Role of Xanthine Oxidase Activation and Reduced Glutathione Depletion in Rhinovirus Induction of Inflammation in Respiratory Epithelial Cells
2008; Elsevier BV; Volume: 283; Issue: 42 Linguagem: Inglês
10.1074/jbc.m805766200
ISSN1083-351X
AutoresAlberto Papi, Marco Contoli, Pierluigi Gasparini, Laura Bristot, Michael R. Edwards, Milvia Chicca, Marilena Leis, Adalberto Ciaccia, Gaetano Caramori, Sebastian L. Johnston, Silvano Pinamonti,
Tópico(s)Respiratory viral infections research
ResumoRhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-κB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential. Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-κB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential. Rhinoviruses (RV) 3The abbreviations used are: RV, rhinoviruses; f-RV, filtered virus; COPD, chronic obstructive pulmonary disease; HBEC, human bronchial epithelial cell; O2•¯, superoxide anion; XD, xanthine dehydrogenase; XO, xanthine oxidase; PMSF, phenylmethylsulfonyl fluoride; Leu, serine and cysteine protease inhibitor leupeptin; Pep, aspartic protease inhibitor pepstatin; Apr, serine protease inhibitor aprotinin; Phe, metalloprotease inhibitor phenanthroline; Oxy, oxypurinol; SOD, superoxide dismutase; HPLC, high performance liquid chromatography; siRNA, small interfering RNA; RT, reverse transcriptase; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; H2S, sulfidric acid; IL, interleukin; ICAM-1, intercellular adhesion molecule 1. are the major cause of the commonest human acute infectious disease, the common cold (1Stanway G. Webster R. Granoff A. Encyclopedia of Virology. Academic Press, London1994: 1253-1259Google Scholar). They are also associated with the majority of acute exacerbations of asthma (2Johnston S.L. Pattemore P.K. Sanderson G. Smith S. Lampe F. Josephs L. Symington P. O'Toole S. Myint S.H. Tyrrell D.A. Holgate S.T. Br. Med. J. 1995; 310: 1225-1229Crossref PubMed Scopus (1710) Google Scholar, 3Grissell T.V. Powell H. Shafren D.R. Boyle M.J. Hensley M.J. Jones P.D. Whitehead B.F. Gibson P.G. Am. J. Respir. Crit. Care Med. 2005; 172: 433-439Crossref PubMed Scopus (174) Google Scholar) and chronic obstructive pulmonary disease (COPD) (4Seemungal T. Harper-Owen R. Bhowmik A. Moric I. Sanderson G. Message S. Maccallum P. Meade T.W. Jeffries D.J. Johnston S.L. Wedzicha J.A. Am. J. Respir. Crit. Care Med. 2001; 164: 1618-1623Crossref PubMed Scopus (875) Google Scholar, 5Papi A. Bellettato C.M. Braccioni F. Romagnoli M. Casolari P. Caramori G. Fabbri L.M. Johnston S.L. Am. J. Respir. Crit. Care Med. 2006; 173: 1114-1121Crossref PubMed Scopus (839) Google Scholar). No licensed effective antiviral is currently available for the treatment of the common cold (6Jefferson T.O. Tyrrell D. Cochrane Database Syst. Rev. 2007; 18: CD002743Google Scholar, 7Heikkinen T. Jarvinen A. The Lancet. 2003; 361: 51-59Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar) and treatment of virus-induced asthma and COPD exacerbations is a major unmeet therapeutic need (8Mallia P. Contoli M. Caramori G. Pandit A. Johnston S.L. Papi A. Curr. Pharm. Des. 2007; 13: 73-97Crossref PubMed Scopus (55) Google Scholar). Understanding the mechanisms of virus-induced exacerbation of airway diseases is required to identify molecular targets for therapeutic intervention. The mechanisms underlying virus-induced exacerbations of airway diseases are poorly understood. However, rhinoviruses are believed to directly infect airway epithelium inducing pro-inflammatory cytokine production (9Papadopoulos N.G. Bates P.J. Bardin P.G. Papi A. Leir S.H. Fraenkel D.J. Meyer J. Lackie P.M. Sanderson G. Holgate S.T. Johnston S.L. J. Infect. Dis. 2000; 181: 1875-1884Crossref PubMed Scopus (454) Google Scholar, 10Edwards M.R. Haas J. Panettieri Jr., R.A. Johnson M. Johnston S.L. J. Biol. Chem. 2007; 282: 15366-15375Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 11Edwards M.R. Hewson C.A. Laza-Stanca V. Lau H.