Green tea polyphenols such as EGCG have potent anti-inflammatory properties. Prior research had shown that EGCG inhibits tumor necrosis factor through a mechanism that was thought to have implications for inflammation generally. Epidemiological studies link regular consumption of tea with decreased cancer risk and a reduction in mortality during the 12 month period following a heart attack. “Considerably less is known regarding the mechanisms by which tea confers these health benefits.” The present research demonstrates one important mechanism in the inhibition of interleukin-1 mediated signal transduction. “Given the long safety record of tea consumption, the use of EGCG and related compounds may represent a novel pharmacological strategy for the modulation of inflammation. EGCG and related compounds could potentially be used as a nutritional supplement in patients with inflammatory disease processes. The next steps to further substantiate these assertions are to test the efficacy of green tea derived polyphenols such as EGCG in animal models of inflammation associated organ injury and to further elucidate the mechanisms by which these compounds modulate no inflammatory signal transduction pathways.”
Epigallocatechin-3-gallate, a Green Tea-Derived Polyphenol, Inhibits IL-1[beta]-Dependent Proinflammatory Signal Transduction in Cultured Respiratory Epithelial Cells1,2
The Journal of Nutrition . May 1, 2004 . Catravas, John D; Denenberg, Alvin; Et al; Odoms, Kelli; Wheeler, Derek S
ABSTRACT Polyphenolic components of green tea, such as epigallocatechin-3-gallate (EGCG), have potent anti-inflammatory properties. We previously showed that EGCG inhibits tumor necrosis factor-[alpha] (TNF-[alpha])-mediated activation of the nuclear factor-[kappa]B (NF-[kappa]B) pathway, partly through inhibition of I[kappa]B kinase (IKK). The NF-[kappa]B pathway may also be activated in response to interleukin-1[beta] (IL-1[beta]) stimulation through a distinct signal transduction pathway. We therefore hypothesized that EGCG inhibits IL-1 [beta]-mediated activation of the NF-[kappa]?B pathway. Because the gene expression of interleukin-8 (IL-8), the major human neutrophil chemoattractant, is dependent on activation of NF-[kappa]B, IL-8 gene expression in human lung epithelial (A549) cells treated with human IL-1[beta] was used as a model of IL-1[beta] signal transduction. The EGCG markedly inhibited IL-1[beta]-mediated IL-1[beta] receptor-associated kinase (IRAK) degradation and the signaling events downstream from IRAK degradation: IKK activation, I[kappa]B[alpha] degradation, and NF-[kappa]B activation. In addition, EGCG inhibited phosphorylation of the p65 subunit of NF-[kappa]B. The functional consequence of this inhibition was evident by inhibition of IL-8 gene expression. Therefore, the green tea polyphenol EGCG is a potent inhibitor of IL-1[beta] signal transduction in vitro. The proximal mechanisms of this effect involve inhibition of IRAK-dependent signaling and phosphorylation of p65. J. Nutr. 134: 1039-1044, 2004.
KEY WORDS: * transcription factors * inflammation * signal transduction * chemokines * polyphenols
During the initial host inflammatory response to an infection or other inciting event, several pro inflammatory cytokines are released into the systemic circulation, which, if left unchecked, can ultimately cause a dysregulated inflammatory cascade that results in significant autoinjury to the host (1). The systemic administration of either recombinant interleukin-1[beta] (IL-1[beta])4 or tumor necrosis factor-[alpha] (TNF-[alpha]) rapidly induces a shocklike state in experimental animals (2,3) and causes fever and hypotension in healthy human volunteers (4-6). These two important proinflammatory cytokines appear to orchestrate the inflammatory response through the activation of transcription factors, such as nuclear factor-[kappa]B (NF-[kappa]B) and activated protein-1, with the subsequent induetion of proinflammatory gene expression. Although these two cytokines share many biologic and physiologic properties, the signaling mechanisms that lead to IL-1[beta]-dependent signal transduction are distinct from that of TNF-[alpha]-dependent signal transduction.
