Thiol Regulation of Pro-Inflammatory Cytokines Reveals a Novel Immunopharmacological Potential of Glutathione in the Alveolar Epithelium

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1 /01/ $3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 296, No. 3 Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 3263/ JPET 296: , 2001 Printed in U.S.A. Thiol Regulation of Pro-Inflammatory Cytokines Reveals a Novel Immunopharmacological Potential of Glutathione in the Alveolar Epithelium JOHN J. E. HADDAD, BARED SAFIEH-GARABEDIAN, NAYEF E. SAADÉ, and STEPHEN C. LAND Oxygen Signaling Group, Center for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, United Kingdom (J.J.H., S.C.L.); and the Departments of Biology (B.S.-G.), Faculty of Arts and Sciences, Human Morphology and Physiology (N.E.S.), Faculty of Medicine, American University of Beirut, Beirut, Lebanon Received August 25, 2000; accepted November 17, 2000 This paper is available online at ABSTRACT The therapeutic immunopharmacological potential of glutathione in the alveolar epithelium is not well characterized. We developed an in vitro model of fetal alveolar type II epithelial cells to investigate the effect of redox disequilibrium on chemioxyexcitation ( po 2 /ROS) induced up-regulation of pro-inflammatory cytokines. Buthionine sulfoximine, an irreversible inhibitor of -glutamylcysteine synthetase, the rate-limiting enzyme in glutathione (GSH) biosynthesis, induced intracellular reactive oxygen species (ROS) and the release of interleukin-1 (IL-1 ), IL-6, and tumor necrosis factor-. Chloroethyl nitrosourea, which blocks the NADPH-dependent recycling of oxidized glutathione (GSSG), reduced ROS-induced cytokine production, similar to pyrrolidine dithiocarbamate, an antioxidant/pro-oxidant thiuram, which elevates GSSG. The antioxidant and GSH precursor, acetylcysteine, abrogated cytokine release concomitant with suppression of ROS, an effect mimicked by -glutamylcysteinyl-ethyl ester, a cell permeant GSH. Cysteine, the rate-limiting amino acid in the de novo synthesis of GSH, administered as oxothiazolidine carboxylate and adenosylmethionine, mitigated the chemioxyexcitation-dependent cytokine release. Ebselen, an anti-inflammatory antioxidant, which mimics the effect of glutathione peroxidase, neutralized ROS by the GSH-peroxidase-coupled reaction, thereby blocking the pathway to cytokine enhancement. Our results indicate that modulating redox equilibrium by pharmacological thiols exhibits differential regulation on pro-inflammatory cytokines, thus bearing clinical consequences for the therapeutic treatment of pediatric distresses in pathophysiology. This work was supported by grants from the Medical Research Council, Anonymous Trust and Tenovus-Scotland (S.C.L.). J.J.H. is a recipient of the George John Livanos prize (London). Part of this work was presented at Experimental Biology-2000, San Diego, CA (Haddad et al., 2000b). The tripeptide L- -glutamyl-l-cysteinyl-glycine, or glutathione (GSH), a ubiquitous thiol, plays a major role in maintaining intracellular redox balance and regulating pathways augmented by oxidative stress (Meister, 1988; Haddad and Land, 2000a; Haddad et al., 2000a). The cysteinyl moiety of GSH provides the reactive thiol as a functional element responsible for the diverse properties of glutathione, whose participation in the physiology of metabolism reflects its importance in intracellular functions. These include: 1) an antioxidant potential mediated by the peroxidase-coupled reaction; 2) regulation of cellular sulfhydryl status and redox equilibrium; 3) governing pathways in neuro-immune-endocrine interactions as a neurotransmitter and an immunopharmacological thiol; and 4) regulation of the expression/ activation of redox-sensitive transcription factors induced by stress-evoked responses (Dröge et al., 1994; Hayes and McLellan, 1999; Haddad et al., 2000a). The pivotal role of redox cycle in maintaining the integrity of the biological system in the face of oxidative stress is, therefore, of particular clinical relevance. The biomarkers of oxidative stress, such as antioxidant inefficiency, redox disequilibrium, and derivation of oxidant radicals, for instance, may arise from conditions other than hyperoxia (oxidizing signals) per se, such as hypoxia/reoxygenation and cytokine-dependent processes (Thom et al., 1997; Haddad and Land, 2000a). In physiological conditions, the intracellular redox status of thiols is highly reductive. GSH, for example, is present in high concentrations in lung ABBREVIATIONS: GSH, L- -glutamyl-l-cysteinyl-glycine; NAC, N-acetyl-L-cysteine; SAM, S-adenosyl-L-methionine; BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea; BSO, L-buthionine-(S,R)-sulfoximine; DHR, dihydrorhodamine; -GCE, -glutamyl-l-cysteinyl-ethyl ester; GSSG, glutathione disulfide oxidized; GSH-PX, glutathione peroxidase; GSSG-RD, glutathione reductase; -GCS, -glutamylcysteine synthetase; GS, glutathione synthase; IL, interleukin; OTC, 2-oxothiazolidine-4-carboxylate; ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygen species; redox, reduction-oxidation; TNF-, tumor necrosis factor- ; X/XO, xanthine/xanthine oxidase; DMEM, Dulbecco s modified Eagle s medium; FCS, fetal calf serum; EU, enzyme unit activity; HIF1-, hypoxia-inducible factor 1 ; NF- B, nuclear factor- B. 996