-T. H. Mukaida N. Hershenson M.B. Johnston S.L. Mol. Immunol. 2007; 44: 1587-1597Crossref PubMed Scopus (13) Google Scholar). This leads to recruitment and activation of inflammatory cells, resulting in airway inflammation (12Fraenkel D.J. Bardin P.G. Sanderson G. Lampe F. Johnston S.L. Holgate S.T. Am. J. Respir. Crit. Care Med. 1995; 151: 879-886Crossref PubMed Scopus (375) Google Scholar, 13Calhoun W.J. Dick E.C. Schwartz L.B. Busse W.W. J. Clin. Investig. 1994; 94: 2200-2208Crossref PubMed Scopus (269) Google Scholar). We have recently demonstrated that bronchial epithelial cells from asthmatic subjects have a deficient innate immune response to rhinovirus infection, responsible for: (i) increased virus replication (14Wark P.A. Johnston S.L. Bucchieri F. Powell R. Puddicombe S. Laza-Stanca V. Holgate S.T. Davies D.E. J. Exp. Med. 2005; 201: 937-947Crossref PubMed Scopus (1039) Google Scholar, 15Contoli M. Message S.D. Laza-Stanca V. Edwards M.R. Wark P.A. Bartlett N.W. Kebadze T. Mallia P. Stanciu L.A. Parker H.L. Slater L. Lewis-Antes A. Kon O.M. Holgate S.T. Davies D.E. Kotenko S.V. Papi A. Johnston S.L. Nat. Med. 2006; 12: 1023-1026Crossref PubMed Scopus (887) Google Scholar) that could account for increased and more persistent inflammatory responses (12Fraenkel D.J. Bardin P.G. Sanderson G. Lampe F. Johnston S.L. Holgate S.T. Am. J. Respir. Crit. Care Med. 1995; 151: 879-886Crossref PubMed Scopus (375) Google Scholar); (ii) increased severity and duration of lower respiratory tract symptoms and reductions in lung function (16Corne J.M. Marshall C. Smith S. Schreiber J. Sanderson G. Holgate S.T. Johnston S.L. Lancet. 2002; 359: 831-834Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar) in rhinovirus-induced asthma exacerbations. Increased oxidative stress is implicated in induction of the acute airway inflammation during exacerbations of asthma and COPD (17Caramori G. Papi A. Thorax. 2004; 59: 170-173Crossref PubMed Scopus (142) Google Scholar). Oxidants are directly involved in inflammatory responses via signaling mechanisms, including the redox-sensitive activation of transcription factors such as NF-κB (18Li N. Karin M. FASEB J. 1999; 13: 1137-1143Crossref PubMed Scopus (792) Google Scholar, 19Janssen-Heininger Y.M. Poynter M.E. Baeuerle P.A. Free Radic. Biol. Med. 2000; 28: 1317-1327Crossref PubMed Scopus (620) Google Scholar). Recent data indicate that rhinovirus and other respiratory viruses can alter cellular redox homeostatic balance toward a pro-oxidative condition (20Peterhans E. J. Nutr. 1997; 127: 962S-965SCrossref PubMed Google Scholar, 21Casola A. Burger N. Liu T. Jamaluddin M. Brasier A.R. Garofalo R.P. J. Biol. Chem. 2001; 276: 19715-19722Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 22Zsengeller Z.K. Ross G.F. Trapnell B.C. Szabo C. Whitsett J.A. Am. J. Physiol. 