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Division of Genomic Medicine, University of Sheffield Medical School, Sheffield S10 2RX, UK and * Coastside BioResources, Stonington, ME 04681
3To whom correspondence should be addressed. E-mail: D.J.Buttle{at}sheffield.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • green tea • catechin • cartilage breakdown • proteoglycan • type II collagen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). They are the most common diseases evaluated in ambulatory care, accounting for 
315 million physician visits per year in the United States; they are the second most common reason for visits to surgeons, the third most common to see a family doctor and the fourth most common reason for appointments to see a consultant (3
). New musculoskeletal complaints are the most time-consuming for physicians to evaluate and manage (4
).
All forms of arthritis are characterized by varying degrees of inflammation in joint tissues. Osteoarthritis (OA)4
was known for many years as an arthrosis, exhibiting a lack of overt inflammation. Although it is now recognized that inflammatory mediators are a part of the disease, it is clear that the degree of inflammation is much greater in other forms of arthritis, such as rheumatoid arthritis (RA). Inflammation is accompanied by destruction of the connective tissue of the joint, particularly to the layer of cartilage covering the ends of bone in diarthrodial joints. This thin mantle is of extreme importance to joint function; if damage is sufficiently advanced, it leads to total joint dysfunction, the only remedy for which is surgical joint replacement. There is now a great deal of evidence to suggest that the inflammatory and destructive components of cartilage are distinct disease processes, and that joint destruction may continue even when inflammation is suppressed (5
). The traditional treatments for arthritis treat the inflammatory component of the disease and lead to reduced pain and swelling. However, none appear to be able to spare cartilage from this ongoing degenerative process, and indeed some may even accelerate it (6
).
The identification of common dietary substances capable of affording protection or modulating the onset and severity of arthritis may have important health implications. One such dietary component, which has been the focus of much attention in the last decade, is green tea. Green tea is made from the leaves of the tea plant, Camellia sinensis. It differs from black tea in that there is no fermentation process involved and therefore none of the associated changes in chemical composition. The potential medical benefits of consuming green tea have received a great deal of attention over the past few years, most of which has been directed at a group of polyphenolic compounds called catechins. These are condensed into tannins in black tea, and are found in sources other than green tea, such as grape skins and seeds. The most abundant of the polyphenolic compounds in green tea is epigallocatechin gallate (EGCG), with other catechins such as epicatechin (EC), epigallocatechin (EGC) and epicatechin gallate (ECG) also present (Fig. 1
). The catechins are antioxidants, and are probably largely responsible for the reported antioxidant effects of green tea (7
). They have also been found to have anti-inflammatory properties, which may be due to their ability to inhibit tumor necrosis factor (TNF) synthesis (8
), possibly by the inhibition of kinase(s) in signaling cascades, leading to activation of certain transcription factors (9
–12
). They are also inhibitors of matrix metalloproteinases (13
,14
). The reported beneficial effects on a number of clinical conditions, including stroke and cerebral hemorrhage (15
), cardiovascular and liver diseases (16
), bacterial infections (17
), stomach ulcers (18
) and cancer (19
) may be related to their antioxidant, anti-inflammatory and antiproteinase properties.
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), with a half-life of a few hours (21
,22
). The catechins also undergo a number of chemical modifications, both in the digestive tract and after adsorption. These include condensation reactions, methylation, glycosylation and sulfation (23
,24
); indeed, some of the variability in reported plasma concentrations may be due to the difficulties in measuring chemically modified as well as unaltered catechins. Evidence to date suggests that dimerization or polymerization does not affect the antioxidative properties of gallate esters because the total numbers of antioxidative catechol and pyrogallol structures are retained (25
,26
). Similarly, glucuronidation of catechins does not appear to affect the electrochemical properties of the molecules, suggesting that their antioxidant properties will not be greatly altered (27
). Very little is known about the effects of derivatization on potential bioactivity other than the antioxidant properties.
It might be expected that compounds that are bioavailable and anti-inflammatory would have beneficial effects on inflammatory diseases such as rheumatoid arthritis, and indeed Haqqi et al. (28
) demonstrated that oral administration of a polyphenolic fraction from green tea can ameliorate inflammation in a murine model of inflammatory arthritis.
