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• W2034696505 abstract "Cigarette smoking is a major factor for the development of pulmonary emphysema because it induces abnormal inflammation and a protease-rich local milieu that causes connective tissue breakdown of the lungs. As a result of its capacity to degrade lung tissue and the high risk of patients lacking α1-antitrypsin to develop emphysema, much interest has focused on neutrophil elastase (NE). Two similar neutrophil serine proteases (NSPs), cathepsin G and proteinase 3, coexist with NE in humans and mice, but their potential tissue-destructive role(s) remains unclear. Using a gene-targeting approach, we observed that in contrast to their wild-type littermates, mice deficient in all three NSPs were substantially protected against lung tissue destruction after long-term exposure to cigarette smoke. In exploring the underlying basis for disrupted wild-type lung air spaces, we found that active NSPs collectively caused more severe lung damage than did NE alone. Furthermore, NSP activities unleashed increased activity of the tissue-destructive proteases macrophage elastase (matrix metalloproteinase-12) and gelatinase B (matrix metalloproteinase-9). These in vivo data provide, for the first time, compelling evidence of the collateral involvement of cathepsin G, NE, and proteinase 3 in cigarette smoke–induced tissue damage and emphysema. They also reveal a complex positive feed-forward loop whereby these NSPs induce the destructive potential of other proteases, thereby generating a chronic and pathogenic protease-rich milieu. Cigarette smoking is a major factor for the development of pulmonary emphysema because it induces abnormal inflammation and a protease-rich local milieu that causes connective tissue breakdown of the lungs. As a result of its capacity to degrade lung tissue and the high risk of patients lacking α1-antitrypsin to develop emphysema, much interest has focused on neutrophil elastase (NE). Two similar neutrophil serine proteases (NSPs), cathepsin G and proteinase 3, coexist with NE in humans and mice, but their potential tissue-destructive role(s) remains unclear. Using a gene-targeting approach, we observed that in contrast to their wild-type littermates, mice deficient in all three NSPs were substantially protected against lung tissue destruction after long-term exposure to cigarette smoke. In exploring the underlying basis for disrupted wild-type lung air spaces, we found that active NSPs collectively caused more severe lung damage than did NE alone. Furthermore, NSP activities unleashed increased activity of the tissue-destructive proteases macrophage elastase (matrix metalloproteinase-12) and gelatinase B (matrix metalloproteinase-9). These in vivo data provide, for the first time, compelling evidence of the collateral involvement of cathepsin G, NE, and proteinase 3 in cigarette smoke–induced tissue damage and emphysema. They also reveal a complex positive feed-forward loop whereby these NSPs induce the destructive potential of other proteases, thereby generating a chronic and pathogenic protease-rich milieu. Pulmonary emphysema represents one of the components of the spectrum of chronic obstructive pulmonary disease.1Tuder R.M. Petrache I. Pathogenesis of chronic obstructive pulmonary disease.J Clin Invest. 