2001; 280: L503-L511Google Scholar). The molecular pathways responsible for such disequilibrium are virtually unknown. A recent study suggested NADPH oxidase involvement in rhinovirus-induced production of reactive oxygen species over a 6-h infection (23Kaul P. Biagioli M.C. Singh I. Turner R.B. J. Infect. Dis. 2000; 181: 1885-1890Crossref PubMed Scopus (84) Google Scholar). In a previous study we documented that rhinovirus infection induces a rapid increase of intracellular super-oxide anion ( O2•¯), which occurs within 15 min after infection. This early pro-oxidative response was found to induce NF-κB activation and downstream pro-inflammatory molecule production (24Papi A. Papadopoulos N.G. Stanciu L.A. Bellettato C.M. Pinamonti S. Degitz K. Holgate S.T. Johnston S.L. FASEB J. 2002; 16: 1934-1936Crossref PubMed Scopus (54) Google Scholar). O2•¯ is a product of cellular metabolism and mainly originates from the activity of two enzyme systems: NADPH oxidase and xanthine dehydrogenase/xanthine oxidase (XD/XO) (25Halliwell B. Gutteridge J. Free Radical Biology & Medicine. Oxford, University Pess, Oxford, UK1999Google Scholar). Here we studied the molecular mechanisms by which rhinovirus induces rapid O2•¯ production in respiratory epithelial cells. We also analyzed the mechanisms by which reducing agents can abolish rhinovirus-induced O2•¯ production and thus can stabilize the intracellular redox state in respiratory epithelial cells following infection. Finally, we demonstrated that blocking the activity of the system responsible for rhinovirus-triggered O2•¯ generation inhibited rhinovirus-induced inflammatory mediator production in respiratory epithelial cells. Ohio HeLa cells were obtained from the MRC Common Cold Unit, Salisbury, UK, and A549 cells, a type II respiratory cell line, were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Primary human bronchial epithelial cells (HBEC) were obtained by bronchial brushing from healthy volunteers, and cultured as previously described (14Wark P.A. Johnston S.L. Bucchieri F. Powell R. Puddicombe S. Laza-Stanca V. Holgate S.T. Davies D.E. J. Exp. Med. 2005; 201: 937-947Crossref PubMed Scopus (1039) Google Scholar, 24Papi A. Papadopoulos N.G. Stanciu L.A. Bellettato C.M. Pinamonti S. Degitz K. Holgate S.T. Johnston S.L. FASEB J. 2002; 16: 1934-1936Crossref PubMed Scopus (54) Google Scholar, 26Papi A. Johnston S.L. J. Biol. Chem. 1999; 274: 9707-9720Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Rhinovirus type 16 (RV16, a major group rhinovirus) was obtained from the MRC Common Cold Unit. Viral stocks were prepared by infection of sensitive cell monolayers (Ohio HeLa, HeLa) as described elsewhere (24Papi A. Papadopoulos N.G. Stanciu L.A. Bellettato C.M. Pinamonti S. Degitz K. Holgate S.T. Johnston S.L. FASEB J. 2002; 16: 1934-1936Crossref PubMed Scopus (54) Google Scholar, 26Papi A. Johnston S.L. J. Biol. Chem. 1999; 274: 9707-9720Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). TCID50/ml values were determined and the rhinovirus serotype was confirmed by neutralization with serotype-specific antibodies (ATCC) (27Johnston S.L. Tyrrell D.A.J. Lennette E.H. Schmidt N.J. Diagnostic Procedures of Viral, Rickettsial and Clamydial Infections. American Public Health Association, Washington, D. C.1997: 553-563Google Scholar). For selected experiments rhinovirus type 1B (RV1B, minor group), obtained from the MRC Common Cold Unit, was used to evaluate whether the results were group/receptor restricted. For selected experiments filtration of the virus from inoculum, to remove viral particles, was