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• W2004999743 abstract "Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o− cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor κB (NFκB) was activated in 16HBE14o− cells stimulated with NE. Western blot analysis demonstrated that activation of NFκB by NE was preceded by phosphorylation and degradation of IκB proteins, principally IκBβ. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o− cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88Δ) or TRAF-6 (TRAF-6Δ) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Δ inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of IκBβ and nuclear translocation of NFκB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition. Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o− cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor κB (NFκB) was activated in 16HBE14o− cells stimulated with NE. Western blot analysis demonstrated that activation of NFκB by NE was preceded by phosphorylation and degradation of IκB proteins, principally IκBβ. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o− cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88Δ) or TRAF-6 (TRAF-6Δ) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Δ inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of IκBβ and nuclear translocation of NFκB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition. cystic fibrosis neutrophil elastase interleukin tumor necrosis factor bronchial epithelial cell nuclear factor κB interleukin-1 receptor- associated kinase tumor necrosis factor receptor-associated factor dominant negative TRAF-6/2 epithelial lining fluid polymerase chain reaction reverse transcriptase-PCR phenylmethylsulfonyl fluoride recombinant secretory leukoprotease inhibitor Toll-like receptor phosphate-buffered saline glyceraldehyde-3-phosphate dehydrogenase Cystic fibrosis (CF)1 is the most common life-threatening hereditary disorder affecting the Caucasian population. It is caused by mutations of the CF transmembrane conductance regulator gene, a 27-exon, 250-kilobase gene on chromosome 7 at q31, whose predicted primary translation product is a 1480-amino acid protein (1Rommens J.M. Iannuzzi M.C. Kerem B.-S. Drumm M.L. Melmer G. Dean M. Rozmahel R. Cole J.L. Kennedy D. Hidaka N. Zsiga M. Buchwald M. Riordan J.R. Tsui L.-C. Collins F.S. Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2531) Google Scholar, 2Riordan J.R. Rommens J.M. Kerem B.-S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.-L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5925) Google Scholar). The major cause of mortality and morbidity in CF is lung disease. Although airway obstruction and infection are clearly major pathogenic factors in CF, there is increasing recognition that the pulmonary inflammatory response is of key importance (3Boat T.F. Welsh M.J. Beaudet A.L. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Diseases. McGraw-Hill Book Co., New York1989: 2649-2680Google Scholar, 4Birrer P. McElvaney N.G. Rudeberg A. Sommer C.W. Liechti-Gallati C. Kraemer R. Hubbard R. Crystal R.G. Am. J. Respir. Crit. Care Med. 1994; 150: 207-213Crossref PubMed Scopus (333) Google Scholar, 5Konstan M.W. Hilliard K.A. Norvell T.M. Berger M. Am. J. Respir. Crit. Care Med. 1994; 150: 448-454Crossref PubMed Scopus (473) Google Scholar, 6Balough K. McCubbin M. Weinberger M. Smits W. Ahrens R. Fick R. Pediatr. Pulmonol. 1995; 20: 63-70Crossref PubMed Scopus (285) Google Scholar, 7Khan T.Z. Wagener J.S. Bost T. Martinez J. Accurso F.J. Riches D.W. Am. J. Respir. Crit. Care Med. 1995; 151: 939-941PubMed Google Scholar). One of the essential components of this inflammation is the neutrophil (8Taggart C. Coakley R.J. Greally P. Canny G. O'Neill S.J. McElvaney N.G. Am. J. Physiol. 