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Abstract

17β-estradiol (E2) is a female sex steroid hormone and exerts a pivotal role not only in female pregnancy but also in organ immune responses. Macrophages, as a kind of antigen-presenting cells, play an important influence on the cellular and humoral immune responses and also express the E2 receptor. In the present study, we explored the effects of E2 on the viability, endocytosis, surface molecule, and inflammatory cytokine expression of RAW264.7 macrophages. Results showed that E2 slightly increased the cell proliferation and endocytosis of RAW264.7 cells, while notably decreasing the mRNA and protein levels of inflammatory cytokines such as tumor necrosis factor (TNF)-α and monocyte chemoattractant protein-1 (MCP-1). As for the expression of surface molecules closely associated with the functions of macrophages, E2 significantly reduced the levels of CD40, CD80, and MHC-II. Interestingly, E2 reduced the levels of CD86 at low dose (10 nM and 1 nM), while enhancing its expression at high doses (1 μM and 0.1 μM). These results suggest that E2 may play an immuno-suppressive role in the inflammatory reactions and some autoimmune diseases partly by influencing the expressions of some important surface molecules and inflammatory cytokines of macrophages.

Keywords

17β-estradiol monocyte chemoattractant protein-1 (MCP-1)phenotype tumor necrosis factor (TNF)-α viability

Introduction

17β-estradiol (E2), as one of the important steroid estrogens, performs an essential role not only in the reproductive system but also in the immune system.¹ Much clinical evidence displays that some physiological processes, such as menstrual cycle,² pregnancy, and menopausal status, are affected by the fluctuation of E2. In addition, E2 has an obvious influence on inflammatory responses and autoimmune diseases.^1,3 E2 plays the biological role in immune regulations through binding its specific receptors. Numerous research has shown that E2 receptors (ER) are expressed by many types of immune cells, for example, T lymphocytes,⁴ B lymphocytes, natural killer cells, dendritic cells (DCs),⁵ and macrophages,⁶ which are involved in organ immune responses.

As pivotal effective and antigen-presenting cells, macrophages initiate, maintain, and modulate the innate and acquired immune responses. The existence of ER on or in macrophages suggests that E2 might regulate organ immune responses partly through regulating the functions of macrophages. Previous studies have focused on the role of E2 in regulating the functions of other immune cells such as T lymphocytes,⁷ B lymphocytes, and DCs,⁸ which seem to be more important in the adaptive immune responses. In our previous works, we studied the influence of the female sex hormone on murine spleen DCs (SDCs).^13,14 However, little is known about the effects of E2 on macrophages which are also a critical and classic membership in the immune system.^11,12 Therefore, this study is designed to characterize the influence of E2 on the viability, phenotype, and inflammatory cytokine expression of RAW264.7 cells in vitro.

Materials and methods

Reagents

E2, trypsin, fetal calf serum (FCS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), saponin, monensin, pipes, neutral red, Dulbecco’s modified Eagle’s medium (DMEM), and antibiotics all were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse anti-mouse FITC-conjugated monoclonal antibodies of IgG1k, MHC-II, CD40, monocyte chemoattractant protein-1 (MCP-1), and PE-labeled monoclonal antibodies of IgG2a, CD80, CD86, and tumor necrosis factor (TNF)-α were bought from Becton Dickinson Company (San Jose, CA, USA). The primers of TNF-α, MCP-1, and GAPDH were synthesized by Shanghai Sangong Biological Engineering Technology and Service Company. E2 was dissolved in 100% ethanol into 10 mM, and then diluted in DMEM medium to a concentration of 10 μM and stored at −20°C until use.

Cell culture

RAW264.7 cell line was kindly donated by Professor Yayi Hou (Medical School, Nanjing University). The cells were grown in DMEM (10% FCS, 1.5 g/L Sodium bicarbonate, 5×10⁻⁵ M β-mercaptoethanol, 100 U/mL penicillin, and 0.1 mg/mL streptomycin) and cultured at 37°C with 5% CO₂.

In this experiment, we designed five groups of RAW264.7 cells in accordance with the concentration of E2 which stimulated the cells for 24 h: Con (control group: add DEME culture media containing 0.1% water-free ethanol without E2), 1 μM, 0.1 μM, 10 nM, and 1 nM.

Viability test

The concentration of cells for each group was adjusted to 5×10⁴ mL⁻¹. A total of 100 μL of cell solution was added into each well of the flat-bottom 96-well microculture plates with five parallel wells of each group and cultured for 24 h. A total of 25 μL of 0.5% MTT was added into each well and incubated at 37°C for 4 h, then 100 μL of DMSO was put into every well. Finally, the plate was shaken slightly for 10 min and OD values were read at 570 nm on a Microplate Reader Bio-Rad 550.

