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• W1981678052 abstract "There is accumulating evidence that following bacterial infection, the massive recruitment and activation of the phagocytes, neutrophils, is accompanied with the extracellular release of active neutrophil elastase (NE), a potent serine protease. Using NE-deficient mice in a clinically relevant model of Pseudomonas aeruginosa-induced pneumonia, we provide compelling in vivo evidence that the absence of NE was associated with decreased protein and transcript levels of the proinflammatory cytokines TNF-α, MIP-2, and IL-6 in the lungs, coinciding with increased mortality of mutant mice to infection. The implication of NE in the induction of cytokine expression involved at least in part Toll-like receptor 4 (TLR-4). These findings were further confirmed following exposure of cultured macrophages to purified NE. Together, our data suggest strongly for the first time that NE not only plays a direct antibacterial role as it has been previously reported, but released active enzyme can also modulate cytokine expression, which contributes to host protection against P. aeruginosa. In light of our findings, the long held view that considers NE as a prime suspect in P. aeruginosa-associated diseases will need to be carefully reassessed. Also, therapeutic strategies aiming at NE inhibition should take into account the physiologic roles of the enzyme. There is accumulating evidence that following bacterial infection, the massive recruitment and activation of the phagocytes, neutrophils, is accompanied with the extracellular release of active neutrophil elastase (NE), a potent serine protease. Using NE-deficient mice in a clinically relevant model of Pseudomonas aeruginosa-induced pneumonia, we provide compelling in vivo evidence that the absence of NE was associated with decreased protein and transcript levels of the proinflammatory cytokines TNF-α, MIP-2, and IL-6 in the lungs, coinciding with increased mortality of mutant mice to infection. The implication of NE in the induction of cytokine expression involved at least in part Toll-like receptor 4 (TLR-4). These findings were further confirmed following exposure of cultured macrophages to purified NE. Together, our data suggest strongly for the first time that NE not only plays a direct antibacterial role as it has been previously reported, but released active enzyme can also modulate cytokine expression, which contributes to host protection against P. aeruginosa. In light of our findings, the long held view that considers NE as a prime suspect in P. aeruginosa-associated diseases will need to be carefully reassessed. Also, therapeutic strategies aiming at NE inhibition should take into account the physiologic roles of the enzyme. Inflammation is a physiological reaction of the host to protect itself from insulting agents such as pathogens. This complex and dynamic process is characterized by an innate immune response, which involves a coordinated expression of inflammatory cytokines and implication of various cell types particularly immune cells aimed at clearing the pathogenic agent. In the setting of respiratory bacterial infections (e.g. bacterial pneumonia), the host innate immune response is characterized by the initial recognition of invading microbes by host “sentinel” cells via Toll-like receptors (TLRs) 3The abbreviations used are: TLRToll-like receptorPMNpolymorphonuclear neutrophilNEneutrophil elastaseAEBSF4-(2-aminoethyl)-benzenesulfonyl fluorideSLPIsecretory leukocyte proteinase inhibitorBALbronchoalveolar lavageMyD88myeloid differentiation primary response protein 88IRAKinterleukin-1 receptor-associated kinaseTRAF6TNF receptor associated factor 6NF-κBnuclear factor κ-light-chain-enhancer of activated B cellsLPSlipopolysaccharideMIP-2macrophage inflammatory protein-2. or other pattern recognition molecules (1Kolls J.K. McCray Jr., P.B. Chan Y.R. Cytokine-mediated regulation of antimicrobial proteins.Nat. Rev. Immunol. 