Nuclear factor-[kappa]B belongs to the Rel family of transcription factors, which share common structural motifs for dimerization and DNA binding. Five known subunits belong to the mammalian NF-[kappa]B/Rel family: c-Rel, NF-[kappa]B1 (p50/p105), NF-[kappa]B2 (p52/p100), Rel A (p65), and Rel B. Nuclear factor-[kappa]B consists of 2 such subunits arranged as either homodimers (e.g., p50/p50) or heterodimers (e.g., p65/p50), although the most common form of activated NF-[kappa]B consists of a p65 (Rel A) and p50 heterodimer. Nuclear factor-[kappa]B activation appears to be a master switch, or control point, for the expression of a large number of proinflammatory genes, including several cytokines, chemokines, and adhesion molecules (7). Nuclear factor-[kappa]B is usually present in the cytoplasm of cells in an inactive state bound to a related inhibitory protein known as 1[kappa]B[alpha], an association that physically masks the nuclear translocation sequence of NF-[kappa]B, thereby retaining it in the cytoplasm.
The regulation of NF-[kappa]B activation following stimulation with IL-1[beta] appears to involve at least 2 independent signal transduction pathways. The best-characterized mechanism for the activation of NF-[kappa]B involves the phosphorylation of the inhibitory protein, I[kappa]B[alpha]. Interleukin-1[beta] binds to its receptor, the IL-1 receptor type 1 (IL-1R1), which forms a complex with a related accessory protein, IL-1 receptor accessory proteins (IL-1RAcP). This interaction between IL-1R1 and IL-1RAcP is possible due to the presence of a shared region of homology in the cytoplasmic domain of each protein called the Toll/ IL-1R domain (8). The cytosolic adaptor protein MyD88 (9) and the Toll-interacting protein (10) interact with this receptor complex, which is a necessary step for association with a serine-threonine kinase, IL-1 receptor-associated kinase (IRAK) (11-13). The IRAK then recruits several additional adaptor proteins, including TNF receptor-associated factor 6 (14), transforming growth factor-[beta]-activated kinase-1 (TAK1), and the TAK1 binding proteins 1 and 2 (14,15). Autophosphorylation of IRAK promotes its dissociation from this complex, which is followed by its polyubiquitination, and subsequent degradation by the 26S proteosome system (16). Interestingly, the kinase activity of IRAK may not be an essential step in the IL-1[beta] signal transduction pathway and may function to terminate signal transduction instead (1719). Nevertheless, this sequence of events is temporally and functionally associated with the downstream activation of I[kappa]B kinase (IKK), which phosphorylates the serine-32 and -36 residues of I[kappa]B[alpha] (20). Phosphorylated I[kappa]B[alpha] is targeted for rapid ubiquitination and degradation by the 26S proteosome system, which unmasks the nuclear translocation sequence of NF-[kappa]B and allows it to enter the nucleus and bind to the NF-[kappa]B consensus sequence to direct the transcription of target proinflammatory genes (20).
An alternative mechanism for the activation of NF-[kappa]B is I[kappa]B[alpha]-independent and involves direct phosphorylation of the p65 subunit of NF-[kappa]B at multiple sites by several candidate kinases (21). In addition, phosphorylation of specific tyrosine residues on I[kappa]B[alpha] causes activation of NF-[kappa]B without the proteolytic degradation of I[kappa]B[alpha] (22). It is likely that further study in this area will yield additional mechanisms of ????independent NF-[kappa]B activation.
Given the important role that NF-kB plays in the regulation of a large number of proinflammatory genes, there is growing interest in targeting NF-kB directly in order to affect the inherent redundancy of the inflammatory cascade. A potential novel, safe, and nontoxic strategy for inhibiting NF-[kappa]B activation involves the polyphenolic compounds found in green tea, especially epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea (23). Apart from their antioxidant properties, the catechins, especially EGCG, inhibit several proteins involved in inflammation, including NF-[kappa]B (23-25). We previously showed that EGCG inhibits the TNF[alpha]-mediated activation of NF-[kappa]B in cultured respiratory epithelial cells, partly through the inhibition of IKK (26). Accordingly, we hypothesized that EGCG would inhibit IL1[beta]-mediated activation of NF-[kappa]B.
