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• W2073978087 abstract "Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normal T-cells expressed and secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study we investigated the role of reactive oxygen species in the induction of RANTES gene expression in human type II alveolar epithelial cells (A549), following RSV infection. Our results indicate that RSV infection of airway epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of 2′,7′-dichlorofluorescein. Pretreatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES gene expression and protein secretion. This effect is mediated through the ability of BHA to inhibit RSV-induced interferon regulatory factor binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3 nuclear translocation. Together, these data indicates that a redox-sensitive pathway is involved in RSV-induced IRF activation, an event necessary for RANTES gene expression. Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normal T-cells expressed and secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study we investigated the role of reactive oxygen species in the induction of RANTES gene expression in human type II alveolar epithelial cells (A549), following RSV infection. Our results indicate that RSV infection of airway epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of 2′,7′-dichlorofluorescein. Pretreatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES gene expression and protein secretion. This effect is mediated through the ability of BHA to inhibit RSV-induced interferon regulatory factor binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3 nuclear translocation. Together, these data indicates that a redox-sensitive pathway is involved in RSV-induced IRF activation, an event necessary for RANTES gene expression. respiratory syncytial virus regulated upon activation, normal T-cells expressed and secreted reactive oxygen species N-acetylcystein tetramethyl thiourea butylated hydroxyanisol interferon-stimulated responsive element interferon regulatory factor extracellular signal-regulated kinase mitogen-activated protein human immunodeficiency virus interleukin small alveolar epithelial cells multiplicity of infection 2,7-dichlorofluorescein diacetate polyacrylamide gel electrophoresis signal transducers and activators of transcription Respiratory syncytial virus (RSV)1 is an enveloped, negative-sense single-stranded RNA virus (1Hall C.B. McCarthy C.A. Mandel G.L. Bennett J.E. Dolin R. Principles and Practice of Infectious Diseases. Churchill Livingston, New York1995Google Scholar). Since its isolation, RSV has been identified as a leading cause of epidemic respiratory tract illness in children in the United States and worldwide. In fact, RSV is so ubiquitous that it will infect 100% of children before the age of 3. It is estimated that 40–50% of children hospitalized with bronchiolitis and 25% of children with pneumonia are infected with RSV, resulting in 100,000 hospital admissions annually in the United States alone (1Hall C.B. McCarthy C.A. Mandel G.L. Bennett J.E. Dolin R. Principles and Practice of Infectious Diseases. Churchill Livingston, New York1995Google Scholar). In addition to acute morbidity, there are long-term consequences of RSV infection in infancy: RSV has been shown to predispose to the development of hyperreactive airway disease (2Webb M.S.C. Henry R.L. Milner A.D. Arch. Dis. Child. 1985; 60: 1064-1067Crossref PubMed Scopus (63) Google Scholar) and recurrent episodes of wheezing in asthmatic children are often precipitated by RSV infection. The mechanisms of RSV-induced airway disease and its long-term consequences are largely unknown, but the delicate balance between immunopathology and immunoprotection in the airway mucosa may be altered by an exuberant and unwanted local inflammatory response. Airway infiltration of monocytes and lymphocytes is typical of RSV infection (1Hall C.B. McCarthy C.A. Mandel G.L. Bennett J.E. Dolin R. Principles and Practice of Infectious Diseases. Churchill Livingston, New York1995Google Scholar), and activation of eosinophil and basophil leukocytes has been shown to correlate with the severity of acute RSV disease (3Garofalo R.P. Welliver R.C. Ogra P.L. Pediatr. Allergy Immunol. 