Green Tea Epigallocatechin-3-Gallate Mediates T Cellular NF-κB Inhibition and Exerts Neuroprotection in Autoimmune Encephalomyelitis¹

Tea (Camellia sinensis) and its constituent polyphenols have been reported to possess anticarcinogenic properties in a wide variety of experimental systems in vitro and in vivo. Drinking tea, especially green tea, is associated with a lower incidence of human cancer (1, 2). Subsequent studies demonstrated that certain fractions of green tea, among them (−)-epigallocatechin-3-gallate (EGCG),³⁴ promote apoptosis and cell cycle arrest of transformed cells (3, 4, 5). Moreover, immunomodulatory properties of green tea extracts such as inhibiting endotoxin-induced TNF-α production (6) and neutrophil-mediated effects have been recently described (7). In a mouse model of stroke, a neuroprotective role was observed (8) and linked in a Parkinson’s disease model to regulation of superoxide dismutase and catalase activity in the brain (9).

Multiple sclerosis (MS) is a multiphasic autoimmune disease of the CNS (10) in which myelin-specific CD4⁺ Th1 cells are thought to orchestrate the effector processes resulting in the destruction of the myelin sheath (11). Recent studies in MS and its animal model, experimental autoimmune encephalomyelitis (EAE) (12), suggest that already during the early phase of inflammation, neuronal pathology involving axonal transection and loss of parental cell bodies plays a pivotal role in disease severity (13, 14). It has become evident that long-term disability in MS correlates better with axonal damage than with the degree of demyelination (13, 14). We have very recently unraveled a process linking demyelination and neuronal damage (15). As a consequence of demyelination, the myelin breakdown product 7-ketocholesterol, which can be detected in the cerebrospinal fluid of MS patients, induces microglia-mediated apoptotic cell death of neurons in the brain stem motor region (15). However, currently approved therapies, which have to be administered parenterally in young disabled adults with neuroinflammatory disorder, primarily focus on the inflammatory aspect of the CNS disease (16), are only partly effective, and are often limited by side effects. In contrast, neuroprotective agents have not yet been established in this autoimmune disorder. In light of the pharmacological profile of EGCG, and its reported properties in immunity and neurodegeneration, we hypothesized a potential beneficial role of EGCG in the treatment of neuroinflammation and subsequently discovered the underlying mechanisms of action.

Female SJL/J mice (6–8 wk; ∼20 g body weight; Charles River Laboratories, Sulzfeld, Germany) were immunized s.c. with 75 μg of proteolipid protein (PLP) peptide p139–151 (purity >95%; Pepceuticals, Leicester, UK) in a 0.2-ml emulsion consisting of equal volumes of PBS and CFA (Difco, Franklin Lakes, NJ) and containing 6 mg/ml Mycobacterium tuberculosis H37Ra (Difco). Bordetella pertussis toxin (200 ng; List Biological Laboratories, Campbell, CA) was administered i.p. at days 0 and 2. For treatment, EGCG (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9% NaCl. EGCG or vehicle (0.9% NaCl) were administered by gavage (100 μl per mouse) twice daily. Mice were scored as follows: 0, no disease; 1, tail weakness; 2, paraparesis; 3, paraplegia; 4, paraplegia with forelimb weakness; and 5, moribund or dead animals (17). All procedures were conducted according to protocols approved by the local animal welfare committee.

Human CD4⁺ T cell lines (TCLs) specific for human myelin basic protein (MBP; FN8 and MA14) or birch pollen (SW21) were established with a modified “split-well” protocol as previously described (17). For proliferation, 0.7 × 10⁵ cells were stimulated with 2 × 10⁵ autologous-irradiated (3000 rad) PBMC as APCs and Ag, or with anti-CD3 (OKT3; coated at 1 μg/ml; American Type Culture Collection, Manassas, VA) and 2.5 μg/ml anti-CD28 (R&D Systems, Minneapolis, MN), respectively. For murine bulk cell cultures, cell suspensions from draining lymph nodes were stimulated in 96-well microtiter plates (2 × 10⁵ cells/well) with peptide. [³H]Thymidine incorporation (0.5 μCi/well; Amersham Biosciences, Piscataway, NJ) was measured 72 h after stimulation with Ag and 48 h with anti-CD3/CD28. Radioactivity was detected using a MicroBeta beta counter (PerkinElmer, Wellesley, MA) after 18 h. For apoptosis, DNA fragmentation of susceptible Jurkat T cells was determined by flow cytometry of hypodiploid DNA as described previously (17).