2008; 8: 829-835Crossref PubMed Scopus (265) Google Scholar). Subsequently, this results in the production of an array of inflammatory mediators including early responsive cytokines. Another hallmark of innate host lung defense, especially when the first lines of defense: epithelial barrier and resident macrophages, are breached is the massive recruitment of polymorphonuclear neutrophils (PMN) to the infected site (2Mizgerd J.P. Acute lower respiratory tract infection.N. Engl. J. Med. 2008; 358: 716-727Crossref PubMed Scopus (324) Google Scholar). PMNs are efficient phagocytes whose main function upon activation is thought to be the clearance of infecting bacteria. To do so, these cells are equipped with a myriad of antimicrobial molecules grouped into oxidative and nonoxidative systems (3Ganz T. Oxygen-independent microbicidal mechanisms of phagocytes.Proc. Assoc. Am. Physicians. 1999; 111: 390-395Crossref PubMed Scopus (22) Google Scholar, 4Hampton M.B. Kettle A.J. Winterbourn C.C. Inside the neutrophil phagosome. Oxidants, myeloperoxidase, and bacterial killing.Blood. 1998; 92: 3007-3017Crossref PubMed Google Scholar). Toll-like receptor polymorphonuclear neutrophil neutrophil elastase 4-(2-aminoethyl)-benzenesulfonyl fluoride secretory leukocyte proteinase inhibitor bronchoalveolar lavage myeloid differentiation primary response protein 88 interleukin-1 receptor-associated kinase TNF receptor associated factor 6 nuclear factor κ-light-chain-enhancer of activated B cells lipopolysaccharide macrophage inflammatory protein-2. Regarding the nonoxidative system, we and others have identified the PMN-specific serine protease, neutrophil elastase (NE), as a key antimicrobial enzyme (5Belaaouaj A. McCarthy R. Baumann M. Gao Z. Ley T.J. Abraham S.N. Shapiro S.D. Mice lacking neutrophil elastase reveal impaired host defense against Gram-negative bacterial sepsis.Nat. Med. 1998; 4: 615-618Crossref PubMed Scopus (539) Google Scholar). NE catalytic activity relies on the His57-Asp102-Ser195 triad (chymotrypsin numbering system), where Ser represents the active residue (6Bode W. Meyer Jr., E. Powers J.C. Human leukocyte and porcine pancreatic elastase. X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors.Biochemistry. 1989; 28: 1951-1963Crossref PubMed Scopus (384) Google Scholar). NE, a cationic glycoprotein, is stored in readily active form in PMN primary granules at concentrations exceeding millimolar range making it a major component of PMN (7Liou T.G. Campbell E.J. Nonisotropic enzyme–inhibitor interactions. A novel nonoxidative mechanism for quantum proteolysis by human neutrophils.Biochemistry. 1995; 34: 16171-16177Crossref PubMed Scopus (94) Google Scholar). Gene targeting of NE in mice revealed that the enzyme contributes significantly to host protection against microbial infections (8Reeves E.P. Lu H. Jacobs H.L. Messina C.G. Bolsover S. Gabella G. Potma E.O. Warley A. Roes J. Segal A.W. Killing activity of neutrophils is mediated through activation of proteases by K+ flux.Nature. 2002; 416: 291-297Crossref PubMed Scopus (896) Google Scholar). In determining NE-mediated defense against Gram-negative bacteria, it was shown that active enzyme degrades the major outer membrane protein (Omp), structural proteins localized on the cell wall (9Belaaouaj A. Kim K.S. Shapiro S.D. Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase.Science. 2000; 289: 1185-1188Crossref PubMed Scopus (287) Google Scholar, 10Hirche T.O. Benabid R. Deslee G. Gangloff S. Achilefu S. Guenounou M. Lebargy F. Hancock R.E. Belaaouaj A. Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.J. Immunol. 2008; 181: 4945-4954Crossref PubMed Scopus (74) Google Scholar). NE also has the capacity to attenuate the pathogenicity of invading microbes by targeting their virulence factors (11López-Boado Y.S. Espinola M. Bahr S. Belaaouaj A. Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity.J. Immunol. 2004; 172: 509-515Crossref PubMed Scopus (77) Google Scholar). In recent years, it was shown that NE binds to PMN-derived chromatin structures, termed neutrophil extracellular traps, and exerts its antimicrobial function (12Weinrauch Y. Drujan D. Shapiro S.D. Weiss J. Zychlinsky A. Neutrophil elastase targets virulence factors of enterobacteria.Nature. 