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Effect of (−)-epigallocatechin-3-gallate on respiratory burst of rat macrophages
International Immunopharmacology 2 (2002) 849 – 855 www.elsevier.com/locate/intimp
Effect of ( )-epigallocatechin-3-gallate on respiratory burst of rat macrophages ´ lvarez a, J. Leiro b,*, F. Orallo a E. A a
Departamento de Farmacologı´a, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain b Departamento de Microbiologı´a y Parasitologı´a, Laboratorio de Parasitologı´a, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain Received 10 October 2001; received in revised form 15 March 2002; accepted 23 March 2002
Abstract The toxic effects derived from overproduction of oxygen radicals [reactive oxygen species (ROS)] by immune cells can be partially abolished by the antioxidant activities of plant polyphenols. In the present study, we investigated the antioxidant action of a catechin, ( )-epigallocatechin-3-gallate (EGCG), on the respiratory-burst responses of rat peritoneal macrophages. EGCG at concentrations of 50 – 200 AM blocked the production of nitric oxide by macrophages stimulated in vivo with sodium thioglycollate then 5 days later in vitro with lipopolysaccharide and gamma-interferon. At 1 – 100 AM, EGCG also inhibited the extracellular liberation of oxygen radicals by resident peritoneal macrophages stimulated with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). At low concentrations (1 – 5 AM), EGCG increased the reduction of nitro blue tetrazolium (NBT) by the superoxide anions generated in the non-enzymatic system NADH/PMS, acting as a pro-oxidant agent, while at concentrations above 10 AM, EGCG acts as a scavenger of superoxide anions. These results show that EGCG is capable of modulating ROS production during the respiratory burst of rat peritoneal macrophages by acting as a superoxide anion scavenger. EGCG may therefore be useful in the prevention and treatment of diseases due to increased free radical production. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rat; Respiratory burst; Peritoneal macrophage; EGCG; Nitric oxide; Superoxide anion
1. Introduction Polyphenols are major components of many traditional herbal remedies, and exhibit several beneficial pharmacological effects [1]. Tea (from Camellia sinensis) and wine (from Vitis vinifera) are two popular beverages that both contain large amounts of phytopolyphenols [2,3], including proanthocyanidines (condensed tannins) which have attracted particular interest *
Corresponding author. Tel.: +34-81-593316; fax: +34-81593316. E-mail address: [email protected] (J. Leiro).
among pharmacologists [4]. In grapes, proanthocyanidines are present at highest concentration in the seeds, and are largely constituted by dihydrolyzed compounds such as (+)-catechin, ( )-epicatechin and ( )-epicatechin-3-gallate (EGCG) [4]. In green tea, the major catechins are EGCG, ( )-epigallocatechin, ( )-epicatechin-3-gallate, ( )-epicatechin, (+)-gallocatechin and (+)-catechin, EGCG being present at the highest concentration [5]. EGCG, like the other catechins, has recently attracted great interest for its potential anticancer and anti-inflammatory activities [3,6,7]. Both activities have been attributed to the notable antioxidant capacity of EGCG [3,8]. Reactive oxygen spe-
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cies (ROS) are generated endogenously by all aerobic cells as products of a number of metabolic reactions [9,10]. Phagocytic cells, such as polymorphonuclear leukocytes and macrophages, generate large quantities of ROS in response to a variety of membrane stimulants, by a coordinated sequence of biochemical reactions known as the oxidative burst [11]. ROS are low-molecular-mass compounds that include reactive oxygen intermediates (ROI) such as superoxide (O2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH), and reactive nitrogen intermediates (RNI) such as nitric oxide (NO) and peroxynitrite (ONOO ) [12]. In addition to possessing microbicidal activity, these compounds have been shown to be important regulatory molecules in diverse physiological processes; furthermore, excessive production of ROS has been implicated in a number of pathological processes, such as the development of cardiovascular diseases, neurodegenerative disorders, and cancer [6]. Several studies have analyzed the effects of EGCG on different stages of RNI production by macrophages [3,13]. To date, however, there has been only a single study of effects on ROS production, specifically on superoxide anion generation induced in mouse macrophages by 12-0-tetradecanoylphorbol-13-acetate [14]. Most of these studies have used mouse macrophage cell lines, which may show alterations in phagocytic functions with respect to normal cells [15]. There have been no previous studies of the effects of EGCG on the respiratory-burst responses of rat macrophages, which constitute a model that is widely employed in biomedical investigation and which present a number of differences with respect to the mouse-macrophage model of the respiratory burst [16 –18]. In the present study, we investigated the in vitro effects of EGCG on the production of ROS by rat peritoneal macrophages. Our results demonstrate that EGCG significantly inhibits LPS-induced RNI production and PMAinduced ROI production in a concentration-dependent manner, probably by acting as a ROS scavenger.
study. They were housed (groups of five) in Macrolon cages (Panlab, Barcelona, Spain) on poplar shaving bedding (B&K Universal, G. Jordi, Barcelona, Spain) in a standard experimental animal bio-clean room, illuminated from 08:00 to 20:00 h (12 h light:12 h dark cycle) and maintained at a temperature of 22– 24 jC. The animals had free access to food pellets (B&K Universal, G. Jordi), and drinking fluid (tap water), and were allowed to acclimatize for 1 week before the experiments. All experiments were carried out in accordance with European regulations on the protection of animals (Directive 86/609), the Declaration of Helsinki and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). In this context, all experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Santiago de Compostela. 2.2. Stimuli, drugs and chemicals Stock solution (2 mg/ml) of phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO, USA) was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 80 jC until use. Stock solution of lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) was made up at 10 Ag/ml in phenol-red-free Dulbecco’s Eagle medium (DMEM; Sigma) and stored at 20 jC until use. Thioglycollate broth (Merck, Germany) was prepared to a concentration of 3% (w/v) in PBS, autoclaved at 121 jC for 10 min, and stored at room temperature until use. Stock solution of the arginine analogue N-monomethyl-L-arginine monoacetate (L-NMA, Calbiochem, USA) was made up at 100 mM in water, and stored until use at 20 jC in the dark. L-Glutamine, sulfanilamine and N-(1-naphthyl)ethylenediamine dihydrochloride were purchased from Sigma. 2.3. Isolation of rat peritoneal-exudate macrophages
2. Materials and methods 2.1. Animals Male Wistar rats (Iffa-Credo), purchased from Criffa (Barcelona, Spain), were used throughout this
For induction of inflammatory responses, rats were injected intraperitoneally with 1 ml of 3% thioglycollate broth, and peritoneal exudate was extracted 5 days later. Rat resident and inflammatory peritoneal macrophages were obtained from rats euthanized by cervical dislocation in a laminar flow chamber to
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ensure sterile conditions. The abdomen of the rat was soaked with 70% ethanol for disinfection, a midline incision was then made with scissors and the abdominal skin retracted. Thirty milliliters of Hanks’ balanced salt solution (HBSS) was then injected into the peritoneal cavity, using a syringe with a 19-G needle. After gentle abdominal massage, about 30 ml of fluid was extracted using the same syringe and transferred to 50-ml sterile polypropylene tubes on ice. A 20-Al aliquot was then extracted for cell counting in a hemocytometer. The remaining cells were washed once by centrifugation at 400g for 10 min at 4 jC and resuspended to a concentration of 106 cells ml 1. The number of viable cells was estimated by the trypan blue exclusion test: trypan blue (0.4% in PBS) was added to wells and incubated for 3 min at room temperature, after which the number of unstained (viable) and stained (non-viable) cells were counted. Aliquots (100 Al) of the cell suspension were added to the wells of 96-well microculture plates (Corning, USA) or placed on microscope slides, and left for 90 min in a humidified incubator at 37 jC, 5% CO2, to allow adhesion. Non-adherent cells were then removed by gently washing with HBSS. More than 97% of the cells plated on microscope showed nonspecific esterase activity, determined as per Ref. [19], indicating that they were macrophages.