2 Thiopharmacological Regulation of Pro-Inflammatory Cytokines 997 epithelial lining fluid (Cantin et al., 1989) and has been reported to maintain the integrity of the airspace epithelium in vitro and in vivo (Li et al., 1997). In contrast, GSH depletion has been linked to the pathophysiology of idiopathic pulmonary fibrosis (Cantin et al., 1989), adult respiratory distress syndrome (Bunnell and Pacht, 1993), bronchopulmonary dysplasia (Saugstad, 1997), and cystic fibrosis (Roum et al., 1993), thus highlighting its central role in maintaining the functional integrity of a physiologically competent epithelium. There is growing evidence, moreover, supporting the notion that oxidative conditions modulate redox-linked pathways by altering the dynamic equilibrium of glutathione homeostasis (Haddad et al., 2000a). Exogenous/endogenous agents, which induce the formation of ROS, for example, can affect redox homeostasis by up-regulating antioxidant enzymes, particularly glutathione peroxidase and enzymes involved in glutathione recycling and biosynthesis (Douglas, 1987; Goss et al., 1997; Li et al., 1997; Haddad and Land, 2000a). Furthermore, ROS signaling could be mediated by cytokines, peptide hormones, and immunoregulators, whose participation in cellular pathways is modulated by redox status (Rovin et al., 1997; Pena et al., 1999; Yamashita et al., 1999; Haddad et al., 2001). Conversely, cytokines, which themselves are mediators of oxidative stress (Nussler et al., 1992; Desmarquest et al., 1998; Yamashita et al., 1999), have the potential to alter redox equilibrium, thereby affecting GSH/oxidized glutathione disulfide (GSSG) shuttling and recycling (Chen et al., 1998). How chemioxyexcitation [ po 2 / reactive oxygen species (ROS)] induction of cytokines modulate signaling pathways in oxidative stress through redox equilibrium in the fetal alveolar epithelium has yet to be ascertained. Furthermore, pharmacological manipulation of glutathione homeostasis in perinatal epithelia and its effects on cytokine-mediated responses are not known; subsequently, unraveling the biochemistry of redox-linked pathways bears a typical clinical approach for diagnosing pathophysiological conditions in the developing lung. The immunopharmacological potential assigned to glutathione (Thompson et al., 1985) stems from established observations. Interleukin-1 (IL-1)-induced responses, for instance, occur through modulating redox equilibrium (Rovin et al., 1997). In addition, ROS signaling regulating the transcription of IL-4 (Jeannin et al., 1995), IL-6, IL-8 (Gosset et al., 1999), and tumor necrosis factor- (TNF- ) (Neuschwander- Tetri et al., 1996; Gosset et al., 1999) occurs through a thioldependent mechanism. Interestingly, antioxidants (Reimund et al., 1998; Barrett et al., 1999) and glutathione precursors (Jeannin et al., 1995; Pena et al., 1999) have been shown to down-regulate cytokine synthesis, activation, and downstream processes. Among several agents that were used for repletion and depletion of GSH, N-acetyl-L-cysteine (NAC) and L-buthionine-(S,R)-sulfoximine (BSO) are, respectively, of particular importance as they exhibit antagonistic effects on a pro-inflammatory signal. NAC, an antioxidant and a GSH precursor (Bernard, 1991; Haddad et al., 2000a), ameliorates cytokine production (Tsuji et al., 1999) and ROSmediated lung injury (Bernard, 1991). In contrast, BSO, which depletes GSH by irreversibly inhibiting -glutamyl-lcysteinyl-ethyl ester ( -GCS), the rate-limiting enzyme in the biosynthesis of glutathione (Griffith and Meister, 1979; Haddad and Land, 2000a), has the potential to enhance cytokine secretion by up-regulating ROS (Gosset et al., 1999). We reasoned that a differential manipulation of glutathione homeostasis and shuttling may antagonistically affect a proinflammatory signal, thus bearing potential consequences for the treatment of respiratory distresses, where cytokines are recognized as major participants in their pathophysiology (Saugstad, 1997). This study elaborates in vitro an immunopharmacological potential of glutathione in the perinatal epithelium. Accordingly, we derive the hypotheses that 1) chemioxyexcitation ( po 2 /ROS) regulation of intracellular redox homeostasis is dependent on the flux kinetics and duration of ROS exposure, and 2) glutathione depletion and repletion differentially manipulate pro-inflammatory cytokines, an effect antagonistically reversed by restoring redox equilibrium. The mechanisms implicated in redox-associated cytokine pathways are thereafter developed in the light of the novel role of glutathione as an immunopharmacological regulatory thiol. Materials and Methods All experimental procedures involving the use of live animals were approved under the Animals Act legislation, 