Review l -Glutamyl-l-cysteinyl-glycine (glutathione;...

Molecular Immunology 42 (2005) 987–1014

Review

l-�-Glutamyl-l-cysteinyl-glycine (glutathione; GSH) and GSH-relatedenzymes in the regulation of pro- and anti-inflammatory cytokines: a

signaling transcriptional scenario for redox(y) immunologic sensor(s)?

John J. Haddada,b,∗, Hisham L. Harbb,c

a Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanonb Departments of Biology and Biomedical Sciences, Faculty of Arts and Sciences, Lebanese International University, Beirut, Lebanon

c Department of Pharmacology, Faculty of Pharmacy, Lebanese International University, Beirut, Lebanon

Received 1 September 2004; accepted 29 September 2004Available online 23 November 2004

Abstract

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Of the antioxidant/prooxidant mechanisms mediating the regulation of inflammatory mediators, particularly cytokines, oxidativerelated pathways remain a cornerstone. It is conspicuous that there is a strong association between free radical accumulation (Roxidative stress) and the evolution of inflammation and inflammatory-related responses. The scenario that upholds a consensaforementioned is still evolving to unravel, from an immunologic perspective, the molecular mechanisms associated with ROS/RNS-deinflammation. Cytokines are keynote players when it comes to defining an intimate relationship among reduction–oxidation (redox)oxidative stress and inflammation. How close we are to identifying the molecular basis of this intricate association should be weighethe involvement of specific signaling molecules and, potentially, transcription factors.l-�-Glutamyl-l-cysteinyl-glycine, or glutathione (GSH),

Abbreviations: NAC, N-acetyl-l-cysteine; AIDS, acquired immune deficiency syndrome; RANTES, activation normal T cell expressed and secAP, activator/activating protein; ARDS, acute respiratory distress syndrome; ARE, antioxidant response element; ARNT, aryl-hydrocarbon receptor nucleartranslocator; bHLH-PAS, basic-helix–loop–helix-PAS; BCNU, 1,3-bis (2-chloroethyl)-1-nitrosourea; BSO, L-buthionine-(S,R)-sulfoximine; BHA, butylatedhydroxyanisole; CAPE, caffeic acid phenethyl ester; CD, cluster of designation/differentiation; ConA, concanavalin A; COX, cyclooxygenase; De, dex-amethasone; DEM, diethyl maleate; DMTU, dimethylthiourea; EC, endothelial cell; EGF, epidermal growth factor; EPO, erythropoietin; ERK, extrallularsignal-regulated kinase; GalN, galactosamine; GR, glucocorticoid receptor; GSH, L-�-glutamyl-l-cysteinyl-glycine;�-GCS,�-glutamyl-l-cysteinyl-ethylester;�-GCS,�-glutamylcysteine synthetase;�-GT, �-glutamyl transpeptidase; GPx, glutathione peroxidase; GSTp, glutathioneS-transferase Pi; GST, glu-tathioneS-agarose transferase; GAP, GTPase activating proteins; GEF, guanine nuclear exchange factor; HSP, heat shock protein; HBMEC, humicrovascular endothelial cells; HEK, human embryonic kidney; HIV, human immunodeficiency virus; H2O2, hydrogen peroxide;•OH, hydroxyl radical;HIFhypoxia-inducible factor; HRE, hypoxia response element; iNOS, inducible nitric oxide synthase; I�B, inhibitory-�B; IDDM, insulin-dependent diabetesmellitus; ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; I/R, ischemia/reoxygenation; JNK, Jun N-terminal kinase; JIP, JNK-interactingproteins; LAT, linker for activation of T cells; LPS, lipopolysaccharide; METH, methamphetamine; NMDA,N-methyl-d-aspartate; MAPK, mitogen-activatedprotein kinase; MKP, MAPK phosphatases; MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase; MLK, mixed lineage kinases; mAb, monocantibody; MCP, monocyte chemoattractant protein; MBP, myelin basic protein; NAL, nacystelyn; NK, natural killer; NGF, nerve growth factor; NOricoxide; SNAP,S-nitroso-N-acetylpenicillamine; GSNO,S-nitrosoglutathione; NF-�B, nuclear factor-�B; NF-IL, nuclear factor-IL; GSSG, oxidized glutathionedisulfide; PAK, p21-activated kinase; PMA, phorbol 12-myristate 13-acetate; PDGF, platelet-derived growth factor; PAEC, porcine aortic endothelial cell; PKA,Protein kinase A; PKC, protein kinase C; PP, protein phosphatase; PTK, protein tyrosine kinase; PDTC, pyrollidine dithiocarbamate; RNS, reactiveitrogenspecies; ROS, reactive oxygen species; RTK, receptor tyrosine kinases; redox, reduction–oxidation; Ref, redox factor; RCC, renal cell carcinoma;SRF, serumresponse factor; Stat, Signal transducer and activator of transcription; SIV, simian immune virus; SAPK, stress-activated protein kinases; O2

•−, superoxideradical anion; SOD, superoxide dismutase; TCR, T-cell receptor; TRX, thioredoxin; TGF, transforming growth factor; TNF, tumor necrosis factor; TNFR, TNFreceptor; UPJ, ureteropelvic junction; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; VCAM, vascular cell adhesion molecule; VHL, vonHippel-Lindau

∗ Corresponding author. In affiliation with Prof. Bared Safieh-Garabedian, Department of Biology, Faculty of Arts and Sciences, American UniversfBeirut, Beirut, Lebanon. Previous address: Severenghaus-Radiometer Research Laboratories, University of California, San Francisco, CA, USA.E-mail address:[email protected] (J.J. Haddad).

0161-5890/$ – see front matter © 2004 Published by Elsevier Ltd.doi:10.1016/j.molimm.2004.09.029

988 J.J. Haddad, H.L. Harb / Molecular Immunology 42 (2005) 987–1014

an antioxidant thiol, has shaped, and still is refining, the face of oxidative signaling in terms of regulating the milieu of inflammatory mediators,ostensibly via the modulation (expression/repression) of oxygen- and redox-responsive transcription factors, hence termed redox(y)-sensitivecofactors. When it comes to the arena of oxygen sensing, oxidative stress and inflammation, nuclear factor-�B (NF-�B) and hypoxia-inducible factor-1� (HIF-1�) are key players that determine antioxidant/prooxidant responses with oxidative challenge. It is the theme thereinto underlie current understanding of the molecular association hanging between oxidative stress and the evolution of inflammation, walkedthrough an elaborate discussion on the role of transcription factors and cofactors. Would that classify glutathione and other redox signalingcofactors as potential anti-inflammatory molecules emphatically remains of particular interest, especially in the light of identifying upstreamand downstream molecular pathways for conceiving therapeutic, alleviating strategy for oxidant-mediated, inflammatory-related diseaseconditions.© 2004 Published by Elsevier Ltd.

Keywords:Antioxidant; AP-1; Cytokine; Glutathione; HIF-1�; Inflammation; MAPK; NF-�B; Pro-oxidant; Redox

1. Introduction and background

The tripeptidel-�-glutamyl-l-cysteinyl-glycine, or glu-tathione (GSH), a ubiquitous thiol, plays a major role in main-taining intracellular reduction–oxidation (redox) balance andregulating signaling pathways augmented by oxidative stress(Meister, 1988; Haddad and Land, 2000a; Haddad et al.,2000a). The cysteinyl moiety of GSH provides the reactivethiol as a functional element responsible for the diverse prop-erties of glutathione, the participation of which in metabolismreflects its importance in physiologic inter- and intracellular

pathways by altering the dynamic equilibrium of glutathionehomeostasis (Haddad et al., 2000a). Exogenous/endogenousagents, which induce the formation of reactive oxygen andnitrogen species (ROS/RNS), for example, can affect redoxhomeostasis by up-regulating antioxidant enzymes, particu-larly glutathione peroxidase (GPx), and enzymes involved inglutathione recycling and biosynthesis (see below) (Douglas,1987; Goss et al., 1997; Li et al., 1997; Haddad and Land,2000a).

Furthermore, ROS/RNS signaling could be mediated bycytokines, peptide hormones and immunoregulators, the par-

-itas,

e

u--es,re-

functions.These mechanisms include: (i) an antioxidant potential

mediated by the peroxidase-coupled reaction; (ii) regulationof cellular sulfhydryl status and redox equilibrium; (iii) gov-erning pathways in neuro-immune–endocrine interactionsas a neurotransmitter and an immunopharmacological thiol;and (iv) regulation of the expression/activation of redox-sensitive transcription factors induced by stress-evoked re-sponses (Droge et al., 1994; Hayes and McLellan, 1999;Haddad et al., 2000a). The pivotal role of redox cycle inmaintaining the integrity of the biological system in the faceof oxidative stress is, therefore, of particular immunologic

ticipation of which in cellular pathways is modulated by redox status (Rovin et al., 1997; Pena et al., 1999; Yamashet al., 1999; Haddad et al., 2001). Conversely, cytokinewhich are mediators of oxidative stress (Nussler et al., 1992;Desmarquest et al., 1998; Yamashita et al., 1999), can al-ter redox equilibrium by affecting GSH/oxidized glutathiondisulfide (GSSG) shuttling and recycling (Chen et al.,1998).

The immunopharmacological potential attributed to gltathione (Thompson et al., 1985), therefore, stems from established observations. Interleukin-1 (IL-1)-induced responsfor instance, has been purported to occur via modulating

aant

i antr thanh ionss t pro-c

tuso enti ta y ofed anyd ht,1i f ap

ortst ked

dox equilibrium (Rovin et al., 1997). In addition, ROS sig-naling regulating the transcription of IL-4 (Jeannin et al.,1 isf sete entm

9a al.,1 the-s verala ,a( ago-n iox-i l.,2a ,wl in

nd physio-clinical relevance.The ‘biomarkers’ of oxidative stress, such as antioxid

nefficiency, redox disequilibrium, and derivation of oxidadicals, for instance, may arise from conditions otheryperoxia (oxidizing signals) per se. This involves condituch as hypoxia/reoxygenation and cytokine-dependenesses (Thom et al., 1997; Haddad and Land, 2000a).

In physiological conditions, the intracellular redox staf thiols is highly reductive. GSH, for example, is pres

n high concentrations in epithelial lining fluid (Cantin el., 1989) and has been reported to maintain the integritpithelia in vitro and in vivo (Li et al., 1997). In contrast, GSHepletion has been linked to the pathophysiology of misease conditions (Cantin et al., 1989; Bunnell and Pac993; Roum et al., 1993; Saugstad, 1997), thus highlighting

ts central role in maintaining the functional integrity ohysiologically active and competent system.

There is growing evidence, moreover, which supphe notion that oxidative conditions modulate redox-lin

995), IL-6, IL-8 (Gosset et al., 1999), and tumor necrosactor-� (TNF-�) (Neuschwander-Tetri et al., 1996; Gost al., 1999) was shown to be mediated by thiol-dependechanisms.Antioxidants (Reimund et al., 1998; Barrett et al., 199)

nd glutathione precursors (Jeannin et al., 1995; Pena et999) have been shown to down-regulate cytokine synis, activation and downstream processes. Among segents that were used for repletion and depletion of GSHN-cetyl-l-cysteine (NAC) andl-buthionine-(S,R)-sulfoximineBSO) are of particular importance as they exhibit antistic effects on a pro-inflammatory signal. NAC, an ant

dant and a GSH precursor (Bernard, 1991; Haddad et a000a), ameliorated cytokine production (Tsuji et al., 1999)nd ROS-mediated injury (Bernard, 1991). In contrast, BSOhich depletes GSH by irreversibly inhibiting�-glutamyl--cysteinyl-ethyl ester (�-GCS), the rate-limiting enzyme

J.J. Haddad, H.L. Harb / Molecular Immunology 42 (2005) 987–1014 989

the biosynthesis of glutathione (Griffith and Meister, 1979;Haddad and Land, 2000a; Haddad, 2002g), has the potentialto enhance cytokine secretion by up-regulating ROS (Gossetet al., 1999). It was reasoned that a differential manipulationof glutathione homeostasis may antagonistically affect a pro-inflammatory signal, thus bearing potential consequences forthe treatment of many diseases where cytokines are poten-tially recognized as major participants in their pathophysiol-ogy (Saugstad, 1997).