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an ELISA reader (Titertek Multiscan, Flow Laboratories, Finland). Nitrite concentration was calculated with reference to a standard curve obtained using NaNO2 (1– 256 AM in culture medium). 2.5. Assay of ROI production The production of ROIs during the respiratoryburst response of macrophages was investigated using OxyBURSTR Green probes (Molecular Probes, The Netherlands) that emit fluorescence when they are oxidized by ROS produced during the respiratory burst [21] as previously described [22]. Briefly, for quantification of extracellular release of ROIs, we used the OxyBURST Green H2HFF BSA reagent [bovine serum albumin (BSA) coupled to dihydro2V,4,4,6,7,7V-hexafluorofluorescein (HsHFF)]. A 1 mg/ ml stock solution of the reagent was made up in HBSS containing 2 mM sodium azide and stored at 4 jC in the dark. For the assay, macrophages were incubated with the reagent at 10 Ag/ml in the wells of 96-well flat-bottom microtiter plates for 2 min at 37 jC, then stimulated by addition of 10 Ag/ml of PMA. Extracellular release of ROIs is expressed as variation in fluorescence per unit time [arbitrary fluorescence units (AFU) s 1]. 2.6. Generation of O2 radicals
2.4. Assay of nitrite production One hundred-microliter aliquots of macrophages that had been prestimulated in vivo with thioglycollate broth 5 days previously were incubated in microtitre plates (Corning) at 37 jC with 5% CO2 for 90 min. After adhesion, the wells were gently washed with HBSS, then incubated for 48 h with 100 Al of phenolred-free DMEM containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Serva, USA), 10 U/ml of recombinant murine gamma interferon (IFNg, Genzyme, USA) and 100 ng/ml of LPS. In some experiments, the arginine analogue L-NMA (1 mM), was included in the medium. Nitrite production in the culture supernatants was assayed by the Griess reaction [20]: 100 Al of culture supernatant was added to 100 Al of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine hydrochloride in 2.5% H3PO4), then incubated for 10 min at room temperature, and absorbance was measured at 530 nm using
O2 radicals were generated in a non-enzymatic system (phenazine methosulphate-NADH) and quantified by the spectrophotometric measurement of the product of the reduction of nitro blue tetrazolium (NBT, Sigma), essentially following the procedure described by Ref. [23] with minor modifications. We used 300-Al test solutions made up in phosphate buffer (50 mM KH2PO4 –KOH, pH 7.4) and containing h-nicotinamide adenine dinucleotide (NADH, Sigma), 166 AM, NBT (Sigma), 43 AM, and SOD (10 U/ml) or EGCG at various concentrations (1, 5, 10 or 20 AM). Control experiments were carried out simultaneously without EGCG. In addition, the possible capacity of the tested compounds to directly reduce NBT was determined by adding the corresponding tested drugs to solutions containing only NBT in a phosphate buffer. Reaction was started with test solutions already in a Cobas Fara 22-3123 (Roche) autoanalyzer, by add-
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ing phenazine methosulphate (PMS, 2.7 AM; freshly diluted in 100 ml of the above phosphate buffer) and continued at 25 jC for 10 min, a period in which absorbance increased linearly from the third minute. The rate of NBT reduction was calculated from the differential absorbance at 560 nm with respect to a blank solution in which phenazine methosulphate was replaced by buffer solution, and was expressed as increment of absorbance per minute. 2.7. Statistical analysis Means were compared by unpaired two-tailed Student’s t-tests when variances were equal (Fisher’s F-test, P > 0.05), or by Welch’s t-tests when variances were different. P values less than 0.05 were considered significant.
3. Results 3.1. Effects of EGCG on ROS production by rat peritoneal macrophages We first investigated the effects of EGCG on PMAstimulated in vitro ROI production by resident peri-
Fig. 1. Effect of EGCG (1 – 100 AM) on extracellular release of reactive oxygen species (ROI), as quantified by the OxyBURST Green H2HFF BSA, by rat resident peritoneal macrophages stimulated with PMA (1 Ag/ml). Values shown are means (n=5) Fstandard errors of fluorescence emission by cells (arbitrary units per second). Asterisks indicate statistical significance ( P<0.01) with respect to the control values (macrophages incubated in the absence of EGCG).
Fig. 2. Effect of EGCG (50 – 200 AM) on nitrite concentration, assayed by the Griess reaction, in cultures of rat peritoneal macrophages stimulated in vivo with thioglycollate then 5 days later in vitro with IFNg (10 U/ml) and LPS (100 ng/ml). The rightmost bar represents assays in which the arginine analogue Nmonomethyl-L-arginine monoacetate (L-NMA; 1 mM) was added to cultures instead of EGCG; this compound abolishes nitrite generation in the Griess reaction. Values shown are means (n=5), Fstandard errors. Asterisks indicate statistical significance ( P<0.01) with respect to the control values (macrophages incubated in the absence of EGCG).
toneal-exudate macrophages. As shown in Fig. 1, EGCG at concentrations of 1– 100 AM significantly and dose-dependently inhibited extracellular ROI production, as quantified fluorometrically using the OxyBURST Green H2HFF BSA reagent. To study the effects of EGCG on RNI production, we prestimulated the macrophages in vivo by intraperitoneal injection of thioglycollate 5 days before extraction of macrophages for the in vitro assay. After stimulation with LPS and IFNg in the presence of different concentrations of EGCG, we then determined accumulated nitrite in the medium, as estimated by the Griess reaction (Fig. 2). EGCG at concentrations of 50– 200 AM significantly and dose-dependently inhibited RNI production. The inhibition obtained with EGCG at the maximum concentration tested (200 AM) was similar to that obtained with 1 mM L-NMA. To rule out the possibility that the observed inhibitions of ROS production were due to effects of the EGCG on macrophage viability, EGCG was added to the culture medium at 200 AM (the maximum concentration used in the assays). Mean macrophage viabilityFstandard error (determined on the basis of
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Fig. 3. Effect of EGCG (1 – 20 AM) on reduction of NBT by superoxide anion generated in the phenazine methosulfate (PMS)/ reduced beta-nicotinamide adenine dinucleotide (NADH) system. The rightmost bar represents assays in which the enzyme superoxide dismutase (SOD; 10 U/ml) was added to cultures instead of EGCG; this enzyme abolishes superoxide generation in the PMS/NADH system. Values shown are mean percentages of the control response (n=5)Fstandard errors. Asterisks indicate significant difference (*P<0.01; **P<0.05) with respect to control.
trypan blue exclusion: see Materials and Methods) was 94F4% after 2 h of incubation with EGCG, versus 97F3% (n=5) in untreated controls. 3.2. O2 scavenging by EGCG We investigated the ability of EGCG to scavenge O2 generated by a non-enzymatic system (PMS/ NADH). EGCG at 1 – 5 AM appeared to enhance O2 production, whereas EGCG at 10 AM or higher had a significant inhibitory effect. EGCG at 20 AM had inhibitory effects close to those obtained with SOD at 10 U/ml. (Fig. 3).
