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Ergothioneine inhibits oxidative stress- and TNF-α -induced NF-κ B activation and interleukin-8 release in alveolar epithelial cells
BBRC Biochemical and Biophysical Research Communications 302 (2003) 860–864 www.elsevier.com/locate/ybbrc
Ergothioneine inhibits oxidative stress- and TNF-a-induced NF-jB activation and interleukin-8 release in alveolar epithelial cells Irfan Rahman,a,* Peter S. Gilmour,a Luis A. Jimenez,a Saibal K. Biswas,a Frank Antonicelli,b and Okezie I. Aruomac,* a
c
ELEGI and Colt Research Laboratory, Respiratory Medicine Unit, Wilkie Building, University of Edinburgh, Medical School, Teviot Place, Edinburgh EH8 9AG, UK b Laboratory of Biochimie, FRE-CNRS 2534, UFR Sciences, 51100 Reims, France Department of Neuroinflammation, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College London, Charing Cross Hospital Campus, Fulham Palace Road, London W6 8RF, UK Received 6 February 2003
Abstract Oxidants and inflammatory mediators such as tumour necrosis factor-a (TNF-a) activate transcription factors such as NF-jB. Interleukin-8 (IL-8) is a ubiquitous inflammatory chemokine that mediates a multitude of inflammatory events in the lung. Ergothioneine is a naturally occurring thiol compound, which possesses antioxidant property. The aim of this study was to determine whether ergothioneine can inhibit the hydrogen peroxide (H2 O2 )- and TNF-a-mediated activation of NF-jB and the release of IL-8 in human alveolar epithelial cells (A549). Treatment of A549 cells with H2 O2 (100 lM) and TNF-a (10 ng/ml) significantly increased NF-jB activation using a reporter assay. Ergothioneine inhibited both H2 O2 - and TNF-a-mediated activation of NF-jB. Both H2 O2 and TNF-a significantly increased IL-8 release, which was inhibited by pre-treatment of A549 cells with ergothioneine compared to the control untreated cells. Ergothioneine also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyltransferase (CAT) reporter system, transfected into A549 cells. This indicates a molecular mechanism for the antiinflammatory effects of ergothioneine. Ó 2003 Published by Elsevier Science (USA). Keywords: Oxidant; IL-8; Ergothioneine; Glutathione; NF-jB; Dietary antioxidants; A549 cells
Airway inflammation is a characteristic of many lung disorders including asthma, chronic obstructive pulmonary disease, adult respiratory distress syndrome, and idiopathic pulmonary fibrosis. All these diseases involve the recruitment of immune and inflammatory cells to the lungs. These cells are activated and produce mediators of inflammation including oxidants and cytokines, such as the pro-inflammatory cytokine tumour necrosis factor-a (TNF-a) [1,2]. TNF-a is a pleiotropic protein that mediates a multitude of inflammatory events in the lung [1]. The induction of inflammatory mediators can be regulated by * Corresponding authors. Faxes: +44-131-651-1558 (I. Rahman), +44-20-8846-7025 (O.I. Aruoma). E-mail addresses: [email protected] (I. Rahman), o.aruoma @imperial.ac.uk (O.I. Aruoma).
the activation of redox-sensitive transcription factors Activator Protein-1 (AP-1), (c-Fos/c-Jun), and nuclear factor-jB (NF-jB) stimulated in response to oxidants and TNF-a [3]. Reactive oxygen species (ROS) and cellular redox status, particularly intracellular thiol status, can be directly involved in the activation of NFjB [4]. TNF-a activates NF-jB via the classical Ij-B kinase pathway [3]. Interleukin-8 (IL-8) is a major chemotactic and activating mediator for polymorphonuclear leukocytes (PMN) in the lungs. Thus modulation of its production may provide a therapeutic target in inflammatory lung diseases. NF-jB has been shown to be involved in the transcriptional activation of IL-8 [5]. Ergothioneine (2-mercaptohistidine trimethylbetaine) (Fig. 1) is a naturally occurring antioxidant found in most plants and animals, with human blood values estimated to be in the range 1–4 mg/100 ml blood (ap-
0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00224-9
I. Rahman et al. / Biochemical and Biophysical Research Communications 302 (2003) 860–864
Fig. 1. Structure of ergothioneine. Chemically, L -ergothioneine corresponds to the betaine of 2-thio-L -histidine. In aqueous solution, the tautomeric 2-thio-imidazole exists predominantly in the thione form.
proximately 46–183 lM) [6,7]. The compound is known to be formed via hercynine from histidine, methionine, and cysteine in microorganisms [7]. Ergothioneine inhibits the peroxynitrite-dependent nitration of nitrotyrosine [8] and also inhibits oxidative DNA and cell death in N-18-RE-105 cells [9]. Ergothioneine also inhibits the formation of xanthine and hypoxanthine (a precursor of xanthine) and this may have many implications for inflammatory conditions such as gout, a condition characterised by overproduction of uric acid (the oxidation product of xanthine). Further, the indication that ergothioneine permitted increased cellular tolerance of N-acetyl-L -cysteine may be beneficial to pulmonary macrophages. It is therefore important to begin to understand the role of ergothioneine in the oxidative stress/ signal transduction mechanisms involved in cellular responses. Thus ergothioneine may regulate the TNF-a and H2 O2 -mediated activation of NF-jB and AP-1 and the release of the pro-inflammatory mediator IL-8. We studied the effects of ergothioneine on TNF-a and H2 O2 -induced activation of NF-jB, GSH levels, and IL-8 release in alveolar epithelial cells (A549). The mechanism by which ergothioneine modulates the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyltransferase (CAT) reporter system, transfected into A549 cells, was also studied.