1986 (UK). Chemicals/ reagents were obtained from Sigma Chemical Co. (St. Louis, MO) and Calbiochem (La Jolla, CA). Primary Cultures of Alveolar Epithelia. Fetal alveolar type II (fatii) epithelial cells were isolated from lungs of rat fetuses on gestation day 19, essentially as described elsewhere (Haddad and Land, 2000a,b). Briefly, fetal rats were removed from pregnant Sprague-Dawley rats by cesarean section at day 19 of gestation (term 22 days), and the lungs were excised, teased free from heart and upper airway tissue, and finely minced then washed free of erythrocytes using sterile, chilled Mg 2 - and Ca 2 -free Hanks balanced salt solution. The cleaned lung tissue was resuspended in 1 ml/fetus Hanks balanced salt solution containing trypsin (0.1 mg/ ml), collagenase (0.06 mg/ml), and DNase I (0.012% w/v), and was agitated at 37 C for 20 min. The solution was then centrifuged at 100g for 2 min to remove undispersed tissue, the supernatant was saved to a fresh sterile tube, and an equal volume of Dulbecco s modified Eagle s medium (DMEM) with 10% (v/v) fetal calf serum (FCS) was added to the supernatant. After passing the supernatant through a 120- m pore sterile mesh, the filtrate was centrifuged at 420g for 5 min, the pellet was resuspended in 20 ml of DMEM/FCS, and the cells were placed into a T-150 culture flask for 1hat37 C to enable fibroblasts and nonepithelial cells to adhere. Unattached cells were washed three times by centrifugation at 420g for 5 min each and then seeded onto 24-mm diameter Transwell-clear permeable supports (0.4- m pore size, Costar, Cambridge, MA) at a density of cells per filter and were allowed to adhere overnight at fetal distal lung po 2 (23 torr 3% O 2 /5% CO 2 ). DMEM/FCS was exchanged for 4 ml of serum-free PC-1 media (BioWhittaker, Walkersville, MD) pre-equilibrated to po 2 23 torr and 37 C 24 h later, and cells were maintained at this po 2 until the experiment. The change in O 2 equilibrium from fetal to postnatal environments constitutes a potential signaling mechanism in the perinatal lung. The choice of this form of oxidative stress was based on its clinical relevance of the situation resembling the birth transition period and beyond (Haddad and Land, 2000a; Haddad et al., 2000a). Consequently, shifts in po 2 were recreated where cells were cultured at fetal po 2 (23 torr), followed by control period at the same po 2 or re-equilibrated to early postnatal po 2 (100 torr), mild (152 torr), and severe hyperoxia (722 torr). In each case, and under conditions of independent pretreatments, the adenylate energy charge, an index of cell viability and competence, remained at 0.7, and transepithelial monolayer resistance was maintained constant at 250 to 350 cm 2 (Haddad and Land, 2000a). The static cellular energy charge of cell cultures pretreated with various drugs rules out any nonspecific

3 998 Haddad et al. cytotoxicity. To further confirm this fact, the total protein content of pretreated cells was determined to be insignificantly different (P 0.05) from control cultures (data not shown). In addition, alveolar pretreatment has been shown to intervene specifically at the level of the cell cycle events (such as with p53), and at the level of factors that are key components of the signaling pathways governing apoptosis (such as with Bax and Bcl-2 proto-oncogenes), suggesting specificity rather than necrotic toxicity (Haddad and Land, 2000b). We have also shown that these drugs selectively interfere with the stabilization, nuclear translocation, and consensus DNA binding activity of the redox-sensitive transcription factors, hypoxia-inducible factor 1 (HIF-1 ) and nuclear factor- B (NF- B), implicating specific intervention in signaling transduction pathways (J. J. E. Haddad, B. Safieh-Garabedian, N. E. Saadé, and S. C. Land, unpublished observations). Enzyme-Linked Immunosorbent Assay Assessment of the Cytokine Profile. Cytokines in cell-free supernatants were measured by sandwich enzyme-linked immunosorbent assay. Polyclonal antibodies (2 g/ml) were used to coat high binding microtiter plates (Safieh-Garabedian et al., 1997). Recombinant and biotinylated immunoglobulins (a generous gift from Dr. Stephen Poole, National Institute for Biological Standards and Control, UK) were used for capturing, followed by color development with streptavidin-polyhorseradish peroxidase and tetramethylbenzidine dihydrochloride. Inter- and intra-assay coefficients of variations at 450 nm were 10%, and the minimum detectable sensitivity was 2 pg/ml. Thiol Regulation of Intracellular ROS with Ascending po 2 Regimen (Oxyexcitation). To determine H 2 O 2 production, cells were coated in microtiter plates (10 5 /well) and incubated overnight at either 23 or 152 torr, followed by pretreatment (24 h) with BSO (50 M), 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU; 100 M), pyrrolidine dithiocarbamate (PDTC; 100 M), NAC (50 mm), -GCE (100 M), or ebselen (100 M). Phenolsulfonphthalein containing horseradish peroxidase (20 U/ml) was added followed by shifting to ascending