Spinal cords and brain stems from mice transcardially perfused with 4% paraformaldehyde were postfixed overnight. Vibratome sections (Thermo Shandon, Pittsburgh, PA) were stained with H&E and evaluated in a blinded manner for inflammation as calculated by number of inflammatory foci (four ocular fields per section and six mice per group) (17). For caspase 3 and β-amyloid precursor protein (β-APP) staining, sections were quenched using 3% H₂O₂, washed, and blocked (10% NGS, 0.5% Triton X-100) before overnight incubation with anti-active caspase 3 (1:100; R&D Systems) and anti-β-APP (1:1000, Zymed Laboratories, San Francisco, CA). Sections were immunolabeled and developed using the avidin:biotinylated enzyme complex technique (ABC-Elite; Vector Laboratories, Burlingame, CA). Neurons positive for active caspase 3 were counted in brain stem sections (four ocular fields per section and six mice per group) (15).

Single-cell suspensions were prepared from draining lymph nodes, and a portion was stained with anti-CD25-APC (BD Pharmingen, San Jose, CA). Before staining, cells were incubated with anti-CD14/CD32 (BD Pharmingen) to prevent unspecific Ab binding. For intracellular staining, cells were activated for 4 h (5 ng/ml PMA, 1 μg/ml ionomycin), and brefeldin A (5 μg/ml) was added for the last 2 h. Cells were fixed, permeabilized with FACS buffer containing saponin (PBS, 0.5% saponin, 0.5% BSA, 0.1% NaN₃), stained with anti-IFN-γ-PE/TNF-α-APC/IL-4-APC Abs (BD Pharmingen), and analyzed on a FACSCalibur using CellQuest software (BD Pharmingen).

Cells were washed with PBS and resuspended in lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 0.5 μg/ml leupeptin). Total protein (30 μg/ml per lane) was separated by 10% SDS-PAGE. After transfer, blots were incubated with anti-IκB-α (Chemicon International, Temecula, CA), anti-cyclin-dependent kinase (CDK4; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p27^kip1 (Santa Cruz Biotechnology), or anti-ubiquitin (Sigma-Aldrich) following HRP-conjugated secondary Ab (DakoCytomation, Carpinteria, CA) and ECL system (Amersham Biosciences) as described (18).

Organotypic entorhinal-hippocampal slice (350 μm thick) cultures from 10-day-old SJL mice were prepared on a tissue chopper (Bachhofer) and cultured as described (19). Neuronal cell death was induced with recombinant TRAIL and N-methyl-d-aspartate (NMDA; Sigma-Aldrich) after 24 h. For TRAIL production, N-terminal truncated human TRAIL (residues 95–281) containing an N-terminal 6-His tag was expressed in Escherichia coli BL21 cells. After purification on Ni²⁺-nitrilotriacetate-agarose (Clontech Laboratories, Palo Alto, CA), untagged proteins were dialyzed against PBS. Damage in brain slices was detected by addition of 5 μg/ml propidium iodide (30 min at 36°C). Slices were examined using a rhodamine filter (488 nm/515 nm) with a dark-field inverse fluorescence microscope (Olympus, Melville, NY). For resectioning, slices were immersed before the preparation of 18-μm thin horizontal cryostat sections, and quantification of PI+ cells was conducted as previously described (15).

For assessment of viability, hippocampal HT22 cells (20) were seeded at 5000 neurons per well in 96-well plates and cultured for 16 h. On the following day, cells received fresh medium and were incubated with EGCG 2 h before incubation with TRAIL with and without cycloheximide (CHX; 0.5 μg/ml). Crystal violet assay was performed 16 h thereafter as previously described (21). Briefly, cells were stained for 30 min with 0.5% crystal violet in 20% methanol. After washing and drying, crystal violet was dissolved in 50 ml/well of 0.1 M sodium citrate solution diluted in 50% ethanol and subsequently quantified photometrically by absorbance at 550 nm (Dynatech Laboratories, Chantilly, VA). Values are expressed as the percentage of survival compared with untreated controls. Total protein extracts for immunoblots were prepared similarly. For detection of intracellular peroxides, intracellular accumulation of reactive oxygen species (ROS) was induced by 100 μM buthionine sulfoximine (BSO) in HT22 cells (20). Cells were incubated with 10 μM 5-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA; Molecular Probes, Eugene, OR) for 30 min at 37°C. Wells without cells were loaded with DCFDA serving as negative controls. Fluorescence measurements were made on a Fluorostar platereader (SLT Labinstruments) at E_xλ 488 nm and E_mλ 530 nm.

For group comparisons, the Mann-Whitney U test was applied. For EAE courses, ANOVA with Bonferroni adjustment was performed as previously described (17).