Materials and methods Unless otherwise stated, all of the biochemical reagents used in this study were purchased from Sigma Chemical (Poole, UK); and the gel shift assay kit was from Promega (Southampton, UK). Ergothioneine was provided by OXIS Health Products, Portland, OR, USA. A549 alveolar epithelial cells. The human type II alveolar epithelial cell line, A549 (ECACC No. 86012804), was maintained in continuous culture at 37 °C, 5% CO2 in DulbeccoÕs modified minimum essential medium (DMEM) containing L -glutamine (2 mM), penicillin (100 U/ ml), streptomycin (100 lg/ml), and 10% foetal bovine serum (FBS). Treatments. Monolayers of A549 and NF-jB transfected A549 cells grown to approximately 80–90% confluency in 6-well plates containing 10% FBS were washed in CMF-PBS and exposed to the treatments in 2% serum-containing media. All treatments were performed in duplicate. The cells were treated with H2 O2 (100 lM) or TNF-a (10 ng/ml) alone or with the addition of ergothioneine (5 mM). The cells were incubated in 2 ml of 2% serum containing medium at 37 °C, 5% CO2
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for 20 h. Following treatments, the cells were washed with cold sterile calcium and magnesium free PBS (Ca2þ =Mg2þ free PBS), lysed, and used for NF-jB transactivation assay. The culture media were used for IL-8 assay. Cell viability was determined by trypan blue exclusion. Enzyme-linked immunosorbent assay for IL-8. An enzyme-linked immunosorbent assay (ELISA) was used to measure IL-8 [10]. All plates were read on a microplate reader (Dynatech MR 5000, Aldermaston, UK) and analysed using a computer-assisted analysis program (Assay ZAP, Blosoft, Cambridge, UK). Typically, standard curves generated with this ELISA were linear in the 50–2500-pg IL-8/ ml range. Only assays having standard curves with a calculated regression line value >0.95 were used for further analysis. Protein assay. Protein levels were measured using the bicinchoninic acid (BCA) kit (Pierce, Chester, UK). Protein standards were obtained by diluting a stock solution of bovine serum albumin (BSA). Linear regression was used to determine the actual protein concentration of the samples. GSH assays. GSH levels were measured by the 5,50 -dithiobis-(2nitro-benzoic acid) DTNB-GSSG reductase recycling method described by Tietze [11] with slight modifications [12]. NF-jB transactivation assay. A549 cells stably transfected with bgalactosidase and a previously described luciferase gene (a gift from Professor Ron Hay, University of St. Andrews, UK) under the control of three synthetic copies of the jB consensus of the immunoglobulin j chain promoter were grown in 6-well plates, serum-starved for 24 h, and exposed to various treatments as described above. Culture media was aspirated off and the cells were washed twice with ice-cold PBS, lysed in 400 ll lysis buffer (25 mM Tris-phosphate buffer, pH 7.8, using phosphoric acid; 8 mM MgC2 , 1 mM DTT, 1% Triton X-100, and 15% glycerol), and centrifuged for 5 min at 13,000 rpm. Fifty microlitres of lysate was added to a cuvette and placed in a Lumac Celcis M2500 (Celsis, Oxford, UK). The luciferase activity was measured by luminescence following the injection of 50 ll luciferin buffer (1 mM ATP, 0.25 mM luciferin, and 1% BSA, all diluted in lysis buffer). Values were expressed as relative percentage of luciferase units per milligram of protein (RLU/mg protein). Transient transfection and chloramphenicol acetyltransferase (CAT) assay. The IL-8 promoter construction was a gift from Professor R. Strieter (Ann Arbour University, Michigan, USA). Briefly, the IL-8 promoter CAT vector is a wild-type IL-8 promoter, which has been PCR amplified to generate a Pst1 restriction site at the 50 end and an Xba site at the 30 end. The Pst–Xba fragment was cloned into PromegaÕs pCAT Basic vector. A549 cells (0:2 106 cells per well) were seeded into 24-well tissue culture plates and cultured at 37 °C to 80% confluency. Using 0.5 lg plasmid DNA transfections were performed using the Lipofectamine reagent (Life Technologies). Following treatment with H2 O2 (100 lM) and TNF-a and the thiol antioxidant compound ergothioneine at 5 mM, cell extracts were prepared and assayed for protein content using the BCA reagent (Pierce, Chester, UK). CAT activity was quantified by a CAT enzyme-linked immunosorbent assay (Boehringer–Mannheim, East Sussex, UK). Statistical analysis. The data are expressed as means SEM. Differences between values were compared by ANOVA and a two-way unpaired t test.
