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Cardiovascular Research 1997 34(3):529-535; doi:10.1016/S0008-6363(97)00060-6
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Copyright © 1997, European Society of Cardiology

Effects of 17β-estradiol and progesterone on migration of human monocytic THP-1 cells stimulated by minimally oxidized low-density lipoprotein in vitro

Mayumi Okada, Akihiko Suzuki, Kimio Mizuno, Yoshimasa Asada, Yasushi Ino, Tomoyuki Kuwayama, Koji Tamakoshi, Shigehiko Mizutani* and Yutaka Tomoda

Department of Obstetrics and Gynecology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan

* Corresponding author. Tel.: +81 (52) 744-2261; fax: +81 (52) 744-2268.

Received 5 July 1996; accepted 28 January 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Many epidemiological studies have shown that postmenopausal hormone replacement therapy (HRT) has a beneficial effect on atherosclerotic cardiovascular disease. The aim of this study was to investigate the effects of estrogen and progestin on the migration of monocytes induced by minimally oxidized low-density lipoprotein (m-ox-LDL) in vitro. Methods: Human monocytic THP-1 cells were used for the study. Migration assay was performed using a modified Boyden chamber. Results: The presence of estrogen receptors was determined in THP-1 cells by Western and Northern blot analysis. Although native LDL had no significant effects on the migration of THP-1 cells, m-ox-LDL increased the migration of THP-1 cells in a dose-dependent manner. Although 17β-estradiol (E2, 10–9 ~10–6 M) inhibited the 10 µg/ml-induced migration of THP-1 cells in a dose-dependent manner, estrone (E1), estriol (E3) and progesterone (P) had no significant effects. The combination of P (10–9~10–6 M) did not show any effect on the inhibitory effect of 10–7 M E2. Preincubation of THP-1 cells with the anti-estrogenic agent, tamoxifen (10–6 M), significantly antagonized the inhibitory effect of 10–7 M E2. m-ox-LDL stimulated MCP-1 secretion from THP-1 cells, which was reduced by E2. Anti-human MCP-1 neutralizing antibody inhibited the migration of THP-1 cells stimulated by m-ox-LDL. E2 also inhibited the 10 ng/ml MCP-1-induced migration of THP-1 cells in a dose-dependent manner. Conclusions: These findings suggest that the inhibitory effect of E2 on the migration of monocytes might be one of the factors involved in the decreased incidence of atherosclerotic cardiovascular disease in premenopausal women and postmenopausal HRT.

KEYWORDS Estrogen; Progestin; Monocytes; Minimally oxidized LDL; Migration; MCP-1; Postmenopausal hormone replacement therapy; Atherosclerosis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Hormone replacement therapy (HRT) with estrogen and progestin is widely used in postmenopausal women. It is known that exogenously administrated estrogen inhibits the progression of experimentally induced atherosclerosis in animal models [1] and also reduces the risk of atherosclerotic cardiovascular disease in postmenopausal women [2, 3]. The significantly low incidence of coronary heart disease in premenopausal women has been noted [2, 3]. However, the mechanisms by which the effects of estrogen are mediated on atherosclerosis are poorly understood. In addition, the effect of progestin, which is usually prescribed with estrogen, on atherosclerosis is also unknown.

At present, it is generally recognized that among the early events in atherogenesis, the focal attachment of monocytes to the endothelium and their subsequent transendothelial migration are important components [4]. Studies in cholesterol-fed animals suggest the increased accumulation of monocytes in the arterial intima in the early stage of atherosclerotic lesions [5]. A subject of great interest in this regard is the role of low-density protein (LDL) in the etiology and the course of atherosclerosis [6]. It is also known that the degree of oxidation of LDL is relatively small in the early stage of atherosclerotic lesions [7, 8], and that minimally oxidized low-density lipoproteins (m-ox-LDL) increase the adhesion of monocytes to endothelial cells (ECs) and induce chemotactic factors such as monocyte chemotactic protein (MCP-1) and interleukin-1 (IL-1), which stimulate the transendothelial migration of monocytes [9]. Therefore, m-ox-LDL is suggested to play a major role not only in adhesion to ECs but also in the migration of monocytes into the subendothelial space. Therefore, the relation between m-ox-LDL and the migration of monocytes is important to the understanding of atherogenesis.

