Phosphatidylinositol 3-Kinase Is Required for Rhinovirus-induced Airway Epithelial Cell Interleukin-8 Expression
2005; Elsevier BV; Volume: 280; Issue: 44 Linguagem: Inglês
10.1074/jbc.m502449200
ISSN1083-351X
AutoresDawn C. Newcomb, Uma S. Sajjan, Suparna Nanua, Yue Jia, Adam M. Goldsmith, J. Kelley Bentley, Marc B. Hershenson,
Tópico(s)Antimicrobial Peptides and Activities
ResumoRhinovirus (RV) is a common cause of asthma exacerbations. The signaling mechanisms regulating RV-induced airway epithelial cell responses have not been well studied. We examined the role of phosphatidylinositol (PI) 3-kinase in RV-induced interleukin (IL)-8 expression. Infection of 16HBE14o- human bronchial epithelial cells with RV39 induced rapid activation of PI 3-kinase and phosphorylation of Akt, a downstream effector of PI 3-kinase. RV39 also colocalized with cit-Akt-PH, a citrogen-tagged fluorescent fusion protein encoding the pleckstrin homology domain of Akt, indicating that 3-phosphorylated PI accumulates at the site of RV infection. Inhibition of PI 3-kinase and Akt attenuated RV39-induced NF-κB transactivation and IL-8 expression. Inhibition of PI 3-kinase also blocked internalization of labeled RV39 into 16HBE14o- cells, suggesting that the requirement of PI 3-kinase for RV39-induced IL-8 expression, at least in part, relates to its role in viral endocytosis. Rhinovirus (RV) is a common cause of asthma exacerbations. The signaling mechanisms regulating RV-induced airway epithelial cell responses have not been well studied. We examined the role of phosphatidylinositol (PI) 3-kinase in RV-induced interleukin (IL)-8 expression. Infection of 16HBE14o- human bronchial epithelial cells with RV39 induced rapid activation of PI 3-kinase and phosphorylation of Akt, a downstream effector of PI 3-kinase. RV39 also colocalized with cit-Akt-PH, a citrogen-tagged fluorescent fusion protein encoding the pleckstrin homology domain of Akt, indicating that 3-phosphorylated PI accumulates at the site of RV infection. Inhibition of PI 3-kinase and Akt attenuated RV39-induced NF-κB transactivation and IL-8 expression. Inhibition of PI 3-kinase also blocked internalization of labeled RV39 into 16HBE14o- cells, suggesting that the requirement of PI 3-kinase for RV39-induced IL-8 expression, at least in part, relates to its role in viral endocytosis. Rhinovirus (RV) 2The abbreviations used are: RV, rhinovirus; ANOVA, analysis of variance; COPD, chronic obstructive pulmonary disease; ELISA, enzyme-linked immunosorbent assay; ICAM, intercellular adhesion molecule; IL, interleukin; IκB, inhibitor of κB; MOI, multiplicity of infection; NF-κB, nuclear factor-κB; PH, pleckstrin homology; PI, phosphatidylinositol; PI(4,5)P2, PI 4,5-bisphosphate; PI(3,4,5)P3, PI 3,4,5-trisphosphate; PBS, phosphate-buffered saline; TCID, tissue culture infectivity dose; TNF, tumor necrosis factor; IKK, IκB kinase. 2The abbreviations used are: RV, rhinovirus; ANOVA, analysis of variance; COPD, chronic obstructive pulmonary disease; ELISA, enzyme-linked immunosorbent assay; ICAM, intercellular adhesion molecule; IL, interleukin; IκB, inhibitor of κB; MOI, multiplicity of infection; NF-κB, nuclear factor-κB; PH, pleckstrin homology; PI, phosphatidylinositol; PI(4,5)P2, PI 4,5-bisphosphate; PI(3,4,5)P3, PI 3,4,5-trisphosphate; PBS, phosphate-buffered saline; TCID, tissue culture infectivity dose; TNF, tumor necrosis factor; IKK, IκB kinase. is a single-stranded RNA virus from the Picornaviridae family responsible for the majority of common colds. Viral infections trigger the majority of asthma exacerbations (1Johnston 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 (1669) Google Scholar, 2Nicholson K.G. Kent J. Ireland D.C. Br. Med. J. 1993; 307: 982-986Crossref PubMed Scopus (947) Google Scholar), and RV accounts for 60% of virus-induced exacerbations (1Johnston 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 (1669) Google Scholar). RV is also an important trigger of chronic obstructive pulmonary disease (COPD) exacerbations (3Seemungal 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 (855) Google Scholar, 4Rohde G. 