1991; 2: 30-37Crossref Scopus (26) Google Scholar, 4Garofalo R.P. Kimpen J.L.L. Welliver R.C. Ogra P.L. J. Pediatrics. 1992; 120: 28-32Abstract Full Text PDF PubMed Scopus (265) Google Scholar). The composition of the cellular response at sites of tissue inflammation is controlled by gradients of chemokines, a family of small chemotactic cytokines, which direct leukocyte transendothelial migration and movement through the extracellular matrix. RANTES is a CC chemokine highly chemoattractant for T lymphocytes, monocytes, eosinophils, and basophils (5Alam R. Stafford S. Forsythe P. Harrison R. Faubion D. Lett-Brown M.A. Grant J.A. J. Immunol. 1993; 150: 3442-3448PubMed Google Scholar), all cell types which are present or activated in the inflammatory infiltrate that follows RSV infection of the lung. Recent in vivo studies have shown elevated RANTES concentrations in nasal washes and bronchoalveolar lavages of children infected with RSV (6Teran L.M. Seminario M.C. Shute J.K. Papi A. Compton S.J. Low J.L. Gleich G.J. Johnston S.L. J. Infect. Dis. 1999; 179: 677-681Crossref PubMed Scopus (77) Google Scholar, 7Sheeran P. Jafri H. Carubelli C. Saavedra J. Johnson C. Krisher K. Sanchez P.J. Ramilio M.O. Pediatr. Infect. Dis. J. 1999; 18: 115-122Crossref PubMed Scopus (223) Google Scholar) and we have recently shown that RANTES is strongly expressed in RSV-infected respiratory epithelial cells (8Saito T. Deskin R.W. Casola A. Haeberle H. Olszewska B. Ernst P.B. Alam R. Ogra P.L. Garofalo R. J. Infect. Dis. 1997; 175: 497-504Crossref PubMed Scopus (149) Google Scholar, 9Olszewska-Pazdrak B. Casola A. Saito T. Alam R. Crowe S.E. Mei F. Ogra P.L. Garofalo R.P. J. Virol. 1998; 72: 4756-4764Crossref PubMed Google Scholar), which are the primary target for viral infection. Therefore, it is likely that RANTES produced by infected epithelial cells plays an important role in the pathogenesis of RSV-induced airway inflammation. Reactive oxygen species (ROS) are ubiquitous, highly diffusable and reactive molecules produced as a result of reduction of molecular oxygen, including species such as hydrogen peroxide, superoxide anion, and hydroxyl radical, and they have been implicated in damaging cellular components like lipids, proteins, and DNA. In the past few years, there has been increased recognition of their role as redox regulators of cellular signaling (reviewed in Refs. 10Allen R.G. Tresini M. Free Radic. Biol. Med. 2000; 28: 463-499Crossref PubMed Scopus (1073) Google Scholar and 11Prasad Gabbita S. Robinson K.A. Stewart C.A. Floyd R.A. Hensley K. Arch. Biochem. Biophys. 2000; 376: 1-136Crossref PubMed Scopus (155) Google Scholar). Inducible ROS generation has been shown following stimulation with a variety of molecules, like cytokines and growth factors, and infection with certain viruses, like HIV, hepatitis B, and influenza (reviewed in Ref. 12Schwarz K.B. Free Radic. Biol. Med. 1996; 21: 641-649Crossref PubMed Scopus (541) Google Scholar). Changes in the level of ROS, generated in response to some of these stimuli, have been shown to modulate the expression of several genes (10Allen R.G. Tresini M. Free Radic. Biol. Med. 2000; 28: 463-499Crossref PubMed Scopus (1073) Google Scholar). Among the different members of the chemokine family, interleukin (IL)-8 is the only one for which redox-sensitive signaling pathways have been identified (13Simeonova P.P. Leonard S. Flood L. Shi X. Luster M.I. Lab. Invest. 1999; 79: 1027-1037PubMed Google Scholar, 14DeForge L.E. Preston A.M. Takeuchi E. Kenney J. Boxer L.A. Remick D.G. J. Biol. Chem. 1993; 268: 25568-25576Abstract Full Text PDF PubMed Google Scholar). The contribution of ROS in RANTES gene expression, as well as in other CC chemokine induction, has not been defined yet. Therefore, the purpose of this study was to investigate the effect of RSV infection on ROS generation in human airway epithelial cells and the role of ROS in RSV-induced RANTES production. Our results indicate that RSV infection of airway epithelial cells induces ROS production, as measured by intracellular oxidation of 2′,7′-dichlorofluorescein, and that treatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well as a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES protein