Phenotype assay by flow cytometry

The level of phenotype molecules such as CD40, CD80, CD86, and MHC-II closely reflected the state and function of murine macrophage. The cells of all groups treated with E2 for 24 h were collected and rinsed in cold FACS buffer (PBS containing 1% fetal calf serum and 0.1% sodium azide). Then, the cells resuspended in FACS buffer were incubated with suitably diluted FITC labeled CD40, FITC labeled MHC-II, PE conjugated CD80, and PE conjugated CD86 monoclonal antibodies, respectively, at 4°C for 30 min according to the standard procedure. After that, the cells were washed three times and fixed by 1% paraformaldehyde. An isotype control (IgG2a) was performed for each antibody and gates were set using it. Fluorescence was analyzed using a FACScan flow cytometry (Becton Dickinson) and data analysis was done using the Cell Quest Software (Becton Dickinson, San Diego, CA, USA). Dead cells were excluded by forward and side scatter characteristics. Statistics presented are based on 10,000 events gated on the population of interest.

Endocytosis test

The cells of each group were adjusted to 5×10⁴ mL⁻¹ and then 200 μL of cell suspension was seeded into a flat-bottom 96-well microculture plate with five parallel wells for each group. After being cultured for 24 h, the supernatant of each well was removed. A total of 200 μL 0.1% neutral red solution was added into each well and cultured at 37°C for 3 h. Subsequently, the cells were lysed using 200 μL cytolysate (50% 0.1 M acetic acid and 50% absolute ethanol) and incubated at 4°C overnight. On the second day, the plate was shaken slightly for 5 min and the absorbance at optical density 490 nm was measured on a Microplate Reader Bio-Rad 550.

Reverse transcript-PCR

To analyze the effect of E2 on the mRNA levels of TNF-α and MCP-1 in RAW264.7 cells, RT-PCR analysis was performed. After 10⁵ cells were seeded in 24-well plates and treated with various concentrations of E2 for 24 h, total RNA was extracted using TRIzol reagent (Sigma) and was reverse transcribed into cDNA. PCR was performed (33 amplification cycles) to amplify TNF-α, MCP-1, and GADPH genes with an initial denaturation step at 94°C for 5 min, then 30 s at 94°C, 30 s at 56°, and 30 s at 72°C. The final elongation was carried out for 5 min at 72°C. Primers for TNF-α, MCP-1, and GADPH were as follows: TNF-α, 5’-CCCTCACACTCAGATCATCTTC -T-3’, and 5’-GCTACGACGTGGGCTACAG-3’ (PCR product: 61 bp); MCP-1, 5’-TAAAAACCTGGATC GGAACCAA-3’ and 5’-GCATTAGCTTCAGATTTACGG -GT-3’ (PCR product: 121 bp); GADPH, 5’-GGTGAAGGTCGGTGTGAACG-3’ and 5’-CTCGCTCCTGGAAGATGGTG-3’ (PCR product: 233 bp). A total of 8 μL of PCR amplifications were electrophoresed on a 1.5% agarose gel (Sigma) in 1×TBE buffer (0.025M EDTA, 0.089M Tris-borate, pH 8.3) under 110 V for 1 h, then the gel was stained with 1 μg/mL ethidium bromide and viewed under UV illumination 302 nm. Images were captured and quantitative densitometric analyses of each mRNA species were performed with Quantity One Software using PCR product of GADPH mRNA as an internal control.

Intracellular cytokine analysis by flow cytometry

RAW264.7 cells treated with different concentrations of E2 for 24 h in the presence of 3 μM monensin were rinsed twice in FACS medium phosphate-buffered PBS supplemented 1% FCS and 0.1% NaN3, and then fixed in 2% formaldehyde and permeabilized with 0.5% saponin for 20 min at 4°C, respectively. Samples were washed with wash buffer containing 0.5% saponin and then incubated with FITC- and PE-labeled monoclonal antibodies of MIP-1 and TNF-α for 30 min. Cells were then washed with 0.5% saponin three times and resuspended in 300 μL of 1% paraformaldehyde in PBS. Finally, cells were analyzed using a FACScan flow cytometry as described above.

Statistical analysis

The results are shown as the mean ± standard deviation (SD). Statistical significance was estimated by one-way analysis of variance (ANOVA) and Student’s t-test. A probability (P) value of <0.05 and 0.01 is considered to be significant and very significant.