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Physiol. 1997; 273 (-L759): L749PubMed Google Scholar). ICAM-1 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. In endothelial cells, ICAM-1 cross-linking increases phosphorylation and activation of pp60 Src (19Wang Q. Pfeiffer G.R. Gaarde W.A. J. Biol. Chem. 2003; 278: 47731-47743Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 20Etienne-Manneville S. Manneville J.-B. Adamson P. Wilbourn B. Greenwood J. Couraud P.-O. J. Immunol. 2000; 165: 3375-3383Crossref PubMed Scopus (267) Google Scholar) and pp125 focal adhesion kinase (21Etienne S. Adamson P. Greenwood J. Strosberg A.D. Cazaubon S. Couraud P.-O. J. Immunol. 1998; 161: 5755-5761PubMed Google Scholar). The p85 regulatory subunit of class 1A PI 3-kinase serves as a substrate for both Src (22Haefner B. Baxter R. Fincham V.J. Downes C.P. Frame M.C. J. Biol. 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Endocytosis of adeno-associated virus, which binds to heparan sulfate proteoglycan, activates PI 3-kinase in HeLa cells (26Sanlioglu S. Benson P.K. Yang J. Atkinson E.M. Reynolds T. Engelhardt J.F. J. Virol. 2000; 74: 9184-9196Crossref PubMed Scopus (198) Google Scholar). Finally, respiratory syncytial virus activates PI 3-kinase in A549 lung carcinoma cells (27Thomas K.W. Monick M.M. Staber J.M. Yarovinsky T. Carter A.B. Hunninghake G.W. J. Biol. Chem. 2002; 277: 492-501Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Taken together, these data strongly suggest that RV binding to ICAM-1 could induce IL-8 expression in part by activation of PI 3-kinases. All PI 3-kinases share a catalytic core domain that interacts directly with ATP, PI, and the PI 3-kinase inhibitor LY294002. Members of the PI 3-kinase family are divided into three classes according to their structure and in vitro substrate specificity, as reviewed in Ref. 28Wymann M.P. Zvelebil M. Laffargue M. Trends Pharmacol. Sci. 2003; 24: 366-376Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar. Class 1A PI 3-kinases α, β, and γ are tightly bound with a regulatory subunit (p85α, p85β, or p55γ) that contains Src homology 2 domains with affinity for phosphotyrosine residues in pYXXM motifs found in growth factor receptors, their substrates, and adaptor proteins. The major substrate for the class I PI 3-kinases in vivo appears to be PI(4,5)P2, and the major product appears to be PI(3,4,5)P3 (29Carpenter C.L. Cantley L.C. Biochemistry. 1990; 29: 11147-11156Crossref PubMed Scopus (296) Google Scholar). When activated, class IA PI 3-kinases regulate cellular functions by recruiting PI(3,4,5)P3-binding proteins to the plasma membrane. Most of these proteins, the prototype of which is the serine threonine kinase Akt, bind to 3-phosphorylated PIs through a pleckstrin homology (PH) domain. We therefore examined the contributions of PI 3-kinase and Akt to RV-induced responses in human airway epithelial cells. We found that infection with RV39 induced rapid activation of PI 3-kinase and phosphorylation of Akt, as well as accumulation of 3-phosphorylated PI at the site of RV infection. Inhibition of PI 3-kinase and Akt attenuated RV39-induced nuclear factor (NF)-κB transactivation and IL-8 expression. Inhibition of PI 3-kinase also blocked internalization of labeled RV39, suggesting that the requirement of PI 3-kinase for RV39-induced IL-8 expression, at least in part, relates to its role in viral endocytosis. Cell Culture—16HBE14o- human bronchial epithelial cells originating from bronchial epithelial tissue transfected with pSVori-containing the origin-defective SV40 genome (30Cozens A. Yezzi M. Kunzelmann K. Ohrui T. Chin L. Eng K. Finkbeiner W. Widdicomb J. Gruenert D. Am. J. Respir. Cell Mol. Biol. 1994; 10: 38-47Crossref PubMed Scopus (772) Google Scholar) were provided by Dr. Steven White (University of Chicago). The cells were grown in minimum essential medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 200 mm of l-glutamine. Differentiated primary human bronchial epithelial cell cultures obtained from lung transplant donor tracheas (University Health Network, Toronto, Canada) were cultured at an air-liquid interface, as described (31Sajjan U. Keshavjee S. Forstner J. Infect. Immun. 