Epigallocatechin-3-gallate suppresses the global interleukin-1beta-induced inflammatory response in human chondrocytes

Abstract

Introduction: Epigallocatechin-3-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1β) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1β-induced expression of proteins associated with OA pathogenesis in human chondrocytes.

Methods: Primary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1β (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1β-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1β-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- κB (MG132) and MAPKs (p38-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059).

Results: Out of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1β enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1β was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-α, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1beta (MIP-1β), granulocyte chemotactic protein-2 (GCP-2), MIP-3alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-κB and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes.

Conclusions: Our results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.

Figure 1

Inhibition of IL-1β induced cytokine expression in human OA chondrocytes by EGCG (a, b). Chondrocytes were pretreated for 2 h with EGCG (100 μM) and then stimulated or not stimulated with IL-1β (5 ng/ml) for 24 h. Values represent Mean ± SE of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. EGCG, epigallocatechin-3-gallate; GCSF, granulocyte colony stimulating factor; GM-CSF, granulocyte-macrophages colony stimulating factor; IFN-γ, interferon-γ; IL, interleukin; LIF, leukemia inhibitory factor; MCSF, macrophages colony stimulating factor; MIF, macrophage migratory inhibitory factor; TNF, tumor necrosis factor.

Figure 2

Effect of EGCG on IL-1β stimulated secretion of chemokines in human OA chondrocytes (a-b). Chondrocytes were pretreated for 2 h with EGCG (100 μM) and then stimulated or not stimulated with IL-1β (5 ng/ml) for 24 h. Values represent Mean ± SE of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. BLC, B-lymphocyte chemoattractant; EGCG, epigallocatechin-3-gallate; ENA-78, epithelial neutrophil activating peptide-78; GCP-2, granulocyte chemotactic protein-2; GRO, growth-related oncogene; IL- interleukin; MCP, monocyte chemotactic protein; MDC, macrophage-derived chemokine; MIG, monokine induced by interferon-gamma; MIP, macrophage inflammatory protein; NAP-2, nucleosome assembly protein-2; RANTES, regulated upon activation normal T-cell expressed and secreted;IP-10,10 kDa interferon gamma-induced protein.

Figure 3

Effect of EGCG on secretion of growth and angiogenic factors in human OA chondrocytes stimulated with IL-1β (a, b). Chondrocytes were pretreated for 2 h with EGCG (100 μM) and then stimulated or not stimulated with IL-1β (5 ng/ml) for 24 h. Values represent Mean ± SE of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. BDNF, brain-derived neurotrophic factor; EGCG, epigallocatechin-3-gallate; EGF, endothelial growth factor; FGF, fibroblast growth factor; GDNF, glial cell derived neurotrophic factor; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; NT, neurotrophin; PDGF-BB, platelet-derived growth factor receptor, beta polypeptide subunit B; PIGF, phosphatidylinositol glycan anchor biosynthesis; TGF, transforming growth factor; TIMP, tissue inhibitor of MMPs; VEGF, vascular endothelial growth factor.

Figure 4

Effect of EGCG IL-6, IL-8 and TNF-α gene expression and production in IL-1β-stimulated human OA chondrocytes. Chondrocytes were pretreated with EGCG (10 to 100 μM) for 2 h and stimulated with IL-1β for (a, b) 8 h or (c, d, e, and f) 24 h. Gene expression for IL-6, IL-8 and TNF-α was determined by quantitative RT-PCR normalized to GAPDH and then compared with the levels present in un-stimulated chondrocytes. Levels of IL-6, IL-8 and TNF-α in the culture supernatants were quantified by sandwich ELISA. Value represents Mean ± SE of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. EGCG, epigallocatechin-3-gallate; IL-interleukin; TNF, tumor necrosis factor.

Figure 5

EGCG mediated inhibition of NF-κB and MAPKs in IL-1β stimulated human OA chondrocytes and regulation of IL-6, IL-8 and TNF-α. (a) Human OA chondrocytes were pretreated with EGCG (10 to 100 μM) for 2 h and stimulated with IL-1β for 24 h. NF-κBp65 was determined in cell lysate by highly sensitive and specific ELISA (Assay design). TNF-α-treated extract of HeLa cells (supplied with the kit) was used as a positive control. The assay is developed with a chemiluminescent substrate and the signal is detected using luminometer (Lumat LB 9507; Berthold Technologies). NF-κB p65 activity was expressed as relative light unit (RLU). (b) After pretreatment with EGCG (10 to 100 μM) for one hour, chondrocytes were stimulated with IL-1β for one half-hour, and then phosphorylation of JNK, ERK and p38-MAPK was determined by Western immunobloting. (c, d, and e) Effect of specific inhibitors for mitogen activated protein kinases and NF-κB on the gene expression of IL-6, IL-8 and TNF-α in IL-1β stimulated human OA chondrocytes. Primary chondrocytes were pretreated with specific inhibitors for 2 h and were stimulated with IL-1β for 6 h. Relative gene expression of IL-6, IL-8 and TNF-α normalized to GAPDH and compared with un-stimulated control, were determined by quantitative RT-PCR. Concentrations of specific inhibitors of p38 (SB202190), JNK (SP600125), ERK (PD98059) and NF-κB (MG132) used in these studies were 100 μM, 10 μM, 50 μM and 100 μM, respectively. Value represents Mean ± SE of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. EGCG, epigallocatechin-3-gallate;IL-interleukin; ERK, extracellular signal-regulated kinases; JNK, cJun-N-termial Kinases; MAPKs, mitogen activated protein kinases; NF-κB, nuclear factor kappa-B; TNF, tumor necrosis factor.