Based on the aforementioned, it is, therefore, the main-stream of this review to discuss current concepts pertainingto glutathione-mediated regulation of cytokines, with partic-ular emphasis on the role of specific transcription factors andcofactors (Haddad, 2002k).

2. Glutathione and the redox cycle as viewed throughthe dynamic GSH/GSSG equilibrium

2.1. Reduction–oxidation biophysics

‘Reduction–oxidation’ (redox) state is a term often widelyadopted in free radical research and oxidative stress signaling(Haddad et al., 2000a; Haddad, 2002d, 2002e, 2002g; Haddadand Fahlman, 2002b; Rahman and MacNee, 2002; Droge,2 rd ione( ana ,2 u eta e,o l.,2f

, isi

G

A ysi-c andt de-t

�

nt(t tion,Fr lts( at2 on-v

�

Similarly, the Nernst equation for the reduction potential oftwo GSH/GSSG couple is:

�E = −240−(

59.1

2

)log

([GSH]2

GSSG

)mV at 25◦C,

pH = 7.0

2.2. Glutathione and glutathione-related enzymes

Glutathione is a ubiquitous, non-essential sulfhydrylamino acid and an antioxidant thiol (Sen, 2000; Del Corsoet al., 2002; Haddad, 2002d, 2004a). It is a tripeptide, con-sisting of glutamic acid, glycine and cysteine, which playscrucial role in maintaining redox equilibrium and establish-ing the mechanisms of cellular defenses augmented by ox-idative stress (Haddad, 2000, 2002g, 2004b; Haddad andLand, 2000a; Rahman, 2002, 2003, 2004; van Meeteren etal., 2004).

Synthesized by the rate-limiting enzyme�-glutamyl-cysteine synthetase (�-GCS) (Fig. 1) (Haddad and Land,2000a,b; Lu, 2000; Rahman and MacNee, 2000b;Soltaninassab et al., 2000; Wild and Mulcahy, 2000; Haddad,2002g; Forman and Dickinson, 2003; Rojas et al., 2003; Starket al., 2003; Watanabe et al., 2004), GSH uniquely providesa na hed olica mM( al.,2

sig-n s ofp 03;H 003a ,2 i-fi ju-g pledr r,2 ndV

ogenp m-d strate(D h isr enseo d,2wi ne

ro-t eg-u r of

003; Poon et al., 2004; Tsukahara et al., 2004). The majoeterminant of redox status in mammalian cells is glutathl-�-glutamyl-l-cysteinyl-glycine) (Rahman, 1999; Rahmnd MacNee, 2000a; Haddad, 2001a, 2002c; Gomez et al.004; Ostergaard et al., 2004; Rebrin et al., 2004; Wl., 2004), a tripeptide thiol that couples with its disulfidxidized form (GSSG) (Barbieri et al., 2004; Jones et a004; Ozturk and Gumuslu, 2004; Yang et al., 2004a,b), thus

orming a redox buffer system.Redox equilibrium, defined by the ratio 2 GSH/GSSG

llustrated by the equation:

SSG+ 2H+ + 2e− → 2GSH

ccording to the theory of Walter H. Nernst, a German phist and chemist mainly known for the Nernst equationhe third law of thermodynamics, redox potential can beermined by the relation:

E = �Eo −(

RT

nF

)lnQ

The terminology defined:R is the gas constaR= 8.314 J/K.mol),T the temperature (in Kelvin{K}), nhe number of moles of electrons involved in the reac

the Faraday constant (F= 9.6485× 104 C/mol) andQ theatio of GSH/GSSG, thereby giving yield results in voSchafer and Buettner, 2001). Thus, the Nernst equation5◦C (298.15 K), taking into consideration 2.303 as a cersion factor for ln into log10, can be written as:

E = �Eo − (59.1 mV/n) log10Q.

functional cysteinyl moiety (Janaky et al., 1999; Dickinsond Forman, 2002), which is responsible for much of tiverse properties of glutathione. GSH is mainly cytost an approximate intracellular concentration of 1–10Sen, 1998; Griffith, 1999; Haddad, 2002g; Noctor et002; Santangelo, 2003).

Glutathione participation in metabolism reflects itsificance in intracellular functions, covering the domainhysiology (Rana et al., 2002; Dringen and Hirrlinger, 20ancock et al., 2003; Jean et al., 2003; Watson et al., 2)nd pathophysiology (Rahman et al., 1999;Awasthi et al.003; Ferrari et al., 2004). GSH is involved in the detoxcation of highly reactive peroxides (ROOH) by the conation of electrophiles and metals through the GPx-coueaction, thus acting as an antioxidant (Coles and Kadluba003; Fujii et al., 2003; Miyamoto et al., 2003; Zelck aon Janowsky, 2004).

Endogenously produced radicals, such as hydreroxide (H2O2), are effectively reduced by the seleniuependent GPx, in the presence of GSH as a subFig. 2A and B) (Lee et al., 2004a,b; Liddell et al., 2004).uring this reaction, GSH is converted to GSSG, whic

ecycled back to 2 GSH by GSSG reductase at the expf NADPH/H+ (Hayes and McLellan, 1999; Hadda002d,e,g,i; Haddad and Land, 2002a,b,c), thus forminghat is known as a redox cycle (seeFig. 1/Fig. 2B), which

s transcriptionally-orientated (Nioi et al., 2003; McMahot al., 2004).

GSH also participates in maintaining intracellular pein integrity by reducing their disulfide linkages and rlating their synthesis, thereby acting as a regulato


Fig. 1. The schematic of the redox cycle shows the relationship between antioxidant enzymes and glutathione. The first step in de novo synthesis of glutathione(GSH) is catalyzed by glutamate-cysteine ligase (GCL). GSH is synthesized from amino acids (l-glutamate andl-cysteine) by the action of�-glutamylcysteinesynthetase (�-GCS), the rate-limiting enzyme, and glutamyl synthase (GS), which ligatesl-�-glutamate-l-cysteine with the amino acid glycine to forml-�-glutamyl-l-cysteinyl-glycine (GSH). This reaction requires energy, is ATP-limited and is specifically inhibited at the level of�-GCS byl-buthionine-(S,R)-sulfoximine (BSO). GSH undergoes the glutathione-peroxidase (GSH-PX) coupled reaction, thereby detoxifying reactive oxygen species (ROS) suchas hydrogen peroxide (H2O2). A major source of H2O2 is the biochemical conversion of superoxide anion (O2

•−) by the action of superoxide dismutase(SOD). During this reaction, GSH is oxidized to generate GSSG, which is recycled back to GSH by the action of glutathione reductase (GSSG-RD) at theexpense of reduced nicotinamide (NADPH/H+), thus forming the redox cycle. The reduction of the glutathione pathway is blocked by the action of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). The major source of NADPH/H+ comes from the conversion of glucose, a reaction blocked by dehydroepiandrosterone(DHEA).

cellular sulfhydryl status (Lash and Jones, 1985; Csala etal., 2003; Dafre et al., 2003; Dalle-Donne et al., 2003;Gough et al., 2003; Lockwood, 2003; O’Malley et al.,2004). In addition, GSH governs signaling pathways inneuroimmune–endocrine interactions as a neurotransmitterand an immunopharmacological reducing thiol (Pereira etal., 1995; Haddad et al., 2001b, 2002a; Gartner et al., 2002;Pastore et al., 2003).

GSH can facilitate the membrane trafficking of reactivechemicals and, in some cases, augment the formation of es-sential biological mediators (Santangelo, 2002; Fernandez-Checa, 2003; Leonoudakis et al., 2004). In addition, GSHhas been shown to regulate the expression and activationof redox-sensitive/responsive transcription factors (Haddadand Land, 2000a,b; Haddad et al., 2000a, 2002d,e; Haddad,2002e,f,g,j, 2003; Rahman, 2002, 2003; Tsuzi et al., 2004),which form key components of defensive/adaptive cellularpathways activated in stress (oxidative)-evoked responses.

It is beyond the scope of this paper, however, to furtherelaborate on the biochemical and physiological mechanismsof glutathione. There are excellent, pertaining reviews inthis regard, as indicated above; that is why, we will moveon to discussing glutathione-related pathways in mediatingthe regulation of cytokines, via the involvement of nuclearfactor (NF)-�B and other transcription factors in variouss

3. Glutathione and glutathione-related enzymes incytokine regulation: involvement of NF-�B

3.1. Role for IL-1

There is increasing evidence indicating that ROS/RNS aremediators in growth factor and cytokine signaling pathways(Haddad, 2000, 2002f; Rogers et al., 2001; Brigelius-Floheet al., 2004; Pawate et al., 2004). Mechanisms by which freeradicals can interfere with signaling cascades include regu-lation of protein activities by the modification of essentialcysteines. Modification can be performed chemically or canbe enzyme-catalyzed. Enzymes catalyzing a reversible thiolmodification within proteins are to be able to react with both,ROS/RNS and protein thiols. If hydroperoxides are involved,likely candidates are peroxiredoxins and GPx, especially thephospholipid hydroperoxide GPx.

IL-1, one of the key players in inflammatory responses,stimulates the production of ROS, but its signaling cascadecan also be influenced by ROS and by thiol modifying agents(Haddad, 2000). Targets are located in early, intermediate andlate events in the signaling cascade. The effects of thiol mod-ifying agents, selenium and GPx, on the assembly of the IL-1receptor signaling complex as an early event, on the activa-tion of NF-�B as an intermediate event and on the expressiono ech-
ignaling mechanisms and scenarios (Haddad, 2002k). f cell adhesion molecules as a late event are potential m


Fig. 2. (A) Selective dismutation of reactive oxygen (ROS) and nitrogen (RNS) species, representing a schematic model of the pathways leading to the generationof ROS/RNS. Pathways mediating the formation of ROS/RNS and their selective dismutation by specific antioxidant enzymes and molecules indicate thatanumber of major cellular enzymes that defend against oxidative stress have been conserved through evolution. H2O2 that has been generated by the actionof superoxide oxidoreductase dismutase (SOD) could be converted to hypochlorous acid (HOCl) by the action of myeloperoxidase (MPO), releasing singletoxygen (1O2) and chloride ions in the process. Another pathway diverges from superoxide anion (O2

•−) to generate nitric oxide (NO), peroxynitrite anion(OONO−) and peroxynitrous acid anion (HOONO). HOONO could be converted to hydroxyl radical (•OH) thereby releasing nitrogen dioxide radical (NO2

•).(B) Schematic representation of the pathways leading to the generation of ROS and their selective dismutation via the glutathione-dependent pathway. O2

•−is metabolized via the dismutation reaction 2O2

•− + 2H+ → O2 + H2O2, which is catalyzed by SOD, a cytoplasmic enzyme that is constitutively expressed,and by a mitochondrial enzyme that is induced in response to oxidant stress. The H2O2 produced by the dismutation of O2

•− is converted by one pathwayto H2O and O2 by catalase (CAT) in peroxisomes and by glutathione peroxidase (GPx) in the cytoplasm, at the expense of reduced glutathione (GSH),leading to the formation of oxidized glutathione disulphide (GSSG) that is recycled back to GSH by glutathione reductase (GSSG-RD). H2O2 could be furtherconverted by another pathway involving iron into the hydroxyl radical (•OH), an injurious ROS causing oxidative stress and the release of cytokines. Thisiron-catalyzed reaction, known as the Fenton-like reaction, is impeded by the iron chelator desferrioxamine, which is also capable of neutralizingthe toxicityof •OH.


anisms in redox-dependent IL-1 regulation (Brigelius-Floheet al., 2004; Pawate et al., 2004).