As a first step, we explored the therapeutic effect of the green tea polyphenol EGCG in EAE. This polyphenol was chosen because it is easily absorbed from the digestive tract and penetrates the brain, reaching levels similar to those found in lung, liver, kidney, and other organs (22). SJL/J mice were started on oral therapy with EGCG (300 μg per mouse in 0.9% NaCl) or vehicle (0.9% NaCl) twice daily at the time of immunization with PLP p139–151, resulting in relapsing-remitting EAE (17). EGCG treatment enhanced animals’ recovery from disease manifestation and significantly protected them from long-term neurological deficits, as indicated by the mean clinical scores over an observation period up to 131 days (Fig. 1,A). EGCG administration started at the onset of acute disease prevented mice from relapses and prolonged neurological sequelae during subsequent relapse with a maximum disease score of 1.4 ± 0.3 in the EGCG group vs 2.7 ± 0.5 in the vehicle group (p < 0.05) and significantly lower mean clinical disease scores (Fig. 1,B). The therapeutic effect of oral EGCG was found to be dose-dependent: a dose of 60 μg per mouse (administered twice daily) was sufficient to attenuate the course of EAE, whereas a lower dosage (12 μg per mouse twice daily) was not (Fig. 1,C). Histological examination revealed large inflammatory lesions throughout the brain stem and spinal cord in vehicle-treated mice, while EGCG-treated animals showed significantly reduced inflammation (Fig. 2 A).

To unravel the underlying mechanisms, we next examined T cell response ex vivo. Draining lymph node cells from immunized animals treated with EGCG for 10 days exhibited a markedly lower proliferative response to the encephalitogenic PLP p139–151 peptide than control animals (Fig. 2,B). Further, oral EGCG led to a reduction in TNF-α synthesis, demonstrated by intracellular FACS staining ex vivo (Fig. 2 C). In contrast, expression of IL-4 or IFN-γ was unaffected. There was no link between successful EAE therapy and expansion of regulatory T cells, determined by comparable fractions of CD4⁺CD25⁺ cells in EGCG- and vehicle-treated animals (data not shown). Similarly, EGCG treatment did not affect the proportion of CD4/CD8 cells.

In human CD4⁺ Th1 MBP-specific T cells, EGCG, in a dose-dependent manner, blocked expansion of cells stimulated with peptide-loaded autologous APCs (Fig. 3,A) and cells proliferating in response to direct TCR engagement by anti-CD3/CD28 stimulation (Fig. 3,B). The micromolar EGCG concentrations used in this and the following in vitro assays are likely to be achieved in humans, as indicated by previous investigations (23). Interestingly, no indication of EGCG-mediated T cell death was observed with trypan blue exclusion assay (data not shown). Even in apoptosis-susceptible Jurkat T cells, no DNA fragmentation was induced (Fig. 3,C), excluding a direct death-promoting role for EGCG in T cells. Western blot analysis showed that inhibition of the T cell proliferation by EGCG can be explained by interference on the cell cycle level, down-regulating expression of the CDK4. This effect was not mediated by p27^kip1, a CDK inhibitor and negative cell cycle regulator (Fig. 4,A). In contrast, we observed that EGCG interfered with NF-κB activation. While in untreated human T cells the crucial NF-κB-inhibitor IκB-α (24) was down-regulated upon stimulation, EGCG inhibited this pathway in a dose-dependent manner, resulting in an intracellular accumulation of IκB-α (Fig. 4,B). Further, TNF-α stimulation of T cells resulted in a strong degradation of IκB-α within a few hours (Fig. 4,C), which was completely reversed in the presence of EGCG. Since degradation of IκB-α, a prerequisite of NF-κB activity, is mediated by the cytosolic proteasome complex (24), intracellular accumulation of IκB-α by EGCG may originate from inhibition of proteasome activity. This assumption was supported by a similar IκB-α accumulation pattern in T cells in the presence of the synthetic 26S proteasome inhibitor MG132 (Fig. 4,C). Indeed, the rapid increase in ubiquitinated proteins, the substrates of the proteasome complex, confirmed the involvement of proteasome inhibition in T cells by EGCG (Fig. 4 D).