4. Discussion The relationship between macrophage cytotoxicity and enhanced production of reactive nitrogen and oxygen intermediates (RNIs and ROIs) is well established [24,25], as is the fact that interaction between RNIs and ROIs generates potentially cytotoxic agents which may have pathological effects [12,26 – 28]. Drugs that inhibit ROS generation may have beneficial effects in the treatment of diseases due to overproduc-
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tion of ROS [29]. Polyphenols are common components in plants, and several studies have demonstrated that they possess diverse pharmacological activities, including anti-inflammatory effects [1,5,8,30,31], prevention of atherosclerosis and cardiovascular diseases [7,13,32 – 34], prevention of oral diseases [35], anti-allergic activities [36], prevention of autoimmune disorders [37] and anticancer properties [3,6]. The results of the present study demonstrate that the polyphenol EGCG dose-dependently inhibits the production of both ROIs and RNIs by rat peritonealexudate macrophages. In previous studies, EGCG (5 and 10 AM) was found to inhibit inducible NOS (iNOS) protein transcription in activated mouse macrophages, by reducing iNOS mRNA expression [5], while the gallic acid moiety of EGCG was found to be essential for its potent anti-inflammatory activity [8]. EGCG also blocks LPS induction of nitric oxide synthase in mouse macrophages, by down-regulating the LPSinduced activity of the transcription factor NF-KB [8]. More specifically, in the mouse macrophage cell line RAW 264.7 and in elicited mouse peritoneal macrophages, EGCG regulates TNFa gene expression by blocking NK-KB activation through inhibition of InBkinase (IKK) activity [3,38]. In addition, it has recently been demonstrated that ROS scavenges, like IL-10 inhibits NF-kappaB activation [39]. Several polyphenols display ROS scavenger activity [40]. For example, pycnogenol (a polyphenolcontaining extract of the bark of Pinus maritima) scavenges ROIs and RNIs, and has effects on NO metabolism in RAW 264.7 macrophages [41]. Phenylethanoids likewise show NO radical-scavenging activity, which possibly contributes to their anti-inflammatory effects [42], while phytolens, polyphenolic compounds from legumes, show significant ROSscavenging ability [37]. In the present study, using a non-enzymatic system (PMS/NADH) with NBT detection, we found that EGCG shows dose-dependent superoxide-scavenging activity. Several previous studies have shown that EGCG protects against NO toxicity by scavenging peroxynitrite (ONOO ), one of the key RNIs in cytotoxic processes [34,43]. In addition, the scavenging of superoxide by EGCG may reduce peroxynitrite levels, since macrophages stimulated with LPS and TNF need superoxide for peroxynitrite production [12]. It has also been shown that the relationship between NO and superoxide levels plays
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important roles in some pathological processes such as atherogenesis [44]. At low concentrations (1– 5 AM), EGCG seems to act as a pro-oxidant, increasing the reduction of NBT in the PMS/NADH system. Several other polyphenols with pro-oxidant effects [45] show anti-cancer activity, associated with an increase in ROS generation by tumor cells, which may ultimately lead to cell lysis [46]. In conclusion, EGCG shows important superoxidescavenging activity, leading to reduced net production of ROS in rat peritoneal macrophage cultures stimulated with PMA (assays of ROI production) or LPS and IFNg (assays of RNI production). These results suggest that EGCG may be useful in the treatment of immune disorders involving ROS production by phagocytic cells, and that EGCG may be able to protect cells from ROS-mediated oxidative modification.
Acknowledgements This work was supported in part by grants from Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT), Spain (SAF2000-0137) and Consellerı´a de Educacio´n e Ordenacio´n Universitaria, Xunta de Galicia, Spain (PGIDT00PXI20314PR and PGIDT99MAR20301).
References [1] Chen YC, Yang LL, Lee TJF. Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX 2 gene expression via suppression of nuclear factor-kappa B activation. Biochem Pharmacol 2000;59:1445 – 57. [2] Carando S, Teissedre PL, Cabanis JC. Estimation de l’apport en catechines et procyanidines relatif a une consommation moderee et reguliere de vins en France. Bulletin-de-l’OIV (France) 1999;72:72 – 86. [3] Pan MH, Liang YC, Lin-Shiau SY, Zhu NQ, Ho CT, et al. Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells. J Agric Food Chem 2000;48:6337 – 46. [4] Souquet JM, Cheynier V, Moutounet M. The proanthocynidins of grape [skin, seed, stem]. Bulletin-de-l’OIV (France) 2000; 73:601 – 9. [5] Lin YL, Lin JK. ( )-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappaB. Mol Pharmacol 1997;52:465 – 72.
