Lung Chitinolytic Activity and Chitotriosidase Are Elevated in Chronic Obstructive Pulmonary Disease and Contribute to Lung Inflammation
2009; Elsevier BV; Volume: 176; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2010.090455
ISSN1525-2191
AutoresS. Létuvé, Alexander Kozhich, Alison A. Humbles, Yambasu A. Brewah, Marie-Christine Dombret, Martine Grandsaigne, Homa Adle, Roland Kolbeck, Michel Aubier, Anthony J. Coyle, Marina Pretolani,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoChronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation and emphysematous alveolar destruction. In this study, we have investigated whether chitotriosidase (ChTRase) and acidic mammalian chitinase, two chitinases with chitinolytic activity, are selectively augmented in COPD and contribute to its pathogenesis. We found that smokers with COPD, but not asthmatics, had higher chitinolytic activity and increased levels of ChTRase in bronchoalveolar lavage, more ChTRase-positive cells in bronchial biopsies, and an elevated proportion of alveolar macrophages expressing ChTRase than smokers without COPD or never-smokers. ChTRase accounted for approximately 80% of bronchoalveolar lavage chitinolytic activity, while acidic mammalian chitinase was undetectable. Bronchoalveolar lavage chitinolytic activity and ChTRase were associated with airflow obstruction and emphysema and with the levels of interleukin (IL)−1β, IL-8, tumor-necrosis factor (TNF)-α, and its type II soluble receptor. Tumor necrosis factor-α stimulated ChTRase release only from alveolar macrophages from smokers with COPD, and exposure of these cells to ChTRase promoted the release of IL-8, monocyte-chemoattractant protein-1, and metalloproteinase-9. Finally, ChTRase overexpression in the lung of normal mice promoted macrophage recruitment and the synthesis of the murine homologue of IL-8, keratinocyte-derived cytokine, and of monocyte-chemoattractant protein-1. We conclude that pulmonary ChTRase overexpression may represent a novel important mechanism involved in COPD onset and progression. Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation and emphysematous alveolar destruction. In this study, we have investigated whether chitotriosidase (ChTRase) and acidic mammalian chitinase, two chitinases with chitinolytic activity, are selectively augmented in COPD and contribute to its pathogenesis. We found that smokers with COPD, but not asthmatics, had higher chitinolytic activity and increased levels of ChTRase in bronchoalveolar lavage, more ChTRase-positive cells in bronchial biopsies, and an elevated proportion of alveolar macrophages expressing ChTRase than smokers without COPD or never-smokers. ChTRase accounted for approximately 80% of bronchoalveolar lavage chitinolytic activity, while acidic mammalian chitinase was undetectable. Bronchoalveolar lavage chitinolytic activity and ChTRase were associated with airflow obstruction and emphysema and with the levels of interleukin (IL)−1β, IL-8, tumor-necrosis factor (TNF)-α, and its type II soluble receptor. Tumor necrosis factor-α stimulated ChTRase release only from alveolar macrophages from smokers with COPD, and exposure of these cells to ChTRase promoted the release of IL-8, monocyte-chemoattractant protein-1, and metalloproteinase-9. Finally, ChTRase overexpression in the lung of normal mice promoted macrophage recruitment and the synthesis of the murine homologue of IL-8, keratinocyte-derived cytokine, and of monocyte-chemoattractant protein-1. We conclude that pulmonary ChTRase overexpression may represent a novel important mechanism involved in COPD onset and progression. Chronic obstructive pulmonary disease (COPD), a debilitating respiratory condition, is a