Decreased Levels of Secretory Leucoprotease Inhibitor in the Pseudomonas-Infected Lung Are Due to 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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 . 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) . 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), , chymo- 2 Address correspondence and reprint requests to Dr. Clifford C. Taggart, Centre for trypsin, , 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- ␬ ␣ ␬ ␤ from BioLegend. SuperSignal West Femto maximum sensitivity substrate tion of key regulatory proteins such as I B ,I B , and IL-1 was purchased from Pierce. All other reagents were of analytical grade. receptor activated kinase (IRAK) (25, 26). Additionally, previ- ous findings from our group have shown that SLPI also exerts CF BALF processing ␬ effects on the NF- B signaling cascade in the nucleus of mono- We studied a total of 26 patients diagnosed with CF (F508del), 15 of whom cytes, where SLPI can compete with p65 for binding to the had P. aeruginosa infection (Table I). CF BALF samples were obtained

NF-␬B binding sites in the promoter regions of proinflamma- from individuals undergoing flexible bronchoscopy for clinical reasons. Downloaded from tory such as IL-8 and TNF-␣ (24). Ethical approval for the use of these samples was obtained from the Insti- tutional Review Board of the Adelaide and Meath Hospital incorporating Although SLPI can inhibit a variety of proteases released during the National Children’s Hospital. Bronchoscopy was performed via a la- inflammation, it appears that SLPI levels and activity may be com- ryngeal mask airway. The bronchoscope was directed to the lingula and promised in numerous disease states where the body is over- right middle lobe. Bronchoalveolar lavage was performed by instilling 1 whelmed by infection and concomitant proteases. Evidence of ml/kg of sterile normal saline per lobe. Return was typically in the region of 40%. Specimens from right and left lobes were pooled. BALF was SLPI cleavage has been reported in individuals with frequent http://www.jimmunol.org/ centrifuged at 4000 rpm for 10 min and the supernatant was aliquoted and chronic obstructive pulmonary disease (COPD) exacerbations stored at Ϫ80°C. Bacteria were cultured from the BALF sample on the day (27), emphysema (28), and CF (29). It has become clear that SLPI of the procedure. and other important host defense proteins are susceptible to pro- teolytic cleavage by a range of proteases of both endogenous and Determination of SLPI levels in Pseudomonas-positive and bacterial origin. We have previously shown that cysteine proteases -negative CF BALF called cathepsins cleave and inactivate SLPI and human ␤-de- SLPI levels were determined by ELISA. Rabbit anti-human SLPI (1/1000 fensins in epithelial lining fluid from individuals with emphysema in Voller’s buffer) was added to Maxisorp 96-well plates (Nunc) and left (28) and CF (30), respectively, while cathepsin-mediated degra- overnight at 4°C. The plate was washed three times with PBS/0.05% (v/v)

Tween 20 (PBST) and blocked in PBST containing 1% BSA for1hat by guest on September 29, 2021 dation of lactoferrin was observed in P. aeruginosa-infected CF room temperature. After washing three times in PBST, SLPI standards (26 sputum and bronchoalveolar lavage fluid (BALF) (31). Addition- pM to 1.66 nM) and CF BALF samples (100 ␮l per well of 1/100 diluted ally, exogenous elastolytic proteases of bacterial origin may play a sample) were added in 100-␮l aliquots to the plate for2hatroom tem- role in the cleavage and inactivation of host defense molecules. For perature. The plate was then washed and biotinylated anti-human SLPI Ab (1:1000 in PBST, R&D Systems) was added to the plate for2hatroom example, Pseudomonas elastase or pseudolysin has been shown to temperature. After washing, the plates were incubated with HRP-conju- cleave SLPI (32), and high concentrations of pseudolysin, Pseudo- gated streptavidin (1/2500 in PBST) for 20 min at room temperature and monas alkaline protease, and NE were also able to inactivate lac- washed with PBST. After the third wash, peroxidase activity was measured toferrin (33). by the addition of substrate (ABTS Single Solution; Zymed Laboratories). Recent findings from our laboratory have demonstrated that re- The absorbance at 405 nm of the wells was measured on a microtiter plate reader (Genios using Magellan software) and results were converted to combinant human elafin (another well-characterized WFDC-con- SLPI concentrations (nM) using GraphPad Prism version 5.1 (GraphPad taining protein found in the lung) is cleaved by NE in CF sputum Software). and that P. aeruginosa infection promotes this degradation (34). In the present study, we expand this research to investigate the hy- Western blot analysis of SLPI in CF BALF pothesis that the high levels of NE present in the P. aeruginosa- Individual P. aeruginosa-positive and -negative CF BALF (20 ␮l) samples infected CF lung correlate with low levels of SLPI and that NE is were separated by denaturing SDS-PAGE using 15% polyacrylamide gels responsible for cleaving and inactivating the multifunctional, host and blotted onto nitrocellulose membrane (Sigma-Aldrich). The membrane was blocked for1hatroom temperature with 3% BSA in PBS containing defense protein SLPI. Our findings show that Pseudomonas infec- 0.1% (v/v) Tween 20. SLPI was detected using a biotinylated anti-SLPI Ab tion increases NE levels, which subsequently cleave SLPI in the (1/500, overnight at 4°C with shaking; R&D Systems) followed by incu- CF lung and may have repercussions on the innate immune func- bation with peroxidase-conjugated streptavidin (1/2500, 20 min at room tions of this protein. temperature). Peroxidase activity was detected using the chemiluminescent SuperSignal West Femto maximum sensitivity substrate and analyzed us- ing the Syngene GeneSnap and GeneTools software on the ChemiDoc Materials and Methods system. Materials Western blot analysis of recombinant SLPI incubated with CF Recombinant human SLPI was obtained from Amgen. Biotinylated anti- BALF and human proteases human SLPI Ab was purchased from R&D Systems. N-(methoxysuccinyl)- Ala-Ala-Pro-Val-chloromethyl ketone (MeOSuc-AAPV-CMK or CMK) Recombinant SLPI (0.166 ␮M) was incubated with 10 ␮l CF BALF in 30 and EDTA were purchased from Sigma-Aldrich. ␣-1-Antichymotrypsin mM TBS (pH 7.5) in a final volume of 20 ␮lfor0,10min,1h,6h,or24h (ACT), N-(methoxysuccinyl)-Ala-Ala-Pro-Val-paranitroanilide (MeOSuc- at 37°C. To test effects of pH on SLPI degradation by CF BALF, some AAPV-pNA), N-(methoxysuccinyl)-Ala-Ala-Pro-Met-paranitroanilide incubations were conducted at pH 5.5 as depicted in the figure legends. In (MeOSuc-AAPM-pNA), CatG, and E64 were purchased from Merck Bio- some cases, CF BALF was preincubated for1hat37°C with the following sciences. Human NE was from Elastin Products. Human proteinase 3 (Pr3) protease inhibitors before adding SLPI: 1 mM PMSF, 13 mM EDTA, 0.4 was from Athens Research. HRP-conjugated streptavidin was obtained mM E64, 1 ␮M ACT, 1 ␮M elafin, or 0.1 mM MeOSuc-AAPV-CMK. 8150 ELASTASE INACTIVATES SLPI IN THE CF LUNG