2004; 72: 4188-4199Crossref PubMed Scopus (50) Google Scholar). Thawed passage 1 cells were seeded into 12-mm collagen-coated Transwell-clear inserts (Costar, Cambridge, MA). The cells were grown under submerged conditions in bronchial epithelial cell growth medium (Clonetics) containing epidermal growth factor (25 ng/ml), bovine pituitary extract (65 ng/ml), all-trans-retinoic acid (5 × 10-8m), bovine serum albumin (1.5 μg/ml), hydrocortisone (0.5 μg/ml), insulin (5 μg/ml), transferrin (10 μg/ml), epinephrine (0.5 μg/ml), triiodothyronine (6.5 ng/ml), gentamycin (50 μg/ml), and amphotericin (50 μg/ml). At confluence, the cells were shifted to a 1:1 mixture of bronchial epithelial cell growth medium and Dulbecco's modified Eagle's medium containing all the above growth factors and hormones and grown at air-liquid interface to promote mucociliary differentiation. The epithelium is pseudostratified with ciliated cells interspersed among mucus secreting cells. Periodic acid Schiff staining shows the presence of goblet cells and luminal glycoproteins, and immunostaining with antibody to tracheo-bronchial mucin has confirmed that the periodic acid Schiff-positive material is mucin. These cultures also show an acceptable transepithelial electrical resistance of 500-540 Ω/cm2. RV Infection—RV39 was obtained from American Type Culture Collection (Manassas, VA). Viral stocks were generated by incubating HeLa cells with RV in serum-free medium until 80% of the cells were cytopathic. For most experiments, HeLa cell lysates were harvested, and cellular debris was pelleted by centrifugation (10, 000 × g for 30 min at 4 °C). To remove soluble factors of HeLa cell origin, RV was concentrated and partially purified by centrifugation with a 100,000 molecular weight cut-off Centricon filter (2,000 rpm for 2 h at 4°C; Millipore, Billerica, MA) (32Papi A. Johnston S.L. J. Biol. Chem. 1999; 274: 9707-9720Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Intact virus fails to go through the filter and is concentrated. For experiments measuring PI 3-kinase activation or phosphorylation of Akt, we harvested HeLa cell supernatants rather than lysates to further decrease the possibility of contamination by HeLa cellular proteins. For fluorescent labeling (see below), partially purified and concentrated RV39 (as above) was overlayered on a sucrose pad consisting of 30% sucrose, 1 m NaCl, and 20 mm Na-Hepes (pH 7.5) and ultracentrifuged (65,000 rpm, 402,000 × gmax for 2 h at 16 °C). The supernatant was carefully removed, and the solid clear pellet was resuspended in 1 ml of phosphate-buffered saline, divided into aliquots, and stored at -80 °C. All titrations were performed by infecting confluent HeLa monolayers with serially diluted RV (range, undiluted to 10-9) and assessing cytopathic effect 5 days after infection. The cells were infected with RV39 for 1 h (or less, depending on experimental design) at 33 °C. Unless otherwise noted, 16HBE14o- cells were infected with RV39 at a multiplicity of infection (MOI) of 1.0. Mucociliary differentiated cultures were infected at a 50% tissue culture infectivity dose (TCID50)/ml of 6 × 105 to 3 × 106. TCID50 values were determined by the Spearman-Karber method (33Johnston S.L. Tyrrell D.A.J. Lennette E.H. Schmidt N.J. Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. 1997: 553-563Google Scholar). For experiments including sham infection, the cells were incubated with an equal volume of HeLa cell lysate or supernatant. In some experiments, purified RV39 was UV-irradiated on ice for 30 min using a UVB CL-1000 cross-linker at 1200 μJ/cm2 (32Papi A. Johnston S.L. J. Biol. Chem. 1999; 274: 9707-9720Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). The effect of UV irradiation was confirmed by the absence of a cytopathic effect on HeLa cell monolayers. Selected cultures were pretreated for 1 h with the chemical PI 3-kinase inhibitor LY294002 in dimethyl sulfoxide (both from Sigma) or carrier alone. In other experiments, the cells were incubated with anti-ICAM-1 antibody (20 