2000; 278: L33-L41Crossref PubMed Google Scholar). Even in young infants with CF, studies have demonstrated elevated numbers of neutrophils and increased neutrophil products on the respiratory epithelial surface (4Birrer P. McElvaney N.G. Rudeberg A. Sommer C.W. Liechti-Gallati C. Kraemer R. Hubbard R. Crystal R.G. Am. J. Respir. Crit. Care Med. 1994; 150: 207-213Crossref PubMed Scopus (333) Google Scholar, 6Balough K. McCubbin M. Weinberger M. Smits W. Ahrens R. Fick R. Pediatr. Pulmonol. 1995; 20: 63-70Crossref PubMed Scopus (285) Google Scholar,9Konstan M.W. Berger M. Pediatr. Pulmonol. 1997; 24: 137-142Crossref PubMed Scopus (2) Google Scholar). While neutrophil accumulation on the airway epithelial surface is an essential component of normal host defense against infection, when exaggerated it can cause progressive damage to the bronchial epithelium. In CF, this damage is mediated most significantly by neutrophil elastase (NE), a powerful proteolytic enzyme released by activated neutrophils (10Nakamura H. Yoshimura K. McElvaney N.G. Crystal R.G. J. Clin. Invest. 1992; 89: 1478-1484Crossref PubMed Scopus (438) Google Scholar, 11McElvaney N.G. Crystal R.G. Crystal R.G. West J.B. Barnes P.F. Weibel E. The Lung. Lippincott-Raven, New York1997: 2205-2218Google Scholar). NE can impair local host defense mechanisms by degrading many extracellular matrix molecules and adversely affecting mucociliary clearance (5Konstan M.W. Hilliard K.A. Norvell T.M. Berger M. Am. J. Respir. Crit. Care Med. 1994; 150: 448-454Crossref PubMed Scopus (473) Google Scholar, 11McElvaney N.G. Crystal R.G. Crystal R.G. West J.B. Barnes P.F. Weibel E. The Lung. Lippincott-Raven, New York1997: 2205-2218Google Scholar). NE also cleaves immunoglobulin and complement proteins, thereby reducing phagocytosis and killing of Pseudomonas aeruginosa by neutrophils (12Breuer R. Christensen T.G. Niles R.M. Stone P.J. Snider G.L. Am. Rev. Respir. Dis. 1989; 139: 779-782Crossref PubMed Scopus (39) Google Scholar, 13Berger M. Soerensen R.J. Tosi M.F. Dearborn D.G. Doring G. J. Clin. Invest. 1989; 84: 1302-1313Crossref PubMed Scopus (166) Google Scholar, 14Fick R.B. Naegel G.P. Squier S. Wood R.E. Gee J.B.L. Reynolds H.Y. J. Clin. Invest. 1984; 74: 236-248Crossref PubMed Scopus (148) Google Scholar, 15Tosi M.F. Zakem H. Berger M. J. Clin. Invest. 1990; 86: 300-308Crossref PubMed Scopus (236) Google Scholar). In CF epithelial lining fluid (ELF), NE has been identified as a major signal capable of inducing the expression of IL-8 in bronchial epithelial cells (BECs) (10Nakamura H. Yoshimura K. McElvaney N.G. Crystal R.G. J. Clin. Invest. 1992; 89: 1478-1484Crossref PubMed Scopus (438) Google Scholar). IL-8, a member of the CXC chemokine family, is a potent activator and chemoattractant of neutrophils (16Leonard E.J. Yoshimura T. Am. J. Respir. Cell Mol. Biol. 1990; 2: 479-486Crossref PubMed Scopus (175) Google Scholar) and has been shown to be expressed in BECs in response to a variety of stimuli (17Baggiolini M. Walz A. Kunkel S.L. J. Clin. Invest. 1989; 84: 1045-1049Crossref PubMed Scopus (1620) Google Scholar, 18Nakamura H. Yoshimura K. Jaffe H.A. Crystal R.G. J. Biol. Chem. 1991; 266: 19611-19617Abstract Full Text PDF PubMed Google Scholar, 19Matsushima K. Oppenheim J.J. Cytokine. 1989; 1: 2-13Crossref PubMed Scopus (545) Google Scholar). In CF, IL-8-induced recruitment of additional neutrophils to the airways results in further release of NE and additional induction of IL-8 gene expression by bronchial epithelial cells, thereby perpetuating a chronic cycle of respiratory inflammation (10Nakamura H. Yoshimura K. McElvaney N.G. Crystal R.G. J. Clin. Invest. 1992; 89: 1478-1484Crossref PubMed Scopus (438) Google Scholar). In vivo studies have demonstrated that NE induction of IL-8 gene expression in the CF respiratory epithelial surface and subsequent neutrophil-dominated inflammation may be attenuated by NE inhibitors (20McElvaney N.G. Nakamura H. Birrer P. Hébert C.A. Wong W.L. Alphonso M. Baker J.B. Catalano M.A. Crystal R.G. J. Clin. Invest. 