po 2. The reaction was terminated with 1 M NaOH and measured at 600 nm (Pick and Mizel, 1981). A standard curve (0 100 M H 2 O 2 ) was developed, and results were converted to nmol mg 1 of protein. To determine O 2 production, cells following pretreatments were covered with 80 M ferricytochrome c suspended in phenol red solution. The amount of O 2 released was measured at 550 nm against blanks containing ferricytochrome c and superoxide oxidoreductase dismutase (300 U/ml) (Pick and Mizel, 1981). Experiments were performed in duplicates, and data are presented as O 2 released based on nanomoles of reduced cytochrome mg 1 of protein. The hydroxyl radical reacts with dihydrorhodamine (DHR) to yield water and a tertiary free radical, which is rather stable. This radical undergoes rearrangement of the electrons, leading to formation of fluorescent rhodamine (Weiss et al., 1978). To determine OH production, cells were pretreated before oxyexcitation in the presence of 50 M DHR. Fluorescence was measured at 485/535 nm excitation and emission wavelengths, respectively. The OH level measured under hypoxia was calibrated to 100%, and variables were plotted against this baseline as logarithmic fluorescence units. Intracellular Redox Homeostasis on Exposure to ROS (Chemiexcitation). To evaluate whether chemioxyexcitation exposure (X/ XO, 100 M/2 mu/ml; H 2 O 2, 250 M) modulates redox potential, we determined the equilibrium ratio GSH/GSSG. Following incubations, filters were treated with 7% perchloric acid, then centrifuged at 10,000g for 5 min. Glutathione concentration (24 h) after neutralization with 3 M KHCO 3 was spectrophotometrically determined (Haddad and Land, 2000a). The assay conditions for determining the activities of enzymes involved in glutathione homeostasis are detailed elsewhere (Haddad and Land, 2000a). Specific activities of glutathione peroxidase (GSH- PX), glutathione reductase (GSSG-RD), -GCS, and glutathione synthase (GS) determined in cytosolic extracts of cells treated with X/XO and H 2 O 2 are expressed as units mg 1 of protein, where 1 unit (U) of enzyme activity is the amount that catalyzes the formation of 1 mol of product/min. All assays were conducted at 30 C. To determine GSH-PX activity, cytosolic extracts were incubated in PBS buffer containing 5 mm EDTA, 10 mm NAD(P)H, GSSG-RD (100 U/ml), M NaN 3 (a catalase inhibitor), and 150 mm GSH in a final volume of 1 ml. The enzymatic reaction was initiated by addition of 100 l of2mmh 2 O 2 (30%; M), and the linear rate of conversion of NADPH/H to NADP at 340 nm between 0 and 5 min after initiation of the reaction, was followed. The rate of oxidation of NAD(P)H by GSSG at 30 C was used as a standard measure of the enzymatic activity of GSSG-RD, by monitoring the rate of formation of NADP at 340 nm between 0 and 5 min after addition of the sample. The enzyme activity of -GCS was determined in a reaction mixture (1 ml) containing Tris-HCl (100 mm, ph 8.2), sodium L-glutamate (10 mm), Na 2 -ATP (5 mm), sodium phosphoenol pyruvate (2 mm), KCl (150 mm), NADH (0.2 mm), pyruvate kinase (5 U; bovine heart type III), and lactate dehydrogenase (10 U; rabbit heart type II). The reaction was initiated by adding the sample, and the rate of NAD formation was followed at 340 nm. GS activity was assayed in a reaction mixture containing Tris-HCl (100 mm; ph 8.2 at 30 C), KCl (50 mm), L- -glutamyl-l- -aminobutyric acid (5 mm), ATP (10 mm), glycine (5 mm), MgCl 2 (20 mm), EDTA (2 mm), and sample (added last) in a final volume of 0.1 ml. Added to this was 0.02 ml of 10% sulfosalicylic acid and 0.9 ml of a buffer containing phosphoenolpyruvate (0.5 mm), NADH (0.2 mm), pyruvate kinase (1 U), MgCl 2 (40 mm), KCl (50 mm), and K 2 HPO 4 (250 mm; ph 7.0). The reaction was initiated with 1 unit of lactate dehydrogenase, and the rate of NAD formation was followed as above. Regression analysis was performed to determine the degree of correlation between enzyme activities (units mg 1 of protein) and chemioxyexcitation. Redox Homeostasis and Chemioxyexcitation-Induced Cytokine Secretion. To determine the effect of redox disequilibrium on cytokine release, cells were pretreated (24 h) with: 1) BSO, a specific and irreversible inhibitor of -GCS (Griffith and Meister, 1979); 2) BCNU, a specific inhibitor of GSSG-RD (Hardwick et al., 1990); and 3) PDTC, an antioxidant/pro-oxidant, which elevates GSSG (Schreck et al., 1992; Haddad et al., 2000a). Cells were exposed to chemioxyexcitation, and supernatants were collected 24 h later and assessed for cytokines. The hypothesis that replenishing GSH would interfere with the capacity to produce cytokines has been investigated with exogenous precursors. Cells were pretreated (24 h) with: 1) -GCE, a membrane-permeating precursor (Okamota et al., 1999); 2) NAC, an antioxidant and GSH precursor (Bernard, 1991; Haddad et al., 2000a); 3) 2-oxothiazolidine-4-carboxylate (OTC; 100 M), a cysteine pro-drug (Anderson and Luo, 1998); and 4) S-adenosyl-L-methionine (SAM; 100 M), an antioxidant and a precursor of GSH (Evans et al., 