significant cause of morbidity, mortality, and health care costs worldwide and its global burden is increasing.1Barnes PJ Chronic obstructive pulmonary disease.N Engl J Med. 2000; 343: 269-280Crossref PubMed Scopus (1167) Google Scholar The defining feature of COPD is irreversible airflow limitation measured during forced expiration, caused by either an increase in airway resistance, or in lung compliance due to emphysematous lung destruction, or both.1Barnes PJ Chronic obstructive pulmonary disease.N Engl J Med. 2000; 343: 269-280Crossref PubMed Scopus (1167) Google Scholar A dominant hallmark of COPD is an abnormal inflammatory response to inhaled particles and this has the potential to produce lung injury.1Barnes PJ Chronic obstructive pulmonary disease.N Engl J Med. 2000; 343: 269-280Crossref PubMed Scopus (1167) Google Scholar, 2Hogg JC Pathophysiology of airflow limitation in chronic obstructive pulmonary disease.Lancet. 2004; 364: 709-721Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, 3Barnes PJ Mediators of chronic obstructive pulmonary disease.Pharmacol Rev. 2004; 56: 515-548Crossref PubMed Scopus (504) Google Scholar Recent data suggest that emphysema results from an exaggerated synthesis, predominantly by neutrophils and macrophages, of serine and cysteine proteases and matrix-degrading metalloproteinases (MMPs), enzymes that promote cell activation and injury and that degrade connective tissue components.3Barnes PJ Mediators of chronic obstructive pulmonary disease.Pharmacol Rev. 2004; 56: 515-548Crossref PubMed Scopus (504) Google Scholar, 4MacNee W Pathogenesis of chronic obstructive pulmonary disease.Proc Am Thorac Soc. 2005; 2: 258-266Crossref PubMed Scopus (271) Google Scholar, 5Barnes PJ Alveolar macrophages as orchestrators of COPD.COPD. 2004; 1: 59-70Crossref PubMed Scopus (236) Google Scholar Recently, a family of enzymes named chitinases has been identified in humans and rodents.6Bleau G Massicotte F Merlen Y Boisvert C Mammalian chitinase-like proteins.EXS. 1999; 87: 211-221PubMed Google Scholar These enzymes are endo-β-1,4-N-acetylglucosamidases that degrade chitin, an abundant wall- and exoskeleton-derived polysaccharide that protects nematodes, fungal cells, and insects from the animal and plant hosts that they invade.7Gooday GW Aggressive and defensive roles for chitinases.EXS. 1999; 87: 158-169Google Scholar, 8Herrera-Estrella A Cher I Chitinases in biological control.EXS. 1999; 87: 171-184PubMed Google Scholar Although chitin does not have a mammalian counterpart, elevated chitinolytic activity and prominent expression of the two chitinases that catalyze the hydrolysis of chitin, namely chitotriosidase (ChTRase) and acidic mammalian chitinase (AMCase), have been associated with pathological conditions that are characterized by tissue inflammation and remodeling, including atherosclerosis and asthma.9Boot RG van Achterberg AE van Aken BE Renkema GH Jacobs MJHM Aerts JM de Vries CJ Strong induction of members of the chitinase family of proteins in atherosclerosis: chitotriosidase and human cartilage gp-39 expressed in lesion macrophages.Arterioscler Thromb Vasc Biol. 1999; 19: 687-694Crossref PubMed Scopus (278) Google Scholar, 10Artieda M Cenarro A Ganan A Jerico I Gonzalvo C Casado JM Vitoria I Puzo J Pocoví M Civeira F Serum chitotriosidase activity is increased in subjects with atherosclerosis disease.Arterioscler Thromb Vasc Biol. 2003; 23: 1645-1652Crossref PubMed Scopus (104) Google Scholar, 11Zhu Z Zheng T Homer RJ Kim Y-K Chen NY Cohn L Hamid Q Elias JA Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation.Science. 