Decreased Levels of Secretory Leucoprotease Inhibitor in the Pseudomonas-Infected Cystic Fibrosis Lung Are Due to Neutrophil Elastase Degradation This information is current as of September 29, 2021. Sinéad Weldon, Paul McNally, Noel G. McElvaney, J. Stuart Elborn, Danny F. McAuley, Julien Wartelle, Abderrazzaq Belaaouaj, Rodney L. Levine and Clifford C. Taggart J Immunol 2009; 183:8148-8156; ; doi: 10.4049/jimmunol.0901716 Downloaded from http://www.jimmunol.org/content/183/12/8148 References This article cites 50 articles, 17 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/183/12/8148.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Decreased Levels of Secretory Leucoprotease Inhibitor in the Pseudomonas-Infected Cystic Fibrosis Lung Are Due to Neutrophil Elastase Degradation1 Sine´ad Weldon,* Paul McNally,† Noel G. McElvaney,† J. Stuart Elborn,* Danny F. McAuley,* Julien Wartelle,‡ Abderrazzaq Belaaouaj,‡ Rodney L. Levine,§ and Clifford C. Taggart2* Secretory leucoprotease inhibitor (SLPI) is a neutrophil serine protease inhibitor constitutively expressed at many mucosal sur- faces, including that of the lung. Originally identified as a serine protease inhibitor, it is now evident that SLPI also has antimi- crobial and anti-inflammatory functions, and therefore plays an important role in host defense. Previous work has shown that some host defense proteins such as SLPI and elafin are susceptible to proteolytic degradation. Consequently, we investigated the status of SLPI in the cystic fibrosis (CF) lung. A major factor that contributes to the high mortality rate among CF patients is Downloaded from Pseudomonas aeruginosa infection. In this study, we report that P. aeruginosa-positive CF bronchoalveolar lavage fluid, which contains lower SLPI levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective at cleaving recombinant human SLPI. Additionally, we found that only NE inhibitors were able to prevent SLPI cleavage, thereby implicating NE in this process. NE in excess was found to cleave recombinant SLPI at two novel sites in ␬ the NH2-terminal region and abrogate its ability to bind LPS and NF- B consensus binding sites but not its ability to inhibit activity of the serine protease cathepsin G. In conclusion, this study provides evidence that SLPI is cleaved and inactivated by NE http://www.jimmunol.org/ present in P. aeruginosa-positive CF lung secretions and that P. aeruginosa infection contributes to inactivation of the host defense screen in the CF lung. The Journal of Immunology, 2009, 183: 8148–8156. ystic fibrosis (CF)3 is an autosomal recessive disease presence of large numbers of neutrophils and ensuing high con- caused by loss of expression/function mutations in the centrations of neutrophil proteases, particularly neutrophil elastase C cystic fibrosis transmembrane conductance regulator (NE), in the airways of CF patients that overwhelm the host’s (CFTR) protein. Lung disease causes 95% of the morbidity and antiprotease screen (1). During the past three decades it has be- mortality in CF patients and is associated with the failure of pul- come clear that a number of proteins involved in defending the by guest on September 29, 2021 monary innate immune functions leading to a vicious cycle of lung against proteases possess multiple, yet seemingly indepen- continual infection, inflammation, and remodeling of lung tissue dent, functions that under normal circumstances serve to protect (1). A major factor that contributes to this mortality rate is infec- the lung from infection and inflammation as well as protease-in- tion with Pseudomonas aeruginosa. Once chronic infection is es- duced degradation. tablished, it is virtually impossible to eradicate and is associated One such protein is human secretory leucoprotease inhibitor with reduced survival (2, 3). Another contributing factor is the (SLPI), a cationic 11.7 kDa serine protease inhibitor constitutively expressed at mucosal surfaces, primarily the upper respiratory tract. SLPI consists of 107 amino acids organized in two whey *Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, Northern Ireland; †Respiratory Re- acidic