3.1.1. Effect of dexamethasoneThe primary report to depict an association between redox

signaling and NF-�B on the one hand, and cytokines, partic-ularly IL-1, on the other hand, emerged with the characteri-zation of mechanisms involved in trans-repression of NF-�Bby activated glucocorticoid receptors (GR) (Scheinman etal., 1995). Glucocorticoids are potent immuno-suppressants,which work in part by inhibiting cytokine gene transcrip-tion (Rahman, 2000; Hermoso and Cidlowski, 2003; Iwasakiet al., 2004). It was shown that NF-�B, an important regu-lator of numerous cytokine genes (Hanada and Yoshimura,2002; Wajant, 2004; Watanabe et al., 2004b), was function-ally inhibited by dexamethasone (Dex). Dex treatment, inthe presence of cotransfected GR, inhibited NF-�B p65-mediated gene expression and p65 inhibited GR activation ofa glucocorticoid response element (Scheinman et al., 1995).

In addition, it was demonstrated that the ability of p65,p50 and c-Rel subunits to bind DNA was impaired by Dexand GR. Furthermore, RU486 treatment of wild-type GR andDex treatment of a transactivation mutant of GR was shownto inhibit p65 activity; this mechanism involved the zincfinger domain of GR, a redox-sensitive cofactor, a require-m lls,D lockTo ta ac siona ni ofc

3own

t e,e1( al -A oml n of� onseo(

cellsta xr taw -sh tionst

Cytokines may also be mediators of�-cell damage ininsulin-dependent diabetes mellitus (IDDM) (Sparre et al.,2003). For instance, IL-1 effect on insulin-producing cellswas reported to involve the activation of NF-�B, an in-creased transcription of the inducible form of nitric ox-ide (NO) synthase (iNOS) and the subsequent productionof NO (Azevedo-Martins et al., 2003; Saldeen and Welsh,2004). Whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a GPx mimetic (Belvisi et al., 2000; Haddad etal., 2002f; Mouithys-Mickalad Mareque et al., 2004), couldcounteract the effects of IL-1�, H2O2 and alloxan in rat pan-creatic islets and in the rat insulinoma cell line RINm5F (RINcells) was further investigated (de-Mello et al., 1996).

Ebselen prevented the increase in nitrite production by ratislets exposed to IL-1� and induced significant protectionagainst the acute inhibitory effects of alloxan or H2O2 expo-sure, as determined by the preserved glucose oxidation rates.Ebselen also prevented the increase in nitrite production byhuman islets exposed to a combination of cytokines (IL-1�,TNF-� and interferon (IFN)-�). In RIN cells, ebselen, more-over, counteracted both the expression of iNOS mRNA andthe increase in nitrite production induced by IL-1� but failedto block IL-1�-induced iNOS expression following exposureto the cytokine. Furthermore, ebselen did not prevent IL-1�-induced NF-�B activation (de-Mello et al., 1996). This sug-g ucedN i-ar y,g di

3r

,2 lialc asale culei IL-1 erewt hib-i aD lth-i ,2 fN withN old ousn ndi-t thatN ngt -(

en-d ytic

ent for the inhibition of p65. Of interest, in HeLa ceex activation of endogenous GR was sufficient to bNF-� or IL-1-dependent activation of NF-�B at the levelsf DNA binding and transcriptional activation (Scheinman el., 1995), suggesting that NF-�B and GR can interact inomplex regulatory network to modulate gene expresnd that cross-coupling of NF-�B and GR may play a

mportant role in glucocorticoid-mediated repressionytokine transcription.

.1.2. Effect of glucoseLong exposure to high glucose concentration was sh

o impair the expression of�-GCS by cytokines. For instancxposing normoglycemic endothelial cells to TNF-� or IL-� increased the activity and the mRNA expression of�-GCSUrata et al., 1996). In addition, inhibitors of NF-�B causedoss of the�-GCS mRNA expression in response to TNF�.

shift of the concentration of glucose in the medium frow (5.5 mM) to high (28 mM) decreased the expressio-GCS. These cells, however, showed no apparent respf �-GCS mRNA or the activity of NF-�B to TNF-� or IL-1�Urata et al., 1996).

Moreover, increase in the GSH concentration of thereated with glucose restored the expression of�-GCS mRNAnd its response to TNF-� or IL-1�, suggesting that redoegulation is involved in the expression of�-GCS (Urata el., 1996). In summary, it seems that the expression of�-GCS,hich is regulated by cytokines, can be mediated by NF�Btimulation, and impairment of the regulation of�-GCS inyperglycemic cells may be a cause of medical complica

hat likely develop in diabetes mellitus.

s

ested that ebselen partially counteracted cytokine-indOS activation in pancreatic�-cells, an effect not assocted with the inhibition of NF-�B. Additionally, IL-1� waseported to regulate NO production and, more importantl�-lutamyl transpeptidase (�-GT) activity, an enzyme involve

n glutathione transport (Meroni et al., 2000).

.1.3. Effect of N-acetyl-l-cysteine: glutathioneepletion

NAC is an antioxidant and GSH derivative (Dekhuijzen004). In cultured human brain microvascular endotheells (HBMEC), it was observed that there was no bxpression of E-selectin, an endothelial adhesion mole

nvolved in binding and targeting of neutrophils, but with�, TNF-�, or lipopolysaccharide (LPS) stimulation thas delayed surface expression (Hess et al., 1996). No-

ably, the cytokine-induced upregulation was partially inted with the glutathione donor, NAC (Haddad et al., 2000;ekhuijzen, 2004), the free radical scavenger, dimethy

ourea (DMTU) (Haddad and Land, 2001b; Krunkosky et al.003) and Dex. In addition, TNF-� induced the activation oF-�B, an effect that was attenuated by prior treatmentAC and Dex (Hess et al., 1996). This suggested that thionors could play a role in inhibiting potentially deleterieutrophil–endothelial interactions in inflammatory co

ions. This was reinforced with a report that indicatedAC can modulate IL-1�B (p65/p50) release by increasi

he levels of the homo- and heterodimeric forms of NF�BParmentier et al., 2000; Renard et al., 2001).

The aforementioned study was followed up, indepently, with the effect of NAC on agonist-induced monoc


cell adhesion to endothelial cells (EC) (Faruqi et al., 1997).NAC, for example, inhibited IL-1�-induced, but not basal,adhesion. In addition, monocytic cell adhesion to EC in re-sponse to TNF-�, LPS,�-thrombin, or phorbol 12-myristate13-acetate (PMA) was similarly inhibited by NAC. NAC alsoinhibited IL-1�-induced mRNA and cell surface expressionof both E-selectin and VCAM-1. However, NAC had no ef-fect on the half-life of E-selectin or vascular cell adhesionmolecule (VCAM)-1 mRNA (Faruqi et al., 1997). AlthoughNAC reduced NF-�B activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding tothe consensus NF-�B binding sites of the VCAM-1 gene,the antioxidant had no sensible effect when an oligomer cor-responding to the consensus NF-�B binding site of the E-selectin gene was used.

NAC also caused an expected dramatic increase in GSHlevels in EC. In vitro studies demonstrated that, whereas thebinding affinity of NF-�B to the VCAM-NF-�B oligomerpeaked at a GSH/GSSG ratio of approximately 200 anddecreased at higher ratios, the binding to the E-selectin-NF-�B oligomer appeared relatively unaffected even at ra-tios > 400, i.e. those achieved in EC treated with NAC (Faruqiet al., 1997). These results indicate that NF-�B bindingto its consensus sequences in the VCAM-1 and E-selectingene exhibits marked differences in redox sensitivity, allow-i amet NACc ssiblem ist-i n ofN -i iono

3g

e thee sR ngm iatedw olarto er e ofpE yn-t ingly,i l-l yex-cT

re-v ngr Al-ta kad-

ing NF-�B activation, it augmented cytokine biosynthesis(Haddad, 2000). These results indicate that glutathione de-pletion is associated with the augmentation of an oxidativestress-mediated pro-inflammatory state in an ROS-dependentmechanism and that the I�B-�/NF-�B pathway is otherwisenot necessarily indispensable for redox-mediated regulationof cytokines. Glutathione depletion, therefore, is associatedwith the augmentation of oxidative stress-mediated inflam-matory state in a ROS-dependent mechanism and that theI�B-�/NF-�B pathway is redox-sensitive but could be differ-entially involved in regulating redox-dependent regulation ofcytokines (Haddad, 2002f).

3.1.5. Effect of pyrollidine dithiocarbamateThe metal chelator and antioxidant pyrollidine dithiocar-

bamate (PDTC) has been extensively used in studies impli-cating ROS with the regulation of NF-�B (Virlos et al., 2003;Jiang et al., 2004; Mukhopadhyay et al., 2004). It was shownthat PDTC can inhibit NF-�B activation in response to IL-1and TNF-�. However, it was also found that the inhibitionwas reversed by treatment of inhibited nuclear extracts withthe reducing agent 2-mercaptoethanol (Brennan and O’Neill,1996). These results suggested that the inhibition was causedby oxidation of NF-�B on a sensitive thiol, possibly on thep50 subunit (which was detected in NF-�B complexes), andn .,2

glu-t thert h di-aa x-iD ade yP in-v

tiont ont ac-t h asG att iox-i ,1 ss tor-a ;C -� ho-l ine(

3e-

s ;C -

ng for differential gene expression regulated by the sranscription factor. These data also demonstrate thatan increase the GSH/GSSG ratio, suggesting a poechanism through which this antioxidant inhibits agon

nduced monocyte adhesion. Of note, supplementatioAC normalized LPS-induced NF-�B activation and pro

nflammatory cytokine production during early rehabilitatf protein malnourished mice (Li et al., 2002).

.1.4. Effect of buthionine-(S,R)-sulfoximine:lutathione depletion

Chemioxyexcitation, a term we have used to describxposure of cells to a combination of�pO2 and exogenouOS (Haddad et al., 2000a), constitutes a potential signaliechanism for regulating an inflammatory signal associth oxidative stress. For instance, exposure of fetal alve

ype II epithelial cells to an ascending�pO2 regimen, withr without the hydroxyl radical (•OH) or the superoxidadical anion (O2•−), induced a dose-dependent releasro-inflammatory cytokines (Haddad, 2000). Similarly, thescherichia coli-derived LPS upregulated cytokine bios

hesis in a dose- and time-dependent manner. Interestrreversible inhibition by BSO of�-GCS, induced intraceular accumulation of ROS and augmented chemioxitation and LPS-mediated release of IL-1�, IL-6 andNF-�.

Analysis of the molecular mechanism implicatedealed an I�B-�/NF-�B-independent pathway mediatiedox-dependent regulation of inflammatory cytokines.hough BSO stabilized the cytosolic inhibitory-�B (I�B)-�nd down-regulated its phosphorylation, thereby bloc

ot by inhibition of the activation pathway (Haddad et al000a).

In addition, PDTC caused an increase in oxidizedathione, indicating that it acts as an oxidizing agent rahan as an antioxidant. Similar results were obtained witmide, a compound designed to oxidize glutathione (Brennannd O’Neill, 1996). Moreover, an increase in the ratio of o

dized to reduced glutathione was shown to inhibit NF-�B-NA binding in vitro (Brennan and O’Neill, 1996; Haddt al., 2000a). While NF-�B activation was unaffected bDTC, DNA binding was inhibited through a mechanismolving a shift towards oxidizing conditions.

The aforementioned was reinforced with the observahat cytokine-mediated NF-�B activation was dependenthe antioxidant potential of the cells, especially on theivity of reduced glutathione-dependent enzymes, sucPx (Renard et al., 1997), thus supporting the notion th

he GSH/GSSG ratio and the activity of intracellular antdant enzymes play a major role in NF-�B tuning (Chapple997; Rovin et al., 1997). Similarly, thiol modulation wahown to inhibit IL-1-mediated activation of an IL-1 recepssociated protein kinase and NF-�B (Tewes et al., 1997handrasekar et al., 2000). Moreover, IL-1-induced NFB activation was inhibited by over-expression of phosp

ipid hydroperoxide GPx in a human endothelial cell lBrigelius-Flohe et al., 1997).