[6] Lin JK, Liang YC, Lin-Shiau SY. Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade. Biochem Pharmacol 1999;58:911 – 5. [7] Hayek T, Fuhrman B, Vaya J, Rosenblat M, Belinky P, et al. Reduced progression of atherosclerosis in apolipoprotein Edeficient mice following consumption of red wine, or its polyphenols quercetin or catechin, is associated with reduced susceptibility of LDL to oxidation and aggregation. Arterioscler, Thromb, Vasc Biol 1997;17:2744 – 52. [8] Lin YL, Tsai SH, Lin-Shiau SY, Ho CT, Lin JK. Theaflavin3,3V-digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-kappaB in macrophages. Eur J Pharmacol 1999;367:379 – 88. [9] Halliwell B. Free radicals and oxidants: a personal view. Nutr Rev 1994;52:253 – 65. [10] Victor VV, Guayerbas N, Puerto M, Medina S, De la Fuente M. Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock. Immunopharmacology 2000;46:89 – 101. [11] Maue´l J. Intracellular survival of Protozoan parasites with special reference to Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi. Adv Parasitol 1996;28:1 – 51. [12] Darley-Usmar V, Wiseman H, Halliwell B. Nitric oxide and oxygen radicals: a question of balance. FEBS Lett 1995;369: 131 – 5. [13] Chan MMY, Mattiacci JA, Hwang HS, Shah A, Fong D. Synergy between ethanol and grape polyphenols, quercetin, and resveratrol, in the inhibition of the inducible nitric oxide synthase pathway. Biochem Pharmacol 2000;60:1539 – 48. [14] Kiyoka HO, Makoto T, Yasuji O. Potent antioxidative activity of non-polyphenolic fraction of green tea (Camellia sinensis)—association with pheophytins a and b. J Sci Food Agric 2000;80:117 – 20. [15] Welborn MB, Christman JW, Shepherd VL. Regulation of tumor necrosis factor-alpha receptors on macrophages: differences between primary macrophages and transformed macrophage cell lines. Reg Immunol 1993;5:158 – 64. [16] Lavnikova N, Drapier JC, Laskin DL. A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity. J Leukocyte Biol 1993;54:322 – 8. [17] Hrabak A, Temesi A, Csuka I, Antoni F. Inverse relation in the de novo arginase synthesis and nitric oxide production in murine and rat peritoneal macrophages in long-term cultures in vitro. Comp Biochem Physiol, Part B 1992;103:839 – 45. [18] Hrabak A, Bajor T, Temesi A. Comparison of substrate and inhibitor specificity of arginase and nitric oxide (NO) synthase for arginine analogues and related compounds in murine and rat macrophages. Biochem Biophys Res Commun 1994;198: 206 – 212. [19] Strober W. Wright – Giemsa and nonspecific esterase staining of cell. In: Coligan JE, Kruissbeek AM, Margulies DH, Shevach EM, Strober W, editors. Current protocols in immunology, vol. 3. Wiley, New York; 1997. p. A.3.C.1 – 2. [20] Green LC, Wagner DA, Glogowki J, Skipper PL, Wishnok JS, et-al. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982;126:131 – 8. [21] Ryan TC, Weil GJ, Newburger PE, Haugland R., Simons ER.
E. A´lvarez et al. / International Immunopharmacology 2 (2002) 849–855
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils. J Immunol Methods 1990;130:223 – 33. Leiro J, Iglesias R, Parama´ A, Sanmartı´n ML, Ubeira FM. Respiratory burst responses of rat macrophages to microsporidian spores. Exp Parasitol 2001;98:1 – 9. Huguet AI, Ma´n˜ez S, Alcaraz MJ. Superoxide scavenging properties of flavonoids in a non-enzymatic system. Z Naturforsch 1990;45c:19 – 24. Nathan CF, Gabay J. Antimicrobial mechanisms of macrophages. In: van Furth R, editor. Mononuclear phagocytes— biology of monocytes and macrophages. Dordrecht: Kluwer Academic Publishers; 1992. p. 259 – 67. Jacquier-Sarlin MR, Fuller K, Dinh-Xuan AT, Richard MJ, Polla BS. Protective effects of hsp70 in inflammation. Experientia 1994;50:1031 – 8. Griot C, Vandevelde M, Richard A, Peterhans E, Stocker R. Selective degeneration of oligodendrocytes mediated by reactive oxygen species. Free Radical Res Commun 1990;11:181 – 93. Bauerova K, Bezek A. Role of reactive oxygen and nitrogen species in etiopathogenesis of rheumatoid arthritis. Gen Physiol Biophys 1999;18:15-20. Watanabe N, Miura S, Zeki S, Ishii H. Hepatocellular oxidative DNA injury induced by macrophage-derived nitric oxide. Free Radical Biol Med 2001;30:1019 – 28. Hassig A, Kremer H, Liang WX, Stampfli K. The role of the Th-1 to Th-2 shift of the cytokine profiles of CD4 helper cells in the pathogenesis of autoimmune and hypercatabolic diseases. Med Hypotheses 1998;51:59 – 63. Martı´nez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound on reactive oxygen species and prostaglandin production. Biochem Pharmacol 2000;59:865 – 70. Srivastava RC, Husain MM, Hasan SK, Athar M. Green tea polyphenols and tannic acid act as potent inhibitors of phorbol ester-induced nitric oxide generation in rat hepatocytes independent of their antioxidant properties. Cancer Lett 2000;153: 1 – 5. Aviram M. Interaction of oxidized low-density lipoprotein with macrophages in atherosclerosis, and the antiatherogenicity of antioxidants. Eur J Clin Chem Clin Biochem 1996;34: 599 – 608. Aviram M, Dornfeld L, Rosenblat M, Volkova N, Kaplan M, et al. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am J Clin Nutr 2000;71:1062 – 76.
855
[34] Paquay JB, Haenen GR, Stender G, Wiseman SA, Tijburg LB, et al. Protection against nitric oxide toxicity by tea. J Agric Food Chem 2000;48:5768 – 72. [35] Sakagami H, Oi T, Satoh K. Prevention of oral diseases by polyphenols (review). In Vivo 1999;13:155 – 71. [36] Cheong H, Ryu SY, Kim KM. Anti-allergic action of resveratrol and related hydroxystilbenes. Planta Med 1999;65:266 – 8. [37] Sandoval M, Ronzio RA, Muanza DN, Clark DA, Miller MJ. Peroxynitrite-induced apoptosis in epithelial (T84) and macrophage (RAW 264.7) cell lines: effect of legume-derived polyphenols (phytolens). Nitric Oxide 1997;1:476 – 83. [38] Yang F, de Villiers WJ, McClain CJ, Varilek GW. Green tea polyphenols block endotoxin-induced tumor necrosis factorproduction and lethality in a murine model. J Nutr 1998;128: 2334 – 40. [39] Dokka S, Shi X, Leonard S, Wang L, Castranova V, et al. Interleukin-10-mediated inhibition of free radical generation in macrophages. Am J Physiol: Lung Cell Mol Physiol 2001; 280:L1196 – 202. [40] Kaul A, Khanduja KL. Plant polyphenols inhibit benzoyl peroxide-induced superoxide anion radical production and diacylglyceride formation in murine peritoneal macrophages. Nutr Cancer Int J 1999;35:207 – 11. [41] Virgili F, Kobuchi H, Packer L. Procyanidins extracted from Pinus maritima (Pycnogenol): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages. Free Radical Biol Med 1998;24:1120 – 9. [42] Xiong Q, Tezuka Y, Kaneko T, Li H, Tran LQ, et al. Inhibition of nitric oxide by phenylethanoids in activated macrophages. Eur J Pharmacol 2000;400:137 – 44. [43] Van-Dyke K, McConnell P, Marquardt L. Green tea extract and its polyphenols markedly inhibit luminol-dependent chemiluminescence activated by peroxynitrite or SIN-1. Luminiscence 2000;15:37 – 43. [44] Luoma J, Stralin P, Marklund SL, Hiltunen TP, Sarkioja T, et al. Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions: colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins. Arterioscler, Thromb, Vasc Biol 1998;18:157 – 167. [45] Ferguson L. Role of plant polyphenols in genomic stability. Mutat Res 2001;475:89 – 111. [46] Padma M, Das UN. Effect of cis-unsaturated fatty acids on the activity of protein kinases and protein phosphorylation in macrophage tumor (AK-5) cells in vivo. Prostaglandins, Leukotrienes Essent Fatty Acids 1999;60:55 – 63.