performed as previously described (24Papi A. Papadopoulos N.G. Stanciu L.A. Bellettato C.M. Pinamonti S. Degitz K. Holgate S.T. Johnston S.L. FASEB J. 2002; 16: 1934-1936Crossref PubMed Scopus (54) Google Scholar, 26Papi A. Johnston S.L. J. Biol. Chem. 1999; 274: 9707-9720Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Filtered virus stocks were used as negative control. Virus at a multiplicity of infection of 1 was used for all the experiments. Confluent A549 or HBEC cells were exposed to rhinovirus, medium alone, or filtered virus (f-RV) inoculum for different time intervals (20 min to 8 h). Cell layers were thereafter washed three times in cold phosphate-buffered saline (PBS) before harvesting by scraping. Harvested cells were centrifuged and the cell pellet was resuspended in phosphate buffer (10 mm, pH 7.2). Cell lysis was obtained by repeated (three times) freezing and thawing. For preparation of cytosolic fractions, the cell homogenate was then ultracentrifuged at 20,000 × g for 30 min, the cell fragments pelleted, and the supernatant (cytosol) collected. Where indicated, to obtain the membrane fraction, the cell homogenates were centrifuged at 800 × g for 10 min to separate nuclei from cell membranes. Supernatants were harvested and again centrifuged at 2,000 × g, supernatants discarded, and membrane pellets diluted in 0.1 m sucrose solution. A final centrifugation at 11,000 × g for 20 min was performed at 4 °C. Pellets containing membranes were diluted in 100 μl of PBS buffer. Protein content was determined photometrically using the Bio-Rad protein assay (Bio-Rad). In selected experiments cells were pretreated, before infection, as follows: 12 h (0.25 to 10 mm) GSH (Sigma), or 12 h (0.25 to 2.5 mm)H2S (Acqua Breta, Riolo Terme SpA, Ravenna, Italy) or 4 h (20 μm) oxypurinol (4, 6-dihydroxyprazol (3, 4-d)pyrimidine, Sigma), a permanent inactivator of xanthine oxidase (28Spector T. Biochem. Pharmacol. 1988; 37: 349-352Crossref PubMed Scopus (64) Google Scholar). Where indicated, a 4-h pretreatment with antiproteases (0.625 μm) serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) or 1.25 μm serine and cysteine protease inhibitor leupeptin (Leu), or 1.25 μm aspartic protease inhibitor pepstatin (Pep), or 1.25 μm serine protease inhibitor aprotinin (Apr), or 1.25 μm metalloprotease inhibitor phenanthroline (Phe) or cysteine protease inhibitor E-64, all from Sigma), or diluent alone was performed (29Stracher A. Ann. N. Y. Acad. Sci. 1999; 884: 52-59Crossref PubMed Scopus (48) Google Scholar, 30Bhattacharya S. Ghosh S. Chakraborty S. Bera A.K. Mukhopadhayay B.P. Dey I. Banerjee A. BMC Struct. Biol. 2001; 1: 4Crossref PubMed Google Scholar, 31Kageyama T. Cell Mol. Life Sci. 2002; 59: 288-306Crossref PubMed Scopus (213) Google Scholar, 32Wegner J. J. Extra Corpor. Technol. 2003; 35: 326-338PubMed Google Scholar, 33Yeh C.T. Lai H.Y. Chu S.P. Tseng I.C. Biochem. Biophys. Res. Commun. 2004; 323: 32-37Crossref PubMed Scopus (2) Google Scholar, 34Tamai M. Matsumoto K. Omura S. Koyama I. Ozawa Y. Hanada K. J. Pharmacobio-Dyn. 1986; 9: 672-677Crossref PubMed Scopus (154) Google Scholar). Diluent was made of phosphate buffer, pH 7.2, with a maximal final concentration of 0.4%. PMSF only was previously resuspended in ethanol before dilution in PBS. Antiproteases were removed immediately before infection. The intracellular production of O2•¯ was spectrophotometrically evaluated by superoxide dismutase (SOD)-inhibitable