.1.6. Effect of heat shock proteinsNO and TNF-� play important role in the pathogen

is of disease during acute inflammation (Dinarello, 2002arlsen et al., 2004; Xanthoulea et al., 2004). For exam


ple, pretreatment of primary cultures of rat hepatocytes withthe NO donor,S-nitroso-N-acetylpenicillamine (SNAP), in-duced the expression of heat shock protein 70 (HSP70)mRNA and protein, which was associated with thermotolerance and cytoprotection from TNF-�/actinomycin D-induced hepatotoxicity and apoptosis (Kim et al., 1997).Furthermore, SNAP transiently changed the intracellular re-dox state by inducing GSH oxidation associated with theformation ofS-nitrosoglutathione (GSNO). HSP70 mRNAwas also induced by the GSH-oxidizing agent diamideand the GSH-conjugating agentN-ethylmaleimide, sug-gesting that NO induces HSP70 expression through GSHoxidation.

Interestingly, the protective effect of SNAP on TNF-�-induced apoptosis correlated with the level of HSP70 expres-sion. SNAP inhibited ROS generation and lipid peroxidation,effects that were reversed by blocking HSP70 expression us-ing an antisense oligonucleotide to HSP70. Endogenous NOformation, induced in hepatocytes stimulated with IFN-� andIL-1�, also led to the formation of GSNO and GSSG, in-duced HSP70, and attenuated TNF-�-mediated cytotoxicity(Kim et al., 1997), demonstrating that NO can induce resis-tance to TNF-�-induced hepatotoxicity, possibly through thestimulation of HSP70 expression.

3

sup-p enti-ae tlw ityo ).M ucedt

fyna sf -rt ptor,f D8m1

L-2-d ple-t n isf t thew tionu defi-c

IV)-i tedr y de-c t wash ome

(AIDS) may be the consequence of a GSSG deficiency (Ailletet al., 1994; Droge et al., 1994).

The first report to suggest redox effect on IL-2 medi-ated by NF-�B emerged with antioxidant treatment of mono-cytes derived from HIV-infected patients (Aillet et al., 1994).For example, antioxidant concentrations (supra-physiologic)high enough to block NK-�B activation were shown tohave a suppressive effect on immune functions in vitro,because NAC-blocked IL-2-induced lymphocyte prolifera-tion. This was reinforced with the inhibition of proliferationand of IL-2 production and utilization in lymphocytes byS-oxalylglutathione (Grove et al., 1996). Similarly, there wasenhancement of T cell receptor signaling by a mild oxidativeshift in the intracellular thiol pool (Hehner et al., 2000).

Evidence suggests that natural killer (NK) cells can con-tribute to the pathogenesis of delayed rejection of vascular-ized xenografts, and NK cells have participated in hyperacutexenograft rejection (Tsuyuki et al., 2001; Yin et al., 2004).Endothelial cells have been shown to be the primary targetof the recipient’s immune responses that mediate both hyper-acute and delayed xenograft rejection. Under conditions ofoxidative stress induced by thiol deprivation, but not undernormal conditions, pretreatment of porcine aortic endothe-lial cells (PAECs) with the NO donor,S-nitroso-N-acetyl-penicillamine, dramatically inhibited killing of PAEC tar-g ,2 rfacee ti-E-s -1,p ellsi sion ofp ECm lvedi

rt thesp sso-c( tiono de-l ularr eneice

ofT re-c ;Gl ra-cR nico mem-b sig-n pho-c niedb on

.2. Role for IL-2

Even a moderate increase in the cellular cysteinely elevates the intracellular GSH/GSSG levels and pottes immunological functions of lymphocytes in vitro (Aillett al., 1994; Droge et al., 1994; Haddad et al., 2000). A

ow GSSG levels, T cells cannot optimally activate NF-�B,hereas high GSSG levels inhibit the DNA binding activf NF-�B (Brennan and O’Neill, 1996; Haddad et al., 2000oreover, the effects of GSSG were antagonized by red

hioredoxin (TRX) (Droge et al., 1994; Jones et al., 2004).As the protein tyrosine kinase activities p56lck and p59

re activated in intact cells by H2O2, they are likely targetor GSSG action (Balamurugan et al., 2002). These redoxegulated enzymes trigger signal cascades for NF-�B activa-ion and transduce signals from the T cell antigen recerom cluster of designation/differentiation (CD)4 and Colecules, and from the IL-2 receptor�-chain (Aillet et al.,994; Droge et al., 1994).

The effector phase of cytotoxic T cell responses and Iependent functions were inhibited even by a partial de

ion of the intracellular GSH pool. As signal transductioacilitated by prooxidant conditions, it was proposed thaell-known immunological consequences of GSH depleltimately may be results of the accompanying GSSGiency (Brennan and O’Neill, 1996; Haddad et al., 2000).

For instance, as human immunodeficiency virus (Hnfected patients and simian immune virus (SIV)-infechesus macaques have, on the average, significantlreased plasma cysteine and intracellular GSH levels, iypothesized that acquired immune deficiency syndr

et cells by IL-2-activated human NK cells (Tsuyuki et al.001). This same combined treatment reduced both suxpression and mRNA levels of E-selectin. Moreover, anelectin monoclonal antibody (mAb), but not Ab to VCAMrotected PAEC from lysis by human IL-2-activated NK c

n a dose-dependent manner, suggesting that the expresorcine E-selectin is important for the cytotoxicity of PAediated by activated human NK cells and may be invo

n the redox-mediated modulation of that cytotoxicity.Since it is known that NF-�B activation is required fo

ranscription of E-selectin, current data indicated thatuppression of E-selectin expression byS-nitroso-N-acetyl-enicillamine pretreatment and thiol deprivation was aiated with reduced NF-�B DNA-binding activity in PAECTsuyuki et al., 2001). These data suggest that the regulaf porcine E-selectin may be important for modulating

ayed xenograft rejection and that manipulation of celledox systems may provide a means to protect xenogndothelial cells from NK cell-mediated cytotoxicity.

The integral membrane protein linker for activationcells (LAT) is a central adapter protein in the T-cell

eptor (TCR)-mediated signaling pathways (Wange, 2000ringhuis et al., 2002; Rudd and Wang, 2003). The cellu-

ar localization of LAT is purported to be sensitive to intellular redox balance alterations (Gringhuis et al., 2000).educed intracellular levels of GSH, a hallmark of chroxidative stress, as stressed above, resulted in therane displacement of LAT, abrogated TCR-mediatedaling and consequently hyporesponsiveness of T lymytes. The membrane displacement of LAT is accompay a considerable difference in the mobility of LAT up


native and non-reducing denaturing polyacrylamide gel elec-trophoresis analysis, a finding indicative of a conformationalchange. In addition, targeted mutation of redox-sensitive cys-teine residues within LAT created LAT mutants which remainmembrane anchored under conditions of chronic oxidativestress.

The expression of redox-insensitive LAT mutants al-lowed for restoration of TCR-mediated signal transduction,whereas CD28-mediated signaling pathways remained im-paired (Gringhuis et al., 2002). These results are indicativethat the membrane displacement of LAT as a result of re-dox balance alterations is a consequence of a conformationalchange interfering with the insertion of LAT into the plasmamembrane. Conclusively, the data suggest a role for LAT asa crucial intermediate in the sensitivity of TCR signaling andhence T lymphocytes toward chronic oxidative stress.

3.3. Role for IL-4

Initially, cytokines (IL-4)-mediated regulation of endothe-lial activation, a redox-sensitive effect, evolved with the effectof NO. For instance, NO decreased IL-4-induced endothe-lial activation and selectively reduced endothelial expressionof adhesion molecules and pro-inflammatory cytokines (DeCaterina et al., 1995). Furthermore, NAC was shown to in-h ane l(

at6)a icha se-q 04I cti-vS ep s, itw edS

othc y ac-tt 293( -i d byt dI -b ites,a ativeb ro-m

3

aug-m IL-6( tive

pathway, since BSO depletion of GSH strengthened IL-6 re-lease and the production of free radicals. Similar report sug-gested that antioxidants can inhibit cytokine (IL-6/IL-8) pro-duction in an NF-�B-dependent mechanism (Blanchard et al.,2001), although Haddad and associates indicated that ROS-mediated increase in IL-6 (and other cytokines) was indepen-dent, at least in part, of the aforementioned NF-�B pathway(Haddad, 2000, 2001a, 2002a,b,h; Haddad and Land, 2001a,2002c; Haddad and Fahlman, 2002a; Haddad et al., 2001d,2002b,c).

This is not in agreement, however, with the suggestionthat ROS-mediated activation of NF-�B can lead to upreg-ulation of cytokine expression (Yu et al., 2002; Song et al.,2004). This discrepancy is perhaps related to the types ofcell cultures being investigated and also has to do with theinvolvement of more than one pathway, such as the mitogen-activated protein kinase (MAPK) pathway (discussed below),in mediating redox-dependent regulation of cytokine produc-tion and expression.

ROS were also reported to play crucial role in mediatingapoptosis (Haddad, 2004b). To understand ROS involvementin Fas-mediated apoptosis of myeloma cells, the effects ofantioxidants were tested. For instance, Fas-mediated apopto-sis was increased in the presence of antioxidants such as NACand glutathione, but it was decreased when H2O2 was added( elw acid,a A).

th,t PP2At th ofa witht lw reasedi fac-t P2At r fors OSa

3

edlye Oic r an-t g thec ctiva-t andw n ofb d byN g-g latedb

ofm via

ibit primary human T cell cultures incubated with IL-4,ffect associated with NF-�B inhibition at the dendritic leveVerhasselt et al., 1999).

Signal transducer and activator of transcription 6 (Stnd NF-�B are widely distributed transcription factors whre induced by different stimuli and bind to distinct DNAuence motifs (Jaruga et al., 2003; Yamamoto et al., 20).

L-4, which was shown to activate Stat6, synergized with aators of NF-�B to induce IL-4-responsive genes (Shen andtavnezer, 1998). Notably, using glutathioneS-transferasulldown assays and coimmunoprecipitation techniqueas demonstrated that NF-�B and tyrosine-phosphorylattat6 can directly bind each other in vitro and in vivo.Moreover, an IL-4-inducible reporter gene containing b

ognate binding sites in the promoter was synergisticallivated in the presence of IL-4 when Stat6 and NF-�B pro-eins were co-expressed in human embryonic kidneyHEK 293) cells (Shen and Stavnezer, 1998). The same IL-4nducible reporter gene was also synergistically activatehe endogenous Stat6 and NF-�B proteins in IL-4-stimulate.29mu B lymphoma cells. Furthermore, Stat6 and NF�Bound cooperatively to a DNA probe containing both snd the presence of a complex formed by their cooperinding correlated with the synergistic activation of the poter by Stat6 and NF-�B.

.4. Role for IL-6

Intracellular GSH depletion has been associated withenting cytokine biosynthesis, including the release of

Haddad, 2000). The mechanism implicated a ROS-sensi

Kang and Choi, 2001). In addition, intracellular ROS levas decreased in myeloma cells treated with okadaicn inhibitor of protein phosphatases 1 and 2A (PP1/PP2

To clarify the direct role of PP2A in myeloma cell growhe PP2A-transfected cell lines, sense- or antisense-ransfectants, were established. Spontaneous cell growntisense-PP2A transfectants was reduced compared

hat of vector transfectants (Kang and Choi, 2001). ROS leveas decreased in antisense-PP2A transfectants but inc

n sense-PP2A transfectants. In addition, anti-apoptoticors such as Bcl-2 and IL-6 were reduced in antisense-Pransfectants, indicating that PP2A is an essential factourvival and growth of myeloma cells via regulation of Rnd anti-apoptotic factors.