Additionally, recombinant SLPI (0.42 ␮M) was incubated with various concentrations of NE, Pr3, or CatG (from 16 nM to 4.2 ␮M) for 24 h in TBS in a 20 ␮l volume at 37°C. All incubations were stopped by adding sample treatment buffer containing reducing agent and boiling for 5 min. Samples were separated by SDS-PAGE using a 15% polyacrylamide gel and blotted onto nitrocellulose membrane (Sigma-Aldrich). SLPI was de- tected as described above. Measurement of elastase activity in CF BALF P. aeruginosa-positive and -negative CF BALF (10 ␮l) were diluted in 0.1 M HEPES, 0.5 M NaCl (pH 7.5) containing 0.13 mM E64, 0.11 mM pepstatin A, and 5.4 mM EDTA and treated with or without NE inhibitor (1 mM MeOSuc-AAPV-CMK) for1hat37°C in a 100 ␮l volume. The chromogenic substrate, MeOSuc-AAPV-pNA (50 ␮l), was mixed with each sample to a final concentration of 1.4 mM and absorbance at 405 nm was measured over the time at 37°C in a 96-well microplate reader (Genios using Magellan software). The activity of NE in samples was determined by comparing the elastase activity (given by the rate of hydrolysis of the substrate) with a standard curve of purified NE. All measurements were performed in duplicate. FIGURE 1. SLPI levels and integrity in CF BALF. A, SLPI levels (nM) in Pseudomonas-infected (Psϩ, n ϭ 11) and noninfected (PsϪ, n ϭ 15) CF HPLC mass spectrometry BALF fluid were quantified by ELISA. B, The integrity of SLPI in indi- Ϫ ϩ Downloaded from Cleavage of SLPI by NE was assessed by incubating recombinant SLPI (2 vidual Ps and Ps BALF fluid samples was compared by Western blot- ␮M) with NE (8 ␮M)for1hin0.1MHEPES/0.5 M NaCl (pH 7.5) in a ting. Twenty microliters of each patient sample was electrophoresed on 40-␮l final volume at 37°C. Elastase activity was neutralized with 1 ␮lof 15% SDS-PAGE, transferred onto nitrocellulose, and probed for SLPI us- PMSF (100 mM) for 30 min at room temperature. Samples were lyophi- ing a biotinylated anti-SLPI Ab. FL SLPI indicates full-length SLPI; C lized until analysis, when they were redissolved in 10 ␮l of 6 M guanidine SLPI, cleaved SLPI. HCl, 100 mM Tris (pH 8.5), 1 mM EDTA. One microliter of 10% triflu- oroacetic acid was added to each sample to bring the pH to Ͻ3. Samples were then analyzed by reverse-phase HPLC coupled to electrospray mass http://www.jimmunol.org/ ␮ spectrometry as described (34). MeOSuc-AAPV-CMK. Samples approximating to 0.166 and 0.666 M SLPI were withdrawn from the mixture and incubated with CatG (0.333 LPS ELISA ␮M) in HEPES/NaCl buffer for 30 min. After this time, CatG substrate MeOSuc-AAPM-pNA (1 mM) was added to the samples and turnover of A Maxisorp plate (Nunc) was coated with 100 ng/well P. aeruginosa LPS substrate was evaluated at 405 nm over a 15 min period. Delta absorbance (P. aeruginosa serotype 10; Sigma-Aldrich) diluted in serum-free RPMI values were converted to the amount of paranitroaniline produced as an 1640 media (SFM) and incubated at 37°C for 3 h. Unbound LPS was indicator of CatG activity. Control reactions included NE-cleaved SLPI washed off the plate with distilled water and the plate was left to air dry (666 nM) incubated with CatG substrate alone and CatG (0.333 ␮M) alone. overnight at room temperature. The next day, the plate was blocked with 1% BSA in PBS for 2 h at room temperature. The plate was washed three times with PBS/0.05% (v/v) Tween. Samples (100 ␮l/well) were added to Results by guest on September 29, 2021 the plate (0.42 ␮M SLPI alone, 0.42 ␮M SLPI incubated with 1.68 ␮M Levels of SLPI in CF BALF NE) and diluted 1/2 in SFM across the plate followed by incubation for 2 h. SLPI levels in BALF from 11 CF patients with Pseudomonas in- Control wells received SFM alone. Again, the plate was washed three times before adding biotinylated anti-SLPI IgG (1/1000) and incubating for 2 h. fection and 15 CF patients not infected with Pseudomonas were After washing, streptavidin peroxidase (1/2500) was added to the plate for determined by ELISA. As shown in Fig. 1A, levels of SLPI were 20 min. After washing, ABTS single solution substrate (Zymed Laborato- found to be significantly lower in P. aeruginosa-positive CF BALF ries) was added and the plate was incubated at room temperature for 20 (Psϩ) compared with P. aeruginosa-negative BALF (PsϪ) (3.99 Ϯ min. The absorbance at 405 nm of wells was measured on a microtiter plate Ϫ 2.11 vs 15.62 Ϯ 5.74 nM, p Ͻ 0.05). Analysis of SLPI in Ps and reader (Genios using Magellan software) and results were analyzed using ϩ GraphPad Prism version 5.1 (GraphPad Software). Ps CF BALF by Western blot revealed distinctive banding pat- terns between the samples. As illustrated in Fig. 1B, in the absence EMSA of Pseudomonas infection, SLPI remains intact and a full-length EMSA was performed as described previously (24). Proteins were incu- protein of ϳ12 kDa is detected. However, in Psϩ BALF samples, bated with double-stranded biotinylated NF-␬B consensus oligonucleotide cleaved SLPI and lower levels of full-length SLPI were found, Ј Ј (5 -AGTTGAGGGGACTTTCCCAGGC-3 ; MWG Biotech) for 30 min at suggesting that lower SLPI levels found in Psϩ BALF are a con- room temperature in binding buffer (4% (v/v) glycerol, 1 mM EDTA, 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM DTT, and 0.1 mg/ml nu- sequence of degradation. clease-free BSA) and 2 ␮g of poly(dI-dC ⅐ dI-dC):poly(dI-dC ⅐ dI-dC) (Sigma-Aldrich). Reaction mixtures were electrophoresed on native 15% Effects of CF BALF incubation on recombinant SLPI integrity ϫ polyacrylamide gels. The gels were transferred to nitrocellulose in 1 TBE The effects of PsϪ and Psϩ CF BALF on the integrity of recom- for 30 min at 380 mA, 100 V, and then cross-linked on a UV transillumi- nator for 10 min. Detection of SLPI:DNA complexes was performed using binant SLPI were investigated to determine and compare their abil- Ϫ a chemiluminescent nucleic acid detection module (Pierce Chemical). In ity to cleave SLPI. Recombinant SLPI was incubated with Ps and brief, after crosslinking, the blot was incubated in blocking buffer for 1 h Psϩ CF BALF for various times at 37°C and analyzed by Western at 37°C. Streptavidin-peroxidase was added for 15 min in blocking buffer blot. As shown in Fig. 2, the levels of recombinant SLPI detected at room temperature, and the blot was then washed six times in wash buffer. ϩ Ϫ The blot was incubated for 5 min in equilibration buffer and developed with decreased in Ps CF BALF but not in Ps BALF over time. This the chemiluminescent reagents provided with the kit. SLPI:DNA binding degradation was visible but not complete after 1 h, with the pres- was analyzed using the Syngene GeneSnap and GeneTools software on the ence of an upper band displaying a similar size to intact SLPI and ChemiDoc system. a lower band corresponding to proteolytic fragments of SLPI. Evaluation of the antiprotease activity of NE-cleaved SLPI However, after 6 and 24 h proteolysis is almost complete, with no intact SLPI detectable in the samples; only the presence of a lower To evaluate the antiprotease activity of NE-cleaved SLPI, we tested the ability of cleaved SLPI to inhibit one of its target proteases, CatG. NE (13.3 band could be visualized. Recombinant SLPI was incubated sep- ␮M) was incubated with SLPI (3.33 ␮M) in HEPES/NaCl buffer for 6 h, arately in the absence of CF BALF under the same conditions and after which NE activity was inactivated with the addition of 1 mM no degradation was detected. SLPI incubated in the presence of The Journal of Immunology 8151