1992; 90: 1296-1301Crossref PubMed Scopus (260) Google Scholar), demonstrating a new potential therapeutic target for the respiratory manifestations of CF. While NE appears to be a major signal for CF bronchial epithelial IL-8 gene expression, the intracellular mechanisms of this response are unknown. NE increases the relative rate of transcription of the IL-8 gene in BECs but does not influence the IL-8 mRNA transcript stability, suggesting that the transcriptional process dominates in the response (10Nakamura H. Yoshimura K. McElvaney N.G. Crystal R.G. J. Clin. Invest. 1992; 89: 1478-1484Crossref PubMed Scopus (438) Google Scholar). With this as background, we have elucidated the intracellular signaling pathways involved in NE induction of IL-8 gene expression in BECs. The data demonstrate that as previously shown BECs are capable of producing IL-8 protein and expressing the IL-8 gene, and these responses are up-regulated by NE. In addition, we have demonstrated that NE-stimulated IL-8 gene expression results in increased activation of NFκB in BECs via a pathway involving IκBβ. We have also identified that interleukin-1 receptor-associated kinase (IRAK) (21Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (770) Google Scholar, 22Martin M. Bol G.F. Eriksson A. Resch K. Brigelius-Flohe R. J. Immunol. 1994; 24: 1566-1571Google Scholar, 23Croston G.E. Cao Z. Goeddel D.V. J. Biol. Chem. 1995; 270: 16514-16517Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 24O'Neill L.A.J. Greene C. J. Leukoc. Biol. 1998; 63: 650-657Crossref PubMed Scopus (497) Google Scholar, 25Greene C. O'Neill L.A.J. Biochim. Biophys. Acta. 1999; 1451: 109-121Crossref PubMed Scopus (18) Google Scholar) is involved in NE-induced signaling events in BECs and provided evidence of the involvement of the signal transducing molecules MyD88 (24O'Neill L.A.J. Greene C. J. Leukoc. Biol. 1998; 63: 650-657Crossref PubMed Scopus (497) Google Scholar, 26Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (982) Google Scholar, 27Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (527) Google Scholar, 28Wesch H. Henzel W.J. Shillinglaw W. Li S. Cao Z.,. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 29Burns K. Martinon F. Esslinger C. Pahl H. Schneider P. Bodmer J.L. Di Marco F. French L. Tschopp J. J. Biol. Chem. 1998; 273: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar) and TRAF-6 (24O'Neill L.A.J. Greene C. J. Leukoc. Biol. 1998; 63: 650-657Crossref PubMed Scopus (497) Google Scholar, 25Greene C. O'Neill L.A.J. Biochim. Biophys. Acta. 1999; 1451: 109-121Crossref PubMed Scopus (18) Google Scholar,30Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1119) Google Scholar) in the NE-induced IL-8 signaling pathway. In summary, these data show that NE activates IL-8 gene expression through a MyD88/IRAK/TRAF-6-dependent pathway, a breakthrough that may enable better targeting of novel anti-inflammatory therapy for the CF condition. 16HBE14o− cells, an SV-40-transformed human bronchial epithelial cell line (31Cozens A.L. Yezzi M.J. Kunzelmann K. Ohrui T. Chin L. Eng K. Finkbeiner W.E. Widdicombe J.H. Gruenert D.C. Am. J. Respir. Cell Mol. Biol. 1994; 10: 38-47Crossref PubMed Scopus (776) Google Scholar), were obtained as a gift from D. Gruenert (University of Vermont). The cells were cultured at 37 °C in Eagle's minimal essential medium (Biowhittaker, Berkshire, United Kingdom) supplemented with 10% fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin (Life Technologies, Inc.). The surface of the culture dishes was coated with a mixture of fibronectin (1 mg/ml; Sigma), collagen (Vitrogen 100, 2.9 mg/ml; Collagen Corp., Palo Alto, CA), and bovine serum albumin (1 mg/ml; Sigma). To eliminate the effect of different factors in the growth media and any effect contributed by trypsin, 24 h prior to agonist treatment cells were washed twice with 1× PBS and placed under serum-free conditions. Before agonist treatment, cells were