1997). Following pretreatments, cells were challenged and supernatants were collected for cytokine assessment. Selective Modulation of Redox-Sensitive Enzymes and Regulation of Cytokines. Glutathione is postulated as a negative modulator of cytokine release, but whether this effect represents an antioxidant property has to be determined in the alveolar epithelium. Cells were pretreated with BSO for 24 h, before simultaneous incubation with NAC and chemioxyexcitation, followed by analysis of cytokines. Separately, we tested the effect of ebselen, a membrane permeant GSH-PX mimetic and an antioxidant (Schewe, 1995), on chemioxyexcitation-induced cytokine release. Statistical Analysis and Data Presentation. Data are the means and the error bars the S.E.M. of at least three independent cell cultures. Statistical evaluation was performed by one-way analysis of variance (ANOVA), followed by post hoc Tukey s test, and the a priori level of significance at 95% confidence level was considered at P Results Thiol Regulation of H 2 O 2 Production. The profile of H 2 O 2 formation in response to oxyexcitation is differentially regulated by glutathione-modulating agents (Fig. 1). The

4 Thiopharmacological Regulation of Pro-Inflammatory Cytokines 999 Fig. 1. Thiol regulation of intracellular H 2 O 2 in response to oxyexcitation. A through E, the formation of H 2 O 2 at 24 h is enhanced with ascending po 2 ( P 0.05, P 0.01, P 0.001, as compared with po 2 23 torr). BSO synergistically potentiated oxyexcitation-induced H 2 O 2 production ( P 0.05, as compared with control). BCNU and PDTC, as agents that elevate GSSG, abrogated the induced production of H 2 O 2. NAC and -GCE, as precursors of GSH, substantially blocked H 2 O 2 formation, an effect mimicked by ebselen (*P 0.05, **P 0.01, ***P 0.001, as compared with control). level of H 2 O 2 is increased with ascending po 2 regimen at 24 h. Although BSO potentiated oxyexcitation-dependent H 2 O 2 production, BCNU, PDTC, NAC, -GCE, and ebselen abrogated this effect. Thiol Regulation of O 2 Production. The variation in O 2 production with thiol-modulating agents is shown in Fig. 2. The level of O 2 is increased with ascending po 2 regimen at 24 h. BSO increased the release of O 2 at 23, , and torr (24 h), whereas BCNU, PDTC, NAC, -GCE, and ebselen abrogated its formation. Thiol Regulation of OH Production. The conversion of DHR to fluorescent rhodamine in the presence of OH is shown in Fig. 3. The profile of OH release in response to thiol-modulating agents is determined with selective inhibitors of enzymes involved in glutathione homeostasis. BSO induced accumulation of OH, an effect potentiated by oxyexcitation. Conversely, BCNU, PDTC, NAC, -GCE, and ebselen mediated suppression of OH with ascending po 2 regimen. Redox Equilibrium and Chemioxyexcitation. We investigated the hypothesis that chemiexcitation synergistically act with oxyexcitation to alter the redox state in favor of a reduction equilibrium. Exposure to H 2 O 2 (Fig. 4A) and X/XO (Fig. 4B) elevated [GSH] with ascending po 2 regimen. [GSSG] was markedly depressed with either treatment, such that the ratio GSH/GSSG is increased 4- to 15-fold (H 2 O 2 ) and 5- to 7-fold (X/XO) relative to controls without ROS exposure. ROS Effect on Redox-Sensitive Enzymes. The transition from fetal to neonatal po 2 attenuates the activity of enzymes involved in maintaining homeostatic levels of intracellular glutathione (Haddad and Land, 2000a). To determine whether exposure to ROS modulates their activities, we evaluated the role of O 2 and OH on GSH-PX, GSSG-RD, -GCS, and GS. The dose-dependent analysis of enzyme activities (EU) is shown in Fig. 5. X/XO exposure induced the activity of GSH-PX (Fig. 5A), -GCS (Fig. 5E), and GS (Fig. 5G), but not that of GSSG-RD (Fig. 5C). Inductive effects are evident at , , and po 2 torr, where the linear regression analysis shows significant correlation between EU and [XO]. Exposure to the OH-generating system significantly induced the activities of GSH-PX (Fig. 5B), -GCS (Fig. 5F), and GS (Fig. 5H) but marginally GSSG-RD (Fig. 5D). The effect of H 2 O 2 is more prominent on enzyme activities than that of X/XO. Linear regression analysis reveals significant correlation between EU and H 2 O 2. GSH Depletion and Chemioxyexcitation-Induced Cytokine Secretion. As shown in Table 1, BSO up-regulated the release of IL-1, IL-6, and TNF-, an effect synergistically enhanced in response to chemiexcitation ( torr). BCNU down-regulated the cytokine profile in a manner similar to PDTC and ebselen. Similar results were reported at other po 2 tensions (data not shown). GSH Repletion and Chemioxyexcitation-Induced Cytokine Secretion. As shown in Table 2, pretreatment with cysteine precursors NAC, OTC, and SAM down-regulated chemioxyexcitation-induced IL-1, IL-6, and TNF- release ( torr). The esterified glutathione-permeating precursor -GCE mimicked the effects of cysteine precursors by inhibiting the release of cytokines. Similar results were reported at other po 2 tensions (data not shown).