FIGURE 2. Effects of Pseudomonas-infected and noninfected BALF fluid on the integrity of recombinant human SLPI. SLPI (0.166 ␮M) was incubated with Psϩ or PsϪ BALF fluid at 37°C and investigated for cleav- age of SLPI over a 24 h time course by Western blotting. Recombinant SLPI was incubated separately under the same conditions as a positive control. FL SLPI indicates full-length SLPI; C SLPI, cleaved SLPI.

PsϪ CF BALF showed no obvious signs of cleavage. These find- ings show that the proteolytic activity directed against SLPI was higher in Psϩ than in PsϪ CF BALF. FIGURE 4. Effects of serine proteases and serine protease inhibitors on Identification of the protease family involved in the cleavage of Psϩ BALF-induced cleavage of SLPI. A, SLPI (0.42 ␮M) was added to SLPI in CF BALF Psϩ CF BALF fluid that had been preincubated for 1 h with various serine

To identify the protease family involved in the cleavage of SLPI, proteases inhibitors (ACT, elafin, and MeOSuc-AAPV-CMK (CMK)) and Downloaded from ϩ analyzed for degradation by Western blot after1hat37°C. B, SLPI (0.42 Ps CF BALF samples were preincubated with different nonspe- ␮M) was incubated with an increasing concentration of purified neutrophil cific protease inhibitors targeting the serine, cysteine, or metallo- serine proteases (NE, CatG, and Pr3) for2hat37°C in TBS. Samples were protease families before adding recombinant SLPI in buffer at pH electrophoresed on a 15% SDS-PAGE and probed for SLPI by Western 7.5. After 24 h of incubation at 37°C, samples were analyzed by blotting. FL SLPI indicates full-length SLPI; C SLPI, cleaved SLPI. Western blot for SLPI. As shown in Fig. 3A, PMSF, a nonspecific ϩ serine protease inhibitor, inhibits SLPI cleavage in Ps CF BALF, http://www.jimmunol.org/ whereas neither the cysteine protease inhibitor E64 nor the met- The ability of purified human neutrophil serine proteases to alloproteinase inhibitor EDTA had any effect. When this incuba- cleave SLPI was evaluated in vitro to clarify which protease is tion was repeated at a lower pH (5.5), SLPI degradation was still mediating the cleavage observed. Recombinant SLPI was incu- prevented by PMSF but not by EDTA or E64 (Fig. 3B). Overall, bated with purified proteases and analyzed by Western blot. A these findings show that nonspecific inhibition of serine proteases dose-response experiment was conducted for1hat37°C using an in Psϩ CF BALF prevented cleavage of SLPI. increasing concentration of protease. As shown in Fig. 4B, incu- bating recombinant SLPI with NE, Pr3, and CatG resulted in the Identification of the serine proteases involved in the cleavage of appearance of SLPI cleavage products in a dose-dependent man-

SLPI in CF BALF ner. However, when the cleavage patterns are compared with those by guest on September 29, 2021 ϩ Given the previous findings, we expanded the number of serine illustrated in Fig. 2 following SLPI/Ps CF BALF incubations, only NE and CatG produce a similar pattern of cleavage products protease inhibitors used to further classify the putative serine pro- ϩ tease that mediates the Psϩ CF BALF cleavage of SLPI. The in- to that found following incubation of SLPI with Ps CF BALF. hibitors chosen target various groups of serine proteases (NE, Pr3, When SLPI was incubated with Pr3, proteolysis is almost com- and CatG) with different efficacies. ACT inhibits CatG and chy- plete, with no intact SLPI detectable in the samples at the higher mase, elafin inhibits elastase and Pr3, and MeOSuc-AAPV-CMK doses used in contrast to the banding pattern found with NE and CatG. However, as shown in Fig. 4A, the CatG inhibitor ACT does specifically targets elastase. As shown in Fig. 4A, ACT did not ϩ prevent SLPI cleavage. In contrast, elafin and MeOSuc-AAPV- not inhibit Ps CF BALF-mediated cleavage of SLPI (Fig. 4A), ϩ indicating that NE is the serine protease involved in the cleavage CMK completely inhibited SLPI cleavage in Ps CF BALF, in- ϩ dicating that NE is most likely to be the serine protease responsible of SLPI by Ps CF BALF. Psϩ for SLPI cleavage in CF BALF. Measurement of NE activity in CF BALF Given that NE is responsible for the pattern of cleavage of SLPI in Psϩ CF BALF, and that the inhibitor is more rapidly degraded in Psϩ than in PsϪ CF BALF, NE activity was measured in both CF BALF samples using the chromogenic substrate MeOSuc-AAPV- pNA. Elastase activity in CF BALF was calculated using a stan- dard curve of purified NE to determine the concentration of free NE in samples. As shown in Fig. 5, the concentration of free NE is increased Ͼ9-fold in Psϩ compared with PsϪ CF BALF (18.76 vs 1.92 ␮M, Fig. 5). Additionally, free elastase activity in both samples is totally abrogated using the NE inhibitor MeOSuc- AAPV-CMK (Fig. 5), thus confirming the specificity of the mea- surements. These results reinforce our finding that NE is respon- sible for SLPI cleavage in Psϩ CF BALF. FIGURE 3. Effects of various protease inhibitors and pH on Psϩ BALF- ϩ induced cleavage of SLPI. SLPI (0.166 ␮M) was added to Ps CF BALF Analysis of NE-SLPI cleavage sites fluid in buffer at pH 7.5 (A)orpH5.5(B) that had been preincubated for 1 h with various proteases inhibitors (PMSF, EDTA, and E64) and ana- To further investigate the elastase-mediated cleavage of SLPI, lyzed for degradation by Western blot for SLPI after 24 h at 37°C. FL SLPI products from NE-SLPI incubations were analyzed by HPLC and indicates full-length SLPI; C SLPI, cleaved SLPI. mass spectrometry. HPLC separation of SLPI products generated 8152 ELASTASE INACTIVATES SLPI IN THE CF LUNG

The measured mass of peak 3 was 11,725 Da, identifying it as full-length SLPI residues Ser1-Ala107 (calculated mass of 11,725.93 Da). These results indicate that 2-fold excess of NE cleaved SLPI at the Ser15-Ala16 and Ala16-Glu17 peptide bonds. In contrast, such cleavages were not detected using conditions with a slight excess of SLPI ([]/[inhibitor] molar ratio of 1:2.5), even after 24 h of incubation (data not shown). Taken together, these findings indicate that NE-mediated cleavage of SLPI occurs only with excess of NE. The cleavage sites generated by NE within SLPI are summarized in Fig. 6B.