again washed twice with 1× PBS and covered with serum-free media for 1 h. Neutrophil elastase was purchased from Elastin Products Company, Inc. (Owensville, MO). Recombinant human TNFα and secretory leukoprotease inhibitor (rSLPI) were purchased from R&D Systems (Oxon, UK), and actinomycin D, MeO-Suc-Ala-Ala-Pro-Val-chloromethyl ketone, and PMSF were all purchased from Sigma. HBE cells were seeded at 1 × 106 on coated 24-well plates (16-mm diameter) 24 h before stimulation. Cells were left untreated or were stimulated with different doses of NE for different time periods or with TNFα (10 ng/ml, 3 h). In some experiments, cells were pretreated with actinomycin D (10 μg/ml) for 1 h before NE or TNFα treatment. The dose dependence studies with NE were carried out using 0–50 nm purified NE for 4 h. The time course studies used 10 nm NE and subsequent incubation of the HBE cells for 0–24 h. IL-8 protein concentrations in the cell supernatants were determined by enzyme-linked immunosorbent assays (R&D Systems). Following the removal of supernatants, the cells were lysed using RIPA buffer (1% Igepal CA-630, 0.5% deoxycholic acid, 0.1% SDS, 1% PMSF (10 mg/ml), 1% sodium orthovanadate (100 mm), and 3% aprotinin) (Sigma), and protein concentrations were determined by the method of Bradford (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar). Cell viability assessed by trypan blue exclusion was >95% in all studies. Total RNA was isolated from 1 × 106 HBE cells that had been left untreated or been stimulated with NE (10 nm, 4 h) or TNFα (10 ng/ml, 3 h) ± actinomycin D (10 μg/ml, 1 h) using TRI reagent (Sigma) according to the manufacturer's instructions. For both quantitative LightCyclerTM (Roche Molecular Biochemicals) PCRs and semiquantitative RT-PCRs, 1 μg of total RNA was reverse-transcribed into cDNA with an oligo(dT)15primer using the first strand cDNA synthesis kit (Roche Molecular Biochemicals) as described in the manufacturer's protocol. cDNA amplifications were done by quantitative PCR using the light cycler and the double-stranded DNA binding dye SYBR Green 1 (Roche Molecular Biochemicals). The samples were continuously monitored during the PCR, and fluorescence was acquired every 0.1 °C. PCR mixtures contained 0.5 μm of either GAPDH- (MWG Biotech, Milton Keynes, UK) or IL-8-specific primers (BIOSOURCE). The samples were denatured at 95 °C for 10 min followed by 45 cycles of annealing and extension at 95 °C for 15 s, 55 °C for 5 s, and 72 °C for 10 s. The melting curves were obtained at the end of amplification by cooling the samples to 65 °C for 15 s followed by further cooling to 40 °C for 30 s. Serial 10-fold dilutions were prepared from known quantities of GAPDH and IL-8 PCR products, which were then used as standards to plot against the unknown samples. Quantification of data was analyzed using the LightCyclerTM analysis software, and values were normalized to GAPDH expression. In semiquantitative RT-PCR, the integrity of RNA extraction and cDNA synthesis was verified by PCR by measuring the amounts of GAPDH cDNA in each sample using GAPDH-specific primers. PCR mixtures contained 10× reaction buffer (Promega, Madison, WI), 2.5 mm MgCl2, 1.25 units of Taqpolymerase, and 0.2 mm of each dNTP (Promega). Thermocycling conditions for IL-8 cDNA were 95 °C for 5 min, 35 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. Twenty cycles were used to amplify the more abundant GAPDH cDNA. A final extension step of 72 °C for 10 min was followed by resolution of the 227-base pair IL-8 products and the 211-base pair GAPDH products on a 1.5% Tris borate-EDTA agarose gel containing 0.5 μg/ml ethidium bromide (Sigma). IL-8 PCR products were quantified by densitometry using the GeneGenius Gel Documentation and Analysis System (Cambridge, UK) and Syngene GeneSnap and GeneTools software. HBE cells (1–4 × 106/ml) were seeded on coated six-well plates (34-mm diameter) 24 h before