2012; 122: 2749-2755Crossref PubMed Scopus (324) Google Scholar Chronic obstructive pulmonary disease currently afflicts approximately 9% to 10% of the population throughout the world, representing a high economic burden (Global Initiative for Chronic Obstructive Lung Disease, http://www.goldcopd.org/guidelines-global-strategy-for-diagnosis-management.html, last accessed April 2, 2014).2Halbert R.J. Natoli J.L. Gano A. Badamgarav E. Buist A.S. Mannino D.M. Global burden of COPD: systematic review and meta-analysis.Eur Respir J. 2006; 28: 523-532Crossref PubMed Scopus (1040) Google Scholar One major risk factor for the development of chronic obstructive pulmonary disease is cigarette smoking, which is increasing worldwide, especially in teens and women.3Hipple B. Lando H. Klein J. Winickoff J. Global teens and tobacco: a review of the globalization of the tobacco epidemic.Curr Probl Pediatr Adolesc Health Care. 2011; 41: 216-230Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 4Amos A. Greaves L. Nichter M. Bloch M. Women and tobacco: a call for including gender in tobacco control research, policy and practice.Tob Control. 2012; 21: 236-243Crossref PubMed Scopus (162) Google Scholar This epidemic disease is expected to rank as the third cause of mortality in the coming years.5Rabe K.F. Hurd S. Anzueto A. Barnes P.J. Buist S.A. 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 (4327) Google Scholar Emphysema is defined as the enlargement of distal air spaces beyond the terminal bronchioles caused by destruction of the walls of these structures.6Hogg J.C. Timens W. The pathology of chronic obstructive pulmonary disease.Annu Rev Pathol. 2009; 4: 435-459Crossref PubMed Scopus (443) Google Scholar According to the protease-antiprotease hypothesis, smoking evokes abnormal inflammation characterized by the presence of unopposed protease-rich milieu. Active proteases degrade lung connective tissue, particularly elastin, leading to emphysema.7Barnes P.J. Mediators of chronic obstructive pulmonary disease.Pharmacol Rev. 2004; 56: 515-548Crossref PubMed Scopus (514) Google Scholar, 8Churg A. Wright J.L. Proteases and emphysema.Curr Opin Pulm Med. 2005; 11: 153-159Crossref PubMed Scopus (114) Google Scholar In support of this, inherited deficiency of α1-antitrypsin, the primary inhibitor of neutrophil elastase (NE), in particular caused by null alleles, predisposes to emphysema. Furthermore, intrapulmonary instillation of proteases in experimental animal models resulted in typical features of the pathologic condition.9Laurell C.B. Eriksson S. The electrophoretic alpha-1globulin pattern of serum in alpha-1-antitrypsin deficiency.Scand J Clin Lab Invest. 1963; 15: 132-140Crossref Google Scholar, 10Senior R.M. Tegner H. Kuhn C. Ohlsson K. Starcher B.C. Pierce J.A. The induction of pulmonary emphysema with human leukocyte elastase.Am Rev Respir Dis. 1977; 116: 469-475Crossref PubMed Scopus (237) Google Scholar, 11Kao R.C. Wehner N.G. Skubitz K.M. Gray B.H. Hoidal J.R. Proteinase 3: a distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters.J Clin Invest. 1988; 82: 1963-1973Crossref PubMed Scopus (344) Google Scholar, 12Wood A.M. Stockley R.A. Alpha one antitrypsin deficiency: from gene to treatment.Respiration. 2007; 74: 481-492Crossref PubMed Scopus (59) Google Scholar Conversely, gene targeting of some specific proteases contributed to the protection of mice against cigarette smoke (CS)–induced emphysema.13Hautamaki R.D. Kobayashi D.K. Senior R.M. Shapiro S.D. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.Science. 1997; 277: 2002-2004Crossref PubMed Scopus (1209) Google Scholar, 14Djekic U.V. Gaggar A. Weathington N.M. Attacking the multi-tiered proteolytic pathology of COPD: new insights from basic and translational studies.Pharmacol Ther. 2009; 121: 132-146Crossref PubMed Scopus (38) Google Scholar Several proteases are stored in neutrophil primary granules, in particular the neutrophil serine proteases (NSPs) NE (encoded by Ela2), cathepsin G (CG), and proteinase 3 (PR3; encoded by Prtn3). CG, NE, and PR3 are structurally highly related and rely on the same His-Asp-Ser triad for their catalytic activities. The enzymes coexist in man and mouse15Grisolano J.L. Sclar G.M. Ley T.J. Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice.Proc Natl Acad Sci U S A. 1994; 91: 8989-8993Crossref PubMed Scopus (52) Google Scholar, 16Belaaouaj A. Moog-Lutz C. Just J. Houzel-Charavel A. Shapiro S.D. Cayre Y. Genomic organization and chromosomal localization of mouse proteinase 3 (Myeloblastin).Mamm Genome. 1999; 10: 210-212Crossref PubMed Scopus (9) Google Scholar and are stored together in primary granules in fully processed, readily active forms at relatively high concentrations.17Liou 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 (90) Google Scholar NSPs are excreted on cell activation and are known to cleave, at least in vitro, a broad range of host proteins.18Lee W.L. Downey G.P. Leukocyte elastase: physiological functions and role in acute lung injury.Am J Respir Crit Care Med. 2001; 164: 896-904Crossref PubMed Scopus (262) Google Scholar, 19Heinz A. Jung M.C. Jahreis G. Rusciani A. Duca L. Debelle L. Weiss A.S. Neubert R.H. Schmelzer C.E. The action of neutrophil serine proteases on elastin and its precursor.Biochimie. 2012; 94: 192-202Crossref PubMed Scopus (41) Google Scholar As a result of its capacity to efficiently degrade elastin and the high risk of patients lacking α1-antitrypsin to develop emphysema, much interest has focused on NE. Unlike NE, the roles of CG and PR3 in emphysema have not been clarified. Rather than investigating the pathogenic roles of individual proteases or combinations of two NSPs, we sought to generate mice deficient in all three NSPs, CG-NE-PR3 [NSP-knockout (KO)], and to expose them to long-term CS. Simultaneous deficiency of the Prtn3 and Ela2 gene cluster (129S6/SvEv), deficiency in CG (129S6/SvEv-C57BL/6J), and deficiency in NE (129S6/SvEv-C57BL/6J) were generated by targeted mutagenesis as described elsewhere.20Belaaouaj 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 (530) Google Scholar, 21MacIvor D.M. Shapiro S.D. Pham C.T. Belaaouaj A. Abraham S.N. Ley T.J. Normal neutrophil function in cathepsin G-deficient mice.Blood. 1999; 94: 4282-4293Crossref PubMed Google Scholar, 22Kessenbrock K. Frohlich L. Sixt M. Lammermann T. Pfister H. Bateman A. Belaaouaj A. Ring J. Ollert M. Fassler R. Jenne D.E. Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin.J Clin Invest. 2008; 118: 2438-2447PubMed Google Scholar Regarding NE-PR3 double deficiency, gene targeting resulted in the deletion of exons 2 to 5 of the Prtn3 gene and exons 1 to 3 of the Ela2 gene, whereas the neighboring genes remained unchanged. NE deficiency was obtained by deleting part of intron 1 and exon 2 of the Ela2 gene, and CG deficiency resulted from the removal of a fragment containing the 3′ end of exon 3, intron 3, and the 5′ end of exon 4. NE-PR3–deficient mice and CG-deficient mice were crossbred to generate heterozygote-deficient progeny (F1). The F1 progeny were intercrossbred to generate mice deficient in NE, PR3, and CG, referred to as NSP-KO. All offspring were genotyped by PCR on tail genomic DNA using specific oligonucleotide primers (Table 1). Mouse strains were subsequently backcrossed (eight generations) on a pure C57BL6/J background. Mice were housed in a pathogen-free facility with food and water ad libitum and a 12-hour light/dark cycle.Table 1Primer Sequences and Expected PCR Product SizesMouse genotypingOligonucleotide sequencesAmplicon (Bp)ELa2-Prtn3 WTF: 5′-CTCGGCCTTATGTGGCATCC-3′WT 1500R: 5′-CCGGCATAGGAAGGTGACCA-3′ELa2-Prtn3 KOF: 5′-AGCTCCCATGCTGTGTTTC-3′KO 1100R: 5′-TCGGTCTTTGGGATGGGTAAG-3′CGF: 5′-AATGCGAGAAAGGACCCAGCAAC-3′WT 550R: 5′-GCAGAAGCATGATGTCATTCCGG-3′NeoR: 5′-AGAAGAACCAGCTGGGGCTC-3′KO 210RT-PCR NEF: 5′-CGTCGTGTGAACGTATGCACTC-3′264R: 5′-TGCAGACAGGTCCTAGTTGGTC-3′ CGF: 5′-GCCAGAGCAGGGGAACAAATG-3′240R: 5′-GCCTGAGACAGGGAGTTAGCAG-3′ PR3F: 5′-ACCTTCCTATGCCGGGAACAC-3′382R: 5′-AGGGAACAGAGCTGACTCCAC-3′ β-actinF: 5′-AGAGGGAAATCGTGCGTGAC-3′83R: 5′-CAATAGTGATGACCTGGCCGT-3′qPCR MMP-9F: 5′-CTGGACAGCCAGACACTAAAG-3′145R: 5′-CTCGCGGCAAGTCTTCAGAG-3′ MMP-12F: 