FIGURE 5. Free neutrophil elastase activity in Pseudomonas-negative Effects of excess NE on SLPI functions and -positive CF BALF. Psϩ and PsϪ CF BALF samples diluted in HEPES In addition to its antiprotease capabilities, SLPI has antibacterial buffer were preincubated alone or with the elastase inhibitor MeOSuc- and anti-inflammatory effects and can interact with the endotoxin AAPV-CMK (CMK) and mixed with the chromogenic substrate MeoSuc- LPS of Gram-negative bacteria such as E. coli and modulate mac- AAPV-pNA. The absorbance of samples was measured at 405 nm over rophage responses after LPS stimulation (21–23). We therefore time at 37°C. The activity of NE in samples was assessed by comparing the investigated the effect of NE-induced cleavage on the ability of NE activity in Pseudomonas-infected and noninfected BALF with that of a standard curve of purified NE. Each measurement was performed in SLPI to bind LPS. Recombinant SLPI was incubated alone or with Downloaded from duplicate. an excess of NE and examined for its capacity to bind to P. aerugi- nosa LPS by ELISA. As illustrated in Fig. 7A, intact SLPI was able to interact with P. aeruginosa LPS in a dose-dependent manner. In within2hbyanexcess of NE resulted in the formation of three contrast, little or no SLPI-LPS binding was detected when SLPI distinct peaks (Fig. 6A, peaks 1–3). Identification of SLPI frag- pretreated with NE or untreated NE were analyzed. Thus, these ments was conducted by analyzing peaks by mass spectrometry. results suggest that NE suppresses the ability of SLPI to bind to P. http://www.jimmunol.org/ The deconvoluted mass of peak 1 was 10,152 Da, identifying it as aeruginosa LPS. Similar findings were obtained when E. coli LPS SLPI residues Glu17-Ala107 (calculated mass of 10,152 Da). Like- was used (data not shown). SLPI can inhibit LPS-induced proin- wise, the measured mass for peak 2 was 10,223 Da, identifying it flammatory responses in monocytes and macrophages, and previ- as SLPI residues Ala16-Ala107 (calculated mass of 10,223.14 Da). ous work from our group has found that SLPI exerts inhibitory by guest on September 29, 2021

FIGURE 6. HPLC analysis of SLPI incubated with NE. A, Human NE (8 ␮M) was incubated with SLPI (2 ␮M) in 0.1 M HEPES/0.5 M NaCl (pH 7.5) for2hat37°C. The samples were neutralized with 5 mM PMSF, dried, and reconstituted in 6 M guanidine, 100 mM Tris (pH 8.5). The samples were then separated by HPLC and three peaks were obtained corresponding to various SLPI products obtained from the incubation. Peaks were analyzed by mass spectrometry. The deconvoluted mass of peak 1 was 10,152 Da, identifying it as SLPI residues Glu17-Ala107; the deconvoluted mass of peak 2 was 10,223 Da, identifying it as SLPI residues Ala16-Ala107; and peak 3 corresponded with full-length SLPI residues Ser1-Ala107 (11,725 Da). B, Schematic repre- sentation of neutrophil elastase cleavage sites in the amino acid sequence of SLPI (Ser1-Ala107). Both WFDC domains present in SLPI are underlined. The lines represent disulfide bridges linking paired cysteine residues (10–39, 18–43, 26–38, 32–47, 64–95, 71–97, 80–92, 86–101), and the asterisk identifies the scissile peptide bond between Leu72 and Met73. The arrows represents the cleavage sites (Ser15-Ala16 and Ala16-Glu17) generated by excess neutrophil elastase for 2 h. The Journal of Immunology 8153

FIGURE 8. NE-cleaved SLPI retains its ability to inhibit activity of the serine protease CatG. NE-cleaved SLPI (C SLPI) was inactivated with 1 mM MeOSuc-AAPV-CMK and incubated with CatG to investigate the antiprotease activity of C SLPI. CatG activity was determined using the MeOSuc-AAPM-pNA substrate and readings taken over a 15 min period at 405 nm. NE-cleaved SLPI (0.666 ␮M) was incubated with CatG substrate to demonstrate that NE present in samples was not involved in the turnover of CatG substrate. Downloaded from

NE-cleaved SLPI reduced CatG activity, and the 2-fold excess sample of cleaved SLPI almost completely abolished CatG activ- ity, similar to that observed with 2-fold excess of native full-length SLPI. Therefore, NE-cleaved SLPI retains its antiprotease activity