There have been no previous reports concerning the effects of estrogen and/or progestin on the migration of monocytes exposed to m-ox-LDL [m-ox-LDL; 2–5 nmol thiobarbituric-acid-reactive substance (TBARS)/mg protein] [7]. In the present study, we examined the presence of estrogen receptors in human monocytic THP-1 cells (human monocytic leukemia cell line), which is generally used as a model of human monocytes in experiments in the field of atherosclerosis research [10, 11], by Western and Northern blot analysis and then investigated whether estrogen influences the m-ox-LDL-stimulated migration of these cells in vitro. In addition, we also examined the effects of m-ox-LDL on the secretion of MCP-1 from THP-1 cells and whether E2 could counteract these effects of m-ox-LDL.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Estrone (E1), 17β-estradiol (E2), estriol (E3), progesterone (P) and tamoxifen were purchased from Sigma Chemical Co. (St. Louis, MO); phenol-red-free RPMI 1640 medium from Cosmo Bio Co. (Tokyo, Japan); Nylon membranes (Hybond N+) and nitrocellulose membrane (Hybond C) from Amersham International (Amersham, UK); anti-human estrogen receptor mouse monoclonal antibody from Medac Co. (Germany); 12-O-tetradecanoylphorbol-13-acetate (TPA) from Wako Pure Chemical Industries (Osaka, Japan); human monocyte chemotactic factor (MCP-1) from Pepro Tech Inc. (Rocky Hill, NJ); anti-human MCP-1 neutralizing antibody from R&D System Inc. (Minneapolis, MN).

In all experiments, E1, E2, E3, P and tamoxifen were dissolved in 99.5% ethanol, and the final concentration of ethanol in each experiment was less than 0.05%. The steroids we used in the following experiments were free from fetal bovine serum.

2.2 Cell culture
THP-1 cells were obtained from the Japanese Cancer Resources Bank which was established from the blood of a 1-year-old boy with acute monocytic leukemia by Tuchiya et al. [10] and has been used in many experiments on atherosclerosis. The cells were incubated in RPMI 1640 medium, containing 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2. MCF-7 cells (estrogen-receptor-rich human breast cancer cell line) were also incubated in RPMI 1640 medium with 10% FBS, and used as positive controls in several experiments. THP-1 cells were differentiated into macrophages by incubation with 10–7 M TPA for 48 h in phenol-red-free RPMI 1640 medium with 10% FBS. We used TPA-untreated and non-adherent cells in this study.

2.3 Determination of estrogen receptor in THP-1 cells and THP-1-derived macrophages
We determined the presence of estrogen receptor by Western blot [12] and Northern blot [13] analyses. THP-1 cells were maintained in RPMI 1640 medium containing 10% FBS, and 48 h prior to the experiments the medium was replaced with phenol-red-free RPMI 1640 containing 10% FBS. THP-1 cells were differentiated into macrophages by incubation with 10–7 M TPA for 48 h prior to experiments in phenol-red-free RPMI 1640 containing 10% FBS. MCF-7 cells were maintained in RPMI 1640 medium containing 10% FBS. Cells were lysed by sonication, and 20 µg of protein from each sample was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred electrically onto a nitrocellulose membrane (Hybond C) and labeled by anti-human estrogen receptor mouse monoclonal antibody and biotinylated anti-mouse immunoglobulin as the second antibody. Labeled proteins were visualized with horseradish peroxidase substrate (Elite ABC Kit, Vectastain, Vector Lab, Inc., CA). For Northern blot analysis, mRNA was extracted using a Quick prep m-RNA purification kit (Amersham, UK). One microgram of mRNA was electrophoresed through 1% denaturing formaldehyde agarose gel, transferred onto a nylon membrane (Hybond N+), hybridized with 32P-labeled 2.1 kb EcoR1 fragment of rat estrogen receptor cDNA (obtained from the Japanese Cancer Resources Bank), and analyzed by autoradiography. Between rat and human estrogen receptor, the homology of the DNA binding domain is 100%, that of the estrogen binding domain is 96%, and the overall homology is 88% at the amino acid sequence level [14]. Protein content was determined according to the method of Lowry et al. [15], using BSA as a standard.

