Epigallocatechin-3-gallate (EGCG) is the major polyphenolic component of green tea. The aim of the current study was to investigate the inhibitory effects of EGCG on anti-β2-glycoprotein I (β2GPI)/β2GPI-induced tissue factor (TF) and tumor necrosis factor-α (TNF-α) expression in the human acute monocytic leukemia cell line, THP-1, as well as the underlying mechanisms. Human THP-1 cells cultured
Antiphospholipid syndrome (APS) is an autoimmune disorder caused by the production of antiphospholipid antibodies (aPLs) which contribute to thrombosis (
The anti-β2GPI/β2GPI complex activates endothelial cells and monocytes upon binding to the surface membrane of endothelial cells and monocytes, promoting tissue factor (TF) activity, thereby increasing the risk of thrombosis, and enhancing the expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, which are beneficial to thrombus formation in APS (
Polyphenols of green tea, which comprise 30% of the dry weight of green tea leaves, include epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epicatechin (EC). Among these, EGCG is the most abundant catechin and has a variety of biological and pharmacological properties, preventing cancer, allergies, oxidation, microbes, thrombosis, inflammation and cardiovascular diseases. Previous studies have demonstrated that EGCG exerts a number of beneficial effects by affecting a wide array of signal transduction pathways, including Notch (
It is known that EGCG has beneficial effects; however, whether it affects the anti-β2GPI/β2GPI complex-stimulated activation of THP-1 cells remains to be determined. In the present study, we investigated the ability of EGCG to block the effects of the anti-β2GPI/β2GPI complex on THP-1 cells and the possible mechanisms involved in this process.
The human acute monocytic leukemia cell line, THP-1, was obtained from Shanghai Institutes Biological Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 1% glutamine, 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) (Gibco-BRL). The cells were cultured at 37°C in a humidified incubator supplemented with 5% CO2 near confluence and deprived of serum for 16 h prior to being used in the experiments. All experimental data were obtained from cells at passages 3–10.
THP-1 cells were seeded at 2×106 cells/well into 6-well plates and serum-starved for 16 h prior to stimulation with monoclonal anti-β2GPI (10 μg/ml; Chemicon, Temecula, CA, USA)/β2GPI (100 μg/ml; US Biological, Swampscott, MC, USA) complex, anti-β2GPI (10 μg/ml)/bovine serum albumin (BSA) (100 μg/ml; Sigma, St. Louis, MO, USA), control rabbit immunoglobulin G isotype (R-IgG) (10 μg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)/β2GPI (100 μg/ml) or 500 ng/ml of LPS (
THP-1 cells were seeded at 2×106 cells/well into 6-well plates and serum-starved for 16 h prior to stimulation with anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml) complex, anti-β2GPI (10 μg/ml)/ BSA (100 μg/ml), R-IgG (10 μg/ml)/β2GPI (100 μg/ml) or 500 ng/ml of LPS for 24 h. The cells in some wells were pre-treated with various concentrations of EGCG (0–50 μg/ml) for 1 h, and EGCG was not removed. TNF-α protein, secreted into the cell culture medium, was measured using the TNF-α ELISA kit (Neobioscience, Shenzhen, China), following the manufacturer’s instructions. The TNF-α protein concentration in the cell culture medium was expressed as pg/ml.