Effect of ( )-epigallocatechin-3-gallate on respiratory burst of rat macrophages ´ lvarez a, J. Leiro b,*, F. Orallo a E. A a
Departamento de Farmacologı´a, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain b Departamento de Microbiologı´a y Parasitologı´a, Laboratorio de Parasitologı´a, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain Received 10 October 2001; received in revised form 15 March 2002; accepted 23 March 2002
Abstract The toxic effects derived from overproduction of oxygen radicals [reactive oxygen species (ROS)] by immune cells can be partially abolished by the antioxidant activities of plant polyphenols. In the present study, we investigated the antioxidant action of a catechin, ( )-epigallocatechin-3-gallate (EGCG), on the respiratory-burst responses of rat peritoneal macrophages. EGCG at concentrations of 50 – 200 AM blocked the production of nitric oxide by macrophages stimulated in vivo with sodium thioglycollate then 5 days later in vitro with lipopolysaccharide and gamma-interferon. At 1 – 100 AM, EGCG also inhibited the extracellular liberation of oxygen radicals by resident peritoneal macrophages stimulated with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). At low concentrations (1 – 5 AM), EGCG increased the reduction of nitro blue tetrazolium (NBT) by the superoxide anions generated in the non-enzymatic system NADH/PMS, acting as a pro-oxidant agent, while at concentrations above 10 AM, EGCG acts as a scavenger of superoxide anions. These results show that EGCG is capable of modulating ROS production during the respiratory burst of rat peritoneal macrophages by acting as a superoxide anion scavenger. EGCG may therefore be useful in the prevention and treatment of diseases due to increased free radical production. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rat; Respiratory burst; Peritoneal macrophage; EGCG; Nitric oxide; Superoxide anion
1. Introduction Polyphenols are major components of many traditional herbal remedies, and exhibit several beneficial pharmacological effects [1]. Tea (from Camellia sinensis) and wine (from Vitis vinifera) are two popular beverages that both contain large amounts of phytopolyphenols [2,3], including proanthocyanidines (condensed tannins) which have attracted particular interest *
Corresponding author. Tel.: +34-81-593316; fax: +34-81593316. E-mail address: [email protected] (J. Leiro).
among pharmacologists [4]. In grapes, proanthocyanidines are present at highest concentration in the seeds, and are largely constituted by dihydrolyzed compounds such as (+)-catechin, ( )-epicatechin and ( )-epicatechin-3-gallate (EGCG) [4]. In green tea, the major catechins are EGCG, ( )-epigallocatechin, ( )-epicatechin-3-gallate, ( )-epicatechin, (+)-gallocatechin and (+)-catechin, EGCG being present at the highest concentration [5]. EGCG, like the other catechins, has recently attracted great interest for its potential anticancer and anti-inflammatory activities [3,6,7]. Both activities have been attributed to the notable antioxidant capacity of EGCG [3,8]. Reactive oxygen spe-
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cies (ROS) are generated endogenously by all aerobic cells as products of a number of metabolic reactions [9,10]. Phagocytic cells, such as polymorphonuclear leukocytes and macrophages, generate large quantities of ROS in response to a variety of membrane stimulants, by a coordinated sequence of biochemical reactions known as the oxidative burst [11]. ROS are low-molecular-mass compounds that include reactive oxygen intermediates (ROI) such as superoxide (O2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH), and reactive nitrogen intermediates (RNI) such as nitric oxide (NO) and peroxynitrite (ONOO ) [12]. In addition to possessing microbicidal activity, these compounds have been shown to be important regulatory molecules in diverse physiological processes; furthermore, excessive production of ROS has been implicated in a number of pathological processes, such as the development of cardiovascular diseases, neurodegenerative disorders, and cancer [6]. Several studies have analyzed the effects of EGCG on different stages of RNI production by macrophages [3,13]. To date, however, there has been only a single study of effects on ROS production, specifically on superoxide anion generation induced in mouse macrophages by 12-0-tetradecanoylphorbol-13-acetate [14]. Most of these studies have used mouse macrophage cell lines, which may show alterations in phagocytic functions with respect to normal cells [15]. There have been no previous studies of the effects of EGCG on the respiratory-burst responses of rat macrophages, which constitute a model that is widely employed in biomedical investigation and which present a number of differences with respect to the mouse-macrophage model of the respiratory burst [16 –18]. In the present study, we investigated the in vitro effects of EGCG on the production of ROS by rat peritoneal macrophages. Our results demonstrate that EGCG significantly inhibits LPS-induced RNI production and PMAinduced ROI production in a concentration-dependent manner, probably by acting as a ROS scavenger.
study. They were housed (groups of five) in Macrolon cages (Panlab, Barcelona, Spain) on poplar shaving bedding (B&K Universal, G. Jordi, Barcelona, Spain) in a standard experimental animal bio-clean room, illuminated from 08:00 to 20:00 h (12 h light:12 h dark cycle) and maintained at a temperature of 22– 24 jC. The animals had free access to food pellets (B&K Universal, G. Jordi), and drinking fluid (tap water), and were allowed to acclimatize for 1 week before the experiments. All experiments were carried out in accordance with European regulations on the protection of animals (Directive 86/609), the Declaration of Helsinki and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). In this context, all experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Santiago de Compostela. 2.2. Stimuli, drugs and chemicals Stock solution (2 mg/ml) of phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO, USA) was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 80 jC until use. Stock solution of lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) was made up at 10 Ag/ml in phenol-red-free Dulbecco’s Eagle medium (DMEM; Sigma) and stored at 20 jC until use. Thioglycollate broth (Merck, Germany) was prepared to a concentration of 3% (w/v) in PBS, autoclaved at 121 jC for 10 min, and stored at room temperature until use. Stock solution of the arginine analogue N-monomethyl-L-arginine monoacetate (L-NMA, Calbiochem, USA) was made up at 100 mM in water, and stored until use at 20 jC in the dark. L-Glutamine, sulfanilamine and N-(1-naphthyl)ethylenediamine dihydrochloride were purchased from Sigma. 2.3. Isolation of rat peritoneal-exudate macrophages
2. Materials and methods 2.1. Animals Male Wistar rats (Iffa-Credo), purchased from Criffa (Barcelona, Spain), were used throughout this
For induction of inflammatory responses, rats were injected intraperitoneally with 1 ml of 3% thioglycollate broth, and peritoneal exudate was extracted 5 days later. Rat resident and inflammatory peritoneal macrophages were obtained from rats euthanized by cervical dislocation in a laminar flow chamber to
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ensure sterile conditions. The abdomen of the rat was soaked with 70% ethanol for disinfection, a midline incision was then made with scissors and the abdominal skin retracted. Thirty milliliters of Hanks’ balanced salt solution (HBSS) was then injected into the peritoneal cavity, using a syringe with a 19-G needle. After gentle abdominal massage, about 30 ml of fluid was extracted using the same syringe and transferred to 50-ml sterile polypropylene tubes on ice. A 20-Al aliquot was then extracted for cell counting in a hemocytometer. The remaining cells were washed once by centrifugation at 400g for 10 min at 4 jC and resuspended to a concentration of 106 cells ml 1. The number of viable cells was estimated by the trypan blue exclusion test: trypan blue (0.4% in PBS) was added to wells and incubated for 3 min at room temperature, after which the number of unstained (viable) and stained (non-viable) cells were counted. Aliquots (100 Al) of the cell suspension were added to the wells of 96-well microculture plates (Corning, USA) or placed on microscope slides, and left for 90 min in a humidified incubator at 37 jC, 5% CO2, to allow adhesion. Non-adherent cells were then removed by gently washing with HBSS. More than 97% of the cells plated on microscope showed nonspecific esterase activity, determined as per Ref. [19], indicating that they were macrophages.