Until now, little information has been available on the effects of green tea catechins on the integrity of joint structure and function. We have now begun this line of investigation by examining the chondroprotective effects of catechins derived from green tea in in vitro models of cartilage breakdown and have found that some, particularly those containing a gallate ester group, are capable of inhibiting both proteoglycan and type II collagen breakdown. The chondroprotective effect is distinct from the ability of some catechins to inhibit inflammatory cytokine synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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EC, EGC, ECG, EGCG, dimethyl sulfoxide (DMSO), gentamicin sulfate, chondroitin sulfate A, papain (E.C 3.4.22.2), proteinase K (EC 3.4.21.64), all-trans retinoic acid (Ret) and the lactate assay kit were obtained from Sigma Chemical, Poole, Dorset, UK. 1,9-Dimethylmethylene blue (DMB) was obtained from Aldrich, Gillingham, Dorset, UK. Cetylpyridinium chloride was from BDH Chemicals, Poole, Dorset, UK. Most reagents for tissue culture were obtained from Gibco Life Technologies, Paisley, UK. Recombinant human interleukin-1
(rhIL-1) was kindly provided by Dr. Craig Reynolds, National Cancer Institute, Frederick, MD. Recombinant human IL-1ß (rhIL-1ß) and recombinant human tumor necrosis factor- (rhTNF) were kindly provided by Dr. Ulf Neumann, Novartis Pharma AG, Basel, Switzerland. [35S]Na2SO4 was from ICN Biomedicals, Thame, UK.
Explant cultures.
Bovine cartilage explants from the nasal septum and metacarpophalangeal joint were obtained from cattle on the day of slaughter as described previously (29
,30
). For the study of proteoglycan breakdown, bovine nasal and articular cartilage explant cultures were maintained in the presence and absence of rhIL-1 (0.3 and 3 nmol/L, respectively), rhTNF (3 or 6 nmol/L, respectively) or Ret (1 µmol/L). The catechins were added to culture media from stock solutions in DMSO to give the appropriate final concentrations while maintaining a 10 g/L final DMSO concentration. All cultures were for 5 d, with a medium change on d 3. For the investigation of type II collagen degradation, bovine nasal cartilage explants were cultured for 28 d with a twice weekly medium change, in the presence and absence of rhIL- (4.5 nmol/L) and the catechins (20 µmol/L). Because 14 d of exposure to rhIL-1 has been shown previously to be sufficient to induce type II collagen release (31
), the cytokine was withdrawn from the cultures after 2 wk, and the cartilage explants were maintained in serum-free Dulbecco’s modified Eagle’s medium (DMEM) with or without the catechins for the remaining 2 wk. Conditioned media and cartilage at the end of the culture period were collected and stored separately at -20°C before assay.
Human cartilage removed during joint replacement or corrective surgery and excess to surgical requirements was obtained with Ethical Committee approval (Barnsley District Hospital, South Yorkshire, UK) from the knee joints of OA and RA sufferers, and also from patients with Marfan’s syndrome. In the last-mentioned case, the cartilage was macrosocopically normal. For the determination of the effect of the catechins on proteoglycan degradation, slices dissected from these cartilage samples were cultured in the presence of a mixture of IL1ß (3 nmol/L) and TNF (6 nmol/L) for 9 d, with medium changes every third day.
Assays of proteoglycan degradation.
The DMB dye-binding assay was used (32
) with adaptation for use with a plate reader (33
). Residual sulfated glycosaminoglycan remaining in the tissue was determined after digestion with papain. Results were then standardized for the size of explant by expressing the amount of proteoglycan released as a proportion of the total (medium plus tissue residue).
Measurement of type II collagen breakdown.
Cartilage explants remaining at the end of the 28-d culture period were digested with 1 g/L proteinase K at 56°C for 15 h. Type II collagen fragments in the conditioned medium and in the proteinase K digests were measured by an inhibition ELISA as described previously (34
). The amount of collagen released into the medium was calculated as the proportion of total type II collagen present in the explant (medium plus tissue residue).
Lactate determination.
The amount of lactate in culture medium was determined with a kit from Sigma Chemical using the lactate oxidase/peroxidase method.
Measurement of proteoglycan synthesis.