2002; 417: 91-94Crossref PubMed Scopus (245) Google Scholar). Although NE has been always regarded as pathogenic in Pseudomonas aeruginosa-associated tissue inflammatory and destructive diseases, we have recently provided compelling in vivo evidence that the enzyme contributes considerably to PMN-mediated host protection in a mouse model of P. aeruginosa-induced pneumonia (10Hirche T.O. Benabid R. Deslee G. Gangloff S. Achilefu S. Guenounou M. Lebargy F. Hancock R.E. Belaaouaj A. Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.J. Immunol. 2008; 181: 4945-4954Crossref PubMed Scopus (74) Google Scholar). There is also accumulating evidence that during P. aeruginosa infections, active NE is released in the extracellular milieu by recruited PMNs (13Birrer P. Proteases and antiproteases in cystic fibrosis. Pathogenetic considerations and therapeutic strategies.Respiration. 1995; 62: 25-28Crossref PubMed Scopus (54) Google Scholar, 14Moraes T.J. Chow C.W. Downey G.P. Proteases and lung injury.Crit. Care Med. 2003; 31: S189-S194Crossref PubMed Google Scholar). A number of in vitro studies have suggested that NE has the potential to change biologic activities of various inflammatory mediators (15Pham C.T. Neutrophil serine proteases. Specific regulators of inflammation.Nat. Rev. Immunol. 2006; 6: 541-550Crossref PubMed Scopus (738) Google Scholar). Altogether, these observations prompted us to hypothesize that the NE role in host defense against P. aeruginosa lung infection may not be only limited to just killing bacteria. Here, we report that in vivo, extracellular active NE has the capacity to induce mRNA expression of the early responsive proinflammatory cytokines, tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2), and interleukin-6 (IL-6); an induction that is mediated at least in part through TLR-4. These findings were further confirmed following exposure of cultured macrophages to purified NE. Our studies reveal for the first time that extracellularly released NE can have physiologic inflammatory properties that contribute to host defense against P. aeruginosa. Purified NE and elastin were obtained from Elastin Products Co. (Owensville, MO). NE activity was determined spectrophotometrically using the specific chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA (Elastin Products Co.) according to the manufacturer's recommendations. Pefabloc SC®, 4-(2-aminoethyl)-benzenesulfonyl-fluoride (AEBSF) and secretory leukocyte proteinase inhibitor (SLPI) were from Roche Applied Science and R&D Systems, respectively. RPMI 1640 medium, DMEM, fetal bovine serum (FBS), penicillin, streptomycin, and PBS were obtained from Invitrogen. Ketamine hydrochloride and medotomidine hydrochloride were obtained from CEVA Santé Animale (Libourne, France). Primers for semiquantitative and real time RT-PCR were purchased from Operon Biotechnologies (Cologne, Germany). SYBR Green for real time RT-PCR was obtained from Invitrogen. Polyclonal anti-murine NE antibody was produced in rabbit (16Kessenbrock K. Fröhlich L. Sixt M. Lämmermann T. Pfister H. Bateman A. Belaaouaj A. Ring J. Ollert M. Fässler R. Jenne D.E. Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating anti-inflammatory progranulin.J. Clin. Invest. 2008; 118: 2438-2447PubMed Google Scholar). Antibody raised against amino acids 198–395 of mouse TLR-4 (sc-293072) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were reagent grade and purchased from Sigma, unless otherwise stated. Of note, glassware used in the study were LPS-free and when appropriate, reagents especially purified for NE were tested for LPS contamination by the Limulus amebocyte-lysate assay (E-toxate, Sigma). NE-deficient (NE−/−) mice were generated by targeted mutagenesis as previously described (5Belaaouaj A. McCarthy R. Baumann M. Gao Z. Ley T.J. Abraham S.N. Shapiro S.D. Mice lacking neutrophil elastase reveal impaired host defense against Gram-negative bacterial sepsis.Nat. Med. 1998; 4: 615-618Crossref PubMed Scopus (539) Google Scholar). C57BL/6 NE−/− mice and their wild-type (WT) littermates and C3H/HeJ (TLR4−/−) and control C3H/HeN mice (Harlan Laboratories, Indianapolis, IN) were used in this study. C3H/HeJ mice have a naturally nonresponsive TLR4. Mice were sex and age (8–10 weeks) matched and maintained in the animal barrier facility with a 12-h light/dark cycle and provided