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an ELISA reader (Titertek Multiscan, Flow Laboratories, Finland). Nitrite concentration was calculated with reference to a standard curve obtained using NaNO2 (1– 256 AM in culture medium). 2.5. Assay of ROI production The production of ROIs during the respiratoryburst response of macrophages was investigated using OxyBURSTR Green probes (Molecular Probes, The Netherlands) that emit fluorescence when they are oxidized by ROS produced during the respiratory burst [21] as previously described [22]. Briefly, for quantification of extracellular release of ROIs, we used the OxyBURST Green H2HFF BSA reagent [bovine serum albumin (BSA) coupled to dihydro2V,4,4,6,7,7V-hexafluorofluorescein (HsHFF)]. A 1 mg/ ml stock solution of the reagent was made up in HBSS containing 2 mM sodium azide and stored at 4 jC in the dark. For the assay, macrophages were incubated with the reagent at 10 Ag/ml in the wells of 96-well flat-bottom microtiter plates for 2 min at 37 jC, then stimulated by addition of 10 Ag/ml of PMA. Extracellular release of ROIs is expressed as variation in fluorescence per unit time [arbitrary fluorescence units (AFU) s 1]. 2.6. Generation of O2 radicals
2.4. Assay of nitrite production One hundred-microliter aliquots of macrophages that had been prestimulated in vivo with thioglycollate broth 5 days previously were incubated in microtitre plates (Corning) at 37 jC with 5% CO2 for 90 min. After adhesion, the wells were gently washed with HBSS, then incubated for 48 h with 100 Al of phenolred-free DMEM containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (Serva, USA), 10 U/ml of recombinant murine gamma interferon (IFNg, Genzyme, USA) and 100 ng/ml of LPS. In some experiments, the arginine analogue L-NMA (1 mM), was included in the medium. Nitrite production in the culture supernatants was assayed by the Griess reaction [20]: 100 Al of culture supernatant was added to 100 Al of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine hydrochloride in 2.5% H3PO4), then incubated for 10 min at room temperature, and absorbance was measured at 530 nm using
O2 radicals were generated in a non-enzymatic system (phenazine methosulphate-NADH) and quantified by the spectrophotometric measurement of the product of the reduction of nitro blue tetrazolium (NBT, Sigma), essentially following the procedure described by Ref. [23] with minor modifications. We used 300-Al test solutions made up in phosphate buffer (50 mM KH2PO4 –KOH, pH 7.4) and containing h-nicotinamide adenine dinucleotide (NADH, Sigma), 166 AM, NBT (Sigma), 43 AM, and SOD (10 U/ml) or EGCG at various concentrations (1, 5, 10 or 20 AM). Control experiments were carried out simultaneously without EGCG. In addition, the possible capacity of the tested compounds to directly reduce NBT was determined by adding the corresponding tested drugs to solutions containing only NBT in a phosphate buffer. Reaction was started with test solutions already in a Cobas Fara 22-3123 (Roche) autoanalyzer, by add-
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ing phenazine methosulphate (PMS, 2.7 AM; freshly diluted in 100 ml of the above phosphate buffer) and continued at 25 jC for 10 min, a period in which absorbance increased linearly from the third minute. The rate of NBT reduction was calculated from the differential absorbance at 560 nm with respect to a blank solution in which phenazine methosulphate was replaced by buffer solution, and was expressed as increment of absorbance per minute. 2.7. Statistical analysis Means were compared by unpaired two-tailed Student’s t-tests when variances were equal (Fisher’s F-test, P > 0.05), or by Welch’s t-tests when variances were different. P values less than 0.05 were considered significant.
3. Results 3.1. Effects of EGCG on ROS production by rat peritoneal macrophages We first investigated the effects of EGCG on PMAstimulated in vitro ROI production by resident peri-
Fig. 1. Effect of EGCG (1 – 100 AM) on extracellular release of reactive oxygen species (ROI), as quantified by the OxyBURST Green H2HFF BSA, by rat resident peritoneal macrophages stimulated with PMA (1 Ag/ml). Values shown are means (n=5) Fstandard errors of fluorescence emission by cells (arbitrary units per second). Asterisks indicate statistical significance ( P<0.01) with respect to the control values (macrophages incubated in the absence of EGCG).
Fig. 2. Effect of EGCG (50 – 200 AM) on nitrite concentration, assayed by the Griess reaction, in cultures of rat peritoneal macrophages stimulated in vivo with thioglycollate then 5 days later in vitro with IFNg (10 U/ml) and LPS (100 ng/ml). The rightmost bar represents assays in which the arginine analogue Nmonomethyl-L-arginine monoacetate (L-NMA; 1 mM) was added to cultures instead of EGCG; this compound abolishes nitrite generation in the Griess reaction. Values shown are means (n=5), Fstandard errors. Asterisks indicate statistical significance ( P<0.01) with respect to the control values (macrophages incubated in the absence of EGCG).
toneal-exudate macrophages. As shown in Fig. 1, EGCG at concentrations of 1– 100 AM significantly and dose-dependently inhibited extracellular ROI production, as quantified fluorometrically using the OxyBURST Green H2HFF BSA reagent. To study the effects of EGCG on RNI production, we prestimulated the macrophages in vivo by intraperitoneal injection of thioglycollate 5 days before extraction of macrophages for the in vitro assay. After stimulation with LPS and IFNg in the presence of different concentrations of EGCG, we then determined accumulated nitrite in the medium, as estimated by the Griess reaction (Fig. 2). EGCG at concentrations of 50– 200 AM significantly and dose-dependently inhibited RNI production. The inhibition obtained with EGCG at the maximum concentration tested (200 AM) was similar to that obtained with 1 mM L-NMA. To rule out the possibility that the observed inhibitions of ROS production were due to effects of the EGCG on macrophage viability, EGCG was added to the culture medium at 200 AM (the maximum concentration used in the assays). Mean macrophage viabilityFstandard error (determined on the basis of
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Fig. 3. Effect of EGCG (1 – 20 AM) on reduction of NBT by superoxide anion generated in the phenazine methosulfate (PMS)/ reduced beta-nicotinamide adenine dinucleotide (NADH) system. The rightmost bar represents assays in which the enzyme superoxide dismutase (SOD; 10 U/ml) was added to cultures instead of EGCG; this enzyme abolishes superoxide generation in the PMS/NADH system. Values shown are mean percentages of the control response (n=5)Fstandard errors. Asterisks indicate significant difference (*P<0.01; **P<0.05) with respect to control.
trypan blue exclusion: see Materials and Methods) was 94F4% after 2 h of incubation with EGCG, versus 97F3% (n=5) in untreated controls. 3.2. O2 scavenging by EGCG We investigated the ability of EGCG to scavenge O2 generated by a non-enzymatic system (PMS/ NADH). EGCG at 1 – 5 AM appeared to enhance O2 production, whereas EGCG at 10 AM or higher had a significant inhibitory effect. EGCG at 20 AM had inhibitory effects close to those obtained with SOD at 10 U/ml. (Fig. 3).