against its target serine protease CatG. http://www.jimmunol.org/

FIGURE 7. Effects of neutrophil elastase on the functional activities of Discussion SLPI. A, Recombinant SLPI (0.42 ␮M) was incubated in the presence or CF is characterized by chronic lung disease and recurrent bacterial absence of an excess of neutrophil elastase (1.68 ␮⌴) in 0.1 M HEPES/0.5 infection. Infection with P. aeruginosa is associated with increased M NaCl (pH 7.5) for1hat37°C. SLPI and NE alone were also incubated morbidity and mortality in patients with CF. Typically respiratory in buffer as controls. The reactions were neutralized by adding the elastase secretions are rich in multifunctional innate immunity proteins and inhibitor MeOSuc-AAPV-CMK. Samples were analyzed for P. aeruginosa peptides such as lactoferrin, LL-37, ␤-defensins, elafin, and SLPI. LPS binding ability by ELISA. The absorbance at 405 nm reflects the binding However, during lung inflammation it appears that a number of P. aeruginosa B ␮ of SLPI to LPS. , Recombinant SLPI (4.2 M) was incubated by guest on September 29, 2021 in the presence or absence of an excess of neutrophil elastase (16.6 ␮M) in 0.1 these host defense components are sensitive to exacerbated pro- M HEPES/0.5 M NaCl (pH 7.5) for1hat37°C. The reaction was neutralized teolytic activity emanating from dysregulated elastolytic by adding the elastase inhibitor MeOSuc-AAPV-CMK. Binding of SLPI to as found in chronic lung diseases such as CF (27, 28, 30, 31, DNA was examined by EMSA. Samples were incubated with biotinylated 33–35). It is important that the cause of this proteolytic activity is NF-␬B consensus oligonucleotide, electrophoresed on a 15% polyacrylamide elucidated to facilitate the development of more efficient and ro- gel, and transferred onto nitrocellulose membrane. SLPI bound to biotinylated bust therapies and to enable our understanding of how CF oligonucleotide was visualized by incubating the blot with streptavidin-HRP progresses. In the upper respiratory tract SLPI is thought to be and detected using chemiluminescence. primarily involved in protecting against human NE-induced dam- age, as it accounts for ϳ90% of the total molar concentration of elastase inhibitors in human bronchial secretions (36) and may in effects on nuclear events of the LPS-induced NF-␬B signaling cas- fact be up-regulated during times of inflammation. Nevertheless, cade by competing with NF-␬B p65 for binding to the NF-␬B there is a growing wealth of evidence purporting that despite its binding sites in the promoter region of genes such as IL-8 and putative proteolytically stable structure due to the number of di- TNF-␣ (24). Therefore, we investigated whether NE-cleaved SLPI sulfide bridges present, SLPI is in fact prone to cleavage by pro- was able to bind NF-␬B consensus oligonucleotides. The results teases of endogenous and bacterial origin. show that while intact SLPI binds biotinylated NF-␬B oligonucle- In this study, we demonstrate the potent ability of P. aerugi- otide, SLPI incubated with an excess of NE or NE alone does not nosa-infected CF bronchial secretions to cleave exogenous recom- bind to the oligonucleotides (Fig. 7B). Overall, these findings sug- binant human SLPI in an NE-dependent manner. In contrast, CF gest that NE abrogates a number of mechanisms by which SLPI BALF from patients not infected with P. aeruginosa was not able exerts its anti-inflammatory functions. to cleave SLPI. In vivo, SLPI concentrations have been found to be higher in upper respiratory airways than in lower airways, with Evaluation of the antiprotease activity of NE-cleaved SLPI reported levels of 0.1–0.2 ␮g/ml (8.33–16.66 nM) in BALF (29) To determine the effect of NE cleavage on the antiprotease activity and between 0.1 and 24 ␮g/ml (8.33 nM to 2 ␮M) in saliva (12, 37, of SLPI, we evaluated the ability of NE-cleaved SLPI to inhibit 38). We have shown that SLPI levels in noninfected CF airways CatG using the CatG-specific substrate MeOSuc-AAPM-pNA. As approximate to the values described previously for BALF (29). before, SLPI was incubated with excess NE and cleavage of SLPI However, when chronic infection has taken hold, a significantly was confirmed by Western blot (data not shown). Samples were lower concentration of SLPI was detected in Pseudomonas-in- withdrawn from the mixture and incubated with a fixed amount of fected BALF (3.99 Ϯ 2.11 nM). CatG (0.333 ␮M): a subinhibitory sample (equivalent of 0.166 ␮M A possible cause of this reduction in SLPI levels was identified cleaved SLPI) and a 2-fold excess sample (equivalent of 0.666 ␮M following Western blotting analysis of BALF samples from PsϪ cleaved SLPI). As illustrated in Fig. 8, the subinhibitory sample of and Psϩ CF patients, which revealed the presence of cleaved SLPI 8154 ELASTASE INACTIVATES SLPI IN THE CF LUNG

fragments of ϳ10 kDa in size in BALF from Psϩ patients. Al- Further analysis of the NE-induced cleavage of SLPI revealed though most PsϪ BALF samples displayed no evidence of SLPI the presence of two cleavage sites at Ser15-Ala16 and Ala16-Glu17