Considering the reported effects of green tea compounds in neurodegenerative models, we analyzed whether the therapeutic efficacy of EGCG in EAE involves protection against neuronal damage. Indeed, we found reduced axonal pathology in EGCG-treated animals in the chronic phase (Fig. 5,A). Quantitative analysis of caspase 3-positive neurons, shown to be present in the brain stem motor area of mice suffering from EAE (15), revealed significantly fewer apoptotic neurons in EGCG-treated as compared with vehicle-treated animals (Fig. 5,B). Accordingly, we observed similar effects of EGCG on neurons in living organotypic hippocampal brain slice cultures from SJL/J mice, a system in which neurons can be maintained in an in vivo-like fashion (19). Damage was induced by exposing slices to neurotoxic NMDA and the apoptosis ligand TRAIL, which is reported to have neurotoxic properties (25) and involve the activation of NF-κB (26). In both constellations, preincubation with EGCG 3 h before damage significantly protected against neuronal cell death (Fig. 5,C). Next, to unravel the underlying mechanisms, we investigated the impact of EGCG on HT22 hippocampal neurons. As in slices, TRAIL induces cell death in HT22 neurons which can be inhibited by EGCG (Fig. 5,D). However, in contrast to our findings in T cells (Fig. 4,B), no accumulation of IκB-α was observed (Fig. 5,E). Therefore, we hypothesized that due to its structure containing two trihydroxybenzoyl groups, EGCG may exert its neuroprotective activity directly as a free radical scavenger (27). In fact, we could show in a model of glutathione depletion-induced neurotoxicity (20) that EGCG is able to inhibit the formation of ROS in neurons. HT22 neurons were incubated with BSO, an inhibitor of glutathione synthetase, thereby inducing glutathione depletion, which subsequently results in neuronal cell death. Treating HT22 neurons with BSO induced a 2-fold increase in ROS, which could be prevented by EGCG application (Fig. 5 F). These results demonstrate that EGCG acts downstream of glutathione synthetase and, therefore, directly targets ROS formation.

We have shown that orally applied EGCG, the major polyphenolic compound of green tea, suppressed inflammation in vivo, inhibiting proliferation and TNF-α synthesis of T cells, and effectively protected against relapsing CNS autoimmune disease, resulting in a favorable long-term clinical outcome. The human dosage equivalent of the most effective EGCG concentration tested here is contained in ∼3 liters of conventionally brewed green tea. Recently, it was reported in collagen-induced experimental arthritis that application of unpurified green tea extracts reduced anti-collagen Ig levels and the expression of the proinflammatory cytokines TNF-α and IFN-γ in arthritic joints (28). Our data provide a mechanistic basis, demonstrating for the first time that the anti-inflammatory properties of EGCG are mediated by down-regulating NF-κB in T cells. This transcription factor, crucial for the development of autoimmune encephalomyelitis (29), regulates the expression of genes encoding proinflammatory cytokines, chemokines, immune receptors, and adhesion molecules that play a key part in the initial recruitment of leukocytes to sites of inflammation (24). EGCG inhibited the catalytic activity of the proteasome in activated T cells, resulting in the intracellular accumulation of ubiquitinated proteins including the NF-κB-suppressor IκB-α. Supporting our conclusion that EGCG acts at the proteasome level, in vitro experiments showed that EGCG inhibited the proteolytic activities of both 20S and 26S proteasome complexes in the presence of Mg²⁺ (data not shown). While the proteasome complex plays a pivotal role in regulating T cell expansion (30), the overexpression of IκB-α is known to suppress proliferation by inhibiting the G₁/S transition (31). Indeed, we found that EGCG directly induced T cell cycle arrest in the G₁/S transition phase, reducing CDK4 expression. We further detected reduced axonal pathology and fewer neurons eventually undergoing cell death in animals with chronic autoimmune encephalomyelitis, revealing an additional mechanism underlying the therapeutic potential of EGCG. The lack of IκB-α accumulation in EGCG-treated neurons indicates that the neuroprotective effect of this polyphenol is mediated by interference with a distinct pathway which differs from the EGCG target in T cells. In fact, we could show a ROS-blocking capacity of EGCG in neurons, providing evidence for the concept that, as the structure of EGCG contains a polyphenol ester bond (32) and two trihydroxybenzoyl groups, both inhibition of catalytic activities of the proteasome and antioxidative properties of EGCG contribute to the therapeutic effects observed in vivo. ROS not only represent a supposed damage process in neurodegeneration, but also in neuroinflammation such as EAE (33).

MS is a multiphasic autoimmune CNS disease, involving injury to neuronal structures, including axons and parent cell bodies. Since the available treatment only consists of immunomodulatory agents, green tea compounds such as epigallocatechin-3-gallate are candidates for the treatment of MS patients, as they are administered orally, are well tolerated (34), and exhibit synergistic beneficial effects, unraveled here, in disorders with both pathological elements, inflammation and neuronal injury (Fig. 6). One may speculate that the biological properties described here contribute to the lower prevalence rates of MS in Asian populations with traditional consumption of green tea (35).

We thank B. Seeger, N. Nowakowski, and O. Riede for technical assistance, and K. Rosegger and A. Mason for carefully reading the manuscript as native speakers.