4. Discussion The relationship between macrophage cytotoxicity and enhanced production of reactive nitrogen and oxygen intermediates (RNIs and ROIs) is well established [24,25], as is the fact that interaction between RNIs and ROIs generates potentially cytotoxic agents which may have pathological effects [12,26 – 28]. Drugs that inhibit ROS generation may have beneficial effects in the treatment of diseases due to overproduc-
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tion of ROS [29]. Polyphenols are common components in plants, and several studies have demonstrated that they possess diverse pharmacological activities, including anti-inflammatory effects [1,5,8,30,31], prevention of atherosclerosis and cardiovascular diseases [7,13,32 – 34], prevention of oral diseases [35], anti-allergic activities [36], prevention of autoimmune disorders [37] and anticancer properties [3,6]. The results of the present study demonstrate that the polyphenol EGCG dose-dependently inhibits the production of both ROIs and RNIs by rat peritonealexudate macrophages. In previous studies, EGCG (5 and 10 AM) was found to inhibit inducible NOS (iNOS) protein transcription in activated mouse macrophages, by reducing iNOS mRNA expression [5], while the gallic acid moiety of EGCG was found to be essential for its potent anti-inflammatory activity [8]. EGCG also blocks LPS induction of nitric oxide synthase in mouse macrophages, by down-regulating the LPSinduced activity of the transcription factor NF-KB [8]. More specifically, in the mouse macrophage cell line RAW 264.7 and in elicited mouse peritoneal macrophages, EGCG regulates TNFa gene expression by blocking NK-KB activation through inhibition of InBkinase (IKK) activity [3,38]. In addition, it has recently been demonstrated that ROS scavenges, like IL-10 inhibits NF-kappaB activation [39]. Several polyphenols display ROS scavenger activity [40]. For example, pycnogenol (a polyphenolcontaining extract of the bark of Pinus maritima) scavenges ROIs and RNIs, and has effects on NO metabolism in RAW 264.7 macrophages [41]. Phenylethanoids likewise show NO radical-scavenging activity, which possibly contributes to their anti-inflammatory effects [42], while phytolens, polyphenolic compounds from legumes, show significant ROSscavenging ability [37]. In the present study, using a non-enzymatic system (PMS/NADH) with NBT detection, we found that EGCG shows dose-dependent superoxide-scavenging activity. Several previous studies have shown that EGCG protects against NO toxicity by scavenging peroxynitrite (ONOO ), one of the key RNIs in cytotoxic processes [34,43]. In addition, the scavenging of superoxide by EGCG may reduce peroxynitrite levels, since macrophages stimulated with LPS and TNF need superoxide for peroxynitrite production [12]. It has also been shown that the relationship between NO and superoxide levels plays
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important roles in some pathological processes such as atherogenesis [44]. At low concentrations (1– 5 AM), EGCG seems to act as a pro-oxidant, increasing the reduction of NBT in the PMS/NADH system. Several other polyphenols with pro-oxidant effects [45] show anti-cancer activity, associated with an increase in ROS generation by tumor cells, which may ultimately lead to cell lysis [46]. In conclusion, EGCG shows important superoxidescavenging activity, leading to reduced net production of ROS in rat peritoneal macrophage cultures stimulated with PMA (assays of ROI production) or LPS and IFNg (assays of RNI production). These results suggest that EGCG may be useful in the treatment of immune disorders involving ROS production by phagocytic cells, and that EGCG may be able to protect cells from ROS-mediated oxidative modification.
Acknowledgements This work was supported in part by grants from Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT), Spain (SAF2000-0137) and Consellerı´a de Educacio´n e Ordenacio´n Universitaria, Xunta de Galicia, Spain (PGIDT00PXI20314PR and PGIDT99MAR20301).
References [1] Chen YC, Yang LL, Lee TJF. Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX 2 gene expression via suppression of nuclear factor-kappa B activation. Biochem Pharmacol 2000;59:1445 – 57. [2] Carando S, Teissedre PL, Cabanis JC. Estimation de l’apport en catechines et procyanidines relatif a une consommation moderee et reguliere de vins en France. Bulletin-de-l’OIV (France) 1999;72:72 – 86. [3] Pan MH, Liang YC, Lin-Shiau SY, Zhu NQ, Ho CT, et al. Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells. J Agric Food Chem 2000;48:6337 – 46. [4] Souquet JM, Cheynier V, Moutounet M. The proanthocynidins of grape [skin, seed, stem]. Bulletin-de-l’OIV (France) 2000; 73:601 – 9. [5] Lin YL, Lin JK. ( )-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappaB. Mol Pharmacol 1997;52:465 – 72.