Results

E2 upregulated the viability of RAW264.7 cells

The effects of E2 on the viability of RAW264.7 cells tested by MTT assays are shown in Figure 1. The results displayed that various concentrations of E2 slightly upregulated the viability of RAW264.7 cells in general compared with control group, but there was a statistical significance only in 0.1 μM group.

Figure 1.

E2 increased the viability of RAW264.7 cells. RAW264.7 cells were treated with various concentrations of E2 (Con, 1 μM, 0.1 μM, 10 nM and 1 nM) at 37°C for 24hr, and the viability of cells was analyzed by MTT assay. Results are shown as mean ± SD (n=5) from three independent experiments. *P < 0.05 vs. the control group.

E2 influenced the expression of surface molecules on RAW264.7 cells

The influence of E2 on the surface molecule expression of RAW264.7 cells was measured by using flow cytometry. As shown in Figure 2, E2 downregulated the expression of CD40, CD80, and MHC-II significantly in all E2-treated groups compared with the control group. However, the expression levels of CD86 increased in groups treated with higher doses (0.1 μM and 1 μM) while decreasing in groups treated with lower doses (10 nM and 1 nM).

Figure 2.

E2 regulated the expression of surface molecules on RAW264.7 cells. The phenotype of RAW264.7 cells were analyzed by flow cytometry after treatment with various concentrations of E2 for 24hr. A, the dot plots represented the double staining results of CD40 and CD80. B and C stood for the CD86 or MHC-II positive cells, respectively. D, the statistical analysis is a quantitative measure for the general expression of surface markers described as bar diagrams. The results shown in these figures are representative of three separate experiments. *P < 0.05, **P < 0.01 vs. the control group.

E2 increased the endocytosis of RAW264.7 cells

Macrophages play a vital role in the immune activities via endocytosis, antigen-presentation, cytokine release, and regulation of T cell signaling,¹³ so endocytosis is often regarded as an important trait of macrophages. To illustrate the influence of E2 on endocytosis of RAW264.7 cells, we used neutral red as the substance of endocytosis. The results showed that the endocytosis activities of RAW264.7 cells in all E2-treated groups increased significantly compared with the control group (Figure 3). Furthermore, the change of 0.1 μM group was the most obvious than other E2-treated groups.

Figure 3.

E2 augmented the endocytosis of RAW264.7 cells. After RAW264.7 cells were exposed to various concentrations of E2, the cell endocytosis was examined by neutral red endocytosis test. All E2-treated groups had a notable increase compared with the control one, while the endocytosis of 0.1μM group appeared to be more significant. Data represent the mean ± SD of 5 parallel wells from three independent experiments. **P < 0.01 vs. the control group.

E2 decreased the mRNA levels of TNF-α and MCP-1 in RAW264.7 cells

The mRNA levels of TNF-α and MCP-1 in RAW264.7 cells were quantified by RT-PCR. The results showed that E2 decreased the expression of TNF-α and MCP-1 significantly compared with the control group (Figure 4). Furthermore, the change appeared to be more obvious in the two terminal groups (i.e. the 1 μM and 1 nM groups).

Figure 4.

The effect of E2 on the mRNA levels of inflammatory cytokines (TNF-α and MCP-1) in RAW264.7 cells. A is the picture of electrophoresis bands which represented the levels of cytokines in RAW264.7 treated with different concentrations of E2 (Con, 1 μM, 0.1 μM, 10 nM, 1 nM) for 24hr, and B was the general presentation using bar diagrams. GADPH was used as a house-keeping gene control for comparison with inflammatory cytokine expression. The relative intensity of the cytokines compared to GADPH band was given for comparison. The results were consistent over three independent experiments. **P < 0.01 vs. the control group.

E2 downregulated the protein levels of TNF-α and MCP-1 in RAW264.7 cells

The protein levels of TNF-α and MCP-1 in RAW264.7 cells were examined by flow cytometry. Results showed that various concentrations of E2 decreased the expressions of TNF-α and MCP-1 significantly compared with the control group (Figure 5). These changes were consistent with the results of PCR.

Figure 5.

E2 suppressed the secretion of TNF-α and MCP-1 in RAW264.7 cells. RAW264.7 cells were treated with different concentrations of E2 (Con, 1 μM, 0.1 μM, 10 nM, 1 nM) for 24hr, then the levels of TNF-α and MCP-1 were analyzed by flow cytometry. A and B represented the percentages of TNF-α and MCP-1 positive cells respectively, and C was the general presentation of TNF-α and MCP-1 using bar diagrams. The results were consistent over three independent experiments. *P < 0.05, **P < 0.01 vs. the control group.