MATERIALS AND METHODS
Cell culture. Epithelium A549 cells (American Type Culture Collection), a human lung adenocarcinoma cell line representative of the distal respiratory epithelium, were maintained in an incubator with room air:CO^sub 2^ (95:5, v:v) at 37Â°C, using DMEM containing 8% FBS and 1% penicillin/streptomycin (Gibco BRL).
Experimental conditions. Cells were treated with either 1 Âµg/L of human IL-1[beta] (Boehringer Mannheim) or vehicle. Epigallocatechin gallate (EGCG; Sigma Chemical) was diluted in filtered PBS to a stock concentration of 10 mmol/L. We noticed an oxidative color change and deterioration in the anti-inflammatory effects noted below when the EGCG stock was used after 24 h (data not shown), and for this reason, EGCG stock was prepared immediately before each use. The EGCG stock was further diluted to experimental concentrations ranging from 3 to 100 Âµmol/L in DMEM. Cells were treated with EGCG for l h before incubation with IL-1[beta]. Cells not treated with EGCG were preincubated in DMEM alone. The concentration of EGCG used and the duration of treatment did not affect the viability of these cells, as previously reported (26).
Western blot analysis for IRAK and I[kappa]B[alpha] degradation. Whole cell lysates of treated cells were prepared and electrophoretically separated as previously described (26) on 8 to 16% Tris-glycine gradient gels (Novex) and subsequently transferred to nitrocellulose membranes using the Novex Xcell Mini-Gel system (Novex).
For IRAK immunoblotting, membranes were blocked with nonfat dried milk:PBS (3:97, v:v) for 30 min. Primary antibody against IRAK (Upstate Biotechnology) was applied at a concentration of 2 mg/L in milk:PBS (3:97) overnight at 4Â°C. After washing twice with distilled H2O, the secondary antibody (peroxidase-conjugated antirabbit IgG; Stressgen) was applied at a 1:5000 dilution in milk:PBS (3:97) for 1 h.
I[kappa]B[alpha] immunoblotting was performed as previously described (26), using a primary antibody directed against human I[kappa]B[alpha] (Santa Cruz Biotechnology). Blots were incubated in commercial enhanced chemiluminescence reagents (ECL; Amersham), and exposed to photographic film (26).
Western blot analysis for phospho-NF-[kappa]B (p65). Treated cells were washed twice in ice-cold PBS. Cells were then lysed in ice-cold lysis buffer containing 50 mmol/L Tris (pH 8.0), 110 mmol/L NaCL, 5 mmol/L EDTA, and 1% Triton X-100, to which 100 mmol/L Na^sub 3^VO^sub 4^, 2 g/L leupeptin, 2 mol/L [beta]-glycerol phosphate, and 100 g/L phenylmethysulfonyl fluoride were added. Electrophoresis and protein transfer were carried out as described above. For immunoblotting, membranes were blocked in nonfat milk:TBS:Tween (5.0:94.9:0.1, by vol) for 1 h. A primary antibody against phospho-p65 (Cell Signaling Technology) was applied at a 1:1000 dilution in milk:TBS:Tween (5:95, v:v) overnight at 4Â°C. After washing 3 times with TBS:Tween (99.9:0.1, v:v), secondary antibody (peroxidase-conjugated antirabbit IgG; Stressgen) was applied at 1:2000 dilution for 1 h. Blots were washed in TBS:Tween twice for 10 min, incubated in commercial enhanced chemiluminescence reagents (ECL; Amersham), and exposed to photographic film.