[6] Lin JK, Liang YC, Lin-Shiau SY. Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade. Biochem Pharmacol 1999;58:911 – 5. [7] Hayek T, Fuhrman B, Vaya J, Rosenblat M, Belinky P, et al. Reduced progression of atherosclerosis in apolipoprotein Edeficient mice following consumption of red wine, or its polyphenols quercetin or catechin, is associated with reduced susceptibility of LDL to oxidation and aggregation. Arterioscler, Thromb, Vasc Biol 1997;17:2744 – 52. [8] Lin YL, Tsai SH, Lin-Shiau SY, Ho CT, Lin JK. Theaflavin3,3V-digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-kappaB in macrophages. Eur J Pharmacol 1999;367:379 – 88. [9] Halliwell B. Free radicals and oxidants: a personal view. Nutr Rev 1994;52:253 – 65. [10] Victor VV, Guayerbas N, Puerto M, Medina S, De la Fuente M. Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock. Immunopharmacology 2000;46:89 – 101. [11] Maue´l J. Intracellular survival of Protozoan parasites with special reference to Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi. Adv Parasitol 1996;28:1 – 51. [12] Darley-Usmar V, Wiseman H, Halliwell B. Nitric oxide and oxygen radicals: a question of balance. FEBS Lett 1995;369: 131 – 5. [13] Chan MMY, Mattiacci JA, Hwang HS, Shah A, Fong D. Synergy between ethanol and grape polyphenols, quercetin, and resveratrol, in the inhibition of the inducible nitric oxide synthase pathway. Biochem Pharmacol 2000;60:1539 – 48. [14] Kiyoka HO, Makoto T, Yasuji O. Potent antioxidative activity of non-polyphenolic fraction of green tea (Camellia sinensis)—association with pheophytins a and b. J Sci Food Agric 2000;80:117 – 20. [15] Welborn MB, Christman JW, Shepherd VL. Regulation of tumor necrosis factor-alpha receptors on macrophages: differences between primary macrophages and transformed macrophage cell lines. Reg Immunol 1993;5:158 – 64. [16] Lavnikova N, Drapier JC, Laskin DL. A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity. J Leukocyte Biol 1993;54:322 – 8. [17] Hrabak A, Temesi A, Csuka I, Antoni F. Inverse relation in the de novo arginase synthesis and nitric oxide production in murine and rat peritoneal macrophages in long-term cultures in vitro. Comp Biochem Physiol, Part B 1992;103:839 – 45. [18] Hrabak A, Bajor T, Temesi A. Comparison of substrate and inhibitor specificity of arginase and nitric oxide (NO) synthase for arginine analogues and related compounds in murine and rat macrophages. Biochem Biophys Res Commun 1994;198: 206 – 212. [19] Strober W. Wright – Giemsa and nonspecific esterase staining of cell. In: Coligan JE, Kruissbeek AM, Margulies DH, Shevach EM, Strober W, editors. Current protocols in immunology, vol. 3. Wiley, New York; 1997. p. A.3.C.1 – 2. [20] Green LC, Wagner DA, Glogowki J, Skipper PL, Wishnok JS, et-al. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982;126:131 – 8. [21] Ryan TC, Weil GJ, Newburger PE, Haugland R., Simons ER.
E. A´lvarez et al. / International Immunopharmacology 2 (2002) 849–855
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils. J Immunol Methods 1990;130:223 – 33. Leiro J, Iglesias R, Parama´ A, Sanmartı´n ML, Ubeira FM. Respiratory burst responses of rat macrophages to microsporidian spores. Exp Parasitol 2001;98:1 – 9. Huguet AI, Ma´n˜ez S, Alcaraz MJ. Superoxide scavenging properties of flavonoids in a non-enzymatic system. Z Naturforsch 1990;45c:19 – 24. Nathan CF, Gabay J. Antimicrobial mechanisms of macrophages. In: van Furth R, editor. Mononuclear phagocytes— biology of monocytes and macrophages. Dordrecht: Kluwer Academic Publishers; 1992. p. 259 – 67. Jacquier-Sarlin MR, Fuller K, Dinh-Xuan AT, Richard MJ, Polla BS. Protective effects of hsp70 in inflammation. Experientia 1994;50:1031 – 8. Griot C, Vandevelde M, Richard A, Peterhans E, Stocker R. Selective degeneration of oligodendrocytes mediated by reactive oxygen species. Free Radical Res Commun 1990;11:181 – 93. Bauerova K, Bezek A. Role of reactive oxygen and nitrogen species in etiopathogenesis of rheumatoid arthritis. Gen Physiol Biophys 1999;18:15-20. Watanabe N, Miura S, Zeki S, Ishii H. Hepatocellular oxidative DNA injury induced by macrophage-derived nitric oxide. Free Radical Biol Med 2001;30:1019 – 28. Hassig A, Kremer H, Liang WX, Stampfli K. The role of the Th-1 to Th-2 shift of the cytokine profiles of CD4 helper cells in the pathogenesis of autoimmune and hypercatabolic diseases. Med Hypotheses 1998;51:59 – 63. Martı´nez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound on reactive oxygen species and prostaglandin production. Biochem Pharmacol 2000;59:865 – 70. Srivastava RC, Husain MM, Hasan SK, Athar M. Green tea polyphenols and tannic acid act as potent inhibitors of phorbol ester-induced nitric oxide generation in rat hepatocytes independent of their antioxidant properties. Cancer Lett 2000;153: 1 – 5. Aviram M. Interaction of oxidized low-density lipoprotein with macrophages in atherosclerosis, and the antiatherogenicity of antioxidants. Eur J Clin Chem Clin Biochem 1996;34: 599 – 608. Aviram M, Dornfeld L, Rosenblat M, Volkova N, Kaplan M, et al. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am J Clin Nutr 2000;71:1062 – 76.
855
[34] Paquay JB, Haenen GR, Stender G, Wiseman SA, Tijburg LB, et al. Protection against nitric oxide toxicity by tea. J Agric Food Chem 2000;48:5768 – 72. [35] Sakagami H, Oi T, Satoh K. Prevention of oral diseases by polyphenols (review). In Vivo 1999;13:155 – 71. [36] Cheong H, Ryu SY, Kim KM. Anti-allergic action of resveratrol and related hydroxystilbenes. Planta Med 1999;65:266 – 8. [37] Sandoval M, Ronzio RA, Muanza DN, Clark DA, Miller MJ. Peroxynitrite-induced apoptosis in epithelial (T84) and macrophage (RAW 264.7) cell lines: effect of legume-derived polyphenols (phytolens). Nitric Oxide 1997;1:476 – 83. [38] Yang F, de Villiers WJ, McClain CJ, Varilek GW. Green tea polyphenols block endotoxin-induced tumor necrosis factorproduction and lethality in a murine model. J Nutr 1998;128: 2334 – 40. [39] Dokka S, Shi X, Leonard S, Wang L, Castranova V, et al. Interleukin-10-mediated inhibition of free radical generation in macrophages. Am J Physiol: Lung Cell Mol Physiol 2001; 280:L1196 – 202. [40] Kaul A, Khanduja KL. Plant polyphenols inhibit benzoyl peroxide-induced superoxide anion radical production and diacylglyceride formation in murine peritoneal macrophages. Nutr Cancer Int J 1999;35:207 – 11. [41] Virgili F, Kobuchi H, Packer L. Procyanidins extracted from Pinus maritima (Pycnogenol): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages. Free Radical Biol Med 1998;24:1120 – 9. [42] Xiong Q, Tezuka Y, Kaneko T, Li H, Tran LQ, et al. Inhibition of nitric oxide by phenylethanoids in activated macrophages. Eur J Pharmacol 2000;400:137 – 44. [43] Van-Dyke K, McConnell P, Marquardt L. Green tea extract and its polyphenols markedly inhibit luminol-dependent chemiluminescence activated by peroxynitrite or SIN-1. Luminiscence 2000;15:37 – 43. [44] Luoma J, Stralin P, Marklund SL, Hiltunen TP, Sarkioja T, et al. Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions: colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins. Arterioscler, Thromb, Vasc Biol 1998;18:157 – 167. [45] Ferguson L. Role of plant polyphenols in genomic stability. Mutat Res 2001;475:89 – 111. [46] Padma M, Das UN. Effect of cis-unsaturated fatty acids on the activity of protein kinases and protein phosphorylation in macrophage tumor (AK-5) cells in vivo. Prostaglandins, Leukotrienes Essent Fatty Acids 1999;60:55 – 63.