THP-1 cells 2×106 cells/well were treated as described above for the indicated periods of time. Cell lysates were collected and assayed using TF activity kits (Assaypro, Greenwich, CT, USA) according to the manufacturer’s instructions. The TF activity in the cells was determined as factor X activation by the TF/VIIa complex as described in our previous studies (
The THP-1 cells were seeded at 2×106 cells/well into 6-well plates and serum-starved for 16 h prior to stimulation with the complex of monoclonal anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml), LPS (500 ng/ml) for 6 h. The cells in some wells were pre-treated with various concentrations of EGCG (0–50 μg/ml) for 1 h, and EGCG was not removed. For the determination of total cellular protein, the cells were collected and lysed with lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 2.5 mM EDTA and 1 mM PMSF. The lysates were centrifuged at 10,000 rpm for 30 min using the Compact High Speed Refrigerated Centrifuge 6930. (Kubota, Tokyo, Japan) to remove unbroken cells, nuclei and other organelles. The supernatant containing plasma membrane was recovered and stored at −70°C for analysis. Equal amounts of protein (5 μg) were electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in fresh 5% dry non-fat milk in Tris-buffered saline/0.05% Tween-20 (TBST) for 1 h at room temperature, washed with TBST 3 times, and then incubated with the primary antibodies against p38 MAPK, phospho-p38 MAPK (p-p38), ERK1/2, phospho-ERK1/2 (p-ERK1/2), JNK, phospho-JNK (p-JNK), NF-κB (p65), phospho-NF-κB (p-p65), IκB-α (1:1,000; Cell Signaling Technology, Beverly, MA, USA), TLR4 (1:500; eBioscience, San Diego, CA, USA) and β-actin (1:2,500; Proteintech Group, Inc., Chicago, IL, USA) overnight at 4°C. Following 3 washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Finally, the immunoblot signals were developed using ECL detection reagents (Millipore, Billerica, MA, USA), imaged and quantified using a Bio-Rad Fluor-S MultiImager (Typhoon 9400; Amersham, Uppsala, Sweden).
Data are expressed as the means ± SEM. The statistically significant differences were calculated by applying analysis of variance (ANOVA) using SPSS software (version 16.0). Values of P<0.05 were considered to indicate statistically significant differences.
Treatment of the THP-1 cells with the anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml) complex significantly enhanced the TF mRNA levels (
In this study, we first investigated whether EGCG decreases the effects of anti-β2GPI/β2GPI complex-induced TF expression in THP-1 cells. The cells were treated with various concentrations of EGCG (0–50 μg/ml) and then stimulated with anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml) complex for the indicated periods of time. Pre-treatment with EGCG (0–50 μg/ml) inhibited anti-β2GPI/β2GPI complex-induced TF expression and activation in a dose-dependent manner, showing statistical significance at 20–50 μg/ml [P<0.05 vs. control (medium only)] (
We then explored the specific effects of EGCG on anti-β2GPI/β2GPI complex-enhanced TF expression in THP-1 cells. Pre-treatment with 50 μg/ml EGCG significantly reduced the anti-β2GPI/β2GPI complex- or LPS-enhanced TF mRNA levels in THP-1 cells (P<0.05 vs. anti-β2GPI/β2GPI complex or LPS stimulation alone) (
In order to investigate the effects of EGCG on the expression of TNF-α induced by the anti-β2GPI/β2GPI complex, the cells were pre-treated with EGCG (0–50 μg/ml) prior to stimulation with the anti-β2GPI/β2GPI complex for the indicated periods of time. Pre-treatment with EGCG (5–50 μg/ml) inhibited the mRNA expression levels of TNF-α in response to the anti-β2GPI/β2GPI complex in a dose-dependent manner, presenting statistical significance at 20–50 μg/ml (P<0.05 vs. control) (
We further determined the specific effects of EGCG on TNF-α expression in THP-1 cells. Pre-treatment with 50 μg/ml EGCG significantly reduced the anti-β2GPI/β2GPI complex-enhanced TNF-α mRNA and protein expression levels (
TLR4, a family of integral membrane proteins, has been reported to mediate aPL-induced endothelial cell or monocytic cell activation (
Previously, we found that the anti-β2GPI/β2GPI complex or LPS stimulated the activation of MAPK pathways in THP-1 cells within 30 min of treatment (