μg/ml; Serotec, Raleigh, NC) for 1 h prior to infection, or tumor necrosis factor (TNF)-α neutralizing antibody (0.1-1.0 μg/ml) (R & D, Minneapolis, MN) immediately after infection. As a control, equal concentrations of irrelevant mouse anti-human IgG (Sigma) were used. Measurement of IL-8 Protein Levels—The cells were grown to 80% confluence, serum-starved for 24 h, and then infected with RV39 for 1 h. Inoculum was then replaced with serum-free medium. Conditioned medium was collected 48 h post-infection, centrifuged to remove cell debris, and then frozen at -80 °C. For mucociliary cultures, RV was applied to the apical surface for 1 h. Forty-eight hours post-infection, apical washings and basolateral supernatants were collected from the basolateral compartment. IL-8 protein levels were measured by enzyme-linked immunosorbent assay (ELISA; R & D Systems). Measurement of IL-8 Promoter Activity and NF-κB Transactivation—The -162/+44 fragment of the human IL-8 promoter subcloned into luciferase (-162/+44 hIL8/Luc) was obtained from Dr. Allan Brasier (34Denning G.M. Wollenweber L.A. Railsback M.A. Cox C.D. Stoll L.L. Britigan B.E. Infect. Immun. 1998; 66: 5777-5784Crossref PubMed Google Scholar). NF-κB reporter plasmid was purchased from Stratagene (La Jolla, CA). pRL family Renilla luciferase plasmid was purchased from Promega (Madison, Wisconsin). pSG5-Myc-Δp85, a dominant-negative class IA PI 3-kinase, and pSG5 p110PI3-KCAAX, an activated PI 3-kinase with a C-terminal farnesylation signal from H-Ras, which causes its membrane localization (35Wennstrom S. Downward J. Mol. Cell. Biol. 1999; 19: 4279-4288Crossref PubMed Scopus (254) Google Scholar), were obtained from Dr. Julian Downward (Cancer Research UK London Research Institute). CS2+myr-Akt K/D, which expresses a constitutively membrane-localized, C-terminally truncated, catalytically inactive Akt1 (amino acids 1-647), and pCS2+PHDDAkt, which expresses an activated Akt1 (amino acids 131-480) in which Ser473 and Thr308 were altered to Asp by PCR mutagenesis, were provided by Dr. Anne Vojtek (University of Michigan) (36Vojtek A.B. Taylor J. DeRuiter S.L. Yu J.-Y. Figueroa C. Kwok R.P.S. Turner D.L. Mol. Cell. Biol. 2003; 23: 4417-4427Crossref PubMed Scopus (87) Google Scholar). Deletion mutants of inhibitor of κB (IκB)-α encoding amino acids 37-317 (IκBαΔN) and 1-242 (IκBαΔC) were provided by Dr. Dean Ballard (Vanderbilt University) (37Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). kinase A dominant-negative IκB (IKK)-β, in which Ser177 and Ser181 were replaced by alanines (38Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar), was obtained from Dr. Anning Lin. 16HBE14o- cells were grown to 50% confluence, washed in Optimem (Invitrogen), and incubated with plasmid DNA, Lipofectamine, and Optimem (Invitrogen). After 4 h, the solution was replaced with minimum essential medium supplemented with 10% fetal bovine serum. The next morning, the cells were serum-starved for 24 h. The cells were then treated with RV39 for 1 h. Inoculum was replaced with fresh serum-free medium, and the were cells harvested for analysis 24 h post-infection. Luciferase activity was measured using a luminometer. Changes in promoter activity were normalized for transfection efficiency by dividing luciferase light units by Renilla luciferase light units. The results were then reported as fold increase over the empty vector/untreated control. Expression of genetic mutants was verified by immunoblotting (data not shown). Northern Analysis—Total RNA was prepared from primary human mucociliary-differentiated tracheal epithelial cell cultures using TRIzol reagent (Sigma), electrophoresed on 1.25% SeaKem Gold agarose gels (Reliant RNA gel system; Cambrex, Rockland, MN), and then transferred onto Immobilin-NY Plus membranes (Millipore, Bedford, MA). Probe template was prepared from RNA extracted from primary cells by reverse transcription-PCR with the IL-8 primers 5′-ATG ACT TCC AAG CTG GCC GTG GCT-3′ (sense) and 5′-TCT CAG CCC TCT TCA AAA ACT TCT C-3′ (antisense). 