Results Effect of TNF-a, H2 O2 , and ergothioneine on IL-8 levels in A549 epithelial cells Increased IL-8 release was observed in A549 cells following 20 h of exposure to TNF-a and H2 O2 compared to untreated cells (p < 0:001) (Fig. 2). Pre-treatment of A549 cells for 2 h with ergothioneine inhibited both TNF-a-
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Fig. 3. The effect of TNF-a (10 ng/ml), H2 O2 (100 lM), and ergothioneine (5 mM) alone or in combinations on pNF-jB luciferase activity in A549 cells. *p < 0:05 and ***p < 0:001, compared to control values. Fig. 2. Effect of TNF-a (10 ng/ml), H2 O2 (100 lM), and ergothioneine for 20 h alone or in combinations on IL-8 release in A549 epithelial cells. Cells were pretreated with ergothioneine for 2 h before the cotreatment of TNF-a or H2 O2 . *p < 0:05 and ***p < 0:001, compared to control values.
and H2 O2 -induced IL-8 release in A549 cells (p < 0:001). Cell viability remained >90% after all of the above treatments as assessed by the trypan blue exclusion test. Effects of ergothioneine on TNF-a- and H2 O2 -mediated decrease in glutathione levels in alveolar epithelial cells TNF-a (10 ng/ml) and H2 O2 (100 lM) significantly decreased GSH levels (nmol/mg protein) after 4 h incubation in A549 cells compared with the control value (TNF-a 110 22, H2 O2 94 18 versus controls 158 11, n ¼ 3, p < 0:01). Co-treatment of A549 cells for 4 h with ergothioneine considerably protected the TNF-a- and H2 O2 -mediated fall in GSH levels (ergothioneine + TNF-a 140 19, ergothioneine + H2 O2 145 31 versus ergothioneine 165 23 or controls 158 11, n ¼ 3).
confer any significant CAT activity to A549 cells under control conditions or in response to H2 O2 . Fig. 4 shows the CAT activity expressed per unit protein. Under basal conditions, measurable amounts of CAT activity were detected in the cell lysate of transfected A549 cells. When transfected A549 cells containing the IL-8 promoter were stimulated by H2 O2 and TNF-a (70% and 116%, respectively) increase in CAT activity over control cells was observed, respectively. Co-treatment of H2 O2 or TNF-a-stimulated cells with ergothioneine abolished this effect significantly (Fig. 4).
Effect of TNF-a, H2 O2 , and ergothioneine on NF-jB transactivation in A549 epithelial cells H2 O2 and TNF-a treatments increased NF-jB transactivation (156% and 320%), compared to the control values (100%) in A549 cells. Pre-treatment of A549 cells with ergothioneine inhibited H2 O2 - and TNFa-induced NF-jB transactivation in A549 cell transfected with the NF-jB-dependent promoter (Fig. 3). Effects of ergothioneine on H2 O2 - and TNF-a-induced IL8 reporter chloramphenicol acetyltransferase activity The transcriptional mechanism by which ergothioneine exerted its effects on H2 O2 - and TNF-a-induced IL-8 release was assessed using an IL-8 promoter construct-chloramphenicol acetyltransferase (CAT) reporter assay system. The promoterless plasmid did not
Fig. 4. Effects of ergothioneine on H2 O2 and TNF-a-induced transcriptional activity of the 50 -flanking region of the IL-8 gene in A549 alveolar epithelial cells. Cells were treated with H2 O2 , TNF-a and ergothioneine. (A) Schematic map of the IL-8 promoter gene. The blocks represent nucleotide positions of the cis-acting DNA binding elements of the promoter region cloned in the pCAT-Basic vector from Promega. The numbers indicate the nucleotide positions from the transcriptional start site of the IL-8 gene, which is indicated by the bent arrow. (B) Transcriptional activity was assessed as the amount of CAT histograms represent the mean and the bars the SEM of three transfection experiments. **p < 0:01, ***p < 0:001 compared with control.
I. Rahman et al. / Biochemical and Biophysical Research Communications 302 (2003) 860–864
Discussion Lung cells, in particular alveolar epithelial type II cells, are susceptible to stimuli such as oxidants and cytokines released into the local airspace environment. GSH is a tripeptide (L -c-glutamyl-L -cysteinyl-glycine) containing a thiol (sulfhydryl) group. The GSH status is critical for various biological events that include transcriptional activation of specific genes and modulation of redox-regulated signal transduction [13]. TNF-a and H2 O2 increased the NF-jB activation which was associated with a decrease in GSH levels in A549 cells. Cotreatment of A549 cells with ergothioneine and TNF-a or H2 O2 inhibited NF-jB activation and protected the cells against fall in GSH levels, suggesting that ergothioneine inhibits NF-jB activation by a mechanism depending on its thiol-mediated antioxidant property. It is well known that ROS and cellular redox status, particularly intracellular thiol status, can be directly involved in the activation of NF-jB [4,5]. TNF-a or H2 O2 increase NF-jB transactivation via the MAP kinase (p38) signalling pathway and/or via the Ij-B