secretion and gene expression. This effect is mediated through the inhibition of RSV-induced interferon regulatory factor (IRF) binding to the RANTES interferon-stimulated responsive element (ISRE), an event that is absolutely required for RSV-stimulated RANTES gene transcription. In infected A549 cells, ISRE binds IRF-1, -3, and -7. IRF-1 and -7 are inducible upon RSV infection of alveolar epithelial cells and treatment with BHA inhibits their gene expression and protein synthesis. In contrast, IRF-3 is constitutively expressed and antioxidant treatment blocks its nuclear translocation. These data strongly indicate that a redox-sensitive pathway is involved in RSV-induced IRF induction and RANTES gene expression. This study provides novel insights on the role of ROS in viral-induced RANTES secretion and IRF protein activation. Identification of the molecular mechanisms involved in RANTES gene expression is fundamental for developing strategies to modulate the inflammatory response associated with RSV infection of the lung. The human Long strain of RSV (A2) was grown in Hep-2 cells and purified by centrifugation on discontinuous sucrose gradients as described elsewhere (15Ueba O. Acta Med. Okayama. 1978; 32: 265-272PubMed Google Scholar). The virus titer of the purified RSV pools was 8–9 log10 plaque forming units/ml using a methylcellulose plaque assay. No contaminating cytokines were found in these sucrose-purified viral preparations (16Patel J.A. Kunimoto M. Sim T.C. Garofalo R. Eliott T. Baron S. Ruuskanen O. Chonmaitree T. Ogra P.L. Schmalstieg F. Am. J. Resp. Cell Mol. 1995; 13: 602-609Crossref PubMed Scopus (120) Google Scholar). Lipopolysaccharide, assayed using the limulus hemocyanin agglutination assay, was not detected. Virus pools were aliquoted, quick-frozen on dry ice/alcohol, and stored at −70 °C until used. A549, human alveolar type II-like epithelial cells (ATCC, Manassas, VA), were maintained in F12K medium containing 10% (v/v) fetal bovine serum, 10 mm glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Small alveolar epithelial cells (SAE) were obtained from Clonetics, San Diego, CA, and grown according to the manufacturer's instructions. Cell monolayers were infected with RSV at a multiplicity of infection (m.o.i.) of 1 (unless otherwise stated), as described (17Garofalo R.P. Sabry M. Jamaluddin M., Yu, R.K. Casola A. Ogra P.L. Brasier A.R. J. Virol. 1996; 70: 8773-8781Crossref PubMed Google Scholar). An equivalent amount of a 20% sucrose solution was added to uninfected A549 cells, as a control. For the antioxidant experiments, cells were pretreated with the antioxidants for 1 h and then infected in the presence of the antioxidants. Since BHA was diluted in ethanol, equal amounts of ethanol were added to untreated cells, as a control. Total number of cells, cell viability, and viral replication, following antioxidant treatment, were measured by trypan blue exclusion and by plaque assay, respectively. Immunoreactive RANTES was quantitated by a double antibody enzyme-linked immunosorbent assay kit (DuoSet, R&D Systems, Minneapolis, MN) following the manufacturer's protocol. Total RNA was extracted from control and infected A549 cells by the acid guanidium thiocyanate-phenol chloroform method (18Salkind A.R. Nichols J.E. Roberts Jr., N.J. J. Clin. Invest. 1991; 88: 505-511Crossref PubMed Scopus (44) Google Scholar). Twenty micrograms of RNA were fractionated on a 1.2% agarose-formaldehyde gel, transferred to nylon membranes, and hybridized to a radiolabeled RANTES, IRF-1, -3, and -7 cDNAs (RANTES cDNA plasmid was a generous gift of Dr A. Krensky, Stanford, CA; IRF-1, -3, and -7 cDNAs were a generous gift of Dr. Lin, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada), as previously described (19Brasier A.R. Jamaluddin M. Casola A. Duan W. Shen Q. Garofalo R.P. J. Biol. Chem. 1998; 273: 3551-3561Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Hybridization temperature for all probes was 55 °C. After washing, membranes were exposed for autoradiography using Kodak X-AR film at −70 °C, using intensifying screens. After exposure, membranes were stripped and rehybridized with a β-actin probe. A549 cells were grown in 96-well tissue culture plates and infected with RSV at 0.1, 0.5 and 1 