.5. Role for IL-8

LPS-induced expression of the IL-8 gene was marknhanced by H2O2 or by deprivation of glutathione by BS

n human astrocytoma U373 cells (Tanaka et al., 1997). Inontrast, it was markedly suppressed by NAC and otheioxidants. Moreover, transient expression analysis usinhloramphenicol acetyltransferase assay revealed that aion of the IL-8 promoter by LPS was stimulated by BSOas suppressed by NAC; likewise LPS-induced activatiooth NF-�B and AP-1 was enhanced by BSO and inhibiteAC (Tanaka et al., 1997). These results unequivocally suest that LPS-induced IL-8 gene expression can be reguy cellular redox via modulation of transcription factors.Helicobacter pyloribacterium induced the expression

RNA and protein for IL-8 and cyclooxygenase (COX)-2


activation of NF-�B and increased the levels of IL-8, an effectthat was inhibited by GSH and PDTC (Kim et al., 2001). Inairway epithelial cells, for example, pre-treatment with NACor glutathione inhibited TNF-�-induced activation of NF-�Btranscriptional activity and IL-8 promoter-mediated reportergene expression (Harper et al., 2001). In contrast, elevatedTRX protein levels in cells enhanced TNF-�-dependent NF-�B transcriptional activity and IL-8 promoter activity (Harperet al., 2001). Furthermore, Nacystelyn (NAL), a recently de-veloped lysine salt of NAC, enhanced intracellular GSH andabolished H2O2-induced IL-8 release from A549 cells, hu-man alveolar epithelial cell line; this was associated withinhibition of NF-�B DNA-binding (Antonicelli et al., 2002,2004). Ergothioneine, a naturally occurring thiol compound,which possesses antioxidant property, also inhibited oxida-tive stress- and TNF-�-induced NF-�B activation and IL-8release in alveolar epithelial cells (Rahman et al., 2003).

3.6. Role for IL-10

The first report to implicate IL-10 redox-sensitivityemerged with the observation that ebselen could protectmice against T cell-dependent, TNF-mediated apoptotic liverinjury. In livers from ebselen-pretreated mice exposed to con-canavalin A (ConA), NF-�B was upregulated (Tiegs et al.,1 ec 10w juryi in Bi elenp inG i.e.t cedh

per-o ownt asesi 0(

3

nsep . Toi statea usec theg . Itw -bis( tedL asa d IL-1

thep gt

and O’Neill, 1996), thus leading to IL-12p40 production(Komatsu et al., 2003). These results indicate that theglutathione-redox couple can play a significant role in theaugmented production of IL-12p40 and in influencing im-mune response patterns.

3.8. Role for TNF-�

A primary report indicated a role for TNF-� in mod-ulating antioxidant equilibrium. For example, secretion ofTNF-� was abolished by butylated hydroxyanisole (BHA), aphenolic, lipid-soluble, chain-breaking antioxidant, in stimu-lated U937 cells (Israel et al., 1992). The antioxidative effectof BHA was accompanied by an increase in thiol, but notglutathione, content in stimulated and unstimulated T cell,whereas TNF-� stimulation itself barely modified the cellu-lar thiol level; this effect was not accompanied with oxidant-mediated activation of NF-�B. It appeared that a basal redoxequilibrium tending toward oxidation is a prerequisite for theactivation of transduction pathways regulating the activityof NF-�B-dependent genes (Aillet et al., 1994; Brennan andO’Neill, 1996; Haddad et al., 2000).

This was corroborated with the observation that raisingglutathione levels with NAC substantially reduced NF-�Binduction by TNF-� in chronic B-leukaemia cells, implicat-i ve tor re anp yb thei p inG ndv

nT oxi-d phen-a di-a anti-c sb I� leadt ,1 acti-v -n ,1N ug-gt ionm SHs nslo-c tt

thatT crip-

998). The release of TNF-� was downregulated, while thirculating amount of the anti-inflammatory cytokine IL-as increased. Moreover, ebselen protected from liver in

nduced by the superantigen staphylococcal enterotoxn galactosamine (GalN)-sensitized mice. Of note, ebsrotected the liver and enhanced circulating IL-10alN-sensitized mice treated with recombinant TNF,

he common distal mediator of ConA and SEB-induepatotoxicity (Tiegs et al., 1998).

In concert with the aforementioned, interestingly, suxide dismutase (SOD)-overexpressing mice were sh

o be resistant to ozone-induced tissue injury and incren NO and TNF-�, ostensibly via the regulation of IL-1Fakhrzadeh et al., 2004).

.7. Role for IL-12

IL-12 plays a key role in determining the immune respoattern that results in maturation of Th0 to Th1 and Th2

nvestigate the correlation between intracellular redoxnd IL-12 production in macrophages, cells from the moell line J774A.1 were treated with reagents modulatinglutathione-redox couple before stimulation with LPSas found that the glutathione reductase inhibitor, 1,3

2-chloroethyl)-1-nitrosourea (BCNU), markedly augmenPS induced IL-12p40 production particularly when it wdded before LPS stimulation, whereas BSO suppresse2p40 production (Komatsu et al., 2003).

Moreover, IL-12p40 augmentation correlated withrofile of GSSG and the activation of NF-�B, suggestin

hat GSSG can play a role in NF-�B activation (Brennan

ng PKC-independent pathways and pathways sensitiedox reagents, in mediating the induction of NF-�B (Jabbat al., 1994). It was also shown that HIV-1 Tat protein cotentiate TNF-induced NF-�B activation and cytotoxicity altering the cellular redox state, thus demonstrating

nduction of a pro-oxidative condition reflected by a droSH (Westendorp et al., 1995). In addition, iron chelatioecreased HIV-1 Tat potentiated TNF-induced NF-�B acti-ation in Jurkat cells (Shatrov et al., 1997).

Inhibition of TNF-�-induced NF-�B activation in huma-cell lines was reported by anetholdithiolthione, an antiant which has been used to protect against acetaminond CCl4-induced hepatotoxicity, lipid peroxidation, ration injury, and also has been used clinically as anholeretic agent (Sen et al., 1996). Of particular interest, it haeen also reported that GPx over-expression can inhibit�B-phosphorylation and degradation, which subsequently

o NF-�B blockade in human T47D cells (Kretz-Remy et al.996). Furthermore, oxidants augmented LPS-inducedation of alveolar macrophages in a TNF-�-dependent maner via the modulation of intracellular GSH (Mendez et al.996). In addition, glutathione modulated TNF-�-inducedF-�B activation in skeletal muscle-derived L6 cells, sesting that the inhibitory effect of PDTC on NF-�B ac-

ivation correlate with its effect on intercellular adhesolecule (ICAM)-1 expression, thus indicating that this G

tatus modifying agent not only can influence nuclear traation of NF-�B proteins but also regulate�B-dependenranscription (Sen et al., 1997; Spiecker et al., 1997).

On the molecular mechanisms, it has been shownNF-� can increase hepatocellular glutathione by trans


tional regulation of the heavy subunit chain of�-GCS(Morales et al., 1997). Moreover, inhibition of LPS-mediatedactivation in rat Kupffer cells by NAC occurred subsequent toNF-�B translocation and required protein synthesis via theregulation of TNF-� (Fox and Leingang, 1998). Similarly,glutathione was reported to down-regulate the phosphory-lation of I�B, involving an autoloop regulation of NF-�B-mediated expression of NF-�B subunits by TNF-� in mousevascular endothelial cells (Cho et al., 1998). Relatively re-cent, a cell protein (FIP-3; 14.7 K interacting protein) wasidentified as a modulator of NF-�B activity and as a tar-get of an adenovirus inhibitor of TNF-�-induced apopto-sis (Li et al., 1999). In concert, over-expression of�-GCSsuppressed TNF-induced apoptosis and activation of NF-�B and activator/activating protein (AP)-1 (Manna et al.,1999).

Similarly, redox manipulation using the thiol-oxidizingagent diethyl maleate (DEM) prevented hepatocellular necro-sis and apoptosis in a rodent endotoxemia model (Jones et al.,1999). This was followed by the suggestion that there wasselective involvement of O2•−, but not the downstream com-pounds H2O2 and peroxynitrite, in TNF-�-induced apopto-sis of rat mesangial cells (Moreno-Manzano et al., 2000). Ofinterest, there was disruption of redox homeostasis in TNF-induced apoptosis in a murine hepatocyte cell line, via theb oxi-d o-t onev(

Brief hypoxic stress was shown to suppress post-bacteremic NF-�B activation and TNF-� bioactivity in per-fused liver (Loftis et al., 2000). The glutathione precursorl-2-oxothiazolidine-4-carboxylic acid was shown to protectagainst liver injury due to chronic enteral ethanol exposure,which led to TNF-� elevation in the rat (Iimuro et al., 2000).Similarly, homocysteine inhibited TNF-�-induced endothe-lial adhesion molecule expression and monocyte adhesion viaNF-�B dependent pathway (Stangl et al., 2001). In addition,it has been reported thatS-nitrosoglutathione inhibited TNF-�-induced NF-�B activation in neutrophils (Fortenberry etal., 2001). On the other hand, glutathione depletion, as re-iterated above, augmented TNF-� secretion in an NF-�B-independent manner (Haddad, 2000, 2001a; Haddad et al.,2002b,c). In correlation, it has been shown that reducedglutathione depletion can cause necrosis and sensitizationto TNF-�-induced apoptosis in cultured mouse hepatocytes(Nagai et al., 2002).

Recently, it has been reported that the expression of thep75 TNF receptor was linked to TNF-induced NF-�B translo-cation and oxyradical neutralization in glial cells (Dopp et al.,2002). Moreover, TNF-� signaling through p55 TNF receptor1 (TNFR1) was shown to play an important role in regulat-ing the expression of antioxidants in knockout mice, thus ex-plaining the fact that reduced generation of antioxidants mayc −/−a

NF-� d inF

F es. Ox ersiG m effe ich regulatet ve the e text forf

lockade of the transcription of genes required for antiant defenses (Pierce et al., 2000). In support, there was p

entiation of tumour apoptosis by human growth hormia glutathione production and decreased NF-�B activityCherbonnier et al., 2003).

ig. 3. Antioxidant/prooxidant mechanisms in the regulation of cytokinSSG, can cause the accumulation of ROS. ROS activates downstrea

he phosphorylation of MKK and I�B/NF-�B complex. Both pathways haurther details).

ontribute to the increased sensitivity of TNFR1 mice tocetaminophen (Chiu et al., 2003).

Schematic pathways signaling the association amongB, cytokines and glutathione redox cycle are depicteig. 3.

idative stress, mainly caused by the depletion of GSH by BSO or convon ontoctor mechanisms involving upstream kinases, such as MEKK/NIK, whspotential to regulate genes implicated with cytokine biosynthesis (se


4. Glutathione and glutathione-related enzymes incytokine regulation: involvement of HIF-1�

4.1. HIF-1�: a redox(y)-sensitive immunologic sensor?

All organisms have mechanisms for sensing oxygen con-centrations and for responding to hypoxia with changes ingene expression (Wenger, 2002; Hopfl et al., 2003; Schu-macker, 2003; Seta and Millhorn, 2004). The transcriptionfactor hypoxia-inducible factor 1 (HIF-1) is one of the mas-ter regulators of oxygen homeostasis (Bracken et al., 2003;Lee et al., 2004a,b; Maxwell, 2004; Maxwell and Salnikow,2004). HIF-1 is also required for the development of keyphysiological systems, such as vasculogenesis and pneu-mogenesis, during fetal and postnatal life (Freeburg andAbrahamson, 2003; Semenza, 2003; Gordeuk et al., 2004;Mukhopadhyay and Datta, 2004).

HIF-1 influences the physiological responses to hypoxia(Fedele et al., 2002; Michiels et al., 2002; Seta et al., 2002;Fung, 2003; Semenza, 2004; Simon, 2004), and the patho-physiology of heart attack, cancer, stroke and chronic lungdisease (Acker and Plate, 2002; Bazan et al., 2002; Cai et al.,2003; Rosenberger et al., 2003; Distler et al., 2004; Hopfl etal., 2004; Ralph et al., 2004).