Figure 6

Gene expression and production of TRAF-6 and IRAK-1 in IL-1β-stimulated human OA chondrocytes. (a) and (c) Effect of EGCG pretreatment on the gene expression of TRAF-6 and IRAK-1 in IL-1β-stimulated OA chondrocytes, respectively. (b) and (d) Effect of EGCG on the production of TRAF-6 and IRAK-1 in IL-1β-stimulated OA chondrocytes, respectively. Value represents Mean ± SD of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. EGCG, epigallocatechin-3-gallate; IL, interleukin; IRAK-1, interleukin-1 receptor-associated kinase 1; TRAF-6, TNF receptor-associated factor 6.

Figure 7

Real-time PCR verification of the expression of IL-1β. (a), LIF (b), IL-7 (c), GM-CSF (d), RANTES (e), ENA-78 (f), GRO (g), GRO-α (h), MCP-1 (i), MCP-2 (j), MCP-3 (k), MIP-1β (l), IP-10 (m), GCP-2 (n), NAP-2 (o). Value represents Mean ± SD of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. ENA-78, epithelial neutrophil activating peptide-78; GCP-2, granulocyte chemotactic protein-2; GM-CSF, granulocyte-macrophages colony stimulating factor; GRO, growth-related oncogene; IL-interleukin; IP-10,10 kDa interferon gamma-induced protein; LIF, leukemia inhibitory factor; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; NAP-2, nucleosome assembly protein-2; RANTES, regulated upon activation normal T-cell expressed and secreted.

Figure 8

Effect of specific inhibitors of MAPKs and NF-κB pathways on the gene expression of pro-inflammatory cytokines and chemokines in IL-1β-stimulated human OA chondrocytes. Primary chondrocytes were pretreated with specific inhibitors for 2 h and were stimulated with IL-1β for 6 h. Relative gene expression of LIF (a), IL-7 (b), GM-CSF (c), ENA-78 (d) and GRO (e) was normalized to GAPDH and compared with un-stimulated control, were determined by quantitative RT-PCR. Concentrations of specific inhibitors of p38 (SB202190), JNK (SP600125), ERK (PD98059) and NF-κB (MG132) used in these studies were 100 μM, 10 μM, 50 μM and 100 μM, respectively. Value represents Mean ± SD of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. ENA-78, epithelial neutrophil activating peptide-78; GM-CSF, granulocyte-macrophages colony stimulating factor; GRO, growth-related oncogene; IL-interleukin; LIF, leukemia inhibitory factor; MAPKs, mitogen activated protein kinases; NF-κB, nuclear factor kappa-B.

Figure 9

Effect of specific inhibitors of MAPKs and NF-κB pathways on the gene expression of pro-inflammatory chemokines in IL-1β-stimulated human OA chondrocytes. Primary chondrocytes were pretreated with specific inhibitors for 2 h and were stimulated with IL-1β for 6 h. Relative gene expression of GRO-α (a), MCP-1 (b), MCP-2 (c), MCP-3 (d) and MIP-1β (e) was normalized to GAPDH and compared with un-stimulated control, were determined by quantitative RT-PCR. Concentrations of specific inhibitors of p38 (SB202190), JNK (SP600125), ERK (PD98059) and NF-κB (MG132) used in these studies were 100 μM, 10 μM, 50 μM and 100 μM, respectively. Value represents Mean ± SD of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. GRO, growth-related oncogene; MAPKs, mitogen activated protein kinases; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; NF-κB, nuclear factor kappa-B.

Figure 10

Effect of specific inhibitors of MAPKs and NF-κB pathways on the gene expression of pro-inflammatory cytokines and chemokines in IL-1β-stimulated human OA chondrocytes. Primary chondrocytes were pretreated with specific inhibitors for 2 h and were stimulated with IL-1β for 6 h. Relative gene expression of IP-10 (a), GCP-2 (b) and NAP-2 (c) was normalized to GAPDH and compared with un-stimulated control, were determined by quantitative RT-PCR. Concentrations of specific inhibitors of p38 (SB202190), JNK (SP600125), ERK (PD98059) and NF-κB (MG132) used in these studies were 100 μM, 10 μM, 50 μM and 100 μM, respectively. Value represents Mean ± SD of three different patients. # represents P < 0.05 Vs control; * P < 0.05 Vs IL-1β stimulated chondrocytes. GCP-2, granulocyte chemotactic protein-2; IP-10, 10 kDa interferon gamma-induced protein; MAPKs, mitogen activated protein kinases; NAP-2, nucleosome assembly protein-2; NF-κB, nuclear factor kappa-B.