multiplicity of infection (m.o.i.). At different times post-infection, cells were washed with Hank's balanced salt solution and loaded with 10 μm 2,7-dichlorofluorescein diacetate (DCF-DA) in Hank's balanced salt solution medium containing 25 mm HEPES, pH 7.4, for 30 min at 37 °C. The cells were then washed twice and fluorescence intensity was determined at 485 nm excitation and 590 nm emission, using an automated fluorescence reader (Flurocount, Hewlett-Packard Instruments, IL). For the experiments in which H2O2 was used as a stimulus for ROS production, cells were preloaded with 10 μm DCF-DA for 30 min, washed, and then fluorescence intensity was measured at different times following addition of H2O2. Measurements were performed in triplicates and results expressed as fluorescence mean ± S.D. of n = 3 independent experiments. A fragment of the human RANTES promoter spanning from −220 to +55 nucleotides (nt), relative to the mRNA start site designated +1 was cloned into the luciferase reporter gene vector pGL2 (Promega, Madison, WI) and defined as pGL2-220. Site mutations of the RANTES ISRE in the context of pGL2-220 plasmid were introduced by polymerase chain reaction using the following upstream and downstream mutagenic primers (mutations in lowercases): 5′-CATATTTCAGTaaaCTaaaCCGT-3′ and 3′-TATAAAGTCAtttGAtttGGCAT-5′. Logarithmically growing A549 cells were transfected in triplicate in 60-mm Petri dishes by DEAE-dextran, as previously described (20Casola A. Garofalo R.P. Jamaluddin M. Vlahopoulos S. Brasier A.R. J. Immunol. 2000; 164: 5944-5951Crossref PubMed Scopus (88) Google Scholar). Cells were incubated in 2 ml of HEPES-buffered Dulbecco's modified Eagle's medium (10 mm Hepes, pH 7.4) containing 20 μl of 60 mg/ml DEAE-dextran (Amersham Pharmacia Biotech) premixed with 6 μg of RANTES-pGL2 plasmids and 1 μg of cytomegalovirus-β-galactosidase internal control plasmid. After 3 h, media was removed and 0.5 ml of 10% (v/v) Me2SO in phosphate-buffered saline was added to the cells for 2 min. Cells were washed with phosphate-buffered saline and cultured overnight in 10% fetal bovine serum/Dulbecco's modified Eagle's medium. The next morning, cells were infected with RSV and at 24 h post-infection cells were lysed to measure independently luciferase and β-galactosidase reporter activity, as previously described (20Casola A. Garofalo R.P. Jamaluddin M. Vlahopoulos S. Brasier A.R. J. Immunol. 2000; 164: 5944-5951Crossref PubMed Scopus (88) Google Scholar). Luciferase was normalized to the internal control β-galactosidase activity. All experiments were performed in duplicate or triplicate. Nuclear extracts of uninfected and infected A549 cells were prepared using hypotonic/nonionic detergent lysis, as previously described (20Casola A. Garofalo R.P. Jamaluddin M. Vlahopoulos S. Brasier A.R. J. Immunol. 2000; 164: 5944-5951Crossref PubMed Scopus (88) Google Scholar). Proteins were normalized by protein assay (Protein Reagent, Bio-Rad, Hercules, CA) and used to bind to a duplex oligonucleotide corresponding to the RANTES ISRE, whose sequences is shown: 5′-GATCCATATTTCAGTTTTCTTTTCCGT-3′, 3′-TATAAAGTCAAAAGAAAAGGCATCTAG-5′. DNA-binding reactions contained 10–15 μg of nuclear proteins, 5% glycerol, 12 mm HEPES, 80 mm NaCl, 5 mm dithiothreitol, 5 mm Mg2Cl, 0.5 mm EDTA, 1 μg of poly(dI-dC), and 40,000 cpm of32P-labeled double-stranded oligonucleotide in a total volume of 20 μl. The nuclear proteins were incubated with the probe for 15 min at room temperature and then fractionated by 6% nondenaturing polyacrylamide gels (PAGE) in TBE buffer (22 mm Tris-HCl, 22 mm boric acid, 0.25 mm EDTA, pH 8). After electrophoretic separation, gels were dried and exposed for autoradiography using Kodak X-AR film at −70 °C using intensifying screens. Microaffinity purification of proteins binding to the RANTES ISRE was performed using a two-step biotinylated DNA-streptavidin capture assay (20Casola A. Garofalo R.P. Jamaluddin M. Vlahopoulos S. Brasier A.R. J. Immunol. 