protein four disulfide core (WFDC) domains, each with four search Division, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, disulfide bridges (4, 5). SLPI is produced by a number of cell ‡ Ireland; Institut National de la Sante´et de la Recherche Me´dicale, Programme Ave- types, including neutrophils, macrophages, and epithelial cells, and nir/EA Inflammation and Immunity of the Respiratory Epithelium, Universite´de Re- ims Champagne-Ardenne, Institut Fe´de´ratif de Recherche 53, Reims, France; and expression can be altered by various stimuli, including LPS (6, 7), §Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National NE (8, 9), and both pro- and anti-inflammatory cytokines (10, 11). Institutes of Health, Bethesda, MD 20892 Overall, the evidence to date suggests that the function of SLPI is Received for publication May 29, 2009. Accepted for publication October 16, 2009. to protect local tissue from the detrimental consequences of in- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance flammation not only as a result of its well-documented antiprotease with 18 U.S.C. Section 1734 solely to indicate this fact. activities but also via its antimicrobial and anti-inflammatory prop- 1 This study was supported in part by funding from the Northern Ireland Chest Heart erties. SLPI can inhibit a variety of proteases released during in- and Stroke Association (CT 2008 107) and the American Alpha One Foundation. flammation such as elastase, cathepsin G (CatG), trypsin, chymo- 2 Address correspondence and reprint requests to Dr. Clifford C. Taggart, Centre for trypsin, chymase, and tryptase (12). Meanwhile, a number of Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland. studies have found that SLPI displays antimicrobial effects against E-mail: [email protected] pathogens such as Escherichia coli, P. aeruginosa, and Staphylo- 3 Abbreviations used in this paper: CF, cystic fibrosis; AAT, ␣-1-antitrypsin; ACT, coccus aureus, and it also inhibits the growth of fungi such as ␣-1-antichymotrypsin; BALF, bronchoalveolar lavage fluid; CatG, cathepsin G; Aspergillus fumigatus and Candida albicans (13–15). COPD, chronic obstructive pulmonary disease; MeOSuc-AAPM-pNA, N-(ethoxysuc- cinyl)-Alam-Ala-Pro-Met-paranitroanilide; MeOSuc-AAPV-CMK or CMK, N-(me- SLPI also possesses immunomodulatory activity both in vivo thoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone; MeOSuc-AAPV-pNA, N-(me- and in vitro. Administration of aerosolized SLPI to CF patients thoxysuccinyl)-Ala-Ala-Pro-Val-paranitroanilide; NE, neutrophil elastase; Pr3, proteinase 3; PsϪ, Pseudomonas-negative; Psϩ, Pseudomonas-positive; SLPI, secre- suppressed levels of both NE and IL-8 in the lung (16, 17). In tory leucoprotease inhibitor; WFDC, whey acidic protein four disulfide core. response to LPS, SLPI-deficient mice show increased mortality www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901716 The Journal of Immunology 8149 from endotoxin shock (18). Furthermore, in a mouse model of Table I. Clinical information for patients included in the studya acute lung injury, prior administration of SLPI decreased lung injury and down-regulated NF-␬B activation by preventing deg- Pseudomonas- Pseudomonas- radation of the NF-␬B inhibitor protein I␬B␤ (19, 20). These Negative Positive p Value findings have been confirmed in vitro and mechanisms of action n 15 11 continue to be elucidated. SLPI binds bacterial LPS extracel- Age (years) 7.75 (1.6) 14 (0.9) 0.0047 lularly, thereby down-regulating the uptake of LPS and subse- BMI (kg/m2) 16.7 (0.6) 17 (0.9) 0.7529 quent production of proinflammatory mediators (21–23). How- FEV1 (% predicted) 71.8 (7.8) 52 (8.7) 0.1080 Neutrophils/ml BALF 2.37 (1.5) 9.01 (1.9) 0.0114 ever, as a consequence of the internalization of SLPI into the cytoplasm and nucleus of cells such as monocytes, it appears a Values represent means (SEM). BMI indicates body mass index; FEV1, forced expiratory volume in 1 s. that SLPI also has intracellular sites of action (24). In the cy- toplasm, SLPI inhibited LPS- and lipoteichoic acid-induced NF-␬B activation in human monocytes, by preventing degrada- ␬ ␣ ␬ ␤