2.4 Preparation of minimally oxidized LDL (m-ox-LDL)
Human LDL was purchased from Chemicon International Inc. (Temecula, CA), which was isolated from fresh human plasma by sequential isopycnic ultracentrifugation. Details of the steps taken to avoid oxidation during preparation are shown in the brochure for Human Low Density Lipoproteins from Chemicon International Inc. (Temecula, CA). Oxidation of LDL was performed by short-wavelength ultraviolet irradiation (Osram lamp, 254 nm, 0.5 m/cm2) for 2 h [16, 17]. The efficiency of the oxidation was determined by analyzing the presence of thiobarbituric-acid-reactive substances (TBARS) and expressed as malondialdehyde (MDA) equivalents as described by Yagi [18].

2.5 Migration assay
Migration assay was performed using the Transwell cell culture apparatus (Costar, Cambridge, MA) described by Capsoni et al. [19] with some modifications [20, 21], using a polycarbonate membrane filter with pores 5 µm in diameter. N-LDL, m-ox-LDL and MCP-1 were dissolved in phenol-red-free RPMI 1640 and placed in the lower compartment. Cell suspensions of 2x106 cells/ml in phenol-red-free RPMI 1640 and various concentrations of E1, E2, E3, and P were loaded in the upper compartment and incubated for 6 h at 37°C in a humidified atmosphere with 5% CO2. Non-migrating cells on the upper surface were scraped gently and washed out with PBS 3 times. The filters were fixed in methanol and stained with Giemsa. The number of cells per x400 high-power field (HPF) which had migrated to the lower surface of the filter was counted microscopically. Five HPFs per filter were counted and the results were averaged. All the data in the following experiments are the mean of triplicate within one experiment and we also repeated 3 or more experiments to confirm these experiments. Blocking assay was performed using anti-human MCP-1 neutralizing antibody, which was dissolved in various concentrations in phenol-red-free RPMI 1640 with or without m-ox-LDL and placed in the lower compartment.

2.6 Effects of tamoxifen
THP-1 cells were incubated with tamoxifen (10–6 M) for 3 h before the addition of 10–7 M E2 in order to block estrogen receptors, and then used in the experiments. This concentration of tamoxifen is that normally used known to antagonize estrogen in breast cancer cell lines [22].

2.7 Measurement of MCP-1 in the cultured medium of THP-1
THP-1 cell suspension (2x106 cells/ml) in phenol-red-free RPMI 1640 with added LDLs with and without E2 (10–7 M) were incubated in the flask for 1 h at 37°C in a humidified atmosphere with 5% CO2. Then the cultured medium was centrifuged for 10 min at 150xg to remove THP-1 cells and stored at –80°C for the measurement of MCP-1. The concentrations of MCP-1 were measured by ELISA kit supplied from HyCult biotechnology (Uden, The Netherlands). The intra-assay and interassay coefficients of variations were 4.65% (n=7) and 8.89% (n=3), respectively. The minimum detection level of this kit is 10 pg/ml. After we confirmed the linearity of the standard curves with the kits which we used, we performed our experiment. Protein content of the THP-1 cells collected by centrifugation was determined according to the method of Lowry previously described [15]. The content of MCP-1 was expressed as ng/mg protein. Experiments were performed in triplicate.

