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

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.

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.

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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).

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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.

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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).

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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.

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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.