Discussion

Estrogens are known as the modulators of macrophage functions, however the underlying mechanism has not been clearly discovered.¹⁴

In this study, E2 slightly increased the viability of RAW264.7 cells. Previous research showed that E2 not only inhibited lymphocyte apoptosis^15,16 but also augmented the viability of plasmacytoid DCs,¹⁷ human monocytes, and macrophages,¹⁸ Furthermore, in our early study, E2 also upregulated the viability of SDCs.⁹

Besides the viability, E2 often affects the expression of some important surface markers on macrophages which are relevant to their functions. Our results showed E2 decreased the levels of CD40, CD80, CD86 (the expressions of CD86 were increased only in higher-dose groups), and MHC-II. Yum and his colleagues found that isoflavones, possessing a similar structure as E2 and exerting estrogenic effects, decreased the expression of CD40, CD80, CD86, and MHC-II on LPS-stimulated DCs.¹⁹ However, some research showed E2 increased the expression of CD40 and MHC-II of murine SDCs^9,10 and the levels of CD40 and CD86 of human DCs.²⁰ Of note, another study showed E2 had no obvious effect on the levels of CD40, CD80, and CD86 of plasmacytoid DCs.¹⁷ Our results suggested that E2 inhibits the antigen-presenting and stimulatory capacity of RAW264.7 cells through decreasing the expression of MHC-II, CD40, CD80, and CD86.

The changes in viability and phenotype are relative to the functions of RAW264.7 cells. Our results displayed that E2 increased the endocytosis of RAW264.7. A previous study showed that E2 upregulated the phagocytic capacity of kupffer cells,²¹ moreover, structural analogues of E2 also increased the endocytosis in LPS-stimulated DCs.¹⁹ Nevertheless, some studies showed that E2 suppressed the endocytosis of SDCs⁹ and a macrophage derived cell line from goldfish.²² Our results suggested that E2 enhances the endocytosis and processing capacity of macrophages so as to protect the host from the injury of extrinsic and intrinsic antigen.

It is well known that estrogen modulates cytokine immune responses.^23,24 Macrophages express functional estrogen receptors and secrete TNF-α, IL-6, IL-10, and many other cytokines which regulate organ immune responses.²⁵ We therefore analyzed the effects of E2 on the mRNA and protein expression of inflammatory cytokines of RAW264.7 cells. Our results showed that E2 decreased the intracellular expression of TNF-α and MPC-1 which are the classic inflammatory cytokines.²⁶ Previous studies showed that E2 decreased TNF-α production of bone marrow-derived DCs in young mice²⁷ and central nervous system-derived DCs,⁵ while the interferent of estrogen increased the expression of TNF-α in human macrophages.²⁸ Moreover, analogues of E2 also decreased the level of TNF-α in dendritic cells.¹⁹ MCP-1 belongs to CC chemokine which is chemotactic for monocytes and lymphocytes. MCP-1 can be secreted by macrophages²⁹ which play a crucial role in the process of the inflammation and rheumatoid arthritis. A previous study displayed that E2 downregulated MCP-1 release by monocytes.³⁰ Previously, studies seemed to support a view that E2 inhibits inflammation. For example, Naugler found that E2 administration markedly diminished the inflammation and injury associated with a chemical carcinogen diethylnitrosamine.³¹ In addition, E2 exerted an anti-inflammatory role in cardioprotection³² and protection of the central nervous system.³³ Moreover, during female pregnancy (during which the level of E2 is high), the symptoms concerning inflammation in rheumatoid arthritis patients were mitigated.^34,35 Our results seemed be consistent with the view that E2 inhibits inflammation.

In summary, E2 influences the viability, phenotype, endocytosis, and inflammatory cytokine expression of RAW264.7 cells and may exhibit immunosuppressive activities on these cells.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 31300707) and Training Program for University Prominent Young and Middle-aged Teachers and Presidents.

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17β-estradiol modulates the viability,phenotype,endocytosis,and inflammatory cytokine expression of RAW264.7 macrophages

Abstract

Keywords

Introduction

Materials and methods

Reagents

Cell culture

Viability test

Phenotype assay by flow cytometry

Endocytosis test

Reverse transcript-PCR

Intracellular cytokine analysis by flow cytometry

Statistical analysis

Results

E2 upregulated the viability of RAW264.7 cells

E2 influenced the expression of surface molecules on RAW264.7 cells

E2 increased the endocytosis of RAW264.7 cells

E2 decreased the mRNA levels of TNF-α and MCP-1 in RAW264.7 cells

E2 downregulated the protein levels of TNF-α and MCP-1 in RAW264.7 cells

Discussion

Footnotes

Declaration of conflicting interests

Funding

References