2004; 304: 1678-1682Crossref PubMed Scopus (649) Google Scholar In the present study, we investigated whether chitinolytic activity and the expression of ChTRase and AMCase are augmented in the airways of patients with COPD and whether they contribute to inflammatory and remodeling responses by activating alveolar macrophages, an important orchestrator of these responses.5Barnes PJ Alveolar macrophages as orchestrators of COPD.COPD. 2004; 1: 59-70Crossref PubMed Scopus (236) Google Scholar We found that chitinolytic activity was elevated in the airways of patients with COPD, and that the majority of this activity was accountable by ChTRase, but not AMCase. Moreover, ChTRase activated the synthesis of pro-inflammatory and remodeling chemokines and metalloproteinases by alveolar macrophages and its overexpression in the airways of normal mice induced macrophage recruitment and up-regulated the levels of pro-inflammatory and fibrogenic chemokines that have been implicated in the pathogenesis of COPD. Taken together, these findings demonstrate that COPD is characterized by elevated levels of ChTRase and suggest that this chitinase contributes to disease pathogenesis and progression. Twenty current heavy smokers without COPD, and 30 smokers (17 current and 13 former) with stable COPD were recruited (Supplementary Table S1, at http://ajp.amjpathol.org). COPD severity was graded into stages II (n = 22), III (n = 4), and IV (n = 4), following the Guidelines of the Global Initiative for Obstructive Lung Disease.12Rabe KF Hurd S Anzueto A Barnes PJ Buist SA Calverley P Fukuchi Y Jenkins C Rodriguez-Roisin R van Weel C Zielinski J Global strategy for the diagnosis. management, and prevention of chronic obstructive pulmonary disease GOLD executive summary.Am J Respir Crit Care Med. 2007; 176: 532-555Crossref PubMed Scopus (4208) Google Scholar Two out of the 30 patients with COPD were treated with ≤1000 μg/day of equivalent beclomethasone, and no maintenance therapy, other than bronchodilators, was required. Heavy smokers without COPD had chronic cough and sputum but normal measurements on spirometry. In parallel, 40 never-smoker asthmatic subjects, mainly atopics and fulfilling the criteria of the Guidelines for the Diagnosis and Management of Asthma of the National Heart, Lung, and Blood Institute/World Health Organization13Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA), 2008.Available from http://www.ginasthma.orgGoogle Scholar were recruited (Supplementary Table S2, at http://ajp.amjpathol.org). None of the subjects had experienced asthma exacerbation within the 2 months preceding the bronchoscopy. The 40 asthmatic subjects were distributed into mild (n = 15), moderate (n = 10), and severe (n = 15) asthmatics (Supplementary Table S2, at http://ajp.amjpathol.org).13Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA), 2008.Available from http://www.ginasthma.orgGoogle Scholar Mild and moderate asthmatics had normal baseline lung function (prebronchodilator forced expiratory volume in 1 second ≥80% and forced expiratory volume in 1 second/forced vital capacity ≥70% of predicted values). Mild asthmatics were treated exclusively with inhaled short-acting β2-agonists, taken as needed, whereas moderate asthmatics received 250 to 1000 μg per day of fluticasone propionate, or equivalent, in association with short (n = 10) or long- (n = 5) acting β2-agonists to achieve control (Supplementary Table S2, at http://ajp.amjpathol.org). Severe asthmatics were defined on the basis of the requirement of high-dose inhaled steroids (>1000 μg of fluticasone propionate or equivalent per day) and a long-lasting β2-agonist, as add-on therapy to achieve control.13Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA), 2008.Available from http://www.ginasthma.orgGoogle Scholar Four out of the 15 severe asthmatics