2.8 Data analysis
Statistical analysis was performed by Student's unpaired and t-test, P<0.05 was accepted as statistically significant. All data are expressed as mean±s.d.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Determination of estrogen receptors in THP-1 cells and THP-1-derived macrophages
The expression of estrogen receptors was shown in THP-1 cells and THP-1-derived macrophages by Western and Northern blot analysis. The 65 kDa immunostained bands on Western blots, and 6.3 kb bands on Northern blot analysis of THP-1 cells, THP-1-derived macrophages and MCF-7 cells corresponded to human estrogen receptor protein and mRNA, respectively [23] (Fig. 1).


Figure 1
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  		Fig. 1 Determination of estrogen receptors in THP-1 cells. Estrogen receptor protein and mRNA were determined by Western (left) and Northern blot analysis (right), respectively, as described in Section 2; 20 µg of protein and 1 µg of mRNA were used (lanes 1 and 4, MCF-7; lanes 2 and 5, THP-1-derived macrophages; lanes 3 and 6, THP-1), respectively. MCF-7 cells were used as positive controls.

 
3.2 Effects of E2 and P on migration of THP-1 cells
All the data in the following experiments are the mean of triplicate assays within one experiment and we also repeated 3 or more experiments to confirm these experiments.

N-LDL contained 0.54±0.03 nmol MDA/mg protein (n=3) and m-ox-LDL contained 3.56±0.02 nmol MDA/mg protein (n=3), respectively. We used these n-LDL and m-ox-LDL preparations in experiments on the effects of E2 and P on the migration of THP-1 cells. As a preliminary experiment, THP-1 cells were incubated in the upper compartment and the various concentrations of n-LDL or m-ox-LDL in the lower compartment for 6 h. Although n-LDL (0.52±0.02 nmol MDA/mg protein) had no significant effect on the migration of THP-1 cells, m-ox-LDL (3.23±0.03 nmol MDA/mg protein) increased the migration of THP-1 cells in a dose-dependent manner (Fig. 2). M-ox-LDL 10 µg/ml induced a 2.5-fold higher migration of THP-1 cells than that in the controls (Fig. 2). Since significant migration of THP-1 cells occurred at 3 h after exposure to 10 µg/ml m-ox-LDL and reached a plateau at 6 h (data not shown), we determined the number of migrated cells after 6 h stimulation throughout the following experiments.


Figure 2
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  		Fig. 2 Effect of n-LDL (solid columns) and m-ox-LDL (hatched columns) on migration of THP-1 cells. The migration responses were determined as described in Section 2. Results are expressed as the number of migrated THP-1 cells stimulated by various concentrations of n-LDL (0.52±0.02 nmol MDA/mg protein) and m-ox-LDL (3.23±0.03 nmol MDA/mg protein). Values are expressed as mean±s.d. (n=3, *P<0.01, **P<0.001).

 
E2 reduced significantly the cell migration in a dose-dependent manner, but E1, E3 and P had no significant effects on the migration of THP-1 cells (Table 1). P at various concentrations (10–9 M~10–6 M) did not change the effects on the migration of THP-1 cells due to 10–7 M of E2 (Fig. 3).


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  		Table 1 Effect of E1, E2, E3 and P on 10 µg/ml m-ox-LDL-induced migration of THP-1 cells

 

Figure 3
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  		Fig. 3 Effect of P in combination with E2 on 10 µg/ml m-ox-LDL (3.56±0.02 nmol MDA/mg protein) induced migration of THP-1 cells. The migration responses were determined as described in Section 2. Values are expressed as mean±s.d. (n=3, *P<0.001).

 
In these migration assays, E2 was placed in the upper compartment only. When E2 (10–7 M) was placed in either the upper or the lower compartment only or in both compartments, we did not find any significant difference (Table 2). Therefore, we need not have taken the concentration gradient of E2 into consideration. We confirmed that there were no significant changes in the migration of THP-1 cells, when the various concentrations of E2 were placed in the upper compartment and only medium in the lower compartment (data not shown).