cytochrome c reduction kinetics, as previously described (24Papi A. Papadopoulos N.G. Stanciu L.A. Bellettato C.M. Pinamonti S. Degitz K. Holgate S.T. Johnston S.L. FASEB J. 2002; 16: 1934-1936Crossref PubMed Scopus (54) Google Scholar, 35Pinamonti S. Muzzoli M. Chicca M.C. Papi A. Ravenna F. Fabbri L.M. Ciaccia A. Free Radic. Biol. Med. 1996; 21: 147-155Crossref PubMed Scopus (71) Google Scholar). Kinetics were carried out in 2-ml quartz cuvettes at 37 °C for 20 min in a Uvikon 860 (Kontron Instruments) spectrophotometer in the presence or absence of SOD (500 IU/ml, Sigma). Concentration of cytochrome c from beef heart (Sigma) was 10-5 m. Absorbance readings were taken at 550 nm (peak of reduced cytochrome). Newly generated O2•¯ was measured in each sample and expressed as micromolar, according to standardized procedures (25Halliwell B. Gutteridge J. Free Radical Biology & Medicine. Oxford, University Pess, Oxford, UK1999Google Scholar). Measurements were based on absorbance differences in the presence or absence of SOD, after 5 min of kinetics, when the kinetic slope of cytochrome c reduction was steepest. Data were normalized per mg of protein. Uric acid kinetics were performed at 1 h infection to evaluate the involvement of XD/XO in rhinovirus-induced O2•¯ generation, as uric acid represents the other end product of xanthine degradation by XO. Uric acid kinetics were spectrophotometrically monitored at 293 nm in a UviKon spectrophotometer (Kontron), according to standard procedures (36Priest D.G. Pitts O.M. Anal. Biochem. 1972; 50: 195-205Crossref PubMed Scopus (30) Google Scholar, 37Bartl K. Brandhuber M. Ziegenhorn J. Clin. Chem. 1979; 25: 619-621Crossref PubMed Scopus (11) Google Scholar), on 500 μl of supernatant collected in PBS, pH 7.4, after addition of xanthine (0.1 mm, Sigma). After 15 min of kinetics, uricase (1.0 units/ml) was added to evaluate the amount of uric acid produced. NADPH oxidase assay was performed at different time intervals (20 min to 3 h) to evaluate the involvement of this system in rhinovirus-induced O2•¯ generation. Cell homogenates were centrifuged as described above to separate nuclei from cell membranes. To reconstitute NADPH oxidase, supernatants containing membranes were centrifuged again at 40,000 × g. The reaction mixture contained 200 μl of supernatant and 50 μl of diluted membrane pellet. After 2 min, 200 μm NADPH and 5 mm MgCl2 were added in the presence or absence of the specific inhibitor of NADPH oxidase diphenylene iodonium chloride (0.92 μg/ml, Sigma) (38Morre D.J. Antioxid. Redox Signal. 2002; 4: 207-212Crossref PubMed Scopus (47) Google Scholar). Cytochrome c was added to a concentration of 0.1 mm and PBS, pH 7.2, to a final volume of 0.5 ml, and the reduction kinetics were monitored for 15 min at 37 °C as previously described. Whole cell proteins were extracted from A549 cells as previously described (39Varani K. Caramori G. Vincenzi F. Adcock I. Casolari P. Leung E. Maclennan S. Gessi S. Morello S. Barnes P.J. Ito K. Chung K.F. Cavallesco G. Azzena G. Papi A. Borea P.A. Am. J. Respir. Crit. Care Med. 2006; 173: 398-406Crossref PubMed Scopus (91) Google Scholar). At least 50 mg/lane of whole cell proteins were subjected to a 4-12% Tris glycine gel electrophoresis, and transferred to nitrocellulose filters by blotting. Filters were blocked for 45 min at room temperature in Tris-buffered saline (TBS), 0.05% Tween 20, 5% nonfat dry milk. The filters were then