with water and food ad libitum. Animal handling and procedures were approved by the Animal Studies Committee at our institution (Health and Animal Protection Office, Châlons-en-Champagne, France, authorization number 51-31) in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) and following the European Directive 2010/63/EU on the protection of animals used in scientific procedures. P. aeruginosa H103 was kindly provided by Dr. Hancock (Vancouver, BC, Canada). This strain is noteworthy in its inability to produce Pseudomonas metalloelastase (17Tamura Y. Suzuki S. Sawada T. Role of elastase as a virulence factor in experimental Pseudomonas aeruginosa infection in mice.Microb. Pathog. 1992; 12: 237-244Crossref PubMed Scopus (41) Google Scholar). An overnight bacterial culture (1 ml) was grown in Luria Bertani broth (10 ml) at 37 °C to late exponential phase (3 h). Bacteria were washed twice with PBS (pH 7.4) and the optical density (OD) of the cultures was determined at 600 nm (1 OD600 nm ≈1 × 109 bacteria/ml). Two types of lung infection experiments were carried out. In the first set of experiments, to monitor the release of free active NE in function of time in the setting of infection, NE−/− and WT mice (n = 24 mice/genotype) were challenged intranasally with bacteria and sacrificed at fixed time points. Briefly, mice were anesthetized by intraperitoneal (intraperitoneal) injection of ketamine hydrochloride (75 mg/kg) and medotomidine hydrochloride (1 mg/kg). Next, mice were challenged intranasally with 50 μl of saline buffer (PBS) containing a predetermined dose of bacteria that killed 50% of WT mice (107 CFUs/per mouse, LD50 ≈ 107 CFUs) (10Hirche T.O. Benabid R. Deslee G. Gangloff S. Achilefu S. Guenounou M. Lebargy F. Hancock R.E. Belaaouaj A. Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.J. Immunol. 2008; 181: 4945-4954Crossref PubMed Scopus (74) Google Scholar). One NE−/− mouse died during intranasal instillation. Control mice (n = 8 mice/genotype) were challenged with 50 μl of sterile PBS alone. WT mice and NE−/− mice that survived bacterial challenge were sacrificed at the designated time points of 4, 12, and 24 h post-infection (4 mice per each time point). Mouse lungs were analyzed for histology, inflammatory cell recruitment, and detection of free active NE. Of note, at the onset all NE−/− appeared clinically worse than WT mice. By 12 h, NE−/− mice exhibited signs of distress, which were markedly less severe in WT mice. At 24 h, both types of mice displayed a morbid state that was more marked in NE−/− mice. At this time point, whereas no death was recorded in WT mice 11 NE−/− mice (∼45%) died. The survival of the remaining mice was monitored up to 72 h later and in accordance with our previous report (10Hirche T.O. Benabid R. Deslee G. Gangloff S. Achilefu S. Guenounou M. Lebargy F. Hancock R.E. Belaaouaj A. Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.J. Immunol. 2008; 181: 4945-4954Crossref PubMed Scopus (74) Google Scholar), ∼50% of WT mice survived against ∼20% of NE−/− mice. In the second set of experiments, groups of WT and NE−/− mice were challenged using experimental conditions of lung infection as above. The surviving mice (24 WT mice and 10 NE−/− mice) were then sacrificed at the 24-h time point, a time that corresponded to maximal PMN infiltration and maximal release of free active NE (Fig. 1) (10Hirche T.O. Benabid R. Deslee G. Gangloff S. Achilefu S. Guenounou M. Lebargy F. Hancock R.E. Belaaouaj A. Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.J. Immunol. 2008; 181: 4945-4954Crossref PubMed Scopus (74) Google Scholar). Control mice were sacrificed 24 h post-saline instillation. Bronchoalveolar lavage fluids (BAL) of control and surviving mice were pooled as the following: 3 pools of control BALs (2 pools of 3 BALs and 1 of 2 BALs), 3 pools of infected WT BALs (4 BALS per pool), and 3 pools of infected NE−/− BALs (2 pools of 3 BALs and 1 of 4 BALs). In all mouse experiments, lungs were lavaged in situ using 1 ml of PBS (pH 7.4), cycled three times (18Hirche T.O. Crouch E.C. Espinola M. Brokelman T.J. Mecham R.P. DeSilva N. Cooley J. Remold-O'Donnell E. Belaaouaj A. Neutrophil serine proteinases inactivate