NF-κB, originally emerged as a major regulator of innate and adaptive immunity and inflammatory responses, and has been shown to be involved in the signal transduction of TLR4/LPS (
APS is defined by one or more episodes of thrombosis or unexplained pregnancy loss in association with persisting positive aPLs, either anticardiolipin (aCL), anti-β2GPI, and/or lupus anticoagulant (LAC) (
Recent improvements in the understanding of the pathogenic mechanisms of APS, including the aPL-induced activation of platelets, endothelial cells, monocytes, complement and coagulation cascade, has led to the identification of potential targets and future therapies for APS. In general, treatment regimens for APS must be individualized according to the current clinical status of the patient and the history of thrombotic events. Low-dose aspirin is used widely in the treatment of patients with APS. However, the effectiveness of low-dose aspirin as the primary prevention therapy for APS remains unproven. Clopidogrel has anecdotally been reported to be helpful in individuals with APS and may be useful in patients allergic to aspirin (
Based on recent advances in the pathophysiology of APS, many new therapeutic modalities for treating and/or preventing thrombosis in patients with APS have been reported, such as B-cell targeted therapies (
Green tea, produced from the tea plant
In our previous studies, we revealed that TLR4 acts as a co-factor for Annexin A2 on the THP-1 cell surface, and contributes to anti-β2GPI/β2GPI complex-enhanced TF expression in THP-1 cells (
In a previous study, EGCG was reported to affect an array of signal pathways through which it exerts its pharmacological activities. EGCG was shown to inhibit the degradation of IRAK induced by IL-1β in A549 cells (
In the present study, we further demonstrate that EGCG (50 μg/ml) significantly suppresses TLR4 mRNA and protein expression in cells stimulated with the anti-β2GPI/β2GPI complex or LPS (
In conclusion, data from our present study, as well as from our previous studies, strongly indicate that EGCG inhibits the anti-β2GPI/β2GPI-induced activation of THP-1 cells by decreasing TF and TNF-α expression levels via blocking the intracellular signal transduction pathway of TLRs-MAPKs-NF-κB axis, and may serve as a preventive and therapeutic agent for APS.
This study was supported by grants from the National Natural Science Foundation of China (no. 81370614) to H.Z. and the Student’s Scientific Research of Jiangsu University (no. CX08 B_16x) to T.W.
Anti-β2-glycoprotein I (β2GPI)/β2GPI complex induces the expression of tissue factor (TF) and tumor necrosis factor-α (TNF-α) in THP-1 cells THP-1 cells (2×106) were treated with anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml) complex, rabbit immunoglobulin G (R-IgG; 10 μg/ml)/β2GPI (100 μg/ml), anti-β2GPI (10 μg/ml)/bovine serum albumin (BSA) (100 μg/ml) and lipopolysaccharide (LPS) (500 ng/ml) for the indicated periods of time. Total RNA of cells (at 2 h) or cell lysates (at 6 h) or cultural supernatants (at 24 h) was collected. (A) TF mRNA and (C) TNF-α mRNA levels were detected by qRT-PCR. (B) TF activity and (D) TNF-α protein levels were analyzed using the commercial kits as described in Materials and methods. Data are presented as the means ± SEM from 3 independent experiments. *P<0.05 vs. control (untreated cells, medium only; medium).
Effects of epigallocatechin-3-gallate (EGCG) on the mRNA expression and activity of tissue factor (TF) in THP-1 cells. Cells (2×106) were pre-treated with or without the indicated concentrations of EGCG for 1 h and then incubated with anti-β2-glycoprotein I (β2GPI) (10 μg/ml)/β2GPI (100 μg/ml) complex for 2 or 6 h, as indicated. On the other hand, other THP-1 cells (2×106) were incubated with anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml) complex or lipopolysaccharide (LPS) (500 ng/ml) in the absence or presence of EGCG (50 μg/l) for the indicated periods of time. EGCG was not removed from the medium. Total RNA of cells (2 h) or cell lysates (6 h) was collected. (A and C) TF mRNA expression and (B and D) activity were detected by qRT-PCR using commercial kits as described in Materials and methods. Data are presented as the means ± SEM from 3 independent experiments. *P<0.05 vs. cells treated with medium only (medium); **P<0.05 vs. anti-β2GPI/β2GPI complex or LPS stimulation alone.