kinase pathway [3,14,15]. The inhibitory effect of ergothioneine on H2 O2 - and TNF-a-mediated activation of NF-jB suggest that ergothioneine may be acting to inhibit these signal transduction pathways. The induction of inflammatory mediators can be regulated by the activation of redox-sensitive transcription factors, such as NF-jB stimulated in response to oxidants and TNF-a [3]. IL-8 is a chemokine released during inflammation and is important in the recruitment and activation of immune and inflammatory cells. Moreover, oxidative stress has been shown to mediate IL-8 synthesis [16]. IL-8 induction is associated with the activation of the nuclear transcription factors, such as NF-jB, AP-1, and NF-IL6, in response to diverse stimuli in various cell types [14,17,18]. However, emerging evidence suggests that NF-jB [19,20], but not AP-1 [21,23] or NF-IL6 [22], is the main transcription factor for the expression of IL-8 in response to oxidative stress and/or TNF-a in A549 epithelial cells. In this study, we show that H2 O2 and TNF-a induced an increase in IL-8 release with a corresponding increase in the activation of NF-jB in A549 cells. Previous studies have shown that the interleukin-8 promoter is transcriptionally activated by IL-1 or TNF-a [23–25] and that the activation of the IL-8 promoter is predominantly through the transcription factor NF-jB [19,23,24,26]. However, the involvement of other transcription factors, such as AP-1 and NF-IL6 along with NF-jB in ergothioneine-mediated inhibition of IL-8 expression, cannot be ruled out. Nevertheless, ergothioneine inhibits NF-jB activation and IL-8 release associated with protection against oxidant-mediated decrease in GSH levels corroborates reports showing the ability of other thiol compounds such N-acetyl-L -cysteine, a known
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antioxidant [27], to inhibit IL-1-induced activation of NF-jB and IL-8 release [21,28]. This strongly suggests that therapeutic thiol antioxidants may have the potential to attenuate lung inflammation. The induction of IL-8 protein release and mRNA synthesis by H2 O2 and TNF-a occur as a result of increased gene transcription in A549 cells and for other cell types [16]. Therefore, we investigated the activation of the IL-8 promoter by measuring the gene activity in a CAT reporter assay following transient transfection into A549 epithelial cells. We showed that H2 O2 and TNF-a up-regulated the promoter region of the IL-8 gene, which was inhibited by co-treatment with ergothioneine in A549 cells. This suggests that ergothioneine inhibits IL-8 release at the transcriptional level in A549 cells. This has an implication in inflammatory lung disease states where IL-8 is increased [2,3]. In these cases, ROS and TNF-a would lead to an augmented inflammatory response from the tissue. In conclusion, this study suggests that ergothioneine inhibits the H2 O2 and TNF-a-mediated NF-jB activation leading to decreased IL-8 release from alveolar epithelial cells. Thus, ergothioneine may be a potential antioxidant/anti-inflammatory therapy to inhibit the chronic inflammatory response, which occurs in the development of chronic inflammatory lung diseases.
References [1] F.M. Brenman, R.N. Maini, M. Feldmann, Cytokine expression in chronic inflammatory disease, Br. Med. Bull. 51 (1995) 368– 384. [2] I. Rahman, W. MacNee, Oxidative stress and regulation of glutathione synthesis in lung inflammation, Eur. Respir. J. 16 (2000) 534–554. [3] I. Rahman, W. MacNee, Role of transcription factors in inflammatory lung diseases, Thorax 55 (1998) 601–612. [4] I. Rahman, Oxidative stress transcription factors and chromatin remodeling in lung inflammation, Biochem. Pharmacol. 60 (2000) 1041–1049. [5] I. Rahman, P. Gilmour, L.A. Jimenez, W. MacNee, Oxidative stress and TNF-a induce histone acetylation and AP-1/NF-jB in alveolar epithelial cells: potential mechanism in inflammatory gene transcription, Mol. Cell Biochem. 234/235 (2002) 239–248. [6] J.A. Moncaster, D.T. Walsh, S.M. Gentleman, L.-S. Jen, O.I. Aruoma, Ergothioneine treatment protects neurons against Nmethyl-D -aspartate excitotoxicity in an in vivo rat retinal model, Neurosci. Lett. 328 (2002) 55–59. [7] D.B. Melville, Ergothioneine, Vitamin Horm. XVII (1959) 155– 204. [8] O.I. Aruoma, M.E. Whiteman, B. Halliwell, Antioxidant action of ergothioneine: assessment of its ability to scavenge peroxynitrite, Biochem. Biophys. Res. Commun. 231 (1997) 389–391. [9] O.I. Aruoma, J.P.E. Spencer, N. Mahmood, Protection against oxidative damage and cell death by the natural antioxidant ergothioneine, Food Chem. Toxicol. 37 (1999) 1043–1053. [10] P.S. Gilmour, I. Rahman, S. Hayashi, J.C. Hogg, K. Donaldson, W. MacNee, Adenoviral E1A primes alveolar epithelial cells to PM10 -induced transcription of interleukin-8, Am. J. Physiol: Lung Cell Mol. Physiol. 281 (2001) L598–L606.
