MMP-9 Cleaves SP-D in a Dose- and Time-dependent Manner.

<p>(A) Dose and (B) time course digestions of SP-D by MMP-9. For the dose course (A), the reaction loaded onto lane 2 contained 62 ng/mL MMP-9 and the concentration was raised 3-fold serially to 5 µg/mL MMP-9 at lane 6. Reactions were incubated for 4 hours. For the time course (B), the length of digestion in minutes is listed above the lane. SP-D concentration was 20 µg/mL for both experiments, and MMP-9 concentration was 5 µg/mL for the time course. For the Native PAGE (C), lane 1 contains intact SP-D and lane 2 contains cleaved SP-D. An arrow indicates the band corresponding to MMP-9.</p

Cleaved SP-D Fails to Increase Phagocytosis by MH-S Cells.

<p>Examination and quantitation of phagocytosis of <i>E. coli</i> by MH-S cells was performed using (A) a gentamicin protection assay and (B and C) flow cytometry. For (A), all conditions are significantly different (p<0.05) from 1 µg/mL intact SP-D. An asterisk (*) denotes p≤0.001 when compared to 1 µg/mL intact SP-D. Columns marked with # are significantly different when compared to 0.5 µg/mL intact SP-D (p<0.05). For (B), intact SP-D causes MH-S cells to have significantly higher mean fluorescence intensity (MFI) than all other conditions (p≤0.001). Cleaved SP-D is significantly different when compared to both MMP-9 and PBS controls (p≤0.001). For (C), flow data was gated on forward and side scatter to select for MH-S cells. For both (A) and (B), error bars represent standard deviation.</p

Cleaved SP-D Retains its Ability to Bind <i>E. coli</i> LPS.

<p>Examination of the ability of SP-D to bind to LPS-coated plates. In (A), the ELISA was performed using 2-fold serially diluted SP-D samples. In (B), 1 µg/mL intact or cleaved SP-D was analyzed in triplicate with MMP-9 as a control and NE-cleaved SP-D as a negative control. The average of PBST alone ran in triplicate was subtracted from all values. While intact and MMP-9-cleaved SP-D in PBST are significantly different from PBST alone (p≤0.001 and p<0.05, respectively) and NE-cleaved SP-D (p≤0.001 and p<0.05, respectively), MMP-9 along with intact and cleaved SP-D in PBST with maltose were not significantly different from PBST or PBST with maltose. For (B), error bars represent standard deviation.</p

Clinical Data of CF Subjects.

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

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

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

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