were also treated with 10 to 40 mg per day of oral prednisone (Supplementary Table S2, at http://ajp.amjpathol.org). A group of 20 never-smoker healthy volunteers, not receiving any medication, was also studied. All of them had normal respiratory function, no bronchodilator response, no chronic illness, no history of allergy or asthma and their skin prick tests were negative. The protocol was approved by the Hôtel Dieu hospital Ethics Committee (CP 02819) and all subjects gave their written informed consent before being enrolled in the study. Bronchoalveolar lavage (BAL) was collected by bronchoscopy14Létuvé S Kozhich A Arouche N Grandsaigne M Reed J Dombret MC Kiener PA Aubier M Coyle AJ Pretolani M YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.J Immunol. 2008; 181: 5167-5173PubMed Google Scholar, 15Benayoun L Druilhe A Dombret MC Aubier M Pretolani M Airway structural alterations selectively associated with severe asthma.Am J Respir Crit Care Med. 2003; 167: 1360-1368Crossref PubMed Scopus (630) Google Scholar and stored as cell-free supernatant at −80°C until use. Cytospin preparations from BAL cells were fixed in 4% paraformaldehyde and stored dried at −20°C until use. Two biopsies were taken from the subcarinae in 10 representative never-smokers and in smokers with and without COPD (n = 12 in each group). In addition, lung tissue specimens were collected at distance of the tumor in two never-smokers, three smokers, and four patients with COPD who underwent lung lobectomy for peripheral lung carcinoma. All tissue samples were fixed immediately in 3.7% formaldehyde and embedded in paraffin wax.14Létuvé S Kozhich A Arouche N Grandsaigne M Reed J Dombret MC Kiener PA Aubier M Coyle AJ Pretolani M YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.J Immunol. 2008; 181: 5167-5173PubMed Google Scholar The 293-F cell line was transfected using 293 fectin with PCDNA 3.1 plasmids (all from Invitrogen, Carlsbad, CA) encoding human ChTRase (NM_003465), or AMCase (NM_021797). To facilitate purification, a His Tag (the sequence of six histidine residues) was expressed at C-terminus of the proteins. Recombinant proteins were purified from cell supernatants using Ni columns and were characterized by Western blot and chitinolytic activity assay, as described below, as well as using SDS-polyacrylamide gel electrophoresis, size exclusion chromatography-high-performance liquid chromatography, and mass spectrometry. Preparations were sterilized by passage through 0.2-μm filters and endotoxin content was below 3 EU/mg of protein (Lymulus Amoebocyte Lysate assay, Associates of Cape Cod, East Falmouth, MA). Polyclonal sera were generated by immunizing rabbits (Covance, Denver, CO) with purified recombinant ChTRase or AMCase in complete Freund's adjuvant. The IgGs were subsequently purified using protein G columns (GE Health care, Piscataway, NJ). The specificity and titers of the sera and of the purified anti-ChTRase and anti-AMCase IgGs were determined by an enzyme-linked immunosorbent assay (ELISA) developed in house and by Western blot analysis, as described below. Nunc Maxisorp plates (VWR, West Chester, PA) were coated with rabbit anti-human ChTRase polyclonal IgG #657 (500 ng/ml in PBS, 100 μl/well) overnight at 2°C to 8°C. Plates were then blocked for 1 hour at room temperature with assay buffer (0.1% Tween 20 in PBS, T-PBS) containing 0.5% casein. After extensive wash, standards (0.001 to 1000 ng/ml), or samples were added for 1 hour at room temperature. After wash, biotinylated anti-human ChTRase Ab #657 (500 ng/ml) was added for 1 hour, followed by incubation for 1 hour with 1:25,000 dilution of streptavidin-horseradish peroxidase conjugate (GE Health care) in assay buffer. 