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[11] F. Tietze, Enzymic method for quantitative determination of nanogram amounts of total and oxidised glutathione: applications to mammalian blood and other tissue, Anal. Biochem. 27 (1969) 502–522. [12] I. Rahman, X.Y. Li, K. Donaldson, D.J. Harrison, W. MacNee, Glutathione homeostasis in alveolar epithelial cells in vitro and lung in vivo under oxidative stress, Am. J. Physiol. (Lung Cell. Mol. Physiol.) 269 (1995) L285–L292. [13] I. Rahman, W. MacNee, Regulation of redox glutathione levels and gene transcription in lung inflammation: therapeutic approaches, Free Radical Biol. Med. 28 (2000) 1405–1420. [14] V. Lakshiminarayanan, D.W. Beno, R.H. Costa, K.A. Roebuck, Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2 O2 and tumor necrosis factor-a in endothelial and epithelial cells, J. Biol. Chem. 272 (1997) 32910–32918. [15] I. Rahman, F. Antonicelli, W. MacNee, Molecular mechanism of the regulation of gluthatione synthesis by tumor necrosis factor-a and dexamethasone in human alveolar epithelial cells, J. Biol. Chem. 274 (1999) 5088–5096. [16] L.E. Deforge, A. Preston, E. Takeuchi, J. Kenney, L.A. Boxer, D.G. Remick, Regulation of interleukin-8 gene expression by oxidant stress, J. Biol. Chem. 268 (1993) 25568–25576. [17] R.M. Strieter, Interleukin-8: a very important chemokine of the human epithelium, Am. J. Physiol. Lung Cell Mol. Physiol. 283 (2002) L688–L689. [18] K.A. Roebuck, Oxidant stress regulation of IL-8 and ICAMgene expression: differential activation and binding of the transcription factors AP-1 and NF-jB, Int. J. Mol. Med. 4 (1999) 223–230. [19] X.Y. Pei, Y. Nakanishi, H. Inoue, K. Takayama, F. Bai, H. Kara, Polycyclic aromatic hydrocarbons induce IL-8 expression through nuclear factor jB activation in A549 cell line, Cytokine 19 (2002) 236–241. [20] G.Y. Park, S. Le, K.H. Park, C.T. Le, Y.W. Kim, S.K. Han, Y.S. Shim, C.G. Yoo, Anti-inflammatory effect of adenovirus-mediated IjBa overexpression in respiratory epithelial cells, Eur. Respir. J. 18 (2001) 801–809.
[21] F. Antonicelli, M. Parmentier, E.M. Drost, N. Hirani, I. Rahman, K. Donaldson, W. MacNee, N Acystelyn inhibits oxidantmediated interleukin-8 expression and NF-jB nuclear binding in alveolar epithelial cells, Free Radical Biol. Med. 32 (2002) 492– 502. [22] L.A. Jimenez, E.M. Drost, P.S. Gilmour, I. Rahman, F. Antonicelli, H. Ritchie, W. MacNee, K. Donaldson, PM(10)-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-a, Am. J. Physiol. Lung Cell Mol. Physiol. 282 (2002) L237–L248. [23] T. Matsusaka, K. Fujikawa, Y. Nishio, N. Mukaida, K. Matsushima, T. Kishimoto, S. Akira, Transcription factors NF-IL6 and NF-jB synergistically activate transcription of the inflammatory cytokines, interleukin-6 and interleukin-8, Proc. Natl. Acad. Sci. USA 90 (1993) 10193–10197. [24] C. Kunsch, R.K. Lang, C.A. Rosen, M.F. Shannon, Synergistic transcriptional activation of the IL-8 gene by NF-jB p65 (RelA) and NF-IL-6, J. Immunol. 153 (1994) 153–164. [25] A.R. Brasier, M. Jamaluddin, A. Casola, W. Duan, Q. Shen, R.P. Garofalo, A promoter recruitment mechanism for tumour necrosis factor-a-induced interleukin-8 transcription in type II pulmonary epithelial cells. Dependence on nuclear abundance of Rel, NF-jB1, and c-Rel transcription factors. J. Biol. Chem. 273 (1998) 3551–3561. [26] J.G. Mastronarde, M.M. Monick, N. Mukaida, K. Matsushima, G.W. Hunninghake, Activator protein-1 is the preferred transcription factor for cooperative interaction with nuclear factor-jB in respiratory syncytial virus-induced interleukin-8 gene expression in airway epithelium, J. Infect. Dis. 5 (1998) 1275–1281. [27] O.I. Aruoma, B. Halliwell, B.M. Hoey, J. Butler, The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid, Free Rad. Biol. Med. 6 (1989) 593–597. [28] K. Matsumoto, S. Hashimoto, Y. Gon, T. Nakayama, H. Takizawa, T. Horie, N-Acetylcysteine inhibits IL-1a-induced IL8 secretion by bronchial epithelial cells, Respir. Med. 92 (1998) 512–515.
Ergothioneine inhibits oxidative stress- and TNF-a-induced NF-jB activation and interleukin-8 release in alveolar epithelial cells Irfan Rahman,a,* Peter S. Gilmour,a Luis A. Jimenez,a Saibal K. Biswas,a Frank Antonicelli,b and Okezie I. Aruomac,* a
c
ELEGI and Colt Research Laboratory, Respiratory Medicine Unit, Wilkie Building, University of Edinburgh, Medical School, Teviot Place, Edinburgh EH8 9AG, UK b Laboratory of Biochimie, FRE-CNRS 2534, UFR Sciences, 51100 Reims, France Department of Neuroinflammation, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College London, Charing Cross Hospital Campus, Fulham Palace Road, London W6 8RF, UK Received 6 February 2003
Abstract Oxidants and inflammatory mediators such as tumour necrosis factor-a (TNF-a) activate transcription factors such as NF-jB. Interleukin-8 (IL-8) is a ubiquitous inflammatory chemokine that mediates a multitude of inflammatory events in the lung. Ergothioneine is a naturally occurring thiol compound, which possesses antioxidant property. The aim of this study was to determine whether ergothioneine can inhibit the hydrogen peroxide (H2 O2 )- and TNF-a-mediated activation of NF-jB and the release of IL-8 in human alveolar epithelial cells (A549). Treatment of A549 cells with H2 O2 (100 lM) and TNF-a (10 ng/ml) significantly increased NF-jB activation using a reporter assay. Ergothioneine inhibited both H2 O2 - and TNF-a-mediated activation of NF-jB. Both H2 O2 and TNF-a significantly increased IL-8 release, which was inhibited by pre-treatment of A549 cells with ergothioneine compared to the control untreated cells. Ergothioneine also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyltransferase (CAT) reporter system, transfected into A549 cells. This indicates a molecular mechanism for the antiinflammatory effects of ergothioneine. Ó 2003 Published by Elsevier Science (USA). Keywords: Oxidant; IL-8; Ergothioneine; Glutathione; NF-jB; Dietary antioxidants; A549 cells
Airway inflammation is a characteristic of many lung disorders including asthma, chronic obstructive pulmonary disease, adult respiratory distress syndrome, and idiopathic pulmonary fibrosis. All these diseases involve the recruitment of immune and inflammatory cells to the lungs. These cells are activated and produce mediators of inflammation including oxidants and cytokines, such as the pro-inflammatory cytokine tumour necrosis factor-a (TNF-a) [1,2]. TNF-a is a pleiotropic protein that mediates a multitude of inflammatory events in the lung [1]. The induction of inflammatory mediators can be regulated by * Corresponding authors. Faxes: +44-131-651-1558 (I. Rahman), +44-20-8846-7025 (O.I. Aruoma). E-mail addresses: [email protected] (I. Rahman), o.aruoma @imperial.ac.uk (O.I. Aruoma).