5 1000 Haddad et al. Fig. 3. Thiol regulation of intracellular OH in response to oxyexcitation. The reference value obtained at static 23 torr was set to 100%, and as such the corresponding variations are set against this baseline (dotted line). A through E, OH kinetics following 24-h exposure to oxyexcitation ( P 0.05, P 0.01, as compared with po 2 23 torr). BSO synergistically potentiated oxyexcitation-induced OH production ( P 0.05, as compared with control). BCNU and PDTC abrogated the induced production of OH. NAC and -GCE substantially blocked the formation of OH, an effect mimicked by ebselen (*P 0.05, **P 0.01, ***P 0.001, as compared with control). torr). NAC significantly reduced BSO/chemiexcitation-induced IL-1 (Fig. 6A), IL-6 (Fig. 6B), and TNF- (Fig. 6C) release. Similar results were reported at other po 2 tensions (data not shown). Fig. 2. Thiol regulation of intracellular O 2 in response to oxyexcitation. A through E, the formation of O 2 at 24 h is enhanced with ascending po 2 ( P 0.05, P 0.01, as compared with po 2 23 torr). BSO synergistically potentiated oxyexcitation-induced O 2 production ( P 0.05, as compared with control). BCNU and PDTC, as agents that elevate GSSG, abrogated the induced production of O 2. NAC and -GCE, as precursors of GSH, substantially blocked the formation of O 2, an effect mimicked by ebselen (*P 0.05, **P 0.01, ***P 0.001, as compared with control). Selective Modulation of Redox-Sensitive Enzymes and Cytokines. The effect of BSO on NAC-induced downregulation of cytokine release is shown in Fig. 6 ( Discussion The present report investigated in vitro regulatory mechanisms of pharmacological thiols on pro-inflammatory cytokines in the perinatal epithelium. This approach bears clinical relevance to the pediatric treatment of respiratory distresses, where cytokines are crucial elements in their pathophysiology. Growing evidence implicates an association between oxidative stress and up-regulation of a pro-inflammatory state, thereby placing more demand on the utilization of intracellular glutathione (Sen, 1998; Alder et al., 1999). As such, the respiratory epithelium becomes more engaged in regulating enzymes involved in maintaining redox homeosta-

6 Thiopharmacological Regulation of Pro-Inflammatory Cytokines 1001 Fig. 4. Reduction-oxidation (redox) potential in fatii cells exposed to oxyexcitation. A, reduced (GSH) and oxidized (GSSG) glutathione variations (24 h) are shown for cells exposed to OH-generating system ( P 0.05, P 0.01, P 0.001, as compared with [GSH] Control or [GSH] H2O2 ). H 2 O 2 induced the elevation of [GSH] as compared with control cultures at po 2 23 or 152 torr (*P 0.05, **P 0.01). B, glutathione homeostasis (24 h) in the presence of an O 2 -generating system. The concentration of GSH is increased in the presence of X/XO (*P 0.05, **P 0.01, ***P 0.001). The levels of GSSG are markedly depressed, as compared with [GSH] ( P 0.05, P 0.01, P 0.001, as compared with [GSH] Control or [GSH] X/XO ). Fig. 5. Kinetics of ROS-mediated effects on redox enzymes involved in maintaining homeostatic levels of glutathione. Dose-dependent analysis of the potential activity of GSH-PX (A, B), GSSG-RD (C, D), -GCS (E, F), and GS (G, H) in the presence of X/XO or H 2 O 2 at various po 2 regimens. Linear regression analysis of fitting curves reveals significant correlation within the range 0.75 r 0.85 (P 0.05, as compared with enzyme activity of control). sis. Although the glutathione biosynthetic machinery is overwhelmed in disease, an up-regulation of cytokines may contribute to acute exacerbation of the clinical symptoms. Although cytokine participation in the pathogenesis of respiratory distress has been considerably recognized, the mechanisms involved have not been clearly defined. The development of an in vitro model enabled us to investigate the immunopharmacological potential of glutathione, whereby the alveolar epithelium is recognized as a major participant in a front-line defense strategy to oxidants and subsequent lung injury. The rate-limiting substrate for GSH biosynthesis is glutamate-cysteine (K m glutamate mm; K m cysteine 0.3 mm). This pathway is selectively blocked by BSO, a specific and irreversible inhibitor of -GCS (Griffith and Meister, 1979). Consequently, the capacity of the epithelium to replenish intracellular stores of GSH is dramatically affected, thereby modulating the optimum equilibrium necessary to evoke a defense strategy in oxidative stress (Haddad and Land, 2000a; Haddad et al., 2000a). This subsequently leads to ROS up-regulation, the inappropriate disposition, accumulation, and intracellular localization of which augment a pro-inflammatory signal through activation of redox-sensitive transcription factors (Schreck et al., 1992; Luster and Simeonova, 1998). This is consistent with the observation that the expression of -GCS was shown to suppress TNF- induced activation of NF- B (Manna et al., 1999). We believe that the pathway mediating BSO-induced up-regulation of cytokines in the alveolar epithelium involves a secondary mediator, the most likely candidate being the hydroxyl radical. This conforms to the evidence that GSH is involved in H 2 O 2 reduction, a major source of OH, through the GSH-PXcoupled reaction, suggesting that, in the case of BSO preincubation, GSH depletion seems involved in its effect. It is possible that GSH depletion by blocking its biosynthesis reduces the capacity of the epithelium to dispose accumulating H 2 O 2, with the resulting increase in OH production. The likely occurrence of this biochemical conversion is extended by others (Yamauchi et al., 1990; Manna et al., 1999) and supported by corollary experiments with diethyl maleate, a depletor of glutathione, thereby leading to OH/O 2 induction