Proteoglycan synthesis in bovine nasal cartilage explants was assessed by measuring the incorporation of 35S into cetylpyridinium chloride precipitates (35
). Briefly, following a 24-h period of culture in DMEM containing 50 g/L newborn calf serum, 92.5 MBq 35SO4/L, with or without EGCG at 2 and 20 µmol/L, the explants were washed in serum-free DMEM, weighed, and together with medium, digested with papain. To 100 µL of the papain digest were added 100 µL of 2g/L chondroitin sulfate A, 100 µL of 100 g/L cetylpyridinium chloride and 50 µL of 0.4 mol/L Na2SO4. This mixture was left for 15 min and then centrifuged (10,000 x g) for 10 min after which the supernatants were discarded. The pellets were washed in 30 g/L cetylpyridinium chloride and 0.1 mol/L Na2SO4, followed by the cetylpyridinium chloride solution alone, and then dissolved in formic acid. Scintillation cocktail (5 mL) was added and 35S was quantified in a scintillation counter.
Statistical analysis.
Values are shown for convenience as means ± SEM. However statistical tests compared medians using the Mann-Whitney two-tailed U test for nonparametric data. Because we consistently found that variation between replicate cultures from a single animal was as great as variation between independent observations, i.e., animals, we treated all observations as being independent. Indeed the variation between so-called replicates may indicate heterogeneity of cartilage within a single joint or nasal septum and may therefore not be random. Values were considered to be significantly different from the null hypothesis when P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As shown in Table 1
, EGCG (20 µmol/L) effectively prevented rhTNF-stimulated cartilage proteoglycan degradation in bovine nasal cartilage, but no significant inhibition of basal or of rhIL1- or Ret-stimulated release was observed. In contrast, both ECG and EC at 20 µmol/L significantly inhibited IL1-stimulated degradation but not that invoked by rhTNF or Ret. EGC did not inhibit bovine nasal cartilage proteoglycan breakdown.
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, EGCG again potently inhibited rhTNF-stimulated proteoglycan loss, and also inhibited basal, rhIL-1- and Ret-mediated breakdown. The other gallate ester, ECG, also inhibited rhTNF-, rhIL-- and Ret-stimulated breakdown. Both EC and EGC were without effect.
In view of the potent inhibition of rhTNF-stimulated cartilage proteoglycan degradation by EGCG, we examined the efficacy of this compound in more detail by means of a dose-response study with bovine nasal cartilage, shown in Figure 2
. Inhibition was significant at 2 µmol/L (47% inhibition) and greater, increasing to 84 and 138% inhibition at 20 and 200 µmol/L, respectively. The value of > 100% indicates inhibition of the basal release of proteoglycan as well as that occurring in response to TNF. The 50% inhibitory concentration (IC50) for the inhibition of TNF-mediated proteoglycan breakdown was 2 µmol/L.
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We examined whether catechins containing a gallate ester group influenced proteoglycan breakdown from human articular cartilage. As shown in Table 2
, ECG (20 µmol/L) significantly inhibited proteoglycan breakdown and release from nonaffected, OA and RA cartilage treated with IL1ß and TNF. At the same concentration, EGCG also tended to inhibit breakdown. OA cartilage was also cultured in the absence of exogenous cytokines. Under these conditions EGCG, but not ECG, inhibited proteoglycan breakdown.
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-stimulated bovine nasal cartilage explants.
As demonstrated previously (36
), the culture of bovine nasal cartilage in the presence of IL1 leads to breakdown and loss of type II collagen and dissolution of the cartilage explants. We therefore used this model to investigate the effects of green tea catechins on cartilage type II collagen breakdown. As shown in Table 3
, after 4 wk of culture, inclusion of rhIL1 for the first 2 wk resulted in the almost total dissolution of type II collagen. EGCG, ECG and EGC (20 µmol/L) significantly reduced this degradation, with the proportion of type II collagen released from the explants decreasing by 50% or more in all three cases. This was further demonstrated by the visible retention of most of the tissue after 4 wk in culture with IL1 and ECG or EGCG, compared with explants cultured with rhIL1 alone in which the explants had decreased substantially in size (Fig. 3
).
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Inhibition of proteoglycan and collagen breakdown can be achieved nonspecifically by agents that affect the viability or metabolic activity of the chondrocytes (29
). We therefore undertook an analysis of the effects of the catechins on general metabolic activity as judged by lactate output and rates of proteoglycan synthesis and incorporation into the matrix.