On its biochemistry, HIF-1� is a basic-helix–loop–helix-P one al.,2 04I asa n,t -1A sb on-t assI3 ucha A-b imerf l.,2

H andr -e ave am ,1 ,2

es-s ra-t .,2n lh al.,2r ro-t gra-

dation in the proteasome (Fig. 4) (Ivan et al., 2002; Berra etal., 2003; D’Angelo et al., 2003a,b; Kim and Kaelin, 2003;Kuznetsova et al., 2003; Li et al., 2003; Masson and Ratcliffe,2003; Metzen et al., 2003; Pugh and Ratcliffe, 2003; Schnellet al., 2003; Czyzyk-Krzeska and Meller, 2004; Mekhail etal., 2004; Turcotte et al., 2004).

Iron chelators and cobalt chloride prevent HIF-1� ubiq-uitination besides inducing its expression (Yuan et al., 2001;Yang et al., 2004; Zhao et al., 2004). HIF heterodimers bind tothe hypoxia response element (HRE), a 5′-RCGTG-3′ con-sensus sequence. Several HIF-1-regulated genes have beenidentified, including genes coding for proteins involved inangiogenesis, energy metabolism, erythropoiesis, cell pro-liferation and viability, vascular remodeling and vasomotorresponses, oxidative stress and inflammation (Hu et al., 2003;Komatsu and Hadjiargyrou, 2004; Kong et al., 2004; Liu etal., 2004; Park et al., 2004; Peyssonaux and Johnson, 2004).

The intricate relationship existing between HIF and oxy-gen sensing indicates a likely potential for the regulation ofimmunologic responses, such as inflammation, especially inlight of the fact that oxidative stress and redox perturbationsare implicated with the evolution of inflammatory-relatedstresses (Semenza, 2000a,b; Haddad, 2002d,f,g,h,i,j, 2003;Cramer and Johnson, 2003; Kojima et al., 2003).

The underlying oxygen sensing mechanisms are being ex-p me-d latedt IFm gu-l apet gens toryr

4f

e-v ;VH -m ateo dentH

4ity

o )p air-m ellb am-ma elialg bu-l F-1t oter,

AS (bHLH-PAS) protein (Semenza et al., 1997; Bact al., 1998; Wiesener et al., 1998; Lavista-Llanos et002; Chapman-Smith et al., 2004; Kewley et al., 20).

t is an obligatory component of HIF-1, which existsheterodimer of HIF-1� and another bHLH-PAS protei

he aryl-hydrocarbon receptor nuclear translocator (HIF�,RNT) (Baby et al., 2003; Isaacs et al., 2004). Both subunitelong to a larger family of transcription factors that c

ain bHLH and PER-ARNT-SIM homology domains. Clmembers of this family, such as HIF-1�, HIF-2� and HIF-�, heterodimerize with one of the class II sub-family ss ARNT1, ARNT2 and ARNT3, resulting in different DNinding and transcriptional properties depending on the d

ormation (Maxwell and Ratcliffe, 2002; Maynard et a003; Sowter et al., 2003; Aprelikova et al., 2004).

With regards to subunit tissue distribution, HIF-1� andIF-1� mRNA are essentially expressed in most human

odent tissues. HIF-2�, HIF-3�, ARNT2 and ARNT3, howver, are restricted to certain tissues, and therefore hore specialized role in oxygen homeostasis (Wang et al.995a,b; Wiener et al., 1996; Semenza, 2000a; Berra et al.001a,b).

Whereas HIF-1� is constitutively expressed, the exprion of HIF-1� is induced by hypoxia (oxygen concention below 5–6%) (Haddad and Land, 2000a; Haddad et al000a; Semenza, 2000b, 2002; Wiesener et al., 2003). Underon-hypoxic conditions, HIF-1� is hydroxylated by a prolyydroxylase enzyme (Kageyama et al., 2004; Koivunen et004; Marx, 2004; Marxsen et al., 2004). This modification isequired for the binding of the von Hippel-Lindau (VHL) pein to HIF-1� and its subsequent ubiquitination and de

lored, but essentially hinge around the ability of HIF toiate transcriptional control over many genes closely re

o the regulation of the inflammatory milieu. Whether Hay well act as an immunologic sensor or is indirectly re

ated by mediators of the immune response will likely shhe identity of this transcription factor not only as an oxyensor but also as a mediator of immunity and inflammaesponses.

.2. Glutathione and glutathione-related enzymes: roleor HIF-1α and cytokines

Redox regulation of HIF-1� has been professionally riewed elsewhere (Wang et al., 1995a,b; Dong et al., 2001aux et al., 2001; Haddad, 2002d,e,g; Elkins et al., 2003;addad, 2003, 2004a). Cytokines are evolving as critical imune regulators of HIF. This section will further elaborn the molecular mechanisms mediating cytokine-depenIF regulation from a redox perspective.

.2.1. Role for IL-1IL-1� was reported to stimulate the DNA binding activ

f HIF-1. IL-1-induced inhibition of erythropoietin (EPOroduction, for example, was not mediated by the impent of HIF-1 function, indicating that HIF-1 may we involved in modulating gene expression during inflation (Hellwig-Burgel et al., 1999). In addition, hypoxiand IL-1� were reported to stimulate vascular endothrowth factor (VEGF) production in human proximal tu

ar cells, ostensibly due to increased DNA binding of HIo hypoxia-responsive elements in the VEGF gene prom


Fig. 4. HIF signaling pathways. The primary molecular mechanism of gene activation during hypoxia is through HIF-1. Several genes involved in cellulardifferentiation are directly or indirectly regulated by hypoxia. These include EPO, LDH-A, ET-1, transferrin, transferrin receptor, VEGF, Flk-1,Flt-1, platelet-derived growth factor-� (PDGF-�), basic fibroblast growth factor (bFGF), and others genes affecting glycolysis. HIF-1 is a member of the basic helix–loop–helix(bHLH)-PAS family of transcription factors known to induce gene expression by binding to a∼50-bp HRE containing a core 5′-ACGTG-3′ sequence. bHLH-PAS proteins heterodimerize to form transcription complexes that regulate O2 homeostasis, circadian rhythms, neurogenesis, and toxin metabolism. ThreebHLH-PAS proteins in vertebrates respond to hypoxia: HIF-1, EPAS (HIF-2), and HIF-3. These dimerize with ARNT (aryl hydrocarbon receptor nucleartranslocator protein), ARNT-2, or ARNT-3. HIF-1 is ubiquitinated and subsequently degraded in less than 5 min under normoxic conditions. Although severalcandidate O2-sensing molecules have emerged in the literature, the molecular basis of how cells sense O2 levels is poorly characterized. pVHL, the proteinproduct of a tumor-suppressor gene responsible for von Hippel-Lindau disease, is implicated in this O2-sensing system by its association with HIF-1, targetingit for ubiquitin-mediated degradation. Similarly, F-box-containing proteins recognize substrates of the ubiquitin ligases, targeting them for phosphorylation-dependent ubiquitination and proteosomal degradation. In addition to F-boxes, most of these proteins also contain a WD40 or a leucine-rich repeat (LLR)domain that presumably functions as a Ser/Thr binding module. A second family of proteins assisting the ubiquitin ligases share a region designated SOCS-box(originally from the suppressor of cytokine signaling proteins SOCS). Under low O2 (<5% O2), HIF-1 is stabilized leading to the formation of a functionaltranscription factor complex with ARNT. This complex is the master regulator of O2 homeostasis and induces a network of genes involved in angiogenesis,erythropoiesis and glucose metabolism. Adapted: courtesy of Dr. Kosi Gramatikoff, Abgent, San Diego, CA, USA.

thereby contributing to microvascular leakage and monocyteextravasation (El Awad et al., 2000). This is in agreementwith another report which indicated that IL-1 induced HIF-1in human gingival and synovial fibroblasts, as noted above(Thornton et al., 2000).

The observation that cytokines, such as IL-1, possess theability to induce the expression and stability of HIF-1 undernormoxic conditions remains of particular interest. For in-stance, it has been reported that the normoxic induction ofHIF-1� by insulin and IL-1� involved the PI 3-kinase path-


way, followed by the down-regulation of EPO production(Stiehl et al., 2002). Recently, Haddad reported that recom-binant human IL-1�-mediated regulation of HIF-1� stabi-lization, nuclear translocation and activation required an an-tioxidant GSH/ROS-sensitive mechanism, indicating that anon-hypoxic pathway is mediating cytokine-dependent reg-ulation of HIF-1� (Haddad, 2002c,g). Similarly, as notedearlier, IL-1�-mediated up-regulation of HIF-1� required anNF-�B/COX-2 pathway that identified HIF-1 as a critical linkbetween inflammation and oncogenesis (Jung et al., 2003),knowing that NF-�B is a major player in the regulation ofgenes encoding cytokines.

Further elaborating on the essential mechanisms, it hasbeen observed that the normoxic induction of HIF-1� byIL-1� involved the redox-responsive extracellular signal-regulated kinase (ERK)1/2 pathway (mitogen-activated pro-tein kinase (MAPK)ERK1/2) in normal human cytotrophoblastcells (Qian et al., 2004). It has also been reported that IL-1� can upregulate the expression of epidermal growth fac-tor (EGF) and VEGF receptor (VEGFR), raising the pos-sibility that IL-1� might play an important role in VEGF-mediated neo-vascularization (Amano et al., 2004). Theaforementioned study demonstrated that IL-1� played akey role in ischemia-induced neo-vascularization essen-tially by mobilizing CD34−/B220−CD3−Flk-1+ endothe-l wella anda ,2

4CR-

m istic)i iningga iono ducts( y-p ines fect-ie a-i wasr tingt

4d

p tedi ionwp di wasi ationo ateN nd

IL-6 transcripts compared with hypoxia-exposed cells in thepresence of NF-�B inhibitors.

This suggested that pro-inflammatory cytokines up-regulated by this nuclear factor can result in fibrosis and affecthealing after endopyelotomy (Ruiz-Deya et al., 2002). Of in-terest, hypoxia, and apparently HIF, appeared to participatein the activation of this transcription factor. Furthermore, theexpression of pro-inflammatory cytokines via HIF-1� andNF-�B activation on desferrioxamine-stimulated mast cellsrevealed that cytokines seem to be under transcriptional reg-ulation in hypoxic conditions (Jeong et al., 2003).

4.2.4. Role for IL-8The regulation of IL-8, a chemokine, by hypoxia in hu-

man macrophages revealed a potential role in the pathogen-esis of many diseases, such as the acute respiratory dis-tress syndrome (ARDS) of the lung (Hirani et al., 2001).Rapidly raised intrapulmonary IL-8 levels were associatedwith ARDS progression in patients with major trauma. In ad-dition, acute hypoxia, a clinically relevant stimulus, rapidlyand selectively up-regulated IL-8 associated with a novelpattern of transcription factor activation (HIF). It was sug-gested that acute hypoxia may represent one of potentiallyseveral pro-inflammatory stimuli responsible for rapid in-trapulmonary IL-8 generation in patients at-risk of ARDS(

owthf turem ando fac-t ,2

4s dif-

fi n in-t entralr ho-g hosec ids,tt at-tt .

orat-i edd al-l rest.F to di-r pes.A inec y fork help-f ta

ial precursor cells in a VEGF-dependent manner ass by up-regulating expressions of VEGF, VEGFRdhesion molecules on endothelial cells (Amano et al.004).

.2.2. Role for IL-2In an interesting study, hypoxic exposure and T

ediated activation were reported to be additive (synergn enhancing levels of hypoxia response element-contaene products in lymphocyte supernatants (Caldwell etl., 2001). In contrast, hypoxia inhibited the accumulatf non-hypoxia response element-containing gene proe.g. IL-2 and IFN-�), suggesting that T cell activation in hoxic conditions may lead to different patterns of lymphokecretion and accumulation of cytokines (e.g. VEGF) afng endothelial cells and vascular permeabilization (Caldwellt al., 2001). Moreover, targeting of the VHL-HIF-hypoxi

nduced gene pathway for renal cell carcinoma therapyeported to implicate IL-2 and other cytokines, suggesherapeutic synergism (Sosman, 2003).