5′-GAGTCCAGCCACCAACATTAC-3′232R: 5′-GCGAAGTGGGTCAAAGACAG-3′ GAPDHF: 5′-CAGCCTCGTCCCGTAGACAA-3′60R: 5′-CCCAATACGGCCAAATTCG-3′F, forward; R, reverse. Open table in a new tab F, forward; R, reverse. NSP-KO mice and their wild-type (WT) littermates were further characterized by mRNA and protein levels using RT-PCR and Western immunoblot analysis.20Belaaouaj 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 (530) Google Scholar Protease activities were tested using elastin zymography or kinetic assay as described in Enzymatic Activities and Zymography. Animal handling and procedures were approved by the Animal Studies Committee at our institution (Health and Animal Protection Office, Châlons-en-Champagne, France, Authorisation number: 51-31) in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations and following the European Directive 2010/63/EU on the protection of animals used in scientific procedures. Twenty-five NSP-KO mice and 27 WT littermates (8 to 10 weeks of age) were subjected to two filtered cigarettes per day [3R4F research cigarettes, University of Kentucky; total particulate matter (means ± SEM): 11.0 (0.33) mg per cigarette] for 5 days per week for 6 months in a specially designed vented nose exposure smoking chamber.23Shapiro S.D. Goldstein N.M. Houghton A.M. Kobayashi D.K. Kelley D. Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.Am J Pathol. 2003; 163: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar Twenty-four sham and non–CS-exposed age-matched littermates were used as controls. Mice tolerated CS exposure, and the average level of serum carboxyhemoglobin after exposure to two cigarettes was approximately 8%. Two NSP-KO mice and one WT mouse died during the smoking period. At the end of the CS exposure period, the mice were sacrificed, and their bronchoalveolar lavage (BAL) fluids and lungs were processed for inflammatory and histologic analyses. Mice were anesthetized using 50 mg/kg of ketamine hydrochloride and 5 mg/kg of xylazine hydrochloride, followed by intranasal (i.n.) administration of 10 μg of Pseudomonas aeruginosa–derived lipopolysaccharide (LPS) per mouse in 50 μL of phosphate-buffered saline (PBS).24Hirche 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: 27688-27698Crossref PubMed Scopus (83) Google Scholar Two groups (n = 6 per genotype) of WT and NSP-KO mice received one dose of LPS and were sacrificed 1 day after challenge. In a parallel experiment, two additional groups of mice were i.n. instilled with LPS twice a week and were sacrificed 3 weeks after. In separate experiments, mice (n = 12 per genotype) were anesthetized, followed by i.n. challenge with 50 μL of PBS containing LPS-free NE (10 μg per mouse) or a mix of LPS-free NSPs (NE, CG, and PR3, each at 10 μg).25Zhang M. Liu N. Park S.M. Wang Y. Byrne S. Murmann A.E. Bahr S. Peter M.E. Olson S.T. Belaaouaj A. Ashton-Rickardt P.G. Serine protease inhibitor 6-deficient mice have increased neutrophil immunity to Pseudomonas aeruginosa.J Immunol. 2007; 179: 4390-4396Crossref PubMed Scopus (8) Google Scholar Control mice (n = 5 per genotype) received sterile PBS. Three weeks later, mice were sacrificed and processed. The mice were sacrificed, and the lungs were gently perfused with saline via the right ventricle. The trachea was exposed through a midline incision and was cannulated using a sterile 22-gauge catheter (BD Biosciences, Franklin Lakes, NJ). In all the experiments, the lungs were lavaged in situ (BAL), with 1 mL of PBS, pH 7.4, cycled in three times. Identical recoveries of BAL (700 μL per mouse) were obtained for each mouse.24Hirche 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: 27688-27698Crossref PubMed Scopus (83) Google Scholar Total cell and differential counts were immediately performed on aliquots of BAL fluids. Regarding mice that were exposed to CS, BAL fluids were prepared as follows: Three pools of equal volumes of cell-free BALs (two pools of eight samples and one pool of seven samples for the NSP-KO genotype and two pools of nine samples and one pool of eight samples for the WT genotype) were prepared. Twelve NSP-KO and WT lavaged lungs were processed for histologic analysis.23Shapiro S.D. Goldstein N.M. Houghton A.M. Kobayashi D.K. Kelley D. Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.Am J Pathol. 2003; 163: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar The remaining 11 NSP-KO and 14 WT lavaged lungs were snap frozen in liquid nitrogen for protein and RNA extraction. Three pools of equal lung protein or total RNA amounts were subsequently prepared (two pools of four aliquots and one pool of three aliquots for NSP-KO samples and two pools of five aliquots and one pool of four aliquots for WT samples). All the samples were aliquoted and stored at −80°C until use. When appropriate and to ensure equal protein loading, the total protein concentrations of the samples were determined using an RC DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). For histologic analysis, lungs were processed as previously described,23Shapiro S.D. Goldstein N.M. Houghton A.M. Kobayashi D.K. Kelley D. Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.Am J Pathol. 2003; 163: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar with the following modifications. After CS exposure, each mouse was sacrificed by an i.p. injection with an overdose of pentobarbital. The trachea was exposed through a midline incision and was cannulated using a sterile 22-gauge catheter (BD Biosciences). The lungs were perfused through the right ventricle to remove blood. Next, they were inflated in situ by instilling 10% formalin at a constant fluid pressure of 25 cm for 5 minutes, ligated through the trachea, and removed. Inflated lungs were fixed for 48 hours before embedding in paraffin. Serial lung tissue sections were processed for hematoxylin and eosin staining and were examined by light microscopy. With respect to protease i.n. instillation experiments, six NSP-KO and WT lavaged lungs were processed for histologic analysis. The remaining lavaged lungs were snap frozen in liquid nitrogen and stored at −80°C. Morphometric assessment of emphysema was performed by determination of the average interalveolar distance estimated by calculating the mean linear intercept (Lm).23Shapiro S.D. Goldstein N.M. Houghton A.M. Kobayashi D.K. Kelley D. Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.Am J Pathol. 2003; 163: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 26Dunnill M.S. Evaluation of a simple method of sampling the lung for quantitative histological analysis.Thorax. 1964; 19: 443-448Crossref PubMed Scopus (61) Google Scholar Briefly, for each mouse, 10 random digitized images of representative hematoxylin and eosin–stained lung tissues were captured in a blinded manner by two investigators (N.G. or L.M.) using a DM750 microscope coupled to a digital camera module ICC50 (Leica Microsystems, Inc., Buffalo Grove, IL) and were analyzed using the image analysis software ImageJ version 1.33 (NIH, Bethesda, MD). Air space enlargement was quantified by measuring Lm. Cell-free WT and NSP-KO BAL fluids of CS-exposed mice were processed to assess the levels of various cytokines using the RayBio mouse cytokine antibody array 6 (RayBiotech, Tebu-Bio, Le Perray-en-Yvelines, France) according to the manufacturer's instructions.27Benabid R. Wartelle J. Malleret L. Guyot N. Gangloff S. Lebargy F. Belaaouaj A. Neutrophil elastase modulates cytokine expression: contribution to host defense against Pseudomonas aeruginosa-induced pneumonia.J Biol Chem. 2012; 287: 34883-34894Crossref PubMed Scopus (54) Google Scholar Cell-free BAL fluids of CS-unexposed mice were analyzed as