the activation of redox-sensitive transcription factors Activator Protein-1 (AP-1), (c-Fos/c-Jun), and nuclear factor-jB (NF-jB) stimulated in response to oxidants and TNF-a [3]. Reactive oxygen species (ROS) and cellular redox status, particularly intracellular thiol status, can be directly involved in the activation of NFjB [4]. TNF-a activates NF-jB via the classical Ij-B kinase pathway [3]. Interleukin-8 (IL-8) is a major chemotactic and activating mediator for polymorphonuclear leukocytes (PMN) in the lungs. Thus modulation of its production may provide a therapeutic target in inflammatory lung diseases. NF-jB has been shown to be involved in the transcriptional activation of IL-8 [5]. Ergothioneine (2-mercaptohistidine trimethylbetaine) (Fig. 1) is a naturally occurring antioxidant found in most plants and animals, with human blood values estimated to be in the range 1–4 mg/100 ml blood (ap-
0006-291X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0006-291X(03)00224-9
I. Rahman et al. / Biochemical and Biophysical Research Communications 302 (2003) 860–864
Fig. 1. Structure of ergothioneine. Chemically, L -ergothioneine corresponds to the betaine of 2-thio-L -histidine. In aqueous solution, the tautomeric 2-thio-imidazole exists predominantly in the thione form.
proximately 46–183 lM) [6,7]. The compound is known to be formed via hercynine from histidine, methionine, and cysteine in microorganisms [7]. Ergothioneine inhibits the peroxynitrite-dependent nitration of nitrotyrosine [8] and also inhibits oxidative DNA and cell death in N-18-RE-105 cells [9]. Ergothioneine also inhibits the formation of xanthine and hypoxanthine (a precursor of xanthine) and this may have many implications for inflammatory conditions such as gout, a condition characterised by overproduction of uric acid (the oxidation product of xanthine). Further, the indication that ergothioneine permitted increased cellular tolerance of N-acetyl-L -cysteine may be beneficial to pulmonary macrophages. It is therefore important to begin to understand the role of ergothioneine in the oxidative stress/ signal transduction mechanisms involved in cellular responses. Thus ergothioneine may regulate the TNF-a and H2 O2 -mediated activation of NF-jB and AP-1 and the release of the pro-inflammatory mediator IL-8. We studied the effects of ergothioneine on TNF-a and H2 O2 -induced activation of NF-jB, GSH levels, and IL-8 release in alveolar epithelial cells (A549). The mechanism by which ergothioneine modulates the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyltransferase (CAT) reporter system, transfected into A549 cells, was also studied.
Materials and methods Unless otherwise stated, all of the biochemical reagents used in this study were purchased from Sigma Chemical (Poole, UK); and the gel shift assay kit was from Promega (Southampton, UK). Ergothioneine was provided by OXIS Health Products, Portland, OR, USA. A549 alveolar epithelial cells. The human type II alveolar epithelial cell line, A549 (ECACC No. 86012804), was maintained in continuous culture at 37 °C, 5% CO2 in DulbeccoÕs modified minimum essential medium (DMEM) containing L -glutamine (2 mM), penicillin (100 U/ ml), streptomycin (100 lg/ml), and 10% foetal bovine serum (FBS). Treatments. Monolayers of A549 and NF-jB transfected A549 cells grown to approximately 80–90% confluency in 6-well plates containing 10% FBS were washed in CMF-PBS and exposed to the treatments in 2% serum-containing media. All treatments were performed in duplicate. The cells were treated with H2 O2 (100 lM) or TNF-a (10 ng/ml) alone or with the addition of ergothioneine (5 mM). The cells were incubated in 2 ml of 2% serum containing medium at 37 °C, 5% CO2
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for 20 h. Following treatments, the cells were washed with cold sterile calcium and magnesium free PBS (Ca2þ =Mg2þ free PBS), lysed, and used for NF-jB transactivation assay. The culture media were used for IL-8 assay. Cell viability was determined by trypan blue exclusion. Enzyme-linked immunosorbent assay for IL-8. An enzyme-linked immunosorbent assay (ELISA) was used to measure IL-8 [10]. All plates were read on a microplate reader (Dynatech MR 5000, Aldermaston, UK) and analysed using a computer-assisted analysis program (Assay ZAP, Blosoft, Cambridge, UK). Typically, standard curves generated with this ELISA were linear in the 50–2500-pg IL-8/ ml range. Only assays having standard curves with a calculated regression line value >0.95 were used for further analysis. Protein assay. Protein levels were measured using the bicinchoninic acid (BCA) kit (Pierce, Chester, UK). Protein standards were obtained