2000; 164: 5944-5951Crossref PubMed Scopus (88) Google Scholar). In this assay, duplex oligonucleotides are chemically synthesized containing 5′-biotin on a flexible linker (Genosys, The Woodlands, TX). Four hundred micrograms of 12-h infected A549 cells nuclear extracts were incubated at 4 °C for 30 min with 50 pmol of biotin ISRE, in the absence or presence of 10-fold molar excess of non-biotinylated ISRE wild type (WT) or mutated (MUT). The binding buffer contained 8 μg of poly(dI-dC) (as nonspecific competitor) and 5% (v/v) glycerol, 12 mmHEPES, 80 mm NaCl, 5 mm dithiothreitol, 5 mm Mg2Cl, 0.5 mm EDTA. One hundred microliters of a 50% slurry of pre-washed streptavidin-agarose beads was then added to the sample, and incubated at 4 °C for an additional 20 min with gentle rocking. Pellets were washed twice with 500 μl of binding buffer, and the washed pellets were resuspended in 100 μl of 1 × SDS-PAGE buffer, boiled, and fractionated on a 10% SDS-polyacrylamide gel. After electrophoresis separation, proteins were transferred to polyvinylidene difluoride membrane for Western blot analysis. Total cell lysates and cytoplasmic and nuclear proteins were prepared as previously described, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (20Casola A. Garofalo R.P. Jamaluddin M. Vlahopoulos S. Brasier A.R. J. Immunol. 2000; 164: 5944-5951Crossref PubMed Scopus (88) Google Scholar). Membranes were blocked with 5% albumin in TBS-Tween and incubated overnight with a rabbit polyclonal antibody to IRF-1, -3, and -7 (Santa Cruz Biotechnology, Santa Cruz, CA). For secondary detection, we used a horseradish-coupled anti-rabbit or anti-mouse antibody in the enhanced chemiluminescence assay (Amersham Pharmacia Biotech, Arlington Heights, IL). Data from experiments involving multiple samples subject to each treatment were analyzed by the Student Newman Keuls t test for multiple pairwise comparisons. Results were considered significantly different at a p value <0.05. To determine whether RSV infection induced ROS production, A549 cells were grown to ∼90% of confluency and infected with RSV. At different time points after infection, cells were loaded with the membrane permeable compound 2′,7′-DCF-DA, which is trapped intracellularly following cleavage by cellular esterases. DCF oxidation was measured by changes in mean fluorescence intensity in control versus infected cells (21Kooy N.W. Royall J.A. Ischiropoulos H. Free Radic. Res. 1997; 27: 245-254Crossref PubMed Scopus (157) Google Scholar,22Marchesi E. Rota C. Fann Y.C. Chignell C.F. Mason R.P. Free Radic. Biol. Med. 1999; 26: 148-161Crossref PubMed Scopus (148) Google Scholar). When cells were infected with m.o.i. of 1, the production of ROS was detectable as early as 2 h post-infection, reaching a plateau around 4 h and declining thereafter, although the level of cellular ROS in infected cells was still higher than in control cells at 24 h post-infection (Fig. 1). When cells were infected with a lower multiplicity of infection, such as 0.1 and 0.5, the kinetic of ROS production was delayed of a few hours, reaching a plateau between 6 and 8 h post-infection, reflecting the lower number of cells infected at the earliest time points. We have recently demonstrated that RSV is a potent stimulus for RANTES production in cultured human nasal, bronchial, and alveolar epithelial cells (8Saito T. Deskin R.W. Casola A. Haeberle H. Olszewska B. Ernst P.B. Alam R. Ogra P.L. Garofalo R. J. Infect. Dis. 1997; 175: 497-504Crossref PubMed Scopus (149) Google Scholar, 9Olszewska-Pazdrak B. Casola A. Saito T. Alam R. Crowe S.E. Mei F. Ogra P.L. Garofalo R.P. J. Virol. 1998; 72: 4756-4764Crossref PubMed Google Scholar). In all epithelial cell types, synthesis of RANTES required replicating virus and was dose- and time-dependent, with increased steady state levels of RANTES mRNA observed between 6 and 12 h after infection (8Saito T. Deskin R.W. Casola A. Haeberle H. Olszewska B. Ernst P.B. Alam R. Ogra P.L. Garofalo R. J. Infect. Dis. 1997; 175: 497-504Crossref PubMed Scopus (149) Google Scholar,9Olszewska-Pazdrak B. Casola A. Saito T. Alam R. Crowe S.E. Mei F. Ogra P.L. Garofalo R.P. J. Virol. 1998; 72: 4756-4764Crossref PubMed Google Scholar). To determine the contribution of RSV-induced ROS generation in RANTES secretion, A549 cells were infected with RSV in the absence or presence of the chemically unrelated antioxidants dimethyl sulfoxide (Me2SO), N-acetyl·cystein (NAC), tetramethyl thiourea (TMTU), and butylated hydroxyanisol (BHA). In preliminary studies different concentrations of antioxidants were used to identify the most effective ones in inhibiting RANTES secretion (data not shown). We found that 2% (v/v) Me2SO, 20 mmNAC, 20 mm TMTU, and 400 μm BHA were sufficient to significantly block RSV-induced RANTES production, with BHA being the most effective (Fig. 2,panel A). To confirm these results in a normal cell type, similar experiments were performed in SAE cells infected with RSV. SAE cells are derived from the small bronchioli of the lung and they show a similar pattern of RANTES induction, following RSV infection, compared with A549 cells (9Olszewska-Pazdrak B. Casola A. Saito T. Alam R. Crowe S.E. Mei F. Ogra P.L. Garofalo R.P. J. Virol. 1998; 72: 4756-4764Crossref PubMed Google Scholar). As in A549 cells, the antioxidants TMTU, NAC, and BHA significantly reduced RSV-induced RANTES production (Fig. 2,panel B), suggesting that indeed inducible RANTES secretion is regulated in a redox-sensitive manner. Antioxidant treatment did not significantly affect cell viability or viral replication (data not shown). Since BHA was the most effective compound in reducing RSV-induced RANTES secretion in both A549 and SAE cells, we selected this antioxidant to perform all the subsequent experiments. To directly confirm the ability of BHA to inhibit ROS, A549 cells were stimulated with 200 μm H2O2 in the absence or presence of 400 μm BHA and the amount of cellular ROS was monitored by oxidation of 2′7′-DCF. As shown in Fig.3, H2O2 was able to induce a high level of ROS production, which was almost completely inhibited by treatment with BHA. These data indicate that BHA function as a potent antioxidant in airway epithelial cells. To determine if the reduction in RSV-induced RANTES secretion by BHA was paralled by changes in the steady-state level of RANTES mRNA, A549 cells were infected with RSV for various lenghts of time, in the absence or presence of the antioxidant, and total RNA was extracted from control and infected cells for Northern blot analysis. A small increase in RANTES mRNA expression was first detected at 6 h post-infection, with maximal induction between 12 and 24 h (Fig.4). There was no further increase in mRNA levels at later time points (data not shown). Treatment with 400 μm BHA completely inhibited RSV-induced RANTES mRNA induction at 6 and 12 h post-infection and greatly reduced it at 24 h (Fig. 4). This dramatic change was not due to a nonspecific effect since total cell number and viability in the group treated with antioxidant were unchanged (data not shown) and levels of the housekeeping gene β-actin were not systematically reduced compared with untreated cells (Fig. 4). Inducible RANTES gene expression is controlled at both transcriptional and post-transcriptional levels (23Koga T. Sardina E. Tidwell R.M. Pelletier M. Look D.C. Holtman M.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5680-5685Crossref PubMed Scopus (59) Google Scholar, 24Nelson P.J. Kim H.T. Manning W.c. Goralski T.J. Krensky A.M. J. Immunol. 1993; 151: 2601-2612PubMed Google Scholar, 25Boehlk S. Fessele S. Mojaat A. Miyamoto N.G. Werner T. Nelson E.L. Schlondorff D. Nelson P.J. Eur. J. Immunol. 2000; 30: 1102-1112Crossref PubMed Scopus (55) Google Scholar). We have previously shown that in A549 cells RSV-induced RANTES promoter activation mirrors the induction of the endogenous RANTES gene mRNA, suggesting that in alveolar epithelial cells RANTES expression, following RSV infection, is controlled mainly at the level of transcription. 2A. Casola, R. P. Garofalo, H. Haeberle, T. F. Elliott, M. Jamaluddin and A. R. Brasier,J. Virol. (in press). To determine whether the antioxidant effect of BHA influenced RANTES gene transcription, A549 cells were transiently transfected with a construct containing the first 220 nucleotides of the human RANTES promoter linked to the luciferase reporter gene, defined as pGL2-220. This fragment of the promoter contains all the necessary regulatory elements that drive regulated luciferase expression in A549 cells following RSV infection.2 The day after, cells were infected with RSV for 24 h in the absence or presence of BHA. Similar to what we have observed for mRNA levels, treatment with BHA almost completely abolished RSV-induced luciferase activity (Fig.5), indicating