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  		Table 2 Effect of a concentration gradient of E2 on the migration of THP-1 cells

 
3.3 Effects of tamoxifen on migration of THP-1 cells
Preincubation of THP-1 cells with 10–6 M tamoxifen for 3 h before the addition of 10–7 M E2 significantly antagonized the inhibitory effect of E2 (Fig. 4).


Figure 4
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  		Fig. 4 Antagonistic effect of tamoxifen (10–6 M) on the inhibitory effect of E2 (10–7 M) in migration assay. The migration was stimulated by 10 µg/ml m-ox-LDL (3.56±0.02 nmol MDA/mg protein). The migration responses were determined as described in Section 2. Values are expressed as mean±s.d. (n=3, *P<0.001; NS = not significant).

 
3.4 MCP-1 measurement in the culture medium
As a preliminary experiment, THP-1 cells (2x106 cells/ml) were incubated for 3 h with 10 µg/ml m-ox-LDL in the flask at various time intervals, and the concentrations of MCP-1 in the culture medium was measured. The concentrations of MCP-1 were increased with advancing incubation times and reached their plateau at 1 h and remained at the plateau until 3 h (data not shown). We therefore measured the concentration of MCP-1 after 1 h incubation in the following experiments. Both m-ox-LDL and n-LDL stimulated the secretion of MCP-1 from THP-1 cells, however, the degree of MCP-1 secretion stimulated by m-ox-LDL was much more potent than that by n-LDL. Although E2 (10–9 M) could not reduce secretion of MCP-1 by m-ox-LDL (data not shown), E2 at 10–7 M reduced it significantly (Fig. 5).


Figure 5
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  		Fig. 5 Effects of LDL on the secretion of MCP-1 from THP-1 cells. The concentrations of MCP-1 in the cultured medium of THP-1 cells (2x106 cells/ml) with 10 µg/ml LDL (3.48±0.02 nmol MDA/mg protein) or 10–7 M E2 were measured as described in Section 2. Results are expressed as pg/mg protein. Values are expressed as mean±s.d. (n=4, *P<0.05, **P<0.01, ***P<0.0001).

 
3.5 Effects of anti-MCP-1 neutralizing antibody on migration of THP-1 cells stimulated by m-ox-LDL
The antibody against MCP-1 inhibited significantly the migration of THP-1 cells stimulated by m-ox-LDL in a dose-dependent manner (Fig. 6).


Figure 6
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  		Fig. 6 Effects of anti-human MCP-1 neutralizing antibody (anti-MCP-1 Ab) on migration of THP-1 cells stimulated by 10 µg/ml m-ox-LDL (3.48±0.02 nmol MDA/mg protein). The migration responses were determined as described in Section 2. Values are expressed as mean±s.d. (n=3, *P<0.005, **P<0.0001).

 
3.6 Effects of E2 on migration of MCP-1-stimulated THP-1 cells
As a preliminary experiment, THP-1 cells (2x106 cells/ml) in phenol-red-free RPMI 1640 in the upper compartment and various concentrations of MCP-1 or ethanol vehicle in the lower compartment, respectively, were loaded and incubated for 6 h at 37°C. MCP-1 (0~10 ng/ml) increased the migration of THP-1 cells in a dose-dependent manner, and 10 ng/ml MCP-1 induced a 2.5-fold greater migration than that in the controls (data not shown). E2 reduced significantly the stimulated cell migration by 10 ng/ml MCP-1 in a dose-dependent manner (Table 3).


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  		Table 3 Effects of E2 on 10 ng/ml MCP-1-induced migration of THP-1 cells

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Albeit qualitative, our data clearly showed the presence of both estrogen receptor proteins and mRNA in THP-1 cells and THP-1-derived macrophages by Western and Northern blot analysis (Fig. 1). Previously, Gulshan et al. suggested the presence of estrogen receptors in both human monocytic cell line (J111)-derived macrophages and rat peritoneal macrophages using a receptor binding assay [24]. Our data confirmed the ubiquitous localization of estrogen receptor not only in reproductive organs but also in monocytes and macrophages.