incubated with rabbit anti-human XD/XO (LS-C26419; from LifeSpan Biosciences) for 1 h at room temperature in TBS, 0.05% Tween 20, 5% nonfat dry milk at dilution of 1:500. Filters were washed three times in TBS, 0.5% Tween 20 and after being incubated for 45 min at room temperature with goat anti-rabbit antibody conjugated to horseradish peroxidase (Dako) in TBS, 0.05% Tween 20, 5% nonfat dry milk, at a dilution of 1:4000. After three further washes in TBS, 0.05% Tween 20 visualization of the immunocomplexes was performed using ECL as recommended by the manufacturer (Amersham Biosciences). As an internal control we reprobed each filter with an anti-human actin antibody (Santa Cruz Biotechnology). The 145- and 85-kDa bands of the XD/XO system ((full-length XD and the post-cleavage fragment containing the active site, respectively (40Hille R. Nishino T. FASEB J. 1995; 9: 995-1003Crossref PubMed Scopus (398) Google Scholar, 41Harrison R. Free Radic. Biol. Med. 2002; 33: 774-797Crossref PubMed Scopus (673) Google Scholar)) and the 43-kDa (actin) band were quantified using densitometry with Vision Works® LS software (UVP) and expressed as the ratio with the corresponding actin optical density value of the same lane. RNA interference was used to specifically suppress expression of XD in A549 cells. Cells were transfected in 6-well plates with small interfering RNA (siRNA) using siPORT™ NeoFX™ Transfection Agent (Applied Biosystem), as described by the manufacturer. The following siRNA (all from Ambion) were used: siRNAs for XD (s14918; target sequence: sense, GCAUCGUCAUGAGUAUGUAtt; antisense, UUUAUAGCAUCCUCAAUUGtg), siRNA for GAPDH (4390849) and nonsilencing siRNA (4390843). Total mRNA was extracted by using the RiboPure™ kit (Ambion) as per the manufacturer's instructions. 1 μg of mRNA was used to perform the reverse transcription assay with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). XD mRNA expression was monitored by Real Time RT-PCR using the TaqMan® Gene Expression Assay (Applied Biosystem) specific for XD (catalog number Hs00166010_m1) following the manufacturer's recommendations. The reaction was carried out in a Rotor-Gene™ 6000 instrument (Corbett Life Science). Results were normalized to 18S rRNA (sense, 5′-CGC CGC TAG AGG TGA AAT TCT-3′; antisense, 5′-CAT TCT TGG CAA ATG CTT TCG-3′ 300 nm each, probe, 5′-FAM ACC GGC GCA AGA CGG ACC AGA TAMRA-3′, 175 nm) and expressed as XD mRNA relative levels as compared with nonsilencing siRNA-transfected cells by using the RotorGene software (Corbett Research) and the two standard curve methods for relative quantitation (42Bustin S.A. J. Mol. Endocrinol. 2000; 25: 169-193Crossref PubMed Scopus (3103) Google Scholar). A549 cells were cultured at 85% confluence, incubated with RV16, medium alone, or f-RV for different periods (20 min to 1 h), then trypsinized, collected, and harvested in cryovials with 1.2 ml of 3% metaphosphoric acid in sterile conditions to avoid GSH oxidation and finally frozen in liquid nitrogen until used. Cell homogenate was obtained and protein content was determined as previously described. Intracellular GSH concentration was evaluated by HPLC in a Kontron Instruments apparatus (Milan, Italy) equipped with a C18 hydrophobic column (5 μm particle size, 4.6 × 250 mm), a 420 pump (range 0.005-10 ml/min), a 425 gradient former, and an injection valve with a 20-μl sampling loop. Elution was carried out at room temperature in isocratic gradient (75% methanol and KH2PO4 buffer, pH 3, 1 ml/min speed). GSH was analyzed at 200 nm by a 432 UV detector (Kontron) with an IBM integrated software PC Pack. Homogenates of cells were centrifuged at 40,000 × g for 20 min at 4 °C and the supernatant collected and concentrated on Amicon Ultra 10,000 centrifugal filter devices (Millipore, Bedford, MA) to a final volume of about 300 μl. Samples were analyzed without derivatization against standards of pure lyophilized GSH (Biomedica Foscama), diluted in 1 ml of normal saline solution. The final concentration was obtained by serial dilutions of the lyophilized product. In selected experiments cells were pretreated, before infection, as previously described, for 12 h with GSH, H2S, (0.25 to 2.5 mm), or 4 h with oxypurinol (20 μm). Where indicated, a 4-h pretreatment with protease inhibitors (PMSF, 0.625 μm; Leu, 1.25 μm; Pep, 1.25 μm; Apr, 1.25 μm; Phe, 1.25 μm; E-64, 1.25 μm) or diluent was performed. All protease inhibitors were removed immediately before infection. Titration Assay in a Sensitive Cell Line—Rhinovirus replication was evaluated by titration assay in a sensitive cell line (HeLa) (9Papadopoulos N.G. Bates P.J. Bardin P.G. Papi A. Leir S.H. Fraenkel D.J. Meyer J. Lackie P.M. Sanderson G. Holgate S.T. Johnston S.L. J. Infect. Dis. 2000; 181: 1875-1884Crossref PubMed Scopus (454) Google Scholar). Cells were seeded in a 96-well plate. Where indicated, subconfluent cells were treated with the highest concentration used in the study for each of the tested compounds for the time intervals previously specified (4 h for Leu, Pep, PMSF, Apr, E-64, oxypurinol; 12 h for H2S and GSH) or diluent alone before the infection. Cells were exposed for 1 h to 10-fold serial dilution of RV16, from not diluted down to 10-8 (4 wells per condition). After a 1-h infection, virus unbound to cultured cells was removed and fresh medium added. The cells were incubated in 4% minimal essential medium (Invitrogen) at 37 °C for 5 days, fixed in methanol, and stained with 0.1% crystal violet. The cytopathic effect was evaluated by visual assessment and assessment of the continuity of the monolayer. For each experiment TCID50/ml values were calculated (27Johnston S.L. Tyrrell D.A.J. Lennette E.H. Schmidt N.J. Diagnostic Procedures of Viral, Rickettsial and Clamydial Infections. American Public Health Association, Washington, D. C.1997: 553-563Google Scholar). TaqMan® Real-time PCR—A549 cells were seeded in 6-well plates at 1.7 × 105 cells/ml. Where indicated, subconfluent cells were treated with the highest concentration used in the study for each of the tested compounds for the time intervals previously specified (4 h for Leu, Pep, PMSF, Apr, E-64, and oxypurinol; 12 h for H2S and GSH) or diluent alone before the infection. Cell lysates were harvested at 4 and 8 h following the infection. Total RNA was extracted from cell lysates by using a commercially available kit (RNeasy Kit, Qiagen) following the manufacturer's recommendations. Viral RNA in cell lysates was measured by TaqMan RT-PCR. For this purpose 2 μg of total RNA were used for cDNA synthesis (Omniscript RT kit, Qiagen). TaqMan quantitative PCR was carried out using primers and probe for rhinovirus (sense, 5′-GTG AAG AGC CSC RTG TGC T-3′ 50 nm; antisense, 5′-GCT SCA GGG TTA AGG TTA GCC-3′ 300 nm; probe, 5′-FAM-TGA GTC CTC CGG CCC CTG AAT G-TAMRA-3′, 175 nm) and 18S rRNA (see above) (15Contoli M. Message S.D. Laza-Stanca V. Edwards M.R. Wark