3,3′,5,5′-Tetramethylbenzidine (KPL, Gaithersburg, MD) was used as a substrate, and reaction was stopped after 10 minutes by adding 0.2 mol/L H2SO4. Optical densities were read at 450 nm using a Spectramax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Assay sensitivity was 100 pg/ml human ChTRase. The same protocol as described above was used for developing AMCase ELISA, except that monoclonal mouse anti-human AMCase Ab #107 (1 mg/ml in bicarbonate buffer, 100 μl/well) was used for capture and biotinylated rabbit anti-human AMCase Ab #655 (500 ng/ml in PBS) was used for detection. Streptavidin-horseradish peroxidase conjugate was used at 1:50,000 dilution in assay buffer. Assay sensitivity was 4 ng/ml human AMCase. Absence of cross-reactivity with AMCase for ChTRase ELISA, and with ChTRase, for AMCase ELISA, was confirmed using concentrations of the purified proteins up to 100 ng/ml (Supplementary Figure S1A, at http://ajp.amjpathol.org). Purified ChTRase or AMCase (10, 25, and 50 ng/ml) were separated on a 10% SDS- polyacrylamide gel electrophoresis and transferred onto polyvinyldifluoride membranes. Membranes were blocked for 1 hour in T-PBS and 5% nonfat milk, and incubated for 1 hour with 1 μg/ml rabbit anti-human ChTRase #657 Ab or rabbit anti-human AMCase #655 Ab in T-PBS. After three washes in T-PBS, membranes were incubated with a 1:4000 dilution of horseradish peroxidase-conjugated goat anti-rabbit Ab (GE Health care), in T-PBS. After extensive washing in T-PBS, immunoblots were developed with enhanced chemiluminescence reagent (GE Health care) (Supplementary Figure S1B, at http://ajp.amjpathol.org). Protein deposition was confirmed by staining the membranes with silver nitrate (Sigma) (data not shown). Specificity of the Abs was also assessed using protein extracts from alveolar macrophages from two smokers with and without COPD (Supplementary Figure S1C, at http://ajp.amjpathol.org). Proteins (20 μg) were separated on a 10% SDS-polyacrylamide gel electrophoresis and membranes were reacted for 1 hour with 1 μg/ml rabbit anti-human ChTRase #657 before chemiluminescent visualization, as described above. Serial 5-μm sections from acetone-fixed, frozen bronchial biopsies and cytospin preparations of BAL cells were subsequently incubated with 2 μg/ml of rabbit polyclonal Abs directed against human ChTRase or AMCase (# 657 and # 655, respectively), biotin-conjugated anti-rabbit Ab and avidin-biotinylated alkaline phosphatase complex from the Vectastain kit (Vector, Burlingame, CA). Reaction was developed using Fast Red (DakoCytomation, Trappes, France), used as a substrate and nuclei were visualized with light Mayer's hematoxylin counterstaining.14Létuvé S Kozhich A Arouche N Grandsaigne M Reed J Dombret MC Kiener PA Aubier M Coyle AJ Pretolani M YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.J Immunol. 2008; 181: 5167-5173PubMed Google Scholar, 15Benayoun L Druilhe A Dombret MC Aubier M Pretolani M Airway structural alterations selectively associated with severe asthma.Am J Respir Crit Care Med. 2003; 167: 1360-1368Crossref PubMed Scopus (630) Google Scholar, 16Chupp GL Lee CG Jarjour N Shim YM Holm CT He S Dziura JD Reed J Coyle AJ Kiener P Cullen M Grandsaigne M Dombret MC Aubier M Pretolani M Elias JA A chitinase-like protein in the lung and circulation of patients with severe asthma.N Engl J Med. 2007; 357: 2016-2027Crossref PubMed Scopus (437) Google Scholar Negative controls included control rabbit IgG (DakoCytomation) instead of the primary Ab, or primary Abs adsorbed with purified ChTRase or AMCase. Positive cells in bronchial biopsy sections were expressed as numbers of cells/mm2 total biopsy areas.14Létuvé S Kozhich A Arouche N Grandsaigne M Reed J Dombret MC Kiener PA Aubier M Coyle AJ Pretolani M YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.J Immunol. 