Decreased levels of secretory leucoprotease inhibitor in the Pseudomonas-infected cystic fibrosis lung are due to neutrophil elastase degradation

Abstract

Secretory leucoprotease inhibitor (SLPI) is a neutrophil serine protease inhibitor constitutively expressed at many mucosal surfaces, including that of the lung. Originally identified as a serine protease inhibitor, it is now evident that SLPI also has antimicrobial 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 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 NH(2)-terminal region and abrogate its ability to bind LPS and NF-kappaB 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 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.

Figure 1. SLPI levels and integrity in CF bronchoalveolar lavage fluid

(A) SLPI levels (nM) in Pseudomonas infected (Ps+, n=11) and non-infected (Ps−, n=15) CF BALF fluid were quantified by ELISA. (B) The integrity of SLPI in individual Ps− and Ps+ BALF fluid samples was compared by Western blotting. 20 μl of each patient sample was electrophoresed on 15% SDS-PAGE, transferred onto nitrocellulose and probed for SLPI using a biotinylated anti-SLPI antibody. FL SLPI = full length SLPI, C SLPI = cleaved SLPI.

Figure 2. Effects of Pseudomonas-infected and non-infected BALF fluid on the integrity of recombinant human SLPI

SLPI (0.166 μM) was incubated with Pseudomonas-infected (Ps+) or Pseudomonas non-infected (Ps−) BALF fluid at 37°C and investigated for cleavage 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 = full length SLPI, C SLPI = cleaved SLPI.

Figure 3. Effects of various protease inhibitors and pH on Ps+ BALF-induced cleavage of SLPI

SLPI (0.166 μM) was added to Ps+ CF BALF fluid in buffer at pH 7.5 (A) or pH 5.5 (B) that had been pre-incubated for 1 h with various proteases inhibitors (PMSF, EDTA and E64) and analysed for degradation by Western blot for SLPI after 24 h at 37°C. FL SLPI = full length SLPI, C SLPI = cleaved SLPI.

Figure 4. Effects of serine proteases and serine protease inhibitors on Ps+ BALF-induced cleavage of SLPI

(A) SLPI (0.42 μM) was added to Ps+ CF BALF fluid that had been pre-incubated for 1 h with various serine proteases inhibitors (ACT, elafin and MeOSuc-AAPV-CMK (CMK)) and analysed for degradation by Western blot after 1 h at 37°C. (B) SLPI (0.42 μM) was incubated with an increasing concentration of purified neutrophil serine proteases (neutrophil elastase, cathepsin G and proteinase 3) for 2 h at 37°C in TBS. Samples were electrophoresed on a 15% SDS-PAGE and probed for SLPI by Western blotting. FL SLPI = full length SLPI, C SLPI = cleaved SLPI.

Figure 5. Free neutrophil elastase activity in Pseudomonas negative and positive CF BALF

P. aeruginosa infected (Ps+) and non-infected (Ps−) CF BALF samples diluted in Hepes buffer were pre-incubated alone or with the elastase inhibitor MeOSuc-AAPV-CMK (CMK) and mixed with the chromogenic substrate MeoSuc-AAPV-pNA. The absorbance of samples was measured at 405 nm over time at 37°C. The activity of neutrophil elastase (NE) in samples was assessed by comparing the NE activity in Pseudomonas infected and non-infected BALF with that of a standard curve of purified NE. Each measurement was performed in duplicate.

Figure 6. HPLC analysis of SLPI incubated with neutrophil elastase

(A) Human neutrophil elastase (8 μM) was incubated with SLPI (2 μM) in 0.1 M Hepes/0.5 M NaCl pH 7.5 for 2 h at 37°C. The samples were neutralised 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 3 peaks (1, 2 and 3) were obtained corresponding to various SLPI products obtained from the incubation. Peaks were analysed 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 representation 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 disulphide 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 Met 73. The arrows represents the cleavage sites (Ser15-Ala16 and Ala16-Glu17) generated by excess neutrophil elastase for 2 h.

Figure 7. Effects of neutrophil elastase on SLPI’s functional activities

(A) Recombinant SLPI (0.42 μM) was incubated in the presence or absence of an excess of neutrophil elastase (1.68 μM) in 0.1 M Hepes/0.5 M NaCl pH 7.5 for 1 h at 37°C. SLPI and NE alone were also incubated in buffer as controls. The reactions was neutralised by adding the elastase inhibitor MeOSuc-AAPV-CMK. Samples were analysed for P. aeruginosa LPS binding ability by ELISA. The absorbance at 405 nm reflects the binding of SLPI to P. aeruginosa LPS. (B) Recombinant SLPI (4.2 μM) was incubated in the presence or absence of an excess of neutrophil elastase (16.6 μM) in 0.1 M Hepes/0.5 M NaCl pH 7.5 for 1 h at 37°C. The reaction was neutralised by adding the elastase inhibitor MeOSuc-AAPV-CMK. Binding of SLPI to DNA was examined by EMSA. Samples were incubated with biotinylated NF-κB consensus oligonucleotide, electrophoresed on a 15% polyacrylamide gel, and transferred onto nitrocellulose membrane. SLPI bound to biotinylated oligonucleotide was visualised by incubating the blot with streptavidin-HRP and detected using chemiluminescence.

Figure 8. NE-cleaved SLPI retains its ability to inhibit activity of the serine protease cathepsin G

NE-cleaved SLPI (C SLPI) was inactivated with 1mM MeOSuc-AAPV-CMK and incubated with cathepsin G to investigate the antiprotease activity of C SLPI. Cathepsin G activity was determined using the MeOSuc-AAPM-pNA substrate and readings taken over a 15 min period at 405nm. NE-cleaved SLPI (0.666 μM) was incubated with cathepsin G substrate to demonstrate that NE present in samples was not involved in the turnover of cathepsin G substrate.