32P-Radiolabeled probe was prepared using Ready-To-Go DNA labeling beads (-dCTP) (Amersham Biosciences). Probe hybridization was performed with Ultrahyb buffer as recommended by the manufacturer (Amersham Biosciences). PI 3-Kinase Assay—After RV infection, 16HBE14o- cells were immunoprecipitated with anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Inc., Waltham, MA). Precipitates were incubated with PI(4,5)P2 and [γ32P]ATP for 30 min at room temperature. The reaction was terminated with 20 μl of 6 m HCl and 150 μl of chloroform/methanol (1:1). The organic phase was then was separated by centrifugation at 8000 rpm for 3 min. Lipids were dissolved in 15 μl of chloroform/methanol (95:5) and subjected to thin layer chromatography. The plates were dried, and activity was assessed by autoradiography. Immunoblotting—After the relevant treatment, 16HBE14o- cells were lysed, cellular proteins were resolved by 10% SDS-PAGE, and proteins were transferred to a nitrocellulose membrane. The membranes were probed with antibodies against Ser473 phospho-Akt, total Akt, Ser32/36 phospho-IκBα total IκBα (Cell Signaling, Beverly, MA), or p85 PI 3-kinase (Upstate Biotechnology, Inc.). The signals were amplified and visualized with horseradish peroxidase-conjugated secondary antibody (Bio-Rad) and chemiluminescence solution (Pierce). Fluorescent Microscopy—For fluorescent labeling of RV39, 0.4 ml of 0.1 m NaHCO3 was added to a 0.1-ml aliquot of purified, concentrated virus to raise pH to 8.5. The virus was then incubated in the dark for 1 h with 250 μg of Alexa-fluor 555 carboxylic acid succinimidyl ester, an amine-reactive probe (Molecular Probes, Eugene, OR). The reaction was quenched with 0.5 ml of 1 m Tris buffer (pH 8.0), and labeled virus was dialyzed three times against Tris buffer (in the dark, 12 h at 4 °C). The virus was concentrated by precipitation with a final 6% (w/v) polyethylene glycol 8000 precipitation in 0.5 m NaCl for 4 h at 4°C, and centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant fluid was completely aspirated, and the virus was resuspended in 0.1 ml of phosphate-buffered saline (PBS) with 0.1% bovine serum albumin for stability. This virus maintained infectivity, with no reduction in TCID50 compared with unlabeled virus. To visualize RV39 internalization into 16HBE14o- cells, cells on collagen-coated slides (Becton Dickson Labware, Bedford, MA) were infected with Alexa fluor-labeled virus (1 × 106 TCID50/ml) or an equal volume of Alexa fluor-labeled cell lysate from uninfected HeLa cells for 15 min at 33 °C. The cells were then washed extensively, fixed in PBS with 1% paraformaldehyde for 20 min at 4 °C, washed three times further with PBS, and mounted with ProLong Antifade reagent (Molecular Probes) and visualized by fluorescent microscopy. We also monitored the infection of 16HBE14o- cells stably transfected with enhanced green fluorescent protein (Clontech, Palo Alto, CA) or cit-Akt-PH (39Marshall J.G. Booth J.W. Stambolic V. Mak T. Balla T. Schreiber A.D. Meyer T. Grinstein S. J. Cell Biol. 2001; 153: 1369-1380Crossref PubMed Scopus (233) Google Scholar, 40Hoppe A.D. Swanson J.A. Mol. Biol. Cell. 2004; 15: 3509-3519Crossref PubMed Scopus (276) Google Scholar), a citrogen-tagged fusion protein encoding the pleckstrin homology domain of Akt (provided by Dr. Joel Swanson, University of Michigan). Fifteen min after infection with labeled RV39, the cells were washed, fixed and mounted as above, and visualized by confocal fluorescent microscopy. Colocalization was quantified by NIH Image software (Carl Zeiss, Jena, Germany). Electrophoretic Mobility Shift Assay—NF-κB consensus oligonucleotide was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and radiolabeled with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs, Inc). Nuclear extracts were obtained from 16HBE14o- cells, and electrophoretic mobility assay was conducted as previously described (41Li J. Johnson X.D. Iazvovskaia S. Tan A. Lin A. Hershenson M.B. Am. J. Physiol. 