2008; 181: 5167-5173PubMed Google Scholar, 16Chupp GL Lee CG Jarjour N Shim YM Holm CT He S Dziura JD Reed J Coyle AJ Kiener P Cullen M Grandsaigne M Dombret MC Aubier M Pretolani M Elias JA A chitinase-like protein in the lung and circulation of patients with severe asthma.N Engl J Med. 2007; 357: 2016-2027Crossref PubMed Scopus (437) Google Scholar ChTRase-positive alveolar macrophages were enumerated in cytospin preparations from BAL after counting at least 200 cells in randomly selected fields.14Létuvé S Kozhich A Arouche N Grandsaigne M Reed J Dombret MC Kiener PA Aubier M Coyle AJ Pretolani M YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.J Immunol. 2008; 181: 5167-5173PubMed Google Scholar Paraffin-embedded lung tissue specimens were subjected to heat-induced antigen retrieval in citrate buffer and reacted with 4 μg/ml of the anti-ChTRase, or the anti-AMCase Ab and binding was visualized using Fast Red as described above, or biotin-conjugated anti-rabbit Ab followed by AlexaFluor 568-conjugated streptavidin (Invitrogen). The localization of ChTRase within alveolar macrophages (in bronchial biopsies and lung specimens), and neutrophils (in bronchial biopsies exclusively) was examined by immunofluorescent double staining using a mouse anti-human CD68 monoclonal Ab (clone PG-M1, 1:50 dilution, DakoCytomation), or a mouse anti-human neutrophil elastase monoclonal Ab (clone NP57, 1:50 dilution, DakoCytomation), or their control isotypes, mouse IgG3κ and mouse IgG1κ, respectively. This was followed by goat anti-mouse AlexaFluor 488 (Molecular Probes), treatment with RNase and incubation with TO-PRO-3 (Molecular Probes) to stain nuclei. Slides were examined using a Plan Apochromat 63 × 1.4 oil differential interference contrast objective on the LSM 510 confocal microscope and fluorescence was analyzed with LSM META Acquisition and Image Browser software (both from Carl Zeiss, Le Pecq, France). For ChTRase staining in mouse tissues, formalin-fixed, paraffin-embedded lungs were subjected to mild heat-induced antigen retrieval and reacted with 10 μg/ml rabbit polyclonal anti-ChTRase Ab. Binding was visualized using biotin-conjugated anti-rabbit Ab and avidin-biotinylated peroxidase complex from the Vectastain kit (Vector) followed by incubation with 3,3′-diaminobenzidine (DakoCytomation) and light Mayer's hematoxylin counterstaining. Alveolar macrophages were isolated from the BAL obtained from 8 healthy never-smokers, 10 smokers without COPD, and 14 smokers with COPD (Global Initiative for Obstructive Lung Disease stages [II], n = 11, and III [n = 3]) who underwent bronchoscopy.14Létuvé S Kozhich A Arouche N Grandsaigne M Reed J Dombret MC Kiener PA Aubier M Coyle AJ Pretolani M YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.J Immunol. 2008; 181: 5167-5173PubMed Google Scholar Smokers with COPD were former (n = 8) or current smokers (n = 6). BAL cells were resuspended in RPMI-1640 medium containing 25 mmol/L Hepes, 2 mmol/L glutamine, 2.5 mmol/L sodium pyruvate, non-essential amino acids, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen, Cergy-Pontoise, France) and were seeded at 500,000 macrophages/ml in 12-well plates (Corning-Costar, Acton, MA). After 2 hours at 37°C in a humidified 5% CO2 incubator, cells were washed and further incubated for 24 hours, when they were either collected for RNA isolation, or stimulated over 24 hours with 0.1, 1, and 10 μg/ml of ChTRase, or with 10 ng/ml of recombinant human tumor-necrosis factor (TNF)-α (R&D Systems, Minneapolis, MN), or with the medium alone. In all experiments, cell-free supernatants were