cleavage, we detected the presence of cleavage products in two in the NH2 terminus WFDC domain of the protein. Identification samples, suggesting that other proteases are present in these BALF of Ser15-Ala16 as a cleavage site is in agreement with previous samples that are capable of cleaving SLPI. The identity of this work by Masuda et al. (41), who reported the ability of excess NE protease activity is unknown; however, we speculate that cathep- to cleave SLPI at at least two sites, Ser15-Ala16 and Thr57-Arg58. sins may play a role given our previous demonstration of the abil- These cleavage sites are in contrast to those we previously found ity of cathepsins to cleave SLPI (28). Overall, the findings suggest for cathepsin-mediated cleavage of SLPI, which occurred at Thr67- that the decrease in BALF SLPI levels as evidenced by ELISA is Tyr68 (28). We have previously demonstrated that cathepsin L in a consequence of degradation by factors present in Psϩ but not COPD BALF can cleave and inactivate SLPI; however, there is PsϪ BALF. We further investigated this phenomenon by compar- little or no NE activity in COPD BALF (28). In contrast, NE and ing the effects of CF BALF from P. aeruginosa-infected and non- cysteinyl cathepsin activity are both present in CF BALF (30). infected patients on the integrity of recombinant human SLPI. Nevertheless, we postulate that SLPI has a much greater affinity Previous work by Vogelmeier et al. (29) demonstrated the pres- for NE than cathepsin L, so in the Pseudomonas-infected lung, ence of cleaved SLPI in BALF from patients with CF, but no SLPI most likely binds preferentially to NE, thus masking the 67 68 details of the status of Pseudomonas infection were provided in Thr -Tyr bond from cleavage by cathepsins. However, under the study. Our findings expand this observation, as we show that these circumstances, excess NE can cleave SLPI at the NH2 ter- only Pseudomonas-infected BALF is capable of degrading minus, as we have shown in this report. SLPI. As Pseudomonas elastase (pseudolysin) has been re- The implications of NE-induced cleavage of SLPI in vivo be- Downloaded from ported to cleave SLPI (32), we examined the involvement of came evident from a number of functional studies in which we this bacterial protease by pretreating P. aeruginosa-infected tested the ability of NE-cleaved SLPI to exert well-known anti- ␬ BALF with EDTA, which inhibits Pseudomonas elastase activ- inflammatory functions such as binding to LPS and NF- B con- ity. Pretreatment with EDTA was unable to inhibit P. aerugi- sensus binding sites. Ding et al. demonstrated that recombinant nosa-positive BALF-mediated cleavage of SLPI. However, we human SLPI binds to E. coli LPS and prevents the formation of found that P. aeruginosa-positive BALF cleavage of SLPI was LPS-CD14 complexes and slows the transfer of LPS from the http://www.jimmunol.org/ complexes to macrophages (23). Following incubation with NE, inhibited by the addition of human NE inhibitors (PMSF, elafin, the ability of SLPI to bind P. aeruginosa and E. coli LPS was and MeOSuc-AAPV-CMK), thus implicating human NE in the dramatically reduced, which may contribute to enhanced cellular process. When recombinant human SLPI was incubated with an responsiveness to LPS. Additionally, we demonstrated that NE- excess of purified NE (molar ratio of [enzyme]/[inhibitor] of cleaved SLPI is no longer capable of binding NF-␬B consensus 2:1), we observed a similar pattern of cleavage products to that binding sites and is therefore unable to prevent NF-␬B p65 found with P. aeruginosa-positive BALF. binding and initiating proinflammatory cytokine expression as Although CatG incubation with SLPI also gave a similar pattern shown previously (24). Given that NE was found to cleave SLPI