.2.3. Role for IL-6It has been reported that NF-�B was up-regulated an

ro-inflammatory cytokines, including IL-6, were activan patients with ureteropelvic junction (UPJ) obstructho failed endopyelotomy (Ruiz-Deya et al., 2002). Thoseatients with increased expression of NF-�B demonstrate

ncreases in IL-6 expression as well. In addition, HIFdentified in all the tissue samples tested and the stimulf the human urothelial cells by hypoxia, known to activF-�B, resulted in an increase in the levels of IL-1 a

Hirani et al., 2001).In another setting, it was reported that increased gr

actor production in a human prostatic stromal cell culodel caused by hypoxia involved the secretion of IL-8ther mediators, indicating that hypoxia might be a key

or contributing to prostate pathophysiology (Berger et al.003).

.2.5. Role for IL-12Metastatic renal cell cancer remains a disease which i

cult to treat medically. Prognosis often depends more orinsic disease features than on treatment choices. The cole of VHL in clear cell renal cell carcinoma (RCC) patenesis is conspicuous. Some clinically applied agents wlinical results were highlighted included 5-FU, retinohalidomide, razoxane and IL-12 (Fishman et al., 2001). Fea-ures of the pathophysiology of VHL were described, withention to potential novel therapies targeting HIF-1�, VEGF,ransforming growth factor (TGF)-�1 and TGF-� pathways

Of interest, most basic are cytokine therapies incorpng new IL-2 and IFN-� schedules. Newer cytokine-basrugs include pegylated forms and IL-12. In addition,

ogeneic mini-transplantation has generated much inteor example, tumor-associated antigens are being usedect therapy using both identified and non-identified epito

variety of tumor-cell vaccine and dendritic-cell vacclinical approaches are discussed. Finally, nephrectomnown metastatic disease has been demonstrated to beul in retrospective and now prospective trials (Fishman el., 2001).


4.2.6. Role for IL-15Expression of angiogenic factors was reported to have

been up-regulated in hyperplastic mucosa adjacent to coloncancer, and that this up-regulation was closely associated withcancer growth and metastasis (Kuniyasu et al., 2002/2003).In the hyperplastic mucosa adjacent to KM12SM tumorin the cecum of athymic mice, VEGF up-regulation wasassociated with HIF-1� induction. The hyperplastic mu-cosa also showed hypoacetylation of histone H4 and reduc-tion of both p53 and VHL proteins (Kuniyasu et al., 2002/2003).

To examine the effects of growth factors and cytokineson histone acetylation and levels of p53, VHL and HIF-1�,the rat intestinal epithelial cell line IEC6 was treated withEGF and IL-15. Notably, acetylated histone H4, p53 proteinand ubiquitinated protein levels were reduced, whereas HIF-1� production was up-regulated in EGF- and IL-15-treatedcells, suggesting that EGF- or IL-15-induced histone H4 hy-poacetylation is associated with repression of p53 and VHLgenes (Kuniyasu et al., 2002/2003). The subsequent suppres-sion of protein ubiquitination can lead to up-regulation ofVEGF production by HIF-1� retention.

4.2.7. Role for TNF-αTNF-� was reported to have a stimulatory effect on HIF-

1 -1( fa hy-p F-t e,2 theu fac-t oxiaa ts maya -r

no -r hiche x-p ibutet iveg F-d

tr thatt sioni entot ep-t type2 con-s glymE

Experimental evidence suggested the involvement ofp75TNFR in endothelial cell activation. Northern blot anal-ysis revealed that p75TNFR mRNA was up-regulated inNIH3T3 cells under hypoxia and reoxygenation. This ob-servation directly originated from transcriptional activationof the p75TNFR gene, as shown by reporter gene analysis(Hehlgans et al., 2001).

In addition, co-transfection experiments clearly showedthat the transcriptional induction of the p75TNFR gene wasindependent of HIF-1� and HIF-2�. Of particular inter-est, using deletion mutants of the 5’-flanking region of thep75TNFR gene, the authors were able to identify a putativeDNA binding site for NF-IL-6 to be responsible for the tran-scriptional up-regulation of the p75TNFR gene under condi-tions of hypoxia and reoxygenation.

Furthermore, Haddad and colleagues suggested anon-hypoxic, ROS-sensitive pathway mediating TNF-�-dependent regulation of HIF-� in alveolar epithelial cells(Haddad and Land, 2001b; Haddad, 2002c). TNF-� activatedthe translocation of HIF-1�, associated with up-regulating itsactivity under normoxia. Moreover, analysis of the mode ofaction of TNF-� revealed the accumulation of H2O2, O2

•−and •OH. Of note, antioxidants purported as prototypicalscavengers of H2O2 and •OH, attenuated TNF-�-inducedHIF-1� activation, and blockading NADPH-oxidase by scav-e •− ,i -d -pH

thyl-N mo-p cellp n bym llsi -i cinf -1e eenr iti-n y,ta beu y-p

HIF,c

5c

SOa es-s nw and

DNA binding activity in a manner similar to that of ILHellwig-Burgel et al., 1999). In addition, the induction odhesion molecules, partakers in local inflammation, withoxia were reported to have been induced by IL-1 and TN�

hat was caused with anoxia/reperfusion (Ohga and Matsus000). This was corroborated with the observation thatp-regulation of redox factor-1 (Ref-1), a transcription

or, promoted endothelial cell survival in response to hypnd TNF through NF-�B-independent and NF-�B-dependenignaling cascades, respectively, indicating that Ref-1ct as a critical cofactor mediating the TNF-induced NF�Besponse in the vascular endothelium (Hall et al., 2001).

Recently,Sandau et al. (2001)reported that the regulatiof the HIF-1� by the inflammatory mediators NO and TNF�equires diverse agonists under normoxic conditions, wmploy different signaling pathways. Similarly, HIF-1 eression in early wounds was reported to possibly contr

o the regulation of iNOS and VEGF, two HIF-1-responsenes intimately related to the process of repair, in a TN�-ependent stream (Albina et al., 2001).

On the mechanisms implicated with TNF-�-dependenegulation of HIF-1�, Hehlgans and colleagues reportedhe hypoxic up-regulation of TNF receptor type 2 expresnvolved nuclear factor-IL-6 (NF-IL-6) and was independf HIF-1 or HIF-2 (Hehlgans et al., 2001). TNF is well known

o exert its biologic activity via two distinct membrane recors: TNF receptor type 1 (p55TNFR) and TNF receptor

(p75TNFR). Whereas the p55TNFR gene is rathertitutively expressed, transcription of p75TNFR is stronodulated by a number of stimulatory agents (Akgul anddwards, 2003).

nging O2 reduced the activity of HIF-1�. Furthermorenhibition of the mitochondrion complex I abrogated TNF�-ependent activation of HIF-1�, while interrupting the resiratory chain reversed the excitatory effect of TNF-� onIF-1� (Haddad and Land, 2001b).In another setup, it was shown that capsaicin (8-me

-vanillyl-6-nonenamide), a known natural dietary chereventive agent, which inhibits malignant melanomaroliferation, has the ability to regulate VEGF expressioodulation of HIF-1� in human malignant melanoma ce

ndependent of IL-1 or TNF-� (Patel et al., 2002), suggestng that the inhibition of cellular proliferation by capsaiollows enhanced VEGF production by enhancing HIF�xpression and binding to HRE. Furthermore, it has beported that TNF-� induced the accumulation of a ubiquated form of HIF-1� through a NF-�B-dependent pathwa

hereby allowing optimum interaction with pVHL (Zhou etl., 2003). Reciprocally, inflammatory cytokines seem tonder HIF-1� or NF-�B transcriptional regulation under hoxia in mast cells (Jeong et al., 2003).

Schematic pathways signaling the association amongytokines and glutathione redox cycle are depicted inFig. 5.

. Glutathione and glutathione-related enzymes inytokine regulation: involvement of AP-1

LPS-induced activation of AP-1 was enhanced by Bnd inhibited by NAC, an effect correlating with the exprion of c-fos (Tanaka et al., 1997). In addition, AP-1 activatioas shown to occur during oxidant-induced cell necrosis


Fig. 5. Signaling transduction pathways mediating cytokine-dependent regulation of HIF and HIF-mediated regulation of cytokine via the glutathione-dependentpathway. Cytokines, such as IL-1� and TNF-�, initiate a cascade of receptor-mediated mechanisms, which diverge at the level of G-coupled receptors (Ras),upstream kinases involved with the phosphorylation of MAPKp38/MAPKJNK, and phosphatidylinositol-3 kinase (PI 3K) pathway. The release of cytokines isaugmented with prooxidant mechanisms, achieved with BSO and PDTC. Subsequent phosphorylation/activation mechanisms upregulate the biosynthesis ofthe� subunit of HIF, which couples with the constitutively expressed� subunit, thereby allowing nuclear translocation and transactivation of this transcriptionfactor. The activation of HIF-1� requires phosphorylation. This scenario is likely provided with the normoxic induction by cytokines, which stabilize the�

subunit, ostensibly via downregulation of VHL and/or upregulation of upstream kinases such as MAPKs.

contributed to cell death in a non-passive manner (Xu et al.,1997). Of interest, TNF-� and Dex modulated GSH levelsand�-GCS-HS mRNA expression by their effects on AP-1(c-Jun homodimer) (Rahman et al., 1999a,b).

Intracellular glutathione redox status was also reportedto modulate monocyte chemoattractant protein-1 (MCP-1)expression in pulmonary granulomatous vasculitis in a man-ner that was dependent on NF-�B and AP-1 (Desai et al.,1999). Moreover, Over-expression of�-GCS suppressedTNF-induced apoptosis and activation of AP-1 (Manna etal., 1999).

Cellular and molecular mechanisms of methamphetamine(METH)-induced neurotoxicity may involve alterations ofcellular redox status and induction of inflammatory genes inendothelial cells. It has been recently reported that METH-induced disturbances in cellular redox status and activation ofAP-1 and NF-�B can play critical roles in the signaling path-ways leading to upregulation of inflammatory genes in humanbrain endothelial cells (Lee et al., 2001). In concert, METH-induced TNF-� gene expression and activation of AP-1 indiscrete regions of mouse brain involved a potential role ofROS and lipid peroxidation (Flora et al., 2002).

Furthermore, reduction of DNA binding activity of con-sensus and potential AP-1 binding sites in mouse Fas ligandpromoter sequence was observed in nuclear extracts fromt aredw /R)( S

play an important role in regulation of cell death during cere-bral I/R by modulating intrinsic neuronal sensitivity as wellas brain inflammatory reactions.

The molecular mechanism of TGF-�1-induced glu-tathione depletion in alveolar epithelial cells involved AP-1.For example, a novel mechanism of�-GCSh down-regulationby TGF-�1 involved the binding of c-Jun and Fra-1 dimersto the distal promoter of the antioxidant response element(ARE) (Jardine et al., 2002). Of note, the formation of a c-Jun/Ski (oncoprotein) complex maintained the repressed stateof Smad2-responsive genes in the absence of ligand and par-ticipated in negative feedback regulation of TGF-� signalingby the MAPKJNK cascade (see below) (Pessah et al., 2002).

Similarly, the regulation of IL-8 by AP-1 involved a ROS-sensitive pathway. For instance, proteasome inhibitors werereported to stimulate AP-1 pathway via ROS production,suggesting that ROS production by proteasome inhibitorswhich led to AP-1 activation, even in the absence of NF-�B activation, still transactivated IL-8 gene expression (Wuet al., 2002). Moreover, intracellular thiols contributed to Th2function via a positive role in IL-4 production in an AP-1-dependent mechanism (Monick et al., 2003).