by diluting a stock solution of bovine serum albumin (BSA). Linear regression was used to determine the actual protein concentration of the samples. GSH assays. GSH levels were measured by the 5,50 -dithiobis-(2nitro-benzoic acid) DTNB-GSSG reductase recycling method described by Tietze [11] with slight modifications [12]. NF-jB transactivation assay. A549 cells stably transfected with bgalactosidase and a previously described luciferase gene (a gift from Professor Ron Hay, University of St. Andrews, UK) under the control of three synthetic copies of the jB consensus of the immunoglobulin j chain promoter were grown in 6-well plates, serum-starved for 24 h, and exposed to various treatments as described above. Culture media was aspirated off and the cells were washed twice with ice-cold PBS, lysed in 400 ll lysis buffer (25 mM Tris-phosphate buffer, pH 7.8, using phosphoric acid; 8 mM MgC2 , 1 mM DTT, 1% Triton X-100, and 15% glycerol), and centrifuged for 5 min at 13,000 rpm. Fifty microlitres of lysate was added to a cuvette and placed in a Lumac Celcis M2500 (Celsis, Oxford, UK). The luciferase activity was measured by luminescence following the injection of 50 ll luciferin buffer (1 mM ATP, 0.25 mM luciferin, and 1% BSA, all diluted in lysis buffer). Values were expressed as relative percentage of luciferase units per milligram of protein (RLU/mg protein). Transient transfection and chloramphenicol acetyltransferase (CAT) assay. The IL-8 promoter construction was a gift from Professor R. Strieter (Ann Arbour University, Michigan, USA). Briefly, the IL-8 promoter CAT vector is a wild-type IL-8 promoter, which has been PCR amplified to generate a Pst1 restriction site at the 50 end and an Xba site at the 30 end. The Pst–Xba fragment was cloned into PromegaÕs pCAT Basic vector. A549 cells (0:2 106 cells per well) were seeded into 24-well tissue culture plates and cultured at 37 °C to 80% confluency. Using 0.5 lg plasmid DNA transfections were performed using the Lipofectamine reagent (Life Technologies). Following treatment with H2 O2 (100 lM) and TNF-a and the thiol antioxidant compound ergothioneine at 5 mM, cell extracts were prepared and assayed for protein content using the BCA reagent (Pierce, Chester, UK). CAT activity was quantified by a CAT enzyme-linked immunosorbent assay (Boehringer–Mannheim, East Sussex, UK). Statistical analysis. The data are expressed as means SEM. Differences between values were compared by ANOVA and a two-way unpaired t test.
Results Effect of TNF-a, H2 O2 , and ergothioneine on IL-8 levels in A549 epithelial cells Increased IL-8 release was observed in A549 cells following 20 h of exposure to TNF-a and H2 O2 compared to untreated cells (p < 0:001) (Fig. 2). Pre-treatment of A549 cells for 2 h with ergothioneine inhibited both TNF-a-
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Fig. 3. The effect of TNF-a (10 ng/ml), H2 O2 (100 lM), and ergothioneine (5 mM) alone or in combinations on pNF-jB luciferase activity in A549 cells. *p < 0:05 and ***p < 0:001, compared to control values. Fig. 2. Effect of TNF-a (10 ng/ml), H2 O2 (100 lM), and ergothioneine for 20 h alone or in combinations on IL-8 release in A549 epithelial cells. Cells were pretreated with ergothioneine for 2 h before the cotreatment of TNF-a or H2 O2 . *p < 0:05 and ***p < 0:001, compared to control values.
and H2 O2 -induced IL-8 release in A549 cells (p < 0:001). Cell viability remained >90% after all of the above treatments as assessed by the trypan blue exclusion test. Effects of ergothioneine on TNF-a- and H2 O2 -mediated decrease in glutathione levels in alveolar epithelial cells TNF-a (10 ng/ml) and H2 O2 (100 lM) significantly decreased GSH levels (nmol/mg protein) after 4 h incubation in A549 cells compared with the control value (TNF-a 110 22, H2 O2 94 18 versus controls 158 11, n ¼ 3, p < 0:01). Co-treatment of A549 cells for 4 h with ergothioneine considerably protected the TNF-a- and H2 O2 -mediated fall in GSH levels (ergothioneine + TNF-a 140 19, ergothioneine + H2 O2 145 31 versus ergothioneine 165 23 or controls 158 11, n ¼ 3).
confer any significant CAT activity to A549 cells under control conditions or in response to H2 O2 . Fig. 4 shows the CAT activity expressed per unit protein. Under basal conditions, measurable amounts of CAT activity were detected in the cell lysate of transfected A549 cells. When transfected A549 cells containing the IL-8 promoter were stimulated by H2 O2 and TNF-a (70% and 116%, respectively) increase in CAT activity over control cells was observed, respectively. Co-treatment of H2 O2 or TNF-a-stimulated cells with ergothioneine abolished this effect significantly (Fig. 4).