Media from eight 5-d cultures of bovine nasal cartilage were pooled and assayed for lactate levels. EGCG (200 µmol/L), with or without rhTNF, had no significant effect on lactate production over this period (results not shown). Even over a 28-d period, neither EGCG nor ECG was associated with any significant change in the levels of lactate produced by explants in the presence of rhIL1 (Table 4
). EGCG at 2 and 20 µmol/L had no significant effects on proteoglycan synthesis in bovine nasal cartilage explants (results not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The chondroprotective mechanism(s) of the catechins has not been elucidated. They are strong antioxidants and scavenge free radicals (25
,37
). Indeed, EGCG has been described as the most powerful antioxidant in plant-derived material (38
), and the consumption of green tea leads to a significant increase in the antioxidant activity of plasma (39
). However, the link between antioxidant activity and chondroprotection remains unclear; only one study has claimed a connection between these two processes in the action of propyl gallate (40
). It has been reported that in some circumstances, experiments in tissue culture medium can lead to artifactual results due to the generation of hydrogen peroxide after the addition of catechins (41
). This can be cytotoxic and can lead to the possibly false conclusion that the catechins are antitumorigenic. In our experiments, we found no evidence of cytotoxicity (measured as lactate output or proteoglycan synthesis by the explants), but we currently cannot rule out rather more specific effects that might result from an oxidative burst.
Some work on the effect of the catechins on matrix-degrading enzymes has been published. ECG and EGCG have been reported to inhibit in a dose-dependent manner the actions of matrix metalloproteinases such as gelatinases A (EC 3.4.24.24) and B (EC 3.4.24.35) and macrophage elastase (EC 3.4.24.65), with the gallate esters providing the best inhibition (13
) and EGCG having IC50 of 20 and 50 µmol/L with gelatinases A and B, respectively (14
). The serine proteinase urokinase-type plasminogen activator (EC 3.4.21.73) has been implicated in cartilage aggrecan breakdown (42
), and it has been reported that EGCG potently inhibits this proteinase (43
). However, studies in our laboratory have shown this not to be the case (C. Adcocks and D. J. Buttle, unpublished data).
In addition to antioxidative and antiproteinase properties, the catechins have been shown to modulate various cytokine and growth factor–signaling pathways (9
–12
,44
,45
). It is likely that this property contributed to the anti-inflammatory effects in a murine model of rheumatoid arthritis (28
). The ability of the catechins to inhibit proinflammatory cytokine synthesis did not contribute to their chondroprotective effects in our in vitro models of cartilage breakdown, however, because in these experiments, the cytokines were added to the cultures; the cartilage cells do not synthesize these in sufficient quantity to set up an autocrine pathway. However, it is possible that the green tea catechins were modulating downstream pathways following TNF or IL1 receptor occupation and in this way inhibiting the chondrocytes’ catabolic response.
It therefore seems possible that the catechins possess two independent actions, both of which may be prophylactic for the development of arthritis and beneficial to arthritis sufferers; an anti-inflammatory and an antiproteolytic chondroprotective effect. Further work is required to determine whether this is the case. It also remains to be shown whether ingestion of green tea gallate ester catechins can lead to sufficiently high concentrations of these molecules and their bioactive derivatives in the joint to afford chondroprotection in vivo.
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FOOTNOTES |
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2 Supported by University funds supplemented with a small grant from Coastside BioResources, Stonington, ME 04681 .
4 Abbreviations used: DMB, dimethylmethylene blue; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; EC, (-)-epicatechin; ECG, (-)-epicatechin gallate; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin gallate; IC50, 50% inhibitory concentration; OA, osteoarthritis; RA, rheumatoid arthritis; Ret, all-trans retinoic acid; rhIL-1, recombinant human interleukin-1; rhTNF, recombinant human TNF; TNF, tumor necrosis factor.
Manuscript received 25 September 2001. Initial review completed 30 October 2001. Revision accepted 6 December 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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 M. Ulrich-Vinther, M. D. Maloney, E. M. Schwarz, R. Rosier, and R. J. O'Keefe Articular Cartilage Biology J. Am. Acad. Ortho. Surg., November 1, 2003; 11(6): 421 - 430. [Abstract] [Full Text] [PDF]
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