surfactant protein D by cleaving within a conserved subregion of the carbohydrate recognition domain.J. Biol. Chem. 2004; 279: 27688-27698Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Identical recoveries of BAL (700 μl/mouse) were obtained for each of the experimental groups. Both BALs and recovered lungs were processed for analysis as described below. These infection experiments were carried out twice for data reproducibility. Mouse lungs corresponding to 24 h post-infection were processed for histology analysis as previously described (18Hirche T.O. Crouch E.C. Espinola M. Brokelman T.J. Mecham R.P. DeSilva N. Cooley J. Remold-O'Donnell E. Belaaouaj A. Neutrophil serine proteinases inactivate surfactant protein D by cleaving within a conserved subregion of the carbohydrate recognition domain.J. Biol. Chem. 2004; 279: 27688-27698Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Total cell counts from BAL fluids were immediately performed by a hemacytometer. For differential counts, cells were cytospun, Wright-stained, and identified using standard morphological criteria. The remaining BAL samples were centrifuged for 5 min at 4 °C and supernatants were snap-frozen and stored at −80 °C until use. J774 mouse macrophage cell line was seeded on a 24-well plate (ATGC Biotechnologies SA, Marne-la-Vallée, France) at 1 × 105 cells/well and grown for 3 days to confluence at 37 °C in 5% CO2 in DMEM supplemented with 1% FBS and 1% penicillin/streptomycin (19Belaaouaj A. Shipley J.M. Kobayashi D.K. Zimonjic D.B. Popescu N. Silverman G.A. Shapiro S.D. Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression.J. Biol. Chem. 1995; 270: 14568-14575Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In separate experiments, primary macrophages were elicited to the peritonea of C57BL/6 WT mice or C3H/HeJ (TLR4−/−) and control C3H/HeN mice following intraperitoneal injection with 3 ml of 3% Brewer thioglycollate broth (Difco Laboratories, Detroit, MI) (20Grandjean-Laquerriere A. Tabary O. Jacquot J. Richard D. Frayssinet P. Guenounou M. Laurent-Maquin D. Laquerriere P. Gangloff S. Involvement of Toll-like receptor 4 in the inflammatory reaction induced by hydroxyapatite particles.Biomaterials. 2007; 28: 400-404Crossref PubMed Scopus (92) Google Scholar). Four days later, the peritonea were lavaged with 10 ml of RPMI 1640 (Invitrogen). Cells were collected and washed twice in RPMI 1640 and resuspended in the medium supplemented with 1% autologous serum. Macrophages represented >95% of the cell population and >98% were viable as judged by differential counting and trypan blue dye exclusion, respectively. Cells were seeded on a 24-well tissue culture plate at 5 × 105 cells/well. Of relevance, to minimize PMN contamination and the potential confounding effect of endogenous NE, cells were cultured for 6 h at 37 °C in 5% CO2 to allow macrophages to adhere and washed twice with 1 ml of medium to remove nonadherent cells including PMNs. Cells were cultured for an additional 24 h to ensure for maximal purity of adherent macrophages and absence of PMN-derived NE as judged by light microscopy of cultured cell cytospins and elastin zymography on concentrated cell-free culture media (Nanosep filter units, Life Sciences, VWR International, Radnor, PA). Prior to NE treatment, cell line or primary macrophages were washed twice with 1 ml of PBS and cultured for 1 h with corresponding serum-free medium. To determine optimal experimental conditions for maximal expression of the mediators, experiments of enzyme dose-response and time course of cell exposure to NE and culture post-treatment were carried out. J774 cells were treated with various concentrations of NE (100, 300, and 500 nm) for different times (1, 2, and 4 h), washed twice with 1 ml of PBS, and cultured at 37 °C in 5% CO2 in fresh complemented medium for different periods of time (2 and 4 h). The identified optimized conditions were applied also to primary macrophages. To ensure maximal removal of NE, culture media was checked for the absence of active NE against its specific substrate (21Hirche T.O. Gaut J.P. Heinecke J.W. Belaaouaj A. Myeloperoxidase plays critical roles in killing Klebsiella pneumoniae and inactivating neutrophil elastase. Effects on host defense.J. Immunol. 