Effects of epigallocatechin-3-gallate (EGCG) on the mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) in THP-1 cells. Cells (2×106) were pre-treated with or without the indicated concentrations of EGCG for 1 h and then incubated with anti-β2-glycoprotein I (β2GPI) (10 μg/ml)/β2GPI (100 μg/ml) complex for 2 or 24 h, as indicated. On the other hand, other THP-1 cells (2×106) were incubated with anti-β2GPI (10 μg/ml)/β2GPI (100 μg/ml) complex or lipopolysaccharide (LPS) (500 ng/ml) in the absence or presence of EGCG (50 μg/l) for the indicated periods of time. EGCG was not removed from the medium. Total RNA was collected from the cells (2 h) or cultural supematants (24 h). (A and C) TNF-α mRNA and (B and D) protein expression was detected by qRT-PCR and ELISA kits. Data are presented as the means ± SEM from 3 independent experiments. *P<0.05 vs. cells treated with medium only (medium); **P<0.05 vs. anti-β2GPI/β2GPI complex or LPS stimulation alone.
Effects of epigallocatechin-3-gallate (EGCG) on Toll-like receptor 4 (TLR4) expression in THP-1 cells. Cells (2×106) were stimulated with anti-β2-glycoprotein I (β2GPI) (10 μg/ml)/β2GPI (100 μg/ml) complex or lipopolysaccharide (LPS) (500 ng/ml) in the absence or presence of EGCG (50 μg/ml) for 2 or 6 h. EGCG was not removed from the medium. Total RNA of cells (2 h) and cell lysates(6 h) were collected for the measurement of (A) TLR4 mRNA and (B) protein levels using qRT-PCR and western blot analysis, respectively. Data are presented as the means ± SEM from 3 independent experiments. *P<0.05 vs. cells treated with medium alone (medium); **P<0.05 vs. anti-β2GPI/β2GPI complex or LPS stimulation alone.
Effects of epigallocatechin-3-gallate (EGCG) on the activation of mitogen-activated protein kinase (MAPK) pathways in THP-1 cells. Cells (2×106) were pre-treated with or without the indicated concentrations of EGCG for 1 h and then incubated with anti-β2-glycoprotein I (β2GPI) (10 μg/ml)/β2GPI (100 μg/ml) complex or lipopolysaccharide (LPS) (500 ng/ml) for 30 min. EGCG was not removed from the medium. Cell lysates were collected and subjected to western blot analysis with antibodies against (A) total (t-p38) and phosphorylated p38 (p-p38), (B) total (t-ERK) and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) (p-ERK) or (C) total (t-JNK) and phosphorylated JNK (p-JNK). Data are presented as the means ± SEM from 3 independent experiments. *P<0.05 vs. cells treated with medium only (medium); **P<0.05 vs. anti-β2GPI/β2GPI complex or LPS stimulation alone.
Effects of epigallocatechin-3-gallate (EGCG) on nuclear factor-κB (NF-κB) activation in THP-1 cells. Cells (2×106) were pre-treated with or without the indicated concentrations of EGCG for 1 h and then incubated with anti-β2-glycoprotein I (β2GPI) (10 μg/ml)/β2GPI (100 μg/ml) complex or lipopolysaccharide (LPS) (500 ng/ml) for 60 min. EGCG was not removed from the medium. Cell lysates were collected and subjected to western blot analysis with antibodies against (A) total and phosphorylated NF-κB, IκB-α and (B) β-actin. Data are presented as the means ± SEM from 3 independent experiments. *P<0.05 vs. cells treated with medium only (medium); **P<0.05 vs. anti-β2GPI/β2GPI complex or LPS stimulation alone.