7 1002 Haddad et al. TABLE 1 The effect of glutathione-modulating agents (depletion/recycling) on cytokines (ng/mg of protein) Data are presented as means S.E.M. (n 4, each). BSO BCNU PDTC Ebselen IL-1 Control Variable H 2 O * Variable H 2 O X/XO * * Variable X/XO , IL-6 Control Variable H 2 O Variable H 2 O , X/XO * Variable X/XO , TNF- Control Variable H 2 O * * * Variable H 2 O * *, X/XO * Variable X/XO , BSO, 50 M; BCNU, 100 M; PDTC, 100 M; ebselen, 100 M; H 2 O 2, 250 M; X/XO, 100 M/2 mu/ml. * P 0.05, P 0.01, P 0.001, as compared with control; P 0.01, as compared with H 2 O 2 or X/XO. TABLE 2 The effect of glutathione modulating agents (repletion/biosynthesis) on cytokines (ng/mg of protein) Data are presented as means S.E.M. (n 4, each). NAC -GCE OTC SAM IL-1 Control Variable * H 2 O * * * * Variable H 2 O *, X/XO * * * * Variable X/XO IL-6 Control Variable H 2 O * * * * Variable H 2 O X/XO * * * * Variable X/XO TNF- Control Variable H 2 O * * * * Variable H 2 O X/XO * * * * Variable X/XO GCE, 100 M; NAC, 50 mm; OTC, 100 M; SAM, 50 mm; H 2 O 2, 250 M; and X/XO, 100 M/2 mu/ml. * P 0.05, as compared with control; P 0.01, as compared with H 2 O 2 or X/XO. and cytokine release (J. J. E. Haddad, B. Safieh-Garabedian, N. E. Saadé, S. A. Kanaan, and S. C. Land, unpublished observations). It remains to be defined, however, whether GSH depletion is implicated in up-regulating cytokines in association with lung disease, since the degree of depletion necessary to evoke cytokines in vitro is higher than that observed in pathophysiology in vivo (Sen, 1998; Alder et al., 1999; Gosset et al., 1999). The ability of NAC to provide cysteine for GSH biosynthesis (Bernard, 1991), along with that of BSO to block de novo synthesis (Haddad and Land, 2000a), provided the criteria to establish whether NAC inhibitory effects on chemioxyexcitation-induced cytokine release is rate-limited by glutathione biosynthesis. Interestingly, BSO did not affect NAC-mediated down-regulation of cytokine secretion, suggesting that its inhibitory effect is independent of its role as a GSH precursor. Since the cysteine provided by NAC eventually cannot feed into the biosynthetic pathway because of irreversible inhibition of -GCS (Griffith and Meister, 1979), it s very likely that NAC is acting either on components of the chemioxyexcitation signaling transduction pathway or through an alternative metabolic machinery. In this respect, the antioxidant, scavenging action against ROS-induced cytokine release is a possible mechanism (Aruoma et al., 1989; Eugui et al., 1994). The cell-permeable glutathione pro-drug, -GCE, was shown to be likewise potently effective in down-regulating chemioxyexcitation-induced cytokine release. -GCE is rapidly de-esterified by intracellular esterase, thereby serving as an effective delivery agent for glutathione, which is a peptide

8 Thiopharmacological Regulation of Pro-Inflammatory Cytokines 1003 Fig. 6. Effect of BSO on NAC-induced down-regulation of chemioxyexcitation-induced cytokine release at torr. NAC reduced BSO-induced up-regulation of IL-1 (A), IL-6 (B), and TNF- (C), suggesting that its effects are independent of GSH biosynthesis (*P 0.05, **P 0.01, as compared with control; P 0.05; P 0.05, as compared with BSO). incapable of crossing membranes in its native form. Although a distinction between the biological effects of -GCE and GSH is indiscriminate, intracellular conversion of -GCE suggests that its effects are mediated by GSH. Exogenous/ endogenous glutathione, therefore, may feed into one of the well characterized pathways of metabolism (Meister, 1988). For instance, GSH plays an important role in determining how readily pro-inflammatory genes can be regulated, and GSH/GSSG equilibrium is a major determinant of the activation of redox-sensitive transcription factors (Dröge et al., 1994; Arrigo, 1999). Exposure to chemioxyexcitation constitutes such a mechanism of modulating redox equilibrium in the alveolar epithelium. Since -GCE is able to suppress intracellular ROS formation, the antioxidant/scavenging effects of this molecule are likely to inhibit the production of cytokines. Further support for the involvement of a glutathione pathway in suppressing pro-inflammatory cytokines was provided with exogenous cysteine, a rate-limiting substrate for GSH biosynthesis. Replenishing GSH is accomplished by administering compounds that increase the level of this amino acid, or by promoting the activity of -GCS. NAC, a cysteine pro-drug, can suppress cytokine production (Peristeris et al., 1992; Jeannin et al., 1995; Gosset et al. 1999) and protect against lung injury (Bernard, 1991). In addition, OTC and SAM are incorporated in glutathione therapy, since they provide cysteine for GSH synthesis (Evans et al., 1997; Anderson and Luo, 1998). Although these agents are effective in suppressing chemioxyexcitation-induced cytokine release, whether they act as antioxidants and/or pro-drugs has yet to be defined. The pathway implicated with cysteine is to complement the biosynthesis process, where GSH can directly scavenge ROS. This mechanism does not exclude the probable action of NAC, OTC, and SAM as antioxidants, which were reported to neutralize excess ROS (Aruoma e al., 1989; Evans et al., 1997; Anderson and Luo, 1998). It is apparently evident, therefore, that either pathway is effectively up-regulated in response to oxidative stress. However, the incapacity of BSO to block NAC-induced suppression of cytokines allows us to discriminate between the antioxidant/gsh precursor properties of NAC. These findings are supported by the unequivocal potency of ebselen, an antioxidant peroxidase mimetic (Schewe, 1995), in mitigating the induced release of cytokines. As such, selective modulation of redox pathways regulates the cytokine network in the alveolar