2005; 174: 1557-1565Crossref PubMed Scopus (94) Google Scholar). In parallel experiments, NE was preincubated with AEBSF (1 mm) for 5 min at 37 °C prior to addition to cells. Controls included cells cultured in the absence of NE. Under these experimental conditions, over 90% of the cells were alive, as judged by morphologic criteria and trypan blue dye exclusion. Each treatment condition was performed in triplicate and cell culture experiments were repeated at least 3 times. Cell lysates were prepared using RIPA buffer (50 mm Tris-HCl, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mm EDTA, 1 mm dithiothreitol (DTT), pH 7.4) (Sigma) and centrifuged at 600 × g for 30 min at 4 °C to remove cell debris. Next, lysates were exposed to a fixed NE concentration (500 nm) for the defined time periods (0.25, 0.5, 1, and 5 min). In parallel experiments, NE was preincubated with SLPI (10 μg) for 5 min at 37 °C or heat inactivated for 10 min at 65 °C prior to addition to cell lysates. Cell-free WT and NE−/− BAL fluids of the 24-h time point post-infection were processed to assess the levels of various cytokines using the Raybio® Mouse Cytokine Antibody Array III according to the manufacturer's instructions (RayBiotech, Tebu-Bio, Le Perray-en-Yvelines, France) (22Velard F. Laurent-Maquin D. Guillaume C. Bouthors S. Jallot E. Nedelec J.M. Belaaouaj A. Laquerriere P. Polymorphonuclear neutrophil response to hydroxyapatite particles, implication in acute inflammatory reaction.Acta Biomater. 2009; 5: 1708-1715Crossref PubMed Scopus (39) Google Scholar). Briefly, equal volumes of cell-free BAL fluids (400 μl) were added to antibody-coated membranes and detection of immunoreactive cytokines was carried out following sequential incubations of the membranes with biotinylated anti-cytokine antibodies and streptavidin-horseradish peroxidase and visualization by enhanced chemiluminescence. Images were obtained with a ChemiDoc XRS imaging system (Bio-Rad). Densitometric analysis was performed on captured images using Quantity One one-dimensional analysis software (version 4.5.2) (Bio-Rad). Spots of interest were normalized to an internal control after subtraction of the representative background sample. Next, to determine the concentration of cytokines of interest (TNF-α, MIP-2, and IL-6), equal volumes of WT and NE−/− cell-free BAL fluids (50 μl) were processed using a multiplex bead-based immunoassay kit (Bio-Rad). Cytokine assays were performed as described by the manufacturer's protocol. Each reaction in the kit was performed in triplicate. Both cytokine antibody array and multiplex assays were performed on all BAL pools. NE or TLR-4 antigens were detected by Western blotting (18Hirche T.O. Crouch E.C. Espinola M. Brokelman T.J. Mecham R.P. DeSilva N. Cooley J. Remold-O'Donnell E. Belaaouaj A. Neutrophil serine proteinases inactivate surfactant protein D by cleaving within a conserved subregion of the carbohydrate recognition domain.J. Biol. Chem. 2004; 279: 27688-27698Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Briefly, samples (cell-free BAL fluids (20 μl) and lung or cell lysates (35 μg)) were resolved by SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA). The membranes were sequentially incubated with primary antibodies to mouse NE (dilution, 1:2,000) or TLR-4 (dilution, 1:500) followed by their respective horseradish peroxidase-conjugated secondary antibodies. When indicated, the membranes were stripped (100 mm β-mercaptoethanol, 2% SDS, and 62.5 mm Tris-HCl, pH 6.7, for 30 min at 50 °C) and immunoblotted using primary rabbit polyclonal anti-mouse albumin antibody (Rockland, Gilbertsville, PA) and its corresponding horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences). NE activity was determined by α-elastin zymography (23Hirche T.O. Atkinson J.J. Bahr S. Belaaouaj A. Deficiency in neutrophil elastase does not impair neutrophil recruitment to inflamed sites.Am. J. Respir. Cell Mol. Biol. 2004; 30: 576-584Crossref PubMed Scopus (64) Google Scholar). Briefly, cell-free BAL fluids (20 μl) were electrophoresed under nonreducing conditions at 4 °C on 12% SDS-PAGE gels containing 1 