that the antioxidant effect occurs mainly by interfering with RANTES gene transcription. We have recently investigated the promotercis-regulatory elements and nuclear factors involved in the regulation of RANTES gene transcription following RSV infection of human airway epithelial cells. The results of that study have indicated that RSV-induced RANTES transcription requires cooperation of multiple response elements, including the ISRE.2 The ISRE is absolutely required for RSV-induced promoter activation, since its mutation completely blocks RSV-induced luciferase activity (Fig.6). To determine if BHA-induced inhibition of RANTES transcription was due to changes in the abundance of DNA-binding proteins recognizing the RANTES ISRE, we performed electrophoretic mobility shift assays using nuclear extracts prepared from A549 cells control or infected with RSV for 12 h, in the absence or presence of BHA. As shown in Fig.7, RSV infection induced a dramatic increase in ISRE binding, which was completely abolished by treatment with BHA.Figure 7Electrophoretic mobility shift assay of RANTES ISRE binding complexes in response to antioxidant treatment. Nuclear extracts were prepared from control and cells infected with RSV for 12 h, in the absence or presence of 400 μm BHA, and used for binding to the RANTES ISRE in electrophoretic mobility shift assay. Shown is the nucleoprotein complex formed on the RANTES ISRE in response to RSV infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The major components of the RSV-induced ISRE binding complex are IRF-1, -3, and -7.2 It has been previously shown that IRF-7 gene expression and protein synthesis is viral inducible, while IRF-3 is constitutively expressed and translocates to the nucleus when phosphorylated in response to a viral infection (26Hiscott J. Pitha P. Genin P. Nguyen H. Heylbroeck C. Mamane Y. Algarte M. Lin R. J. Interferon Cytokine Res. 1999; 19: 1-13Crossref PubMed Scopus (197) Google Scholar). We have previously shown that RSV infection of A549 cells induces de novo synthesis of IRF-1 (20Casola A. Garofalo R.P. Jamaluddin M. Vlahopoulos S. Brasier A.R. J. Immunol. 2000; 164: 5944-5951Crossref PubMed Scopus (88) Google Scholar). To determine if RSV infection of A549 cells induced IRF-7 synthesis and IRF-3 activation, we performed Western blot analysis of cytoplasmic and nuclear proteins extracted from A549 cells uninfected or infected for various lengths of time. As shown in Fig. 8, RSV infection inducedde novo synthesis of IRF-7 and its nuclear translocation starting around 12 h post-infection. By contrast, IRF-3 was constitutively expressed and RSV infection ind" @default.
• W2073978087 created "2016-06-24" @default.
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• W2073978087 date "2001-01-01" @default.
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• W2073978087 title "Oxidant Tone Regulates RANTES Gene Expression in Airway Epithelial Cells Infected with Respiratory Syncytial Virus" @default.
• W2073978087 cites W1480920464 @default.
• W2073978087 cites W1485705546 @default.
• W2073978087 cites W1546629881 @default.
• W2073978087 cites W1968985837 @default.
• W2073978087 cites W1971068712 @default.
• W2073978087 cites W1976545475 @default.
• W2073978087 cites W1993910598 @default.
• W2073978087 cites W1994888948 @default.
• W2073978087 cites W1997569654 @default.
• W2073978087 cites W2001804120 @default.
• W2073978087 cites W2009080092 @default.
• W2073978087 cites W2028850807 @default.
• W2073978087 cites W2029285210 @default.
• W2073978087 cites W2029366428 @default.
• W2073978087 cites W2031760220 @default.
• W2073978087 cites W2032064870 @default.
• W2073978087 cites W2033800625 @default.
• W2073978087 cites W2036557115 @default.
• W2073978087 cites W2040730598 @default.
• W2073978087 cites W2043372033 @default.
• W2073978087 cites W2048235174 @default.
• W2073978087 cites W2063841059 @default.
• W2073978087 cites W2084503577 @default.
• W2073978087 cites W2100829376 @default.
• W2073978087 cites W2104001835 @default.
• W2073978087 cites W2106491281 @default.
• W2073978087 cites W2117981959 @default.
• W2073978087 cites W2121284065 @default.
• W2073978087 cites W2125888328 @default.
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