Our present data showed that the oxidation of LDL is related to the migration of monocytes; m-ox-LDL directly induced the migration of monocytes without the existence of ECs or vascular smooth muscle cells (SMCs) (Fig. 2). It was also shown that both m-ox-LDL and n-LDL stimulated the secretion of MCP-1 from THP-1 cells and the degree of MCP-1 secretion stimulated by m-ox-LDL was much more potent than that by n-LDL (Fig. 5). In addition, we showed that antibody against MCP-1 inhibited the migration of THP-1 cells stimulated by m-ox-LDL (Fig. 6). Therefore our in vitro data suggested strongly that the stimulated migration of monocytes by m-ox-LDL is due to the increased secretion of MCP-1 from monocytes themselves. Berliner et al. reported that m-ox-LDL increased the binding of monocytes to endothelium and increased production of a monocyte chemotactic factor from ECs [7]. Cushing et al. showed that m-ox-LDL induced the increase in monocyte chemotactic activity in co-cultures of ECs and SMCs, and this was attributable to monocyte chemotactic protein 1 (MCP-1) [9]. Zhou et al. showed also that phosphatidic acid, which plays a crucial role in lipid biosynthesis, induced the migration of monocytes [25]. Our present data showed that E2 also inhibited the migration of THP-1 cells stimulated by exogenous MCP-1 (Table 3).

Among 3 major endogenous estrogen tested, E2 at its physiological concentration (10–9 M) counteracted the migration of THP-1 cells stimulated by both m-ox-LDL (Table 1) and MCP-1 (Table 3). Although the concentration was much higher than that of physiological range, E2 at 10–7 M counteracted also the secretion of MCP-1 from THP-1 cells stimulated by m-ox-LDL (Fig. 5). In addition, our data with the antiestrogenic agent, tamoxifen, suggested that the inhibitory effects of E2 on the migration of THP-1 cells stimulated by m-ox-LDL are via estrogen receptors (Fig. 4).

Taken together, these data showed that E2 might counteract the action of m-ox-LDL, which plays a major role in the etiology and course of atherosclerosis [6], by inhibiting the secretion of MCP-1 from monocytes. In this respect, it is interesting to note that Frazier-Jessen et al. reported that, not P, but E2 at physiological level inhibited lipopolysaccharide (LPS)-stimulated MCP-1 mRNA expression in two murine macrophage cell lines (ANA-1 and J774A) and murine peritoneal macrophages via estrogen receptor [26]. Shanker et al. also reported that estrogen at its physiological level modulated IL-1 gene expression in activated THP-1-derived macrophages [11]. Furthermore, Mazière et al. showed also that E2 (10–5 M) has preventive effects on the production of m-ox-LDL in vitro [27]. A detailed analysis to delineate the interrelationship among these various factors on monocyte migration was left for future studies.

Our present data showed that P did not affect the inhibitory effects of E2 on the migration of m-ox-LDL-stimulated THP-1 cells (Fig. 3). Although there have been no previous reports concerning the presence of P receptors in monocytes and macrophages, some reports suggested the possibility that P has an effect on the expression of IL-1 in monocytes [28]. The use of progestin with estrogen is usually recommended in HRT for the prevention of endometrial and breast cancer due to the fact that the anti-estrogenic effects of progestin on the uterine endometrium and the mammary glands are beneficial for the treatment of these cancers [29]. Therefore, further studies about the effects of P are warranted.

Our present study showed inhibitory effects of E2 on the migration of monocytes, one of the initial steps in the development of atherosclerotic plaque. E2 inhibited the migration of monocytes stimulated by m-ox-LDL, possibly via inhibition of MCP-1 secretion. Further studies on the effects of HRT on the migration of monocytes, including in vivo experiments, are needed to clarify the beneficial effects of HRT on atherosclerosis.

Time for primary review 28 days.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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