mg/ml of elastin. Following electrophoresis, gels were soaked in 2% Triton X-100 for 30 min (twice), rinsed briefly, and incubated at 37 °C for 72 h in 50 mm Tris-HCl (pH 8.2), containing 5 mm CaCl2. The gels were then stained with Coomassie Blue and destained in 5% acetic acid and 10% methanol. Active NE appears as a transparent lysis band at ∼29 kDa. NE activity in cell-free BAL fluids was further confirmed using conventional chromogenic peptide assays as previously described (21Hirche T.O. Gaut J.P. Heinecke J.W. Belaaouaj A. Myeloperoxidase plays critical roles in killing Klebsiella pneumoniae and inactivating neutrophil elastase. Effects on host defense.J. Immunol. 2005; 174: 1557-1565Crossref PubMed Scopus (94) Google Scholar). Total RNA isolation was performed using MasterPureTM RNA Purification kit as described by the manufacturer's protocol (Epicenter, Biotechnologies, Madison, WI) (19Belaaouaj A. Shipley J.M. Kobayashi D.K. Zimonjic D.B. Popescu N. Silverman G.A. Shapiro S.D. Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression.J. Biol. Chem. 1995; 270: 14568-14575Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Briefly, mouse lung tissues (half-lobe) or cultured macrophage cell pellets were lysed and RNA was extracted. Purified RNA was resuspended in RNase inhibitor-containing TE Buffer. RNA concentration and purity were determined by spectrophotometry (A260/A280 ratio). Integrity of RNA samples was verified by electrophoresis on 2% agarose gels and visualization under UV light. RNA aliquots of individual mice were pooled in the same manner as for BAL fluids at equal concentrations. Total RNA samples (1 μg) were reverse-transcribed using the SuperScript First Strand Synthesis System (Invitrogen). Next, cDNAs were amplified by PCR using specific primers (40 cycles starting with DNA denaturation for 2 min at 94 °C; each cycle corresponded to denaturation for 15 s at 94 °C, primer annealing at 60 °C for 30 s and extension at 72 °C for 30 s). RT-PCR products were analyzed by electrophoresis on 1.5% agarose gels and quantified by densitometry using Quantity One Software (Bio-Rad). Levels of cytokine mRNA transcripts were normalized to the internal control 28 S mRNA. RT-PCR was performed in quadruplicate within each pool. Forward and reverse primers for TNF-α, MIP-2, IL-6, and 28 S were designed as follows: TNF-α forward, 5′-GGG-ACA-GTG-ACC-TGG-ACT-GT-3′; TNF-α reverse, 5′-CTC-CCT-TTG-CAG-AAC-TCA-GC-3′; MIP-2 forward, 5′-CCA-CTC-TCA-AGG-GCG-GTC-AA-3′; MIP-2 reverse, 5′-CCC-CTT-ATC-CCC-AGT-CTC-TTT-CAC-3′; IL-6 forward, 5′-GAT-GCT-ACC-AAA-CTG-GAG-ATA-AAT-C-3′; IL-6 reverse, 5′-GGT-CCT-TAG-CCA-CTC-CTT-CTG-TG-3′; 28 S forward, 5′-CGG-AAT-TCG-CCA-CCA-GCC-GCC-TG-3′; 28 S reverse, 5′-CGT-CTA-GAC-TTT-CTC-CGT-TTA-CTT-GC-3′. Briefly, total RNA of cultured cells was reverse-transcribed into cDNA using the SuperScript First Strand Synthesis System (Invitrogen). Next, real time-PCR amplification was performed on the ABI PRISMTM 7500 Sequence Detection System (PE Applied Biosystems, Carlsbad, CA) using Platinum SYBR® Green qPCR SuperMix-UDG with ROX (Invitrogen). PCR amplification conditions were as follows: initial DNA denaturation for 5 min at 94 °C, primer annealing at 60 °C for 1 min, and extension at 72 °C for 1 min for a total 40 cycles. Data analysis was performed with the SDS Software (Applied Biosystems). Real time RT-PCR was performed in triplicate and repeated at least 3 times in each experimental condition for data reproducibility. Forward and reverse primers for TNF-α, MIP-2, IL-6, and GAPDH were designed as follows: TNF-α forward, 5′-GGC-AGG-TTC-TGT-CCC-TTT-CA-3′; TNF-α reverse, 5′-CTG-TGC-TCA-TGG-TGT-CTT-TTC-TG-3′; MIP-2 forward, 5′-GTG-AAC-TGC-GCT-GTC-AAT-GC-3′; MIP-2 reverse, 5′-ACT-CAA-GCT-CTG-GAT-GTT-CTT-GAA-3′; IL-6 forward, 5′-CAA-CCA-CGG-CCT-TCC-CTA-CTA-3′; IL-6 reverse, 5′-GTT-GGG-AGT-GGT-ATC-CTC-TGT-GA-3′;" @default.
• W1981678052 created "2016-06-24" @default.
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• W1981678052 date "2012-10-01" @default.
• W1981678052 modified "2023-10-14" @default.
• W1981678052 title "Neutrophil Elastase Modulates Cytokine Expression" @default.
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