This was reinforced with another report indicating thatglutathione modulation in stretch-induced cytokine releasefrom alveolar type-2 like cells involved AP-1 (Jafari et al.,2 P-1D P70i

ransgenic mice over-expressing intracellular GPx compith normal animals following ischemia/reoxygenation (I

Ishibashi et al., 2002), indicating that GPx-sensitive RO

004). In addition, acute cadmium exposure enhanced ANA binding and induced cytokines expression and HS

n HepG2 cells, an effect that was attenuated by NAC (Souza


Fig. 6. Signaling pathways mediated by AP-1 in the regulation of cytokines,with particular involvement of GSH/GSSG (see text for further details).

et al., 2004). In jibe with the aforementioned, it was alsoshown that the hypoxic suppression ofE. coli-induced NF-�B and AP-1 transactivation, and cytokine production, re-quired oxyradical signaling mechanism (Matuschak et al.,2004).

Schematic pathways signaling the association among AP-1, cytokines and glutathione redox cycle are depicted inFig. 6.

6. Glutathione and glutathione-related enzymes incytokine regulation: involvement of MAPKs

Since MAPK signaling pathways have been extensivelyreviewed elsewhere (Haddad et al., 2003; Haddad, 2004c,d;Saklatvala, 2004), this topic will not be discussed here. How-ever, we elaborate on the relationship that governs the as-sociation between glutathione and cytokines, on one hand,and MAPKs, on the other hand. For clarity, MAPK signal-ing pathways and their ramifications have been depicted inFig. 7.

Cytokines, including pro-inflammatory TNF-� and IL-1,and anti-inflammatory IL-10, are known to induce theactivation of MAPKs (LoGrasso et al., 1998; Bogatchevaet al., 2003; Haddad et al., 2003; Saklatvala et al., 2003;H .,2 esw gedn of an withn

iso sedc vedha nd

SAPK using myelin basic protein (MBP) and human c-Jun(1-169) glutathioneS-agarose transferase (GST) fusionprotein as substrates. For comparison, neuroma fibroblastcultures were also stimulated with PMA and PDGF-AB,a potent activator for MAPK. Interestingly, TNF-� andboth isoforms of IL-1 produced a rapid activation ofMAPK.

Moreover, TNF-� combined with either IL-1� or IL-1�produced a synergistic effect on the activation of MAPK. Theincreases in MAPK induced by TNF-alpha and IL-1 weresimilar to the increases induced by PMA and PDGF-AB (Luet al., 1997). TNF-� and IL-1 also increased activationof SAPK, but to a lesser extent than MAPK. PMA andPDGF-AB were also much less effective in stimulatingactivation of SAPK. These findings indicate that cytokinesactivate parallel signal transduction pathways, and that theyare relatively stronger activators of MAPK than of SAPK.

As mentioned earlier on, expression of�-GCS play a ma-jor role cytokine-mediated regulation of transcription fac-tors and MAPKs (seeFig. 3). For example,�-GCS abolishedthe activation of AP-1 induced by TNF and inhibited TNF-induced activation of MAPKJNK and MAPKK (Manna et al.,1999). Moreover, it was reported that NAC attenuated TNF-�-induced MAPKp38 activation and regulated on activationnormal T cell expressed and secreted (RANTES) productionie

a tiont dBp -e UrMN NF-�8 cells(a -i par-t

F-� izedt ingLs ants( e-l pho-r iono thes )a rh -s enceo

ofman, 2004; Shakoory et al., 2004; Watanabe et al004). For instance, activation of MAPKs by cytokinas shown to stimulate fibroblast proliferation in damaerves and thereby played a role in the formationeuroma, a disorganized mass of tissue that interfereseural regeneration and repair (Lu et al., 1997).

To investigate the possibility that this mechanismperative in neuroma formation, Lu et al. (1997) uultured, serum-starved fibroblasts from surgically remouman neuromas stimulated with TNF-� and/or IL-1�nd IL-1�, and measured the activation of MAPK a

n human pulmonary vascular endothelial cells (Hashimotot al., 2000).

In addition, BSO facilitated TNF-�-induced MAPKp38

ctivation and RANTES production, with the observahat SB-203580, a specific inhibitor of MAPKp38, attenuateSO-mediated augmentation of TNF-�-induced RANTESroduction (Hashimoto et al., 2000, 2001a). In concert, lowring the intracellular GSH/GSH disulfide ratio by BCNesulted in the culminant enhancement of MAPKJNK andAPKp38but not MAPKERK (Hehner et al., 2000). Similarly,AC and the GSH/GSSG ratio were shown to attenuate T-induced MAPKp38 activation and MAPKp38-mediated IL-production by human pulmonary vascular endothelial

Hashimoto et al., 2001b), and IL-12 production (Utsugi etl., 2002). In addition, MAPKJNK negatively regulated LPS

nduced IL-12 production in human macrophages, withicular involvement of glutathione (Utsugi et al., 2003).

Redox and ROS regulation of MAPK-mediated TNbiosynthesis was recently reported. It was hypothes

hat the involvement of the MAPK pathway in regulatPS-mediated TNF-� secretion is redox-dependent, NF-�B-ensitive and attenuated by NAC and other antioxidHaddad and Land, 2002b). For instance, in alveolar epithial cells, LPS induced a time- and dose-dependent phosylation of MAPKp38. This was associated with the activatf MAPK-activated protein kinase, which phosphorylatedmall Hsp27. Moreover, MAPKp38 inhibition (SB-203580brogated LPS-induced TNF-� production. On the otheand, MAPKERK blockade (PD-98059) attenuated TNF�ecretion, an effect synergistically amplified in the presf SB-203580 (Fig. 8).


Fig. 7. MAPK signaling pathways and their ramifications. The ever evolving MAPK pathways consist of four major groupings and numerous related proteinswhich constitute interrelated signal transduction cascades activated by stimuli such as growth factors, stress, cytokines and inflammation. The four majorgroupings are the Erk, JNK or SAPK, p38 and the Big MAPK or ERK5 cascades. Signals from cell surface receptors such as GPCRs and growth factorreceptors are transduced, directly or via small G proteins such as ras and rac, to multiple tiers of protein kinases that amplify these signals and/or regulateeach other. The diagram is organized to illustrate the cascades by the background colors and also the tiers of kinases as indicated down the left hand side andseparated by the horizontal dashed lines. In some cascades, the first activation tier involves the MAPKKKKs, MAP kinase kinase kinase kinases or MAP4Kproteins. The next tier are the serine/threonine MAPKKKs, MAP kinase kinase kinase or MAP3Ks such as RAF, TAK, ASK, and MEKK1. This level has thegreatest amount of cross-communication currently known. The serine/threonine/tyrosine MAPKKs, MAP Kinase kinases or MAP2Ks, such as the MKK andMEK kinases, are one step up from the MAP kinase cascade, phosphorylating and activating these kinases. The focal tier, the MAPKs or MAP kinases includesJNK1, p38, and ERKs, and are the kinases that give each cascade its name. The endpoints of these cascades, shown in the bottom tier, include the MAPKactivated protein kinases (MAPKAPK) and some of the numerous transcription factors that regulate genes involved in apoptosis, inflammation, cell growth anddifferentiation. Adapted, courtesy of Biocarta. Compare with Fig. 3 (Haddad, 2004d).

Of interest, regulation of NF-�B by selective inhibitorsrevealed that this pathway is partially involved in regulat-ing LPS-mediated TNF-� secretion, supported by previ-ous observations (Haddad, 2002b,c,d,e). Whereas the pro-teasome inhibitor, MG-132, had no effect on LPS-mediatedTNF-� production, caffeic acid phenethyl ester (CAPE), sul-fasalazine and SN-50, a cell-permeant NF-�B inhibitor, at-tenuated but did not abrogate TNF-� biosynthesis (Haddadand Land, 2002b).

Furthermore, LPS up-regulated ROS, an effect abro-gated by 4′-hydroxy-3′-methoxy-acetophenone and NAC,

which reduced TNF-� secretion, induced the accumulationof GSH, reduced the concentration of GSSG, and block-aded the phosphorylation/activation of MAPKp38 pathway.ROS also induced MAPKp38 phosphorylation and selec-tive antioxidants, including the permeant GSH precursor,�-GCE, reduced ROS-dependent MAPKp38 phosphorylation,indicating that the MAPK pathway and MAPK-mediatedregulation of TNF-� production is redox-dependent, GSH-mediated and requires, at least in part, a NF-�B/ROS-sensitive mechanism (Haddad, 2002a; Haddad and Land,2002b).


Fig. 8. The effect of selective inhibition of MAPKERK on LPS-induced TNF-� biosynthesis in alveolar epithelial cells. (A) Pre-treatment with PD-98059 (2 h),a selective inhibitor of MAPKERK, prior to exposure to LPS (1�g/ml), reduced LPS-induced TNF-� production in a dose-dependent manner. LPS up-regulatedthe secretion of TNF-� approximately four-fold relative to cells incubated in medium alone (control). Pre-treatment with PD-98059 (100�M) on its own did notaffect TNF-� synthesis. PD-98059 reduced LPS-induced TNF-� secretion at doses≥0.1�M, with maximum inhibition at 100�M (IC50 = 5.12± 0.47�M).(B) Pre-treatment with a combination of SB-203580 and PD-98059 synergistically reduced, in a dose-dependent manner, the induction of TNF-�, effective atdoses≥ 0.01�M, with maximum and complete suppression at 100�M (IC50 = 0.023± 0.003�M). �P< 0.05, as compared with control;*P< 0.05,** P< 0.01,*** P< 0.001, as compared with LPS (1�g/ml).n= 3, which represents the number of independent experiments run in duplicate with separate cell preparations(Haddad and Land, 2002b).

7. Conclusion, summary and prospects

Oxidative signaling and free radical research in relation toimmunity and inflammation continues apace—a well defined,yet burgeoning, field in molecular immunology (Haddad,2001b; Haddad, 2004e,f). The association that is evolvingbetween an oxidative signal and inflammation leverages aspecific pathway(s) that involves the targeting of potentialtranscription factors. MAPK signaling ramifications are nointruders to be closely motivated in defining this association,given the fact that phosphorylation/dephosphorylation mech-anisms are crucial redox-sensitive pathways for transcrip-tional regulation. The immunopharmacology of antioxidants,particularly glutathione, shapes redox equilibrium in regulat-ing cytokines and associated cofactors, thereby keeping ox-idative stress offensive at bay. These defensive mechanismsare crucial for survival, and the imbalance that may destabi-lize this intricate oxidant-inflammation association can beardetrimental consequences.

In recap, antioxidant and antioxidant-related enzymesin the regulation of inflammatory mediators entail hierarchi-cally arranged mechanisms: (i) an antioxidant/prooxidantsignal is perceived in the context of modulating redox equilib-rium; (ii) when a prooxidant signal prevails, an inflammatoryresponse is augmented and, in the absence of an orchestrateda ation;( olst tors,n ac-t an-s ract,g hal-

lenge, with the prospects of limiting the debilitating effectsof free radicals.

A promising avenue is to further decipher the un-derlying molecular mechanisms associated with oxidativestress—immunologic and oxygen sensors have just been de-fined; what remains to be elucidated is the thread-like con-nection that brings together immunology and redox(y)ology,an association that may well uncover therapeutic strategiesfor alleviating oxidant stress and oxidant-associated inflam-matory diseases.

Acknowledgements

The first author’s publications are, in part, financially sup-ported by the Anonymous Trust (Scotland), the National In-stitute for Biological Standards and Control (England), theTenovus Trust (Scotland), the UK Medical Research Council(MRC, London), the Wellcome Trust (London) and the Na-tional Institutes of Health (NIH; USA). Dr. John J. Haddadheld the Georges John Livanos fellowship (London, UK) andNIH postdoctoral fellowship (UCSF, CA, USA). We thankour colleagues at the American University of Beirut for help-ful comments and critical reading of the manuscript.

R

A ible75.

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ntioxidant defense, cytokines may exacerbate the situiii) this delicate antioxidant/prooxidant equilibrium contrhe routes that lead to the activation of downstream effecotably MAPKs, which in turn, along with upstream cof

ors, signal the regulation of transcriptional entities; (iv) trcription factor regulation ensues to conceive a counteenetically-defined response in the face of oxidative c

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