stimulation. Cells were activated with NE (10 nm) or TNFα (10 ng/ml) for different time periods ± actinomycin D (10 μg/ml, 1 h), and nuclear and cytoplasmic extracts were isolated. Briefly cells were washed and resuspended in 1 ml of ice-cold PBS and kept on ice for 5 min. Cells were lifted from plates with a cell scraper and pelleted by centrifugation at 10,000 rpm for 5 min at 4 °C. The supernatant was removed, and the cell pellet was resuspended in 1 ml of hypotonic buffer (10 mm Hepes (pH 7.9), 1.5 mmMgCl2, 10 mm KCl, 0.5 mm PMSF, and 0.5 mm dithiothreitol) (Sigma). Cells were pelleted by centrifugation at 14,000 rpm for 10 min at 4 °C and then lysed for 10 min on ice in 20 μl of hypotonic buffer containing 0.1% Igepal CA-630. Lysates were centrifuged as before, and the cytoplasmic extract was removed. The remaining nuclear pellet was lysed in 15 μl of lysis buffer (20 mm Hepes (pH 7.9), 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% (v/v) glycerol, 0.5 mm PMSF) (Sigma) for 15 min on ice. After centrifugation at 14,000 rpm for 10 min at 4 °C, nuclear extracts were removed into 35 μl of storage buffer (10 mmHepes (pH 7.9), 50 mm KCl, 0.2 mm EDTA, 20% (v/v) glycerol, 0.5 mm PMSF, and 0.5 mmdithiothreitol). Protein concentrations of cytoplasmic and nuclear extracts were determined (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar), and extracts were stored at −80 °C until required for use. Nuclear extracts (5 μg of protein) were incubated with 10,000 cpm of a 22-base pair oligonucleotide containing the NFκB consensus sequence (Santa Cruz Biotechnology, Santa Cruz, CA) that previously had been labeled with [γ-32P]ATP (10 mCi/mmol) (Amersham Pharmacia Biotech) by T4 polynucleotide kinase (Promega). Incubations were performed for 30 min at room temperature in binding buffer (4% (v/v) glycerol, 1 mm EDTA, 10 mm Tris-HCl (pH 7.5), 100 mm NaCl, 5 mm dithiothreitol, and 0.1 mg/ml nuclease-free bovine serum albumin) and 2 μg of poly(dI-dC)·poly(dI-dC) (Sigma). In some experiments, unlabeled mutant or wild-type oligonucleotides or antibodies to p50, p65, or c-Rel (Santa Cruz Biotechnology) were added to the extracts before incubation with the labeled oligonucleotide. All incubations were subjected to electrophoresis on native 4% (w/v) polyacrylamide gels that were subsequently dried, analyzed on a Molecular Dynamics Storm 820 Phosphorimagery Scanner (Amersham Pharmacia Biotech) for quantification, and autoradiographed. Cytoplasmic extracts (10 μg of protein) were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Sigma) in 20 mm Tris, 150 mm glycine, 0.01% SDS, and 20% (v/v) methanol at 75 mA for 2 h using a semidry electrophoretic blotting system. Nonspecific binding was blocked with 0.2% I-Block (Tropix, Bedford, MA) and PBS containing 0.1% Tween 20 (Sigma). Immunoreactive proteins were detected by incubating the membrane with specific antibodies (IκBα and IκBβ from Santa Cruz Biotechnology and IRAK from Transduction Laboratories, Lexington, KY). Following six 5-min washes with PBS containing 0.1% Tween 20, immunoreactive proteins were detected using alkaline phosphatase-conjugated anti-mouse IgG (Promega) (IRAK and IκBα) or goat anti-rabbit IgG (Tropix) (IκBβ) and CDP-Star chemiluminescent substrate solution (Sigma) according to the manufacturer's instructions. To construct the IL-8 luciferase reporter plasmid, the human IL-8 promoter was PCR-amplified using Pfu polymerase (Stratagene, Cambridge, UK) from genomic DNA with 5′-GCACTCGAGTAACCCAGGCATTATT-3′ (forward) and 5′-GCTAAGCTTAGTGCTCCGGTGGCTTTT-3′ (reverse) and was cloned on a XhoI-HindIII fragment (underlined) into pGL3-PV (Promega). HBE cells were seeded at 5 × 105 on coated six-well plates 24 h before transfection. Transfections were performed with TransFast Transfection Reagent (Promega) in a 1:1 ratio according to the manufacturer's instructions using 500 ng of IL-8 luciferase and 500 ng of either pRK5, TRAF-6Δ, TRAF-2Δ (a gift from Tularik, San Francisco, CA), pCDNA3, or MyD88Δ (a gift from M. Muzio (26Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (982) Google Scholar, 27Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (527) Google Scholar)). Uniform transfection efficiencies were achieved by initially optimizing transfection conditions using a constitutive luciferase expression vector, pGL3-control (Promega). Transfections were incubated for 1 h at 37 °C. Cells were then supplemented with additional growth medium (4 ml/well) for 48 h at 37 °C before being left untreated or stimulated with NE or TNF as indicated. Cells were lysed with Reporter Lysis buffer (Promega) (250 μl/well), protein concentrations were determined (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar), and IL-8 reporter gene activity was quantified by luminometry (Wallac Victor2, 1420 multilabel counter) using the Promega luciferase assay system according to the manufacturer's instructions. Reporter gene expression was expressed as light units/μg of total protein. Data were analyzed with the GraphPad Prism 3.0 software package (GraphPad Software, San Diego, CA). Results are expressed as mean ± S.E. and were compared by Mann-Whitney test or analysis of variance with post hoc analysis. Differences were considered significant when the p value was ≤0.05. Basal and NE-induced IL-8 protein levels in cell supernatants from HBE cells were quantified by enzyme-linked immunosorbent assay. HBE cells produced a mean basal level of IL-8 of 267 ± 17 pg/mg of protein. Time course and dose-response experiments (data not shown) demonstrated that 10 nm NE for 4 h induced maximal IL-8 protein production from HBE cells, increasing IL-8 levels to 538 ± 24 pg/mg of protein (p ≤ 0.001) and confirming a previous report using a Bet1A cell line (10Nakamura H. Yoshimura K. McElvaney N.G. Crystal R.G. J. Clin. Invest. 1992; 89: 1478-1484Crossref PubMed Scopus (438) Google Scholar). It was not feasible to investigate whether doses higher than 10 nm NE could further potentiate IL-8 protein production because at higher concentrations NE degrades IL-8 protein (33Leavell K.J. Peterson M.W. Gross T.J. J. Leukoc. Biol. 1997; 61: 361-366Crossref PubMed Scopus (41) Google Scholar) and renders the enzyme-linked immunosorbent assay inaccurate. NE did not induce apoptosis in HBE cells at doses up to 50 nm NE over 4 h, and the cells retained >90%viability (data not shown). Control TNFα stimulations (10 ng/ml for 3 h) increased IL-8 protein to 4134 ± 125 pg/mg of protein (p ≤ 0.007). Preincubation of NE with the synthetic serine protease inhibitor PMSF (1 μm), a recombinant form of the naturally occurring serine protease inhibitor, rSLPI (500 nm), or the specific NE inhibitor MeO-Suc-Ala-Ala-Pro-Val-chloromethyl ketone (1 μm) abolished its ability to induce IL-8 production (Fig.1 A) (p < 0.005, analysis of variance). This indicated that the elastase activity of NE is required for IL-8 up-regulation. In Fig. 1 B IL-8 gene expression in response to NE and TNFα was measured in HBE cells by quantitative LightCyclerTMPCRs and also by semiquantitative RT-PCRs using IL-8-specific primers and comparison with expression of GAPDH mRNA. To evaluate whether NE increased IL-8 by a transcriptional mechanism, HBE cells were pretreated with actinomycin D (10 μg/ml, 1 h). Real time RT-PCR demonstrated that in HBE cells, NE induced a 4-fold increase in IL-8 mRNA that was inhibited by actinomycin D pretreatment (p ≤ 0.05) (Fig. 1 B). TNF strongly induced IL-8 mRNA, an effect that was also blocked by actinomycin D (p ≤ 0.02). Resolution of the semiquantitative RT-PCR samples on a 1.5% Tris borate-EDTA agarose gel qualified the real time RT-PCR data. Actinomycin D also significantly decreased NE- and TNFα-induced IL-8 protein levels to lower than basal levels (171 ± 21 pg/mg of protein (p ≤ 0.0001) and 191 ± 11 pg/mg of protein (p ≤ 0.0001), respectively). The