Three Wavelength Substrate System of Neutrophil Serine Proteinases

Neutrophil serine proteases, including elastase, proteinase 3, and cathepsin G, are closely related enzymes stored in similar amounts in azurophil granules and released at the same time from triggered neutrophils at inflammatory sites. We have synthesized new fluorescence resonance energy transfer (FRET) substrates with different fluorescence donor–acceptor pairs that allow all three proteases to be quantified at the same time and in the same reaction mixture. This was made possible because the fluorescence emission spectra of the fluorescence donors do not overlap and because the values of the specificity constants were in the same range. Thus, similar activities of proteases can be measured with the same sensitivity. In addition, these substrates contain an N-terminal 2-(2-(2-aminoethoxy)­ethoxy)­acetic acid (PEG) moiety that makes them cell permeable. Using the mixture of these selected substrates, we were able to detect the neutrophil serine protease (NSP) activity on the activated neutrophil membrane and in the neutrophil lysate in a single measurement. Also, using the substrate mixture, we were in a position to efficiently determine NSP activity in human serum of healthy individuals and patients with diagnosed Wegener disease or <i>microscopic polyangiitis</i>

CP and HNE activities, and CP/CPI balance in CF sputum.

<p>(+): <i>P. aeruginosa</i>-colonized CF sputum; (−): <i>P. aeruginosa</i>-negative CF sputum. Cathepsins B, H, K, L and S and HNE activities were quantified as reported in details in the experimental section. Data are shown as individual points and statistically significant P values are shown. The horizontal bars indicate medians. (A) Cathepsins B, L, S, K and H. (B) CP/CPI balance. CPI: expressed as inhibitory site (cystatin-like) equivalent. (C) Elastase activities: the horizontal bars indicate medians. The western blot analysis was performed using a rabbit anti-HNE antibody (representative samples are shown). ◂, unbound HNE; ★, bound HNE.</p

Maturation of human procathepsins in CF sputum.

<p>(A) Autocatalytic maturation of procathepsins B and S. Supernatants were incubated for up to 6 h in 100 mM sodium acetate buffer pH 4.3, 10 µg/ml dextran sulfate, 4 mM DTT, at 37°C. The maturation products were separated by 12% SDS/PAGE under reducing conditions, transferred to a nitrocellulose membrane and analyzed by western blotting using polyclonal antibodies specific for (a) human cathepsin B and (b) human cathepsin S. One representative sample is shown. ★, proforms; ◂, mature double-chain cathepsin B; ←, mature cathepsin S. (B) Elastase-dependent maturation of procathepsin B. Supernatants were incubated for up to 4 h in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% NP40 at 37°C and the maturation products analyzed by immunoblotting using a polyclonal anti-cathepsin B antibody. Pefabloc was used as control (4 h). Pef, Pefabloc (AEBSF). (+): <i>P. aeruginosa</i>-positive CF sputum (one representative sample); (−): <i>P. aeruginosa</i>-negative CF sputum (one representative sample). ◃, single-chain cathepsin B; ◂, double-chain cathepsin B; ★, procathepsin B.</p

Cysteine cathepsins and their inhibitors in supernatants of CF sputum.

<p>Only representative samples are shown. (A) Proteins (30 µg/well) were separated by 15% SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, and analyzed with polyclonal antibodies against human cathepsins B, H, L and S. (+): <i>Pseudomonas aeruginosa</i>-colonized CF sputum; (−): <i>Pseudomonas aeruginosa</i>-negative CF sputum.◃, single-chain cathepsin B; ◂, double-chain cathepsin B; ←, mature cathepsins S and H; ★, proforms. (B) Immunostaining with polyclonal anti-cystatin C antibody and anti-kininogen antibody. (+): <i>Pseudomonas aeruginosa</i>-colonized CF sputum; (−): <i>Pseudomonas aeruginosa</i>-negative CF sputum. Control: Cyst, Cystatin C; HK, HMWK. Recombinant human cystatin C (R&D systems) has an additional C-terminal 10 His-tag and an apparent molecular mass of 17 kDa, according to the supplier. (C) Supernatants of CF sputum incubated with Biot-LVG-CHN₂ (30 µM), for 1 h at 37°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025577#pone.0025577-Florent1" target="_blank">[25]</a>. Other samples were pre-incubated with E-64, CA-074, and Mu-Leu-Hph-VSPh prior to adding the biotinylated activity-based probe. Samples were separated by 12% SDS-PAGE, electroblotted and incubated with extravidin-peroxidase conjugate. The peroxidase activity was revealed by chemiluminescence. WB: individual cathepsins B, H, L and S immunoblotted as control. Control: (−), no pre-incubation with E-64; (+), pre-incubation with E-64 prior to adding Biot-LVG-CHN2. Sputum: E-64, pre-incubation with E-64; CA, pre-incubation with CA-074; VS, pre-incubation with Mu-Leu-HphVSPh.</p

Degradation of human HMWK by CF sputum.

<p>Exogenous HMWK was incubated in the activity buffer with supernatants of CF sputum at 30°C for 0–5 hours. Hydrolysis products were separated by 12.5% SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with rabbit polyclonal anti-kininogen antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025577#pone.0025577-Lalmanach3" target="_blank">[24]</a>. Lane 1, 0-h incubation; lane 2, 2-h incubation; lane 3, 5-h incubation; lane 4, 5-h incubation in the presence of CA-074; lane 5, 5-h incubation in the presence of E-64. For clarity, one representative sample is shown.</p

Clinical follow-up, blood biochemistry and C-reactive protein in pigs after <i>P</i>. <i>aeruginosa</i> infection.

<p>Clinical follow-up, blood biochemistry and C-reactive protein in pigs after <i>P</i>. <i>aeruginosa</i> infection.</p

Cytokine concentrations in the BAL and serum of pigs after <i>P</i>. <i>aeruginosa</i> infection.

<p>A. IL-6, IL-8 and TNF-α in BAL fluid. B. IL-6, IL-8 and TNF-α in serum. Data were analysed by two-way ANOVA followed by Bonferroni's <i>post hoc</i> test. Data are means ± S.E.M. * indicates p<0.05. *** indicates p<0.001.</p

Anatomy of the pig lung.

<p>Diagram from C.L. Pavaux [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168577#pone.0168577.ref026" target="_blank">26</a>]. The black circles correspond to areas where samples were taken on each pig (cranial left lung lobe, caudal left lung lobe, cranial right lung lobe, middle right lung lobe, accessory right lung lobe and caudal right lung lobe), trachea (d: bronchial crossroads) and tracheal lymph nodes.</p

Histological evaluation of the <i>P</i>. <i>aeruginosa-</i>induced inflammatory response in the lungs of control and infected pigs.

<p>Histological evaluation of the <i>P</i>. <i>aeruginosa-</i>induced inflammatory response in the lungs of control and infected pigs.</p

Neutrophil serine proteases in BAL fluids of pigs infected with <i>P</i>. <i>aeruginosa</i> and purified blood neutrophils.

<p>Confocal microscopy showing DNA (blue) and elastase (NE; green). Arrows show NET filaments.</p