of cleavage products to that obtained with NE and P. aeruginosa- by guest on September 29, 2021 at two sites in the NH -terminal, these findings suggest that this positive BALF, ACT (which inhibits CatG and chymase) was un- 2 region of the SLPI protein plays a vital role in at least some of able to prevent this cleavage. Consequently, we concluded that the anti-inflammatory functions of SLPI. Previous work has neither CatG nor chymase mediated the cleavage we observed. highlighted the importance of the NH2-terminal of SLPI in me- Belkowski et al. (39) have reported the ability of chymase to diating its antimicrobial activities (14, 43). Consequently, it is cleave exogenous recombinant human SLPI at residues Leu72- 73 reasonable to hypothesize that in addition to inactivating im- Met , the reactive site bond present in the second WFDC domain portant anti-inflammatory effects of SLPI, NE is also capable of at the COOH-terminal of the protein. However, although chymase dampening the antibacterial ability of SLPI. It is interesting to is known to be a major protease involved in and allergic note, however, that although NE can inactivate a number of responses in the airway, the role of such mast cell proteases in CF SLPI’s anti-inflammatory mechanisms of action, it fails to sig- has yet to be fully elucidated (40). Also, in contrast to our findings, nificantly alter the ability of SLPI to inhibit one of its target Belkowski et al. (39) state that neither CatG nor Pr3 cleaved re- serine proteases, CatG. The preservation of antiprotease prop- combinant human SLPI, but a minor digestion product was re- erties of SLPI fragments may be a result of its structure. The ported following elastase incubation. A possible reason for this antiprotease active site of SLPI is located in a loop (residues discrepancy is the molar ratios of [enzyme]/[inhibitor] used. Sim- 67–74) in the COOH-terminal WFDC domain. It has been re- ilar to Masuda et al. (41), we found that incubation of an equimolar ported that the recombinant COOH-terminal domain of SLPI ratio of [SLPI]/[NE] resulted in little or no cleavage of SLPI. How- retains its elastase inhibitory activity (41, 44). As NE cleaves ever, once the concentration of NE was increased to a 2:1 molar SLPI at the NH2 terminus, we would expect the resulting frag- ratio of [NE]/[SLPI], significant cleavage of SLPI was observed. ment to retain its antiprotease activity as confirmed by our find- Furthermore, in agreement with previous findings (42), incubation ings above. of SLPI with purified human Pr3 also cleaved SLPI. However, The susceptibility of SLPI to proteolytic degradation is a major Western blot analysis revealed that no lower molecular mass hindrance in treating diseases characterized by a protease burden cleaved SLPI fragments could be visualized, in contrast to the such as CF. Given the vicious cycle of infection, inflammation, and cleavage pattern found when SLPI was incubated with NE and proteolytic degradation that exists in CF, and the multifunctional- ϩ CatG or Ps CF BALF, where the presence of a lower molecular ity of SLPI, this protein is an obvious choice as a therapeutic for mass band was detected. Additionally, we have shown that NE such a disease, but findings to date reveal only limited success. ϩ activity is significantly elevated in Ps CF BALF compared with Intravenous administration of recombinant SLPI to animals re- PsϪ CF BALF and this activity is completely inhibited by the sulted in rapid renal clearance hindering their use as i.v. agents MeOSuc-AAPV-CMK, which is specific for NE, once again con- (45). As an alternative, aerosolized recombinant versions have firming that excess NE levels in Psϩ CF BALF are responsible for been evaluated and were found to be effective both in terms of cleaving SLPI. delivery and in mediating therapeutic effects (16). McElvaney The Journal of Immunology 8155 et al. (16) demonstrated suppression of pulmonary NE and IL-8 12. Doumas, S., A. Kolokotronis, and P. Stefanopoulos. 2005. Anti-inflammatory and levels following administration of aerosolized SLPI to CF patients. antimicrobial roles of secretory leukocyte protease inhibitor. Infect. Immun. 73: 1271–1274. However, the use of SLPI in a clinical setting has been limited by 13. Wiedow, O., J. Harder, J. Bartels, V. Streit, and E. Christophers. 1998. Antileu- enzymatic cleavage and failure to deposit efficiently in poorly ven- koprotease in human skin: an antibiotic peptide constitutively produced by ker- atinocytes. Biochem. Biophys. Res. Commun. 