I[kappa]B kinase assay. The I[kappa]B kinase assay was performed as previously prescribed (26). Briefly, cell extracts were immunoprecipitated using anti-IKK[gamma] antibody (Santa Cruz Biotechnology). The kinase reaction was performed using ATP, GST-I[kappa]B[alpha], and [gamma]-[^sup 32^P]ATP as substrate, and the resulting proteins were separated electrophoretically using a Novex Mini-Cell System. Gels were dried, exposed overnight, and analyzed using a Phosphorlmager screen and Image-Quant software (Molecular Dynamics).
Electromobility gel shift assay. All nuclear extraction procedures were performed on ice with ice-cold reagents as previously described (26). Nuclear proteins were stored at – 70Â°C until used for electromobility gel shift assays (EMSAs). The NF-[kappa]B oligonucleotide probe used for EMSA (5′-GTGGAATTTCCTCTGA-3′) corresponds to the NF-[kappa]B site in the interleukin-8 (IL-8) promoter and was synthesized at the University of Cincinnati DNA Core Facility (26). The probe was labeled with [gamma]-[^sup 32^P]adenosine triphosphate using T4 polynucleotide kinase (Gibco BRL) and purified in Bio-Spin chromatography columns (BioRad). The EMSA procedure was as previously described (26).
Transient transfection and luciferase assay. Interleukin-8 promoter activity was measured using a plasmid containing the fulllength promoter region of the IL-8 gene cloned into a luciferase reporter plasmid (pGL2; Promega). Cells were transiently transfected with the IL-8 promoter-luciferase reporter plasmid as previously described (26). After transfection, cells were washed once with PBS, pretreated with EGCG for 1 h, and subsequently treated with human IL-1[beta] for 6 h. Cellular proteins were extracted and analyzed for luciferase activity according to the manufacturer’s instructions (Promega), using a Berthold AutoLumat LB953 luminometer. Luciferase activity was corrected for total cellular protein and reported as fold induction over control cells (cells that were transfected and treated with medium alone).
Northern blot analysis. Total cellular RNA was electrophoretically separated and subsequently transferred to nylon membranes (MicroSeparations) and UV autocrosslinked (UV Stratalinker 1800; Stratagene) as previously described (26). After 4 h of prehybridization at 42Â°C, membranes were hybridized overnight with a radiolabeled human IL-8 cDNA probe. The cDNA was labeled with a y-[^sup 32^P]deoxycytidine triphosphate (specific activity = 3000 Ci/mmol; New England Nuclear Research Products) by random priming (Pharmacia). After washing, the hybridized filters were exposed overnight and analyzed using a Phosphorlmager screen and ImageQuant software (Molecular Dynamics).
Enzyme-linked immunosorbent assay. Immunoreactive IL-8 concentrations in the media of treated cells were measured using a commercially available sandwich ELISA (Biosource). All procedures were performed as recommended by the manufacturer.
Statistical analysis. Differences in immunoreactive IL-8 level, luciferase activity, and cell viability among the experimental groups were evaluated by one-way ANOVA and Student-Newman-Keuls test. Values of P < 0.05 were considered significant.
Interleukin-1[beta]-mediated degradation of IRAK. Treatment with IL-1[beta] caused nearly complete degradation of IRAK compared to control cells, whereas preincubation with 30 and 100 Âµmol/L EGCG inhibited this degradation (Fig. 1).
Interleukin-1[beta]-induced IKK activation. Treatment with IL-1[beta] increased IKK activity compared to untreated control cells. Consistent with the IRAK degradation data above, 30 and 100 Âµmol/L EGCG almost completely suppressed IL-1[beta]induced activation of IKK (Fig. 2).
Interleukin-1 [beta]-mediated degradation of I[kappa]B[alpha]. Treatment with IL-1[beta] caused marked degradation of I[kappa]B[alpha] compared to control cells. Consistent with the previous data involving IRAK degradation and IKK activation, 30 and 100 Âµmol/L EGCG inhibited IL-1[beta]-mediated I[kappa]B[alpha] degradation (Fig. 3).