collected and stored at −80°C until use. Total RNA was extracted from alveolar macrophages using the Nucleospin RNA II kit (Macherey-Nagel, Hoerdt, France) and reverse transcribed using Moloney Murine Leukemia Virus enzyme (Invitrogen). The levels of the transcripts encoding ChTRase were assessed by quantitative real-time PCR (Mx 3000P apparatus, Stratagene Europe, Amsterdam, The Netherlands), and their expression was normalized to that of ubiquitin C, using GeNorm software.17Pégorier S Wagner LA Gleich GJ Pretolani M Eosinophil-derived cationic proteins activate the synthesis of remodeling factors by airway epithelial cells.J Immunol. 2006; 177: 4861-4869PubMed Google Scholar Primers (ChTRase, GenBank Identifier NM_003465, sense 5′-CTGCATCATGGTGCGGTC-3′, antisense, 5′-GTGCTCAGCTGGTGGTTG-3′; ubiquitin C (GenBank Idenfier NM_004168), sense, 5′-CACTTGGTCCTGCGCTTGA-3′, antisense, 5′- TTTTGGGAATGCAACAACTTT-3′) were designed using Primer Express 2 Software (Applied Biosystems, Framingham, MA) and were synthesized by Genosys (Sigma). Their sequences were blasted against Basic Local Alignment Search Tool database (http://www.ncbi.nlm.nih.gov/BLAST). BAL levels of interleukin (IL)−1β, IL-8, TNF-α, soluble type II receptor of TNF-α (TNF-RII), granulocyte-macrophage colony-stimulating factor, and interferon-γ were assessed using the multianalyte LabMAP Luminex technology (Luminex Corp., Austin, TX), which allows simultaneous assessment of multiple protein markers in biological samples.18Vignali DA Multiplexed particle-based flow cytometry assays.J Immunol Methods. 2000; 243: 243-255Crossref PubMed Scopus (785) Google Scholar Sensitivities were of 0.5 pg/ml (IL-1β), 0.7 pg/ml (IL-8), 0.5 pg/ml (TNF-α), 0.6 pg/ml (TNF-RII), 10 pg/ml (granulocyte-macrophage colony-stimulating factor), and 0.9 pg/ml (interferon-γ). Chitinolytic activity of purified human ChTRase and of BAL fluid was tested using 4-methylumbelliferyl-β-D-N,N′,N′ ′-triacetylchitotriose as a substrate and the fluorescence of released 4-methyl-umbelliferone was measured.9Boot RG van Achterberg AE van Aken BE Renkema GH Jacobs MJHM Aerts JM de Vries CJ Strong induction of members of the chitinase family of proteins in atherosclerosis: chitotriosidase and human cartilage gp-39 expressed in lesion macrophages.Arterioscler Thromb Vasc Biol. 1999; 19: 687-694Crossref PubMed Scopus (278) Google Scholar Reactions were performed at two different pH values (7.0 and 3.5). For inhibition experiments, BAL samples, or human purified ChTRase (0.005 to 10 ng/ml) was pre-incubated for 15 minutes at room temperature with the polyclonal rabbit anti-ChTRase Ab, before the addition of the substrate and chitinolytic activity was assessed, as described above. Human ChTRase and AMCase were measured by the corresponding specific ELISA assays, which were developed on site (Supplementary Figure S1A at http://ajp.amjpathol.org). The levels of IL-8 and monocyte-chemoattractant protein (MCP)−1 in the supernatant of alveolar macrophages were assessed by ELISA (R&D Systems) and MMP-9 was measured by the specific Biotrak activity assay (GE Health Care). The detection levels were 6.5 pg/ml (IL-8), 12.5 pg/ml (MCP-1), and 250 pg/ml (MMP-9). Adenoviral vector carrying full-length ChTRase (ChTRase AdV) and control empty vector adenovirus (Null Adv) were generated using the AdEasy system using protocols previously described by Stratagene.19Luo J Deng ZL Luo X Tang N Song WX Chen J Sharff KA Luu HH Haydon RC Kinzler KW Vogelstein B He TC A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.Nat Protoc. 