248: 904–909. tilated, highly inflamed areas of the lung (46) and, as such, clinical 14. Hiemstra, P. S., R. J. Maassen, J. Stolk, R. Heinzel-Wieland, G. J. Steffens, and studies have yet to progress to the next stage. Recent work by J. H. Dijkman. 1996. Antibacterial activity of antileukoprotease. Infect. Immun. Gibbons et al. (47) may present an effective alternative to remedy 64: 4520–4524. 15. Tomee, J. F., P. S. Hiemstra, R. Heinzel-Wieland, and H. F. Kauffman. 1997. some of these obstacles. By encapsulating recombinant human Antileukoprotease: an endogenous protein in the innate mucosal defense against SLPI in biocompatible liposomes, the stability of SLPI was im- fungi. J. Infect. Dis. 176: 740–747. proved in response to cysteine protease exposure; however, 16. McElvaney, N. G., H. Nakamura, P. Birrer, C. A. He´bert, W. L. Wong, M. Alphonso, J. B. Baker, M. A. Catalano, and R. G. Crystal. 1992. Modulation whether encapsulation protects from NE-induced degradation re- of airway inflammation in cystic fibrosis: in vivo suppression of interleukin-8 mains to be determined. levels on the respiratory epithelial surface by aerosolization of recombinant se- Overall, these novel findings broaden our understanding of the cretory leukoprotease inhibitor. J. Clin. Invest. 90: 1296–1301. 17. Vogelmeier, C., A. Gillissen, and R. Buhl. 1996. Use of secretory leukoprotease destruction caused by P. aeruginosa infection and how it contrib- inhibitor to augment lung antineutrophil elastase activity. Chest 110: 261S–266S. utes to chronic damage seen in the CF lung. Despite aggressive 18. Nakamura, A., Y. Mori, K. Hagiwara, T. Suzuki, T. Sakakibara, T. Kikuchi, T. Igarashi, M. Ebina, T. Abe, J. Miyazaki, T. Takai, and T. Nukiwa. 2003. antibiotic treatments, pulmonary infection with P. aeruginosa re- Increased susceptibility to LPS-induced endotoxin shock in secretory leukopro- mains a leading cause of morbidity and mortality in CF patients. tease inhibitor (SLPI)-deficient mice. J. Exp. Med. 197: 669–674. Pseudomonas infection is associated with higher elastase concen- 19. Lentsch, A. B., J. A. Jordan, B. J. Czermak, K. M. Diehl, E. M. Younkin, V. Sarma, and P. A. Ward. 1999. Inhibition of NF-␬B activation and augmen- trations, and proteases, particularly NE, contribute to the pathology tation of I␬B␤ by secretory leukocyte protease inhibitor during lung inflamma- Downloaded from of CF by impairing mucociliary clearance, interfering with innate tion. Am. J. Pathol. 154: 239–247. immune functions, and perpetuating neutrophilic inflammation (1, 20. Mulligan, M. S., A. B. Lentsch, M. Huber-Lang, R. F. Guo, V. Sarma, C. D. Wright, T. R. Ulich, and P. A. Ward. 2000. Anti-inflammatory effects of 48, 49). Our results confirm and expand previous work demon- mutant forms of secretory leukocyte protease inhibitor. Am. J. Pathol. 156: strating that excessive NE activity in the Pseudomonas-infected 1033–1039. CF lung can cause the degradation of elafin, another serine anti- 21. McMichael, J. W., A. Roghanian, L. Jiang, R. Ramage, and J. M. Sallenave. 2005. The antimicrobial antiproteinase elafin binds to lipopolysaccharide and protease produced locally in the lung (34). In vivo, SLPI appears modulates macrophage responses. Am. J. Respir. Cell. Mol. Biol. 32: 443–452. http://www.jimmunol.org/ to be constitutively expressed and may provide a baseline host 22. Yang, J., J. Zhu, D. Sun, and A. Ding. 2005. Suppression of macrophage re- sponses to bacterial lipopolysaccharide (LPS) by secretory leukocyte protease defense shield that can be up-regulated at times when inflamma- inhibitor (SLPI) is independent of its anti-protease function. Biochim. Biophys. tion or infection are anticipated (50). However, given its suscep- Acta 1745: 310–317. tibility to proteolytic degradation by not only cysteinyl proteases 23. Ding, A., N. Thieblemont, J. Zhu, F. Jin, J. Zhang, and S. Wright. 1999. Secretory leukocyte protease inhibitor interferes with uptake of lipopolysaccharide by mac- like cathepsins, but also the serine protease NE, the levels and rophages. Infect. Immun. 67: 4485–4489. biological activities may be compromised in vivo. 24. Taggart, C. C., S. A. Cryan, S. Weldon, A. Gibbons, C. M. Greene, E. Kelly, T. B. Low, S. J. O’Neill, and N. G. McElvaney. 2005. Secretory leucoprotease inhibitor binds to NF-␬B binding sites in monocytes and inhibits p65 binding. Disclosures J. Exp. Med. 202: 1659–1668. 25. Taggart, C. C., C. M. Greene, N. G. McElvaney, and S. O’Neill. 2002. 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