The blockade of ERK1/2 MAPK activation inhibited Ac-PGP production.

<p>Human peripheral blood PMNs (5.25×10⁵) were pretreated with U0126 (10 and 20 µM) versus vehicle for 30 minutes and then incubated for 30 minutes with labeled Ac-PGP. The supernatants collected from incubated cells were placed on type I collagen (1.0 mg/ml) for 24 hours at 37°C. Ac-PGP and C¹³N¹⁵ labeled Ac-PGP were analyzing by <i>ESI-LC-MS/MS</i>. Ac-PGP values of the samples on PBS were subtracted from Ac-PGP values of samples incubated on type I collagen to determine Ac-PGP production. <b>A</b>. Detection of Ac-PGP and C¹³N¹⁵ Ac-PGP via mass spectrometry. <b>B</b>. The detection of gelatinolytic activity in culture supernatants from human PMNs stimulated with labeled Ac-PGP by g<i>elatin zymography</i> representative of three gels. <b>C</b>. The measurement of Ac-PGP production by <i>ESI-LC-MS/MS.</i> The bar graph represents the percent of relative Ac-PGP production normalized to labeled Ac-PGP control. *p<0.05 compared to labeled Ac-PGP without inhibitor pretreatment, n = 4 wells/condition.</p

Clinical Data of CF Subjects.

<p>Clinical Data of CF Subjects.</p

The effect of ERK1/2 MAPK inhibitor on the Ac-PGP mediated MMP-9 release in PMNs.

<p>PMNs were pretreated with ERK1/2 MAPK inhibitor U0126 or vehicle for 30 minutes, and then stimulated with Ac-PGP (1.0 mg/ml) for 30 minutes. Cell-free supernatants were collected for MMP-9 assay. The levels of ERK1/2 MAPK were determined by western blot analysis of lysates from stimulated PMNs with actin controls which paralleled total ERK 1/2. Phosphorylated ERK was determined using an anti-ERK antibody that recognizes phosphorylated threonine and tyrosine residues (Thr202/Tyr204). <b>A</b>. Total and phosphorylated level of ERK1/2 MAPK representative of three gels. <b>B</b>. Fold change of phosphorylation of ERK1/2 MAPK versus total ERK1/2 MAPK normalized to PMN control. <b>C</b>. The detection of gelatinolytic activity by g<i>elatin zymography</i> representative of three gels. <b>D</b>. The quantification of specific MMP-9 activity by ELISA-based assay. <b>E</b>. Total and phosphorylated level of ERK1/2 MAPK representative of three gels. <b>F</b>. Fold change of phosphorylation of ERK1/2 MAPK versus total ERK1/2 MAPK normalized to PMN control. *p<0.05 compared to Ac-PGP without inhibitor pretreatment.</p

Increased MMP-9 activity in culture supernatants from human PMNs stimulated with Ac-PGP.

<p>Human PMNs isolated from peripheral blood were stimulated with Ac-PGP and IL-8 for different times and then supernatants were collected for MMP-9 assay. <b>A</b>. The detection of gelatinolytic activity in a time-dependent manner by g<i>elatin zymography</i> representative of three gels. <b>B</b>. The quantification of specific MMP-9 activity in a time-dependent manner by ELISA-based assay. <b>C</b>. The detection of gelatinolytic activity in a dose-dependent manner by g<i>elatin zymography</i> representative of three gels. <b>D</b>. The quantification of MMP-9 activity in a dose-dependent manner by ELISA-based assay. <i>*p<0.05</i>, compared with PMN only within same time point.</p

The effect of CXCR1 and CXCR2 inhibitor on the Ac-PGP mediated MMP-9 release in PMNs.