Interleukin-1[beta]-mediated activation of NF-[kappa]B. Treatment with IL-1[beta] increased the activation of NF-[kappa]B compared with control cells, as determined by EMSA. Consistent with the effects of EGCG on IL-1[beta]-induced degradation of IRAK and activation of IKK, pretreatment with 30 and 100 Âµmol/L EGCG inhibited activation of NF-[kappa]B (Fig. 4). However, lower concentrations of EGCG (3 and 10 /xmol/L) also moderately inhibited NF-[kappa]B activation, suggesting that additional, I[kappa]B[alpha]-independent mechanisms of NF-[kappa]B inhibition may play a role.
Phosphorylation of p65. Phosphorylated p65 was detected within 30 min after stimulation with IL-1[beta], wheras preincubation with EGCG caused a dose-dependent decrease in phosphorylatcd p65 concentration (Fig. 5).
Interleuicin-1[beta]-mediated expression of the IL-8 gene. Treatment with IL-1[beta] induced nearly 5-fold the luciferase activity in cells transfected with an IL-8 promoter-luciferase reporter plasmid, compared to control cells that were transfected and treated with media alone. Pretreatment with EGCG inhibited luciferase activity in a dose-dependent manner, with significant inhibition at 30 and 100 Âµmol/L EGCG (Fig. 6). Furthermore, IL-1[beta] treatment alone increased IL-8 mRNA expression (measured by Northern blot analysis) compared to control cells treated with media alone, whereas pretreatment with EGCG inhibited the expression of IL-8 mRNA in a dose-dependent manner (Fig. 7). The effects noted for IL-8 mRNA were corroborated by measurement of IL-8 peptide levels by ELISA. Treatment with IL-1[beta] alone markedly increased the production of immunoreactive IL-8 compared to control cells treated with media alone, whereas pretreatment with EGCG decreased the production of immunoreactive IL-8 in a dose-dependent manner (Fig. 8). Collectively, these data demonstrate that the inhibitory effects of EGCG on IL-1[beta]mediated NF-[kappa]B activation are associated with the inhibition of IL-8 gene expression.
A large body of indirect and direct evidence links the NF-[kappa]B pathway to the dysregulated inflammation that is characteristic of diseases such as sepsis and acute respiratory distress syndrome. Several of the genes that comprise the complex network contributing to this dysregulated inflammation are regulated at the transcriptional level by NF-[kappa]B, including the cytokines IL-1[beta] and TNF-[alpha] chemokines such as IL-6, IL-8, and macrophage chemotactic protein-1; cell adhesion molecules such as vascular cell adhesion molecule 1 and intercellular adhesion molecule 1; growth factors such as granulocytemacrophage colony-stimulating factor and granulocyte colonystimulating factor; as well as additional proinflammatory genes such as inducible nitric oxide synthase. There appears to be a correlation between increased NF-[kappa]B activity and the severity of illness and mortality in critically ill patients (27-29). In addition, studies with in vivo animal models of lethal septic shock demonstrate that inhibition of NF-[kappa]B activation reduces mortality (30,31). These data support the general hypothesis that increased NF-[kappa]B-dependent inflammation directly contributes to the outcome of inflammation-mediated organ injury and strongly support the concept of therapeutic strategies targeting the NF-[kappa]B pathway. An attractive feature of this strategy is the fact that NF-[kappa]B activation appears to be a master switch, or control point, for the expression of a large number of proinflammatory genes. Thus, targeting NF-[kappa]B may potentially affect the inherent redundancy of the inflammatory cascade.