Effect of TNF-a, H2 O2 , and ergothioneine on NF-jB transactivation in A549 epithelial cells H2 O2 and TNF-a treatments increased NF-jB transactivation (156% and 320%), compared to the control values (100%) in A549 cells. Pre-treatment of A549 cells with ergothioneine inhibited H2 O2 - and TNFa-induced NF-jB transactivation in A549 cell transfected with the NF-jB-dependent promoter (Fig. 3). Effects of ergothioneine on H2 O2 - and TNF-a-induced IL8 reporter chloramphenicol acetyltransferase activity The transcriptional mechanism by which ergothioneine exerted its effects on H2 O2 - and TNF-a-induced IL-8 release was assessed using an IL-8 promoter construct-chloramphenicol acetyltransferase (CAT) reporter assay system. The promoterless plasmid did not
Fig. 4. Effects of ergothioneine on H2 O2 and TNF-a-induced transcriptional activity of the 50 -flanking region of the IL-8 gene in A549 alveolar epithelial cells. Cells were treated with H2 O2 , TNF-a and ergothioneine. (A) Schematic map of the IL-8 promoter gene. The blocks represent nucleotide positions of the cis-acting DNA binding elements of the promoter region cloned in the pCAT-Basic vector from Promega. The numbers indicate the nucleotide positions from the transcriptional start site of the IL-8 gene, which is indicated by the bent arrow. (B) Transcriptional activity was assessed as the amount of CAT histograms represent the mean and the bars the SEM of three transfection experiments. **p < 0:01, ***p < 0:001 compared with control.
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Discussion Lung cells, in particular alveolar epithelial type II cells, are susceptible to stimuli such as oxidants and cytokines released into the local airspace environment. GSH is a tripeptide (L -c-glutamyl-L -cysteinyl-glycine) containing a thiol (sulfhydryl) group. The GSH status is critical for various biological events that include transcriptional activation of specific genes and modulation of redox-regulated signal transduction [13]. TNF-a and H2 O2 increased the NF-jB activation which was associated with a decrease in GSH levels in A549 cells. Cotreatment of A549 cells with ergothioneine and TNF-a or H2 O2 inhibited NF-jB activation and protected the cells against fall in GSH levels, suggesting that ergothioneine inhibits NF-jB activation by a mechanism depending on its thiol-mediated antioxidant property. It is well known that ROS and cellular redox status, particularly intracellular thiol status, can be directly involved in the activation of NF-jB [4,5]. TNF-a or H2 O2 increase NF-jB transactivation via the MAP kinase (p38) signalling pathway and/or via the Ij-B kinase pathway [3,14,15]. The inhibitory effect of ergothioneine on H2 O2 - and TNF-a-mediated activation of NF-jB suggest that ergothioneine may be acting to inhibit these signal transduction pathways. The induction of inflammatory mediators can be regulated by the activation of redox-sensitive transcription factors, such as NF-jB stimulated in response to oxidants and TNF-a [3]. IL-8 is a chemokine released during inflammation and is important in the recruitment and activation of immune and inflammatory cells. Moreover, oxidative stress has been shown to mediate IL-8 synthesis [16]. IL-8 induction is associated with the activation of the nuclear transcription factors, such as NF-jB, AP-1, and NF-IL6, in response to diverse stimuli in various cell types [14,17,18]. However, emerging evidence suggests that NF-jB [19,20], but not AP-1 [21,23] or NF-IL6 [22], is the main transcription factor for the expression of IL-8 in response to oxidative stress and/or TNF-a in A549 epithelial cells. In this study, we show that H2 O2 and TNF-a induced an increase in IL-8 release with a corresponding increase in the activation of NF-jB in A549 cells. Previous studies have shown that the interleukin-8 promoter is transcriptionally activated by IL-1 or TNF-a [23–25] and that the activation of the IL-8 promoter is predominantly through the transcription factor NF-jB [19,23,24,26]. However, the involvement of other transcription factors, such as AP-1 and NF-IL6 along with NF-jB in ergothioneine-mediated inhibition of IL-8 expression, cannot be ruled out. Nevertheless, ergothioneine inhibits NF-jB activation and IL-8 release associated with protection against oxidant-mediated decrease in GSH levels corroborates reports showing the ability of other thiol compounds such N-acetyl-L -cysteine, a known
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antioxidant [27], to inhibit IL-1-induced activation of NF-jB and IL-8 release [21,28]. This strongly suggests that therapeutic thiol antioxidants may have the potential to attenuate lung inflammation. The induction of IL-8 protein release and mRNA synthesis by H2 O2 and TNF-a occur as a result of increased gene transcription in A549 cells and for other cell types [16]. Therefore, we investigated the activation of the IL-8 promoter by measuring the gene activity in a CAT reporter assay following transient transfection into A549 epithelial cells. We showed that H2 O2 and TNF-a up-regulated the promoter region of the IL-8 gene, which was inhibited by co-treatment with ergothioneine in A549 cells. This suggests that ergothioneine inhibits IL-8 release at the transcriptional level in A549 cells. This has an implication in inflammatory lung disease states where IL-8 is increased [2,3]. In these cases, ROS and TNF-a would lead to an augmented inflammatory response from the tissue. In conclusion, this study suggests that ergothioneine inhibits the H2 O2 and TNF-a-mediated NF-jB activation leading to decreased IL-8 release from alveolar epithelial cells. Thus, ergothioneine may be a potential antioxidant/anti-inflammatory therapy to inhibit the chronic inflammatory response, which occurs in the development of chronic inflammatory lung diseases.
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