<p>PMNs were pretreated with the CXCR1 and CXCR2 inhibitor repertaxin at different dose for 20 minutes and then stimulated with Ac-PGP (1.0 mg/ml) for 30 minutes. Cell-free supernatants were collected for MMP-9 assay. The levels of ERK1/2 MAPK activation were determined by western blot analysis of lysates from stimulated PMNs with actin controls which paralleled total ERK 1/2. Phosphorylated ERK1/2 MAPK was determined using the anti-ERK antibody that recognizes phosphorylated threonine and tyrosine residues (Thr202/Tyr204). <b>A</b>. The detection of gelatinolytic activity by g<i>elatin zymography</i> representative of three gels. <b>B</b>. The quantification of MMP-9 activity by ELISA-based assay. <b>C</b>. Total and phosphorylated level of ERK1/2 MAPK representative of three gels <b>D</b>. Fold change of phosphorylation of ERK1/2 MAPK versus total ERK1/2 MAPK normalized to PMN control. *p<0.05 compared to Ac-PGP without inhibitor pretreatment.</p

Ac-PGP correlated to the MMP-9/MPO ratio in CF patients.

<p>CF (<i>n = </i>14) sputum samples were collected to determine gelatinolytic activity by g<i>elatin zymography</i> (each lane represents an individual patient) (<b>A</b>) and for the measurement of Ac-PGP, MPO and MMP-9, and correlation analysis was conducted between Ac-PGP and MMP-9/MPO ratio (<b>B</b>). The CF sputum samples demonstrated a correlation coefficient (<i>r</i>) of 0.63 (p = 0.017).</p

A model of persistent matrikine production and neutrophilic inflammation.

<p>During chronic PMN inflammation, collagen is hydrolyzed releasing Ac-PGP causing ongoing neutrophilic influx (<b>A</b>). In addition to causing PMN influx, Ac-PGP ligation of CXCR1 and CXCR2 leads to intracellular ERK phosphorylation and activation (<b>B</b>) and degranulation of MMP-9 from PMN tertiary granules (<b>C and D</b>). This MMP-9 acts on exposed collagen leading to Ac-PGP generation (<b>E</b>) and a feed-forward inflammatory response on PMNs (<b>F</b>).</p

Effect of VPA on the secondary structure of rhPE.

<p>(<b>A</b>) Three VPA concentrations (6, 12 and 24 mM) were titrated into 2.5 µM rhPE. VPA caused a significant change in the secondary structure of the enzyme. (<b>B</b>) A high concentration of ZPP (1 µM) was added to the 2.5 µM rhPE/24 mM VPA mixture. ZPP did not cause any change in the secondary structure of the enzyme. (<b>C</b>) Adding 1 µM ZPP to 2.5 µM rhPE does not cause any secondary protein structure change. Adding 24 mM VPA on top of that mixture doesn't cause any structure change.</p

Effect of VPA on the activity of rhPE.

<p>(<b>A</b>) Activity of 10 nM purified rhPE was measured in presence of ten doses of VPA or lithium ranging from 0.2–10 mM. Relative activity of rhPE in the presence of VPA or lithium is shown as a percentage of activity in the absence of VPA and lithium. VPA showed a <i>K_i</i> of approximately 1 mM. lithium showed no effect on PE activity. (<b>B</b>) Inhibition curves of three VPA concentrations (0.8, 1.6 and 3.5 mM) were obtained by incubating VPA with 10 nM rhPE during 90 min at 37 °0. (<b>C</b>) A Lineweaver-Burk plot was made based on the rhPE activity assays with 0.8 and 1.6 mM VPA as inhibitor. Enzyme activity was measured with increasing substrate (Suc-Gly-Pro-pNA) concentrations, ranging from 0.2–10 mM. The velocity was calculated as mM * min⁻¹ * ml⁻¹. Data are shown as the mean ± S.E.M. (n = 3 per group).</p

NMR spectra of the carbobenzoxy-group of ZPP.

<p>(<b>A</b>) 10 µM ZPP was added to 10 µM rhPE, 10 mM VPA was added to that mixture. A shift of the free ZPP peaks at 7.28 and 7.20 ppm to the left is seen (indicated in the red and green bars respectively). (<b>B</b>) 10 µM ZPP was added to 10 µM rhPE. A shift of the free ZPP peaks at 7.28 and 7.20 ppm to the left is seen. (<b>C</b>) 10 µM ZPP was measured without enzyme. The peaks at 7.28 and 7.20 ppm are the free ZPP fractions. (<b>D</b>) 10 mM VPA was added to 10 µM rhPE, 10 µM ZPP was added to that mixture. Note that the free ZPP peaks at 7.28 and 7.20 ppm do not show a shift to the left; there are no peaks in the highlighted areas.</p