epithelium in response to chemioxyexcitation. Although the involvement of GSSG in pathways governing the induction of cytokines in the alveolar epithelium is not well characterized, its role in determining redox equilibrium is established (Meister, 1988; Schreck et al., 1992; Dröge et al., 1994; Haddad et al., 2000a). Replenishing GSH is not only -GCS rate-limited but also determined by the degree of NADPH-dependent GSSG recycling. Thus, favoring an oxidation equilibrium by elevating GSSG has been reported to activate signaling pathways that down-regulate the activation of transcription factors (Sen, 1998; Schreck et al., 1992; Haddad et al., 2000a). Inhibition of GSSG recycling by BCNU negatively attenuates the activation of NF- B in vitro (J. J. E. Haddad, R. E. Olver, and S. C. Land, unpublished observations). Intriguingly, GSSG, like GSH, has the potential to down-regulate chemioxyexcitation-dependent cytokine release. It s likely, therefore, that BCNU mitigates a proinflammatory signal by suppressing the activation of NF- B, through elevation of [GSSG] (Shakhov et al., 1990). To confirm this hypothesis, we used PDTC, a dithiocarbamate that exerts antioxidant/pro-oxidant effects (Schreck et al., 1992; Brennan and O Neill, 1996; Wild and Mulcahy, 1999; Haddad et al., 2000a). Dithiocarbamates inhibit the phosphorylationdependent release of NF- B from its cytosolic inhibitory subunit, I B (Brennan and O Neill, 1996), suggesting that the mechanism of ROS-induced activation of this transcription factor involves a redox-sensitive kinase (Kanakaraj et al., 1998; Li et al., 1999). However, GSSG also promotes the formation of a NF- B/disulfide complex, directly inhibiting DNA binding (Schreck et al., 1992; Brennan and O Neill,

9 1004 Haddad et al. 1996; Haddad et al., 2000a). PDTC elevates [GSSG] at the expense of [GSH], suggesting that GSSG contribution to suppression of chemioxyexcitation-induced cytokine release occurs through NF- B, a transcription factor essentially involved in regulating pro-inflammatory genes (Haddad et al., 2000a). The mechanism involved is that BCNU (GSSG) has the potential to retard NF- B nuclear translocation and subsequent activation, a pathway that in turn switches off the expression of genes encoding pro-inflammatory cytokines (Pfeilschifter and Mühl, 1999). These pathways, however, do not account for GSSG-induced suppression of cytokines in hypoxia, where NF- B activation state is depressed in a reducing environment (Brennan and O Neill, 1996; Haddad and Land, 2000a, Haddad et al., 2000a). Since the activation of hypoxia-inducible factor-1 (HIF-1 ) increases exponentially on lowering po 2, it s possible that this factor is involved in regulating pro-inflammatory genes (Wenger et al., 1996; Hellwig-Bürgel et al., 1999; Yan et al., 1999). BCNU was shown to downregulate HIF-1 activation in vitro by reducing GSH/GSSG, thereby favoring an oxidation equilibrium (J. J. E. Haddad, R. E. Olver, and S. C. Land, unpublished observations). We are currently extending these observations to investigate the kinetics of HIF-1 -mediated regulation of cytokines in hypoxia. Taken together in hand with selective modulation of glutathione pathways as directly affecting the cytokine profile, our results have demonstrated a novel mechanism of thiol regulation mediated by glutathione in the alveolar epithelium. Thiol-mediated pathways simulating functional Fig. 7. Schematic model of thiol regulation of chemioxyexcitation-induced cytokine secretion in the alveolar epithelium. The predominant form of intracellular glutathione is GSH, which is synthesized by -GCS. OTC, SAM, and NAC are major precursors of cysteine, the rate-limiting substrate in the biosynthesis of GSH, a pathway that is selectively blocked by BSO. BSO up-regulates the formation of intracellular ROS, which are major inducers of cytokine secretion. GSH is either rapidly exported, where the membrane-bound -glutamyl transpeptidase ( -GT) degrades it into its subcellular components to be used for resynthesis or is converted by GSSG by GSH-PX and its mimetic ebselen, at the expense of ROS, which are promptly detoxified (reduced) into ROOH, a hydroperoxide, according to the equation ROOH 2GSH 3 ROH H2O GSSG. The formation of GSSG and/or reduction of ROS down-regulate the chemioxyexcitation-dependent cytokine release. GSSG is rapidly recycled back into GSH by GSSG-RD, a pathway, which is selectively blocked by BCNU, leading to accumulation of GSSG, which along with pyrrolidine dithiocarbamate, a precursor of GSSG, down-regulates the formation of ROS and abrogates the cytokine-dependent sequelae. Cytokines act as major participants in the pathophysiology and aggravation of the clinical symptoms of respiratory distresses (RD). mechanisms controlling pro-inflammatory cytokines are schematized in Fig. 7. This report has elaborated in vitro an immunopharmacological potential of glutathione and subsequent regulation of pro-inflammatory cytokines. These findings are highlighted as follows: 1) selective inhibition of -GCS up-regulates cytokines via the formation of ROS; 2) blockage of glutathione recycling uncouples the ROS/cytokine pathway, an effect closely mimicked by PDTC an antioxidant/pro-oxidant agent that elevates [GSSG]; 3) exogenous precursors of [GSH] and cysteine suppress ROS production and the down-stream cytokine-dependent pathway; and 4) shifting redox potential in favor of a reduction equilibrium negatively interferes with the capacity to up-regulate a pro-inflammatory signal. Our results indicate that modulating redox status by pharmacological thiols has potential clinical consequences for the therapeutic treatment of pediatric distresses in which cytokines act as major participants in their pathophysiology. 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