well. Briefly, equal volumes of cell-free BAL fluids (400 μL) were added to antibody-coated membranes, and detection of immunoreactive cytokines was performed after sequential incubations of the membranes with biotinylated anticytokine antibodies and streptavidin–horseradish peroxidase and visualization by enhanced chemiluminescence. Images were obtained using a ChemiDoc XRS imaging system (Bio-Rad Laboratories). Semiquantitative analysis by densitometry was performed on captured images using Quantity One 1-D analysis software version 4.5.2 (Bio-Rad Laboratories). Spots of interest were normalized to an internal control after subtraction of representative background sample. Cytokine antibody array assays were performed on all BAL fluid pools. Lungs of mice that were sacrificed 1 day after i.n. LPS instillation were lavaged with saline, and cells (predominantly neutrophils) were counted and aliquoted (1 × 106). Lung tissue extracts of control and CS-exposed mice were prepared by homogenizing perfused and frozen lungs in 5 mL of 50 mmol/L Tris, 10 mmol/L CaCl2, and 750 mmol/L NaCl buffer using a Tissue-Tearor (BioSpec Products Inc., Bartlesville, OK). After three successive freezing/thawing periods, centrifugation (13,000 × g, 5 minutes, 4°C), and lyophilization, aliquots with equal total lung protein concentrations were prepared.23Shapiro S.D. Goldstein N.M. Houghton A.M. Kobayashi D.K. Kelley D. Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.Am J Pathol. 2003; 163: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar Aliquots of equal volumes of cell-free BAL fluids from control and LPS-challenged mice were prepared as well. Aliquots of equal volumes of cell-free BAL fluids or lung tissue extracts were subjected to 10% SDS-PAGE using 3 mg/mL of elastin or 1.5 mg/mL of gelatin as substrates for zymography.24Hirche 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: 27688-27698Crossref PubMed Scopus (83) Google Scholar The gels were then stained with Coomassie blue and destained in 5% acetic acid and 10% methanol. Active proteases appear as transparent lysis bands at their respective molecular sizes. Aliquots of neutrophils with equal numbers were further activated using LPS and formyl-methionyl-leucyl-phenylalanine24Hirche 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: 27688-27698Crossref PubMed Scopus (83) Google Scholar and were incubated next with 0.2 mmol/L specific substrates for CG, NE, and PR3 at 37°C in a total volume of 1 mL of Tris-NaCl buffer (0.1 mol/L Tris, 1 mol/L NaCl, pH 7.4). Changes in the absorbances of the reactions were recorded every 2 minutes for 10 minutes at λ 410 nm. Absorbance values were corrected for nonspecific activity by subtracting the values of NSP-KO reactions from those of WT reactions. The chromogenic peptide substrates were N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, and N-Boc-Ala-ONp for CG, NE, and PR3, respectively (Elastin Products Company Inc., Owensville, MO). NE, CG, or PR3 or pooled NSP (NE, CG, and PR3, each at 10 μg) was incubated overnight with 3 mg of Congo red elastin (Elastin Products Company Inc.) in 1 mL of Tris buffer (100 mmol/L, pH 8.0).24Hirche 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: 27688-27698Crossref PubMed Scopus (83) Google Scholar After centrifugation (1 minute/14,000 rpm), the elastolytic activity was measured as the absorbance of the supernatant at 495 nm. Absorbances were corrected for nonspecific activity by subtracting the value of controls (Congo red elastin alone) from those of protease-treated Congo red elastin reactions. Immunoblotting experiments were performed on aliquots with equal total protein amounts of bone marrow or lung tissue protein pools.20Belaaouaj 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 (530) Google Scholar, 23Shapiro S.D. Goldstein N.M. Houghton A.M. Kobayashi D.K. Kelley D. Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.Am J Pathol. 2003; 163: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar The membranes were sequentially incubated with designated anti-mouse primary antibodies [anti-NE, anti-PR3, anti-CG (dilution 1:2000), and anti-myeloperoxidase (MPO; dilution 1:2000)20Belaaouaj 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 (530) Google Scholar, 24Hirche 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: 27688-27698Crossref PubMed Scopus (83) Google Scholar or anti–matrix metalloproteinase (MMP)-12 (dilution 1:1000)] followed by their respective secondary horseradish peroxidase–conjugated antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). When indicated, the membranes were stripped (100 mmol/L β-mercaptoethanol, 2% SDS, and 62.5 mmol/L Tris-HCl, pH 6.7, for 30 minutes at 50°C) and immunoblotted using primary rabbit polyclonal anti-mouse albumin antibody (Rockland Immunochemicals Inc., Gilbertsville, PA) and its corresponding horseradish peroxidase–conjugated secondary antibody. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ). Total RNA isolation from bone marrow cells or lung tissues was performed using the MasterPure RNA purification kit (Epicentre, Illumina, Madison, WI), as described by the manufacturer's protocol. Total bone marrow–derived RNA samples (1 μg) were reverse transcribed, and cDNAs were amplified by PCR using specific primers for Ela2 (NE), CG (CG), Prtn3 (PR3), and β-actin (internal control) (Table 1)20Belaaouaj 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 (530) Google Scholar (40 cycles starting with cDNA denaturation for 2 minutes at 94°C; each cycle corresponded to denaturation for 15 seconds at 94°C, primer annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds). RT-PCR products were analyzed by electrophoresis on 1.5% agarose gels and a gel documentation system using Quantity One software version 4.6.1 (Bio-Rad Laboratories). Total lung tissue RNA was reverse transcribed into cDNA using the same procedure as used in RT-PCR. Next, real-time PCR amplification was performed using specific primers for MMP-9 and MMP-12 and normalized to glyceraldehyde-3-phosphate dehydrogenase amplicon (Table 1). PCR amplification conditions were as follows: initial DNA denaturation for 5 minutes at 94°C, primer annealing at 60°C for 1 minute, and extension at 72°C for 1 minute, for a total 40 cycles. Data analysis was performed using the SDS software version 2.4.1 (Applied Biosystems, Foster City, CA).28Velard F. Laurent-Maquin D. Braux J. Guillaume C. Bouthors S. Jallot E. Nedelec J.M. Belaaouaj A. Laquerriere P. The effect of zinc on hydroxyapatite-mediated activation of human polymorphonuclear neutrophils and bone implant-associated acute inflammation.Biomaterials. 2010; 31: 2001-2009Crossref PubMed Scopus (60) Google Scholar WT and NSP-KO mice were i.p. instilled with glycogen (15%) and were sacrificed 4 hours later. Aliquots of the peritoneal lavage were centrifuged, the supernatants removed, and the cells fixed in 2% glutaraldehyde and processed for electron microscopy.20Belaaouaj 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 (530) Google Scholar WT and NSP-KO mice were i.p. injected first with glycogen and 4 hours later with P. aeruginosa H103. After 30 minutes, cells were collected in gentamicin-containing Hanks’ balanced salt solution, washed, and cytospins were prepared and Wright stained.29Hirche T" @default.
• W2034696505 created "2016-06-24" @default.
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• W2034696505 date "2014-08-01" @default.
• W2034696505 modified "2023-10-18" @default.
• W2034696505 title "Unopposed Cathepsin G, Neutrophil Elastase, and Proteinase 3 Cause Severe Lung Damage and Emphysema" @default.
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