2003; 284 (-L315): L307Crossref PubMed Scopus (50) Google Scholar). Briefly, nuclear extract (4 μg) was incubated at room temperature for 10 min in binding buffer (4% glycerol, 1 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 50 mm NaCl, 10 mm Tris, pH 7.5, 2.5 μg of dI-dC). Radiolabeled probe (75,000 disintegrations/s) was added, and the reaction was allowed to continue for 20 min. Samples were loaded on a 4% polyacrylamide nondenaturing gel and electrophoresed at room temperature for 4 h in 0.5% Tris-boric acid-EDTA buffer. The gels were dried, and binding was assessed by autoradiography. IKK Assay—Endogenous IKK was immunoprecipitated from cell extracts with an anti-IKKγ antibody (Santa Cruz Biotechnology). Activity of the immune complex was assayed in 30 μl of kinase buffer in the presence of 10 μm ATP, 5 μCi of [γ-32P]ATP, and glutathione S-transferase-IκBα (3 μg/sample) as a substrate (30 °C for 15 min), as described (38Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar, 41Li J. Johnson X.D. Iazvovskaia S. Tan A. Lin A. Hershenson M.B. Am. J. Physiol. 2003; 284 (-L315): L307Crossref PubMed Scopus (50) Google Scholar). The reactions were terminated with 4× Laemmli sample buffer. Samples were resolved by 10% SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane by semidry transfer. After Ponceau staining, the membrane was exposed to film, and substrate phosphorylation was assessed by optical scanning. Equal levels of immunoprecipitated IKK were confirmed by immunoblotting using anti-IKKγ antibody. Measurement of RV39 Binding and Internalization—35S-Labeled virus was generated by infecting HeLa cells with RV39 in the presence of [35S]methionine and methionine-free Dulbecco's modified Eagle's medium (Sigma) for 24 h, lysing the cells by freeze-thaw, and concentrating by centrifugation with a Centricon filter, as above. 16HBE14o- cells were then infected [35S]methionine-labeled RV39 for either 1 h at 4 °C (binding) or 30 min at 33 °C (internalization) (42Baravalle G. Brabec M. Snyers L. Blaas D. Fuchs R. J. Virol. 2004; 78: 2729-2737Crossref PubMed Scopus (15) Google Scholar). To determine virus binding, 16HBE14o- cells grown in 12-well plates were incubated with 5 × 104 cpm [35S]methionine-labeled RV39/well in 0.5 ml of minimum essential medium for 1 h at 4°C. The cells were washed at 4 °C with PBS, and cell-associated radioactivity was measured in cell lysates by liquid scintillation counting. To quantify internalization, 16HBE14o- cells in 12-well plates were detached with 2 mm EDTA, washed, and incubated with 5 × 104 cpm [35S]methionine-labeled RV39/2 × 105 cells in minimum essential medium supplemented with 0.5% bovine serum albumin at 4 °C for 1 h. The cells were warmed to 33 °C for 30 min, pelleted by centrifugation, washed with ice-cold PBS to remove noninternalized virus, and lysed for measurement of cell-associated radioactivity. Data Analysis—Statistical significance was assessed by repeated measures analysis of variance (ANOVA). Differences identified by ANOVA were pinpointed by the Student Newman-Keuls' multiple range text. RV39 Infection Increases IL-8 Expression in Cultured Airway Epithelial Cells—16HBE14o- human bronchial epithelial cells were infected with RV39 (1 h at 33 °C). Forty-eight hours after infection, cell supernatants were collected for measurement of IL-8 protein by ELISA. RV39 increased IL-8 in a concentration-dependent manner (Fig. 1A). Equal volumes of HeLa cell lysate from uninfected cells had no effect. Concentrations of RV39 as low as 0.1 MOI increased IL-8 protein secretion (data not shown). To test whether IL-8 expression was transcriptionally regulated, cells were transfected with the -162/+44 fragment of the full-length human IL-8 promoter subcloned into a luciferase reporter, infected with RV39 (1 h at 33 °C), and incubated for an additional 24 h (Fig. 1B). RV39 increased IL-8 promoter activity by about 5-fold, consistent with the notion that IL-8 is transcriptionally regulated by RV39 infection. We tested whether RV39 infection increases IL-8 expression in differentiated human tracheal epithelial cells. Passage 1 cells, isolated from the tracheas of lung transplant donors, were grown at air-liquid interface for 2 weeks. After RV39 infection, washings from the apical surface and
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