Recent epidemiological studies link the regular consumption of tea with a decreased risk of cancer (32). In addition, a recent study indicates that consumption of as little as 2 cups (473 mL) of tea per day is associated with a reduction in mortality during the 12-mo period following an acute myocardial infarction (33). Considerably less is known regarding the mechanisms by which tea confers these health benefits. We previously showed that one of the active ingredients in green tea, EGCG, is a potent inhibitor of TNF-[alpha]-induced IL-8 gene expression in A549 cells, at least partially through a mechanism involving the inhibition of NF-[kappa]B signaling (26). The present study shows that EGCG inhibits IL-1[beta]-mediated signal transduction in A549 cells as well. First, our study shows that EGCG inhibits the degradation of IRAK, which appears to be a crucial event in the IL-1 signal transduction pathway, in that cells derived from IRAK-knockout mice do not respond to IL-1 stimulation (34-36). The mechanism by which EGCG affects IRAK degradation may involve the direct inhibition of the proteolytic activity of the 26S proteosome by EGCG itself (37). Similarly, EGCG may inhibit I[kappa]B[alpha] degradation via a similar mechanism (38). However, based on the current experiments, it is also possible that EGCG affects events proximal to IRAK degradation. For example, EGCG appears to disrupt the binding of epidermal growth factor to its receptor in A431 epidermoid carcinoma cells (39,40). Similarly, EGCG may modulate IL-1[beta] signaling at the receptor level, by interfering with the binding of IL-1[beta] to the IL-1R, although further experiments are needed to investigate this hypothesis. In combination, inhibition of the IRAK to I[kappa]B[alpha] pathway accounts in part for the mechanism by which EGCG inhibits NF-[kappa]B activation and subsequent NF-[kappa]B-dependent gene expression. The experimental data also demonstrate that EGCG inhibits phosphorylation of p65, thus providing an additional mechanism for the inhibition of NF-[kappa]B activation. This effect on p65 phosphorylation could be the result of IKK inhibition, because a recent report indicates that IKK can phosphorylate the p65 subunit in vitro (41).
The pharmacokinetics of the green tea polyphenols in humans are well-described (42-44), and the maximum achievable EGCG concentration in vivo is significantly less than the concentrations reported in the current in vitro study. For example, 1 cup (240 mL) of green tea contains 200 mg of EGCG (45), and a single, 200-mg dose of EGCG produces a plasma EGCG concentration of ~0.1 Âµmol/L (46). However, the consumption of pharmaceutically prepared formulations of green tea polyphenols produces plasma EGCG concentrations approaching 2 Âµmol/L (44,46,47). Moreover, little is known regarding the effective EGCG concentration required to modulate these proinflammatory signaling pathways in vivo.
In summary, the green tea-derived polyphenol EGCG appears to be a potent inhibitor of IL-1 [beta]-mediated signal transduction in A549 cells. The mechanism of this effect involves in part inhibition of IRAK degradation and the subsequent activation of the well-characterized I [kappa]B[alpha]-dependent pathway of NF-[kappa]B activation. An additional mechanism appears to involve the inhibition of p65 phosphorylation. Given the long safety record of tea consumption, the use of EGCG and related compounds may represent a novel pharmacological strategy for the modulation of inflammation dependent on the NF-[kappa]B pathway. Alternatively, EGCG and related compounds could potentially be used as a nutritional supplement in patients with inflammatory disease processes. The next steps to further substantiate these assertions are to test the efficacy of green tea-derived polyphenols such as EGCG in animal models of inflammation-associated organ injury and to further elucidate the mechanisms by which these compounds modulate proinflammatory signal transduction pathways.
0022-3166/04 $8.00 Â© 2004 American Society for Nutritional Sciences.
Manuscript received 3 December 2003. Initial review completed 5 January 2004. Revision accepted 3 February 2004.
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Derek S. Wheeler,3 John D. Catravas,* Kelli Odoms,[dagger] Alvin Denenberg,[dagger] Vivek Malhotra,[dagger] and Hector R. Wong[dagger]
Section of Critical Care Medicine, Children’s Medical Center, and * Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912; and [dagger] Division of Critical Care Medicine, Cincinnati Children’s Hospital Medical Center, and Children’s Hospital Research Foundation, Cincinnati, OH 45229
3 To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.