transcription factor NFκB regulates IL-8 gene expression (34Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Abstract Full Text PDF PubMed Google Scholar). NFκB activation in nuclear extracts from HBE cells was measured by electrophoretic mobility shift assay. Cells were left untreated or were stimulated with either NE or TNFα. Time course studies (data not shown) demonstrated that 10 nm NE induced maximum NFκB activation at 5 min, while TNFα (10 ng/ml) induced a similar effect at 10 min. NFκB nuclear translocation was increased 3- and 4-fold compared with control for NE and TNFα, respectively, as measured by phosphorimagery (Fig.2 A). Concomitant Western blotting of cytoplasmic extracts was performed using anti-IκBα and anti-IκBβ antibodies. Stimulation with either NE or TNFα led to degradation of IκBβ. TNF also degraded IκBα, however, NE had only a modest effect on IκBα degradation. In Fig. 2 B competition studies with unlabeled mutant and wild-type NFκB probes demonstrated that NE activated NFκB specifically. Antisera to NFκB components identified the subunit composition of the NE-induced NFκB complexes as heterodimers of p50 and p65 but not c-Rel (Fig. 2 C). Given our interest in Toll/IL-1 receptor signaling (24O'Neill L.A.J. Greene C. J. Leukoc. Biol. 1998; 63: 650-657Crossref PubMed Scopus (497) Google Scholar, 25Greene C. O'Neill L.A.J. Biochim. Biophys. Acta. 1999; 1451: 109-121Crossref PubMed Scopus (18) Google Scholar), we examined a role for IRAK in the activation of NFκB in response to NE. Cytoplasmic extracts from control HBE cells or cells stimulated with either NE (10 nm, 5 min) or TNFα (10 ng/ml, 10 min) were Western blotted to measure IRAK degradation (Fig.3). An immunoreactive band of 80 kDa was detected in extracts from control cells. This was degraded following stimulation with NE but not TNFα, implicating IRAK in the NE pathway. To further elucidate signaling events activated in response to stimulation with NE, IL-8 promoter activity using an IL-8 promoter luciferase reporter gene was measured in HBE cells. NE time course (data not shown) and dose-response studies demonstrated that NE stimulation (50 nm, 4 h) induced optimal IL-8 promoter activity (Fig.4 A) and indicated that increasing doses of NE can potentiate IL-8 expression. However, at higher doses (100 nm) NE caused cell detachment and necrosis and thereby prevented further IL-8 promoter activity. The induction of IL-8 promoter activity was significantly down-regulated by transfection of HBE cells with dominant negative TRAF-6 (TRAF-6Δ) but not dominant negative TRAF-2 (TRAF-2Δ) (Fig.4 B), indicating that TRAF-6, but not TRAF-2, is a component of the NE/IL-8 signaling cascade. TNFα (10 ng/ml, 3 h) also increased IL-8 promoter activity. The TNFα effect was not affected by transfection with TRAF-6Δ but was significantly inhibited by TRAF-2Δ (p ≤ 0.0001). To elucidate additional upstream components in the NE/IL-8 signaling pathway in HBE cells, the effect of dominant negative MyD88 (MyD88Δ) on NE-induced IRAK degradation was determined (Fig.5 A). HBE cells were left untreated or were stimulated with NE (10 nm, 5 min) in the absence or presence of MyD88Δ. Western blotting using an anti-IRAK antibody showed, as before, that NE could induce IRAK degradation. However, this effect was blocked by MyD88Δ, indicating that MyD88 is positioned upstream from IRAK and both components are involved in intracellular events activated by NE. This result was further verified in Fig. 5 B by measuring the effect of MyD88Δ on IL-8 promoter activity in HBE cells in response to NE. IL-8 reporter gene activity was significantly up-regulated in HBE cells following NE stimulation. However, this response was inhibited when the cells were co-transfected with MyD88Δ (p < 0.0001), providing further evidence for the involvement of MyD88 in the NE/IL-8 signaling pathway. W" @default.
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