2007; 2: 1236-1247Crossref PubMed Scopus (628) Google Scholar Briefly, murine ChTRase was cloned into the pCMVShuttle plasmid and recombination performed in the bacterial E. coli strain BJ5183-Ad1, to obtain adenoviral genome plasmid. This plasmid was then transfected into Ad 293 cells to generate virus. Virus was scaled-up in 293F cells (Invitrogen) and purified by cesium chloride gradient centrifugation. Titer was determined by Tissue Culture Infective Dose 50 on Ad 293 cells. Null AdV with no insert was produced in the same way. Female BALB/c mice, aged 8–10 weeks (Harlan Corporation, Indianapolis, IN) were anesthetized with isoflurane (Baxter Health Care Corporation, Deerfield, IL) and instilled intranasally with 50 μl PBS alone, or supplemented with 107 Plaque-Forming Units of ChTRase Adv, or Null Adv. Two or 4 days thereafter, mice were sacrificed with a barbiturate overdose and bled by cardiac puncture. BAL was performed with 3 × 0.6 ml aliquots of Hepes-buffered saline, containing 10 mmol/L HEPES and EDTA, via a tracheal catheter. BAL fluid was centrifuged (400 × g, 10 minutes, 4°C) and the cells removed. Total cell counts were determined using a Coulter Z1 particle counter (Beckman Coulter Corporation, Brea, CA) and differential cell counts (of at least 500 cells per slide) were performed on cytospin preparations stained with eosin and methylene blue (Diff-Quik, Dade Diagnostics, Duedingen, Switzerland). Lung tissue was homogenized (100 mg/ml) in Hepes-buffered saline containing protease inhibitors (Complete EDTA-free tablets, Roche, Nutley, NJ) and centrifuged, (800 × g, 10 minutes, 4°C), and the supernatant collected and stored alongside with BAL fluid supernatants at −80°C until the assessment of the murine homologue of IL-8, keratinocyte-derived cytokine (KC) and MCP-1 by ELISA (R&D Systems). The minimum detection levels for both assays were 2 pg/ml. In separate experiments, mice were terminally anesthetized and the left lung lobe was dissected, fixed in 4% formaldehyde, and embedded in paraffin, and 5-μm serial sections were used to examine the localization of ChTRase by immunohistochemistry. The right lobe was kept for RNA purification using the RNAeasy Plus mini kit (Qiagen, Valencia, CA). Complementary DNA was synthesized using Sprint Power Script Double Preprimed 96 kit (Clontech Laboratories, Mountain View, CA) and the levels of the transcripts encoding ChTRase, AMCase, KC, MCP-1 and the reference gene, glyceraldehyde-3-phosphate dehydrogenase, were measured by TaqMan real-time PCR (Applied Biosystems). Data were analyzed statistically using the GraphPad Prism software version 4.0 for MacIntosh (GraphPad Software, Inc. San Diego, CA). The results are expressed as median (interquartile range), except those obtained with purified alveolar macrophages and with Adv-treated mice, which represent means ± SEM. Since the data were not normally distributed, non-parametric Kruskal-Wallis test was used to compare the different groups of subjects. When an overall significant difference was detected (P ≤ 0.05), pair wise group tests were performed using the Mann-Whitney U-test. Univariate regression analyses were performed by the Spearman's rank-order method and correlation coefficients (r2) were calculated. To account for multiple tests, a Benjamini and Hochberg correction with a family-wise error rate of 0.05 was used.14Létuvé S Kozhich A Arouche N Grandsaigne M Reed J Dombret MC Kiener PA Aubier M Coyle AJ Pretolani M YKL-40 is elevated in patients with chronic obstructive pulmonary disease and activates alveolar macrophages.J Immunol. 2008; 181: 5167-5173PubMed Google Scholar In all cases, P values ≤0.05 were considered significant. Smokers with COPD had higher chitinolytic activity in their BAL fluid than smokers without COPD and never-smokers (Figure 1A). Chitinolytic ac
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