P.A. Bartlett N.W. Kebadze T. Mallia P. Stanciu L.A. Parker H.L. Slater L. Lewis-Antes A. Kon O.M. Holgate S.T. Davies D.E. Kotenko S.V. Papi A. Johnston S.L. Nat. Med. 2006; 12: 1023-1026Crossref PubMed Scopus (887) Google Scholar). Reactions consisted of 2 μl of cDNA (cDNA for 18S was diluted 1:100), 12.5 μl of 2× QuantiTect Probe PCR Master Mix (Qiagen), primers, and probes at the final concentrations listed above and RNase-free water to a total volume of 25 μl. Reactions were performed on a Rotor-Gene™ 6000 instrument (Corbett Life Science). Viral RNA expressions were normalized to 18S rRNA and compared with standard curves and expressed as copies per μg of RNA. Subconfluent A549 cells were pretreated for 4 h with oxypurinol (20 μm) before RV16 or f-RV inoculum or medium alone treatment. After a 1-h infection unbounded virus was removed and fresh medium added. Supernatants were harvested at 4 h and levels of IL-8 and GRO-α were assessed using commercially available enzyme-linked immunosorbent assay kits (R&D System) following the manufacturer's instructions. Detection limits for IL-8 and GRO-α enzyme-linked immunosorbent assay were ∼10 and 15 pg/ml, respectively. Nuclear extracts were prepared from A549 cells using the Nuclear Extract Kit (Active Motif). NF-κB activation was assessed in A549 cell nuclear extracts using the TransAM™ p65 Transcription Factor Assay Kit (Active Motif) following the manufacturer's recommendations. Nuclear extract of Jurkat cells provided by the manufacturer (Active Motif) were used as positive controls. Group data were expressed as mean ± S.E. Analysis of variance was used to determine differences between groups. Paired or unpaired Student's t tests were performed after the analysis of variance when appropriate. All experiments were carried out at least 5 times. Bonferroni adjustment was applied where indicated. A probability value of <0.05 was considered significant. Rhinovirus-induced O2•¯ Production in Respiratory Epithelial Cells Is Cytosolic Not Membrane Associated— O2•¯ production was evaluated by SOD-inhibitable cytochrome c reduction kinetics. In our previous study we found that RV16 infection rapidly induced intracellular O2•¯ production, which was maximal at 1 h in A549 cells, a type II respiratory epithelial cell line (24Papi A. Papadopoulos N.G. Stanciu L.A. Bellettato C.M. Pinamonti S. Degitz K. Holgate S.T. Johnston S.L. FASEB J. 2002; 16: 1934-1936Crossref PubMed Scopus (54) Google Scholar, 35Pinamonti S. Muzzoli M. Chicca M.C. Papi A. Ravenna F. Fabbri L.M. Ciaccia A. Free Radic. Biol. Med. 1996; 21: 147-155Crossref PubMed Scopus (71) Google Scholar). That study evaluated intracellular O2•¯ generation, i.e. cell membranes were precipitated before SOD-inhibitable cytochrome c reduction assay was performed. Because other workers had implicated NADPH oxidase in RV induction of reactive oxygen species (23Kaul P. Biagioli M.C. Singh I. Turner R.B. J. Infect. Dis. 2000; 181: 1885-1890Crossref PubMed Scopus (84) Google Scholar) and because NADPH oxidase is a membrane bound system, we first sought to identify the cellular site of O2•¯ production. In the search of cellular sources of RV16-induced O2•¯ production, we first confirmed our previous findings of rapid induction of O2•¯ by RV16 in membrane-free cytosolic fractions (Fig. 1A). We next investigated total cell homogenates, which included cell membranes (Fig. 1B) and found that O2•¯ production was agai
Referência(s)