometrium known as the zona basalis. Walmer et al. reported that significantly more glands expressing LF protein are positive in the region of the zona basalis than in the zona functionalis of the endometrium both in proliferative and secretory phase. In our preliminary study, LF expression was also localized in epithelial cells but not in stroma cells. Studies to confirm that LF derived from epithelial cells can exert an effect on stroma cells will have to be performed in the future.
The process of induction and intracellular localization of LF in mice has been studied (Yamashita, 1995), and it was shown that LF was rapidly induced in 1 h after addition of oestrogen and was exclusively localized to the nucleoli of surface and glandular epithelium. Following 7 h of oestradiol stimulation, LF became localized to the stroma cell.
It has been reported that LF expression was induced by oestrogen and localized to the epithelium (McMaster et al., 1992; Kelver et al., 1996). These results suggest the possibility that LF could be related to proliferation of the endometrium. In the present study, human endometrial stroma cell culture was used as a model to study the proliferative effect of LF. It is interesting to note that LF possesses a proliferative effect on cultured cells without FBS. This result suggests that LF does not require additional cytokines and other factors present in serum to exert its effect. When FBS was added to the medium, the stimulatory effect of LF on cell proliferation was found to be greater than that without FBS.
Interestingly, the rate of proliferation induced by 10 nmol/l oestradiol was the same as that induced by a physiological concentration of LF (100 ng/ml). Furthermore, when anti-LF monoclonal antibody was added with LF to the culture medium, the stimulatory effect of LF on cell proliferation was inhibited. These results clearly demonstrate for the first time that LF possesses a cell proliferation effect in the endometrium. In the present study, stroma cells in proliferative phase were used. The question arises as to whether there is the same effect on stroma cells in secretory phase. Experiments using stroma cells in secretory phase may clarify the characteristic effect of lactoferrin on stroma cells in proliferative phase. As a preliminary study we have performed similar experiments using tubal cells and Ishikawa cells. However, a proliferation effect of lactoferrin could not be observed (data not shown).
It has been reported that the cell proliferation effect of LF is due to the transport of iron into cells in a manner similar to the action of transferrin. However, earlier studies (Nichols et al., 1989, 1990; Hagiwara et al., 1995) suggested that the cell proliferation action of LF could not merely consist of transporting iron into the cell. According to these results, the effect of LF alone on the proliferation of a rat intestinal epithelial cell line (IEC-18) is much greater than that of EGF alone. Therefore the proliferation effect of LF may be to have a modulatory effect on the action of growth factors (Hagiwara et al., 1995). However, further studies are needed to investigate the mechanism of human endometrium cell proliferation.
As mentioned above, the fact that LF expression was localized in human endometrial epithelium cells (McMaster et al., 1992; Kelver et al., 1996), but not in stroma cells, cannot explain why LF derived from epithelial cells would exert its action to stimulate endometrium proliferation in a paracrine fashion on the stroma. The physiological role of LF as an autocrine stimulator of endometrial epithelium cell proliferation has been shown previously in the mouse (Yamashita, 1995). Experiments to evaluate the similar effect of LF on human epithelial cells will have to be performed.
It was demonstrated (Kawakami et al., 1990) that bovine LF binds to rat transferrin receptor on the brush-border membrane. However, while LF receptors have been found in the colon, they have not yet been found in endometrium. Further study on the LF receptor may help to elucidate the mechanism of LF involvement in endometrial proliferation.
Acknowledgments
The authors are indebted to Dr Philip Troen, Professor of Medicine, Montefiore University Hospital, Pittsburgh, PA, USA for reviewing the manuscripts. We also wish to thank Miss H.Kurihara for her technical assistance.
Notes
1 To whom correspondence should be addressed 
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Submitted on October 11, 1999; accepted on February 4, 2000.
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