The neutrophil chemoattractant peptide proline-glycine-proline is associated with acute respiratory distress syndrome

Abstract

Acute respiratory distress syndrome (ARDS) is characterized by unrelenting polymorphonuclear neutrophil (PMN) inflammation and vascular permeability. The matrikine proline-glycine-proline (PGP) and acetylated PGP (Ac-PGP) have been shown to induce PMN inflammation and endothelial permeability in vitro and in vivo. In this study, we investigated the presence and role of airway PGP peptides in acute lung injury (ALI)/ARDS. Pseudomonas aeruginosa-derived lipopolysaccharide (LPS) was instilled intratracheally in mice to induce ALI, and increased Ac-PGP with neutrophil inflammation was noted. The PGP inhibitory peptide, arginine-threonine-arginine (RTR), was administered (it) 30 min before or 6 h after LPS injection. Lung injury was evaluated by detecting neutrophil infiltration and permeability changes in the lung. Pre- and posttreatment with RTR significantly inhibited LPS-induced ALI by attenuating lung neutrophil infiltration, pulmonary permeability, and parenchymal inflammation. To evaluate the role of PGP levels in ARDS, minibronchoalveolar lavage was collected from nine ARDS, four cardiogenic edema, and five nonlung disease ventilated patients. PGP levels were measured and correlated with Acute Physiology and Chronic Health Evaluation (APACHE) score, $P{a}_O{}_{{}_2}$ to $F{I}_{O_2}$ (P/F), and ventilator days. PGP levels in subjects with ARDS were significantly higher than cardiogenic edema and nonlung disease ventilated patients. Preliminary examination in both ARDS and non-ARDS populations demonstrated PGP levels significantly correlated with P/F ratio, APACHE score, and duration on ventilator. These results demonstrate an increased burden of PGP peptides in ARDS and suggest the need for future studies in ARDS cohorts to examine correlation with key clinical parameters.

Keywords: ARDS; LPS; PGP; acute lung injury.

Fig. 1.

Acetylated proline-glycine-proline (Ac-PGP) induced acute lung injury (ALI). C57BL/6 mice were administered it with 250 μg Ac-PGP for 8 h, and the bronchoalveolar lavage (BAL) fluid was collected for total infiltrated cells (A), neutrophils (B), immunoglobulin M (IgM, C) and total matrix metalloproteinase-9 (MMP-9, D) measurement. Statistical analysis was performed using Mann-Whitney test; n = 4–5 mice/group. All values represent means ± SD.

Fig. 2.

The application of arginine-threonine-arginine (RTR) attenuates neutrophil infiltration in the lung in lipopolysaccharide (LPS)-induced acute lung injury (ALI). A: C56BL/6 mice were administered it with 100 μg LPS (Pseudomonas aeruginosa). After LPS exposure (24 h), bronchoalveolar lavage (BAL) fluid samples were collected and analyzed for proline-glycine-proline (PGP) by electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI-LCMS/MS). Statistical analysis was performed using Mann-Whitney test; n = 5–7 mice/group. B and C: 24 h after initiation of LPS-induced ALI, neutrophils in BAL were determined in mice that received RTR (250 μg) at 30 min before (pre) or 6 h after (pos) induction of ALI and in control mice with ALI but no RTR. Statistical analysis was performed using 1-way ANOVA (P < 0.0001) with Tukey’s multiple-comparison posttest, n = 3–7 mice/group. All values represent means ± SD.

Fig. 3.

The application of arginine-threonine-arginine (RTR) reduces airway vascular leakage and matrix metalloproteinase (MMP)-9 release in lipopolysaccharide (LPS)-induced acute lung injury (ALI). After initiation of LPS-induced ALI (24 h), immunoglobulin M (IgM, A) and matrix metalloproteinase-9 (MMP-9, B) expression in BAL fluid was determined in mice that received RTR (250 μg) and in control mice with ALI only, RTR only, or PBS only. Statistical analysis was performed using 1-way ANOVA (P < 0.0001) with Tukey’s multiple-comparison posttest; n = 3–5 mice/group. All values represent means ± SD.

Fig. 4.

The application of arginine-threonine-arginine (RTR) attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI). After initiation of LPS-induced ALI (24 h), lung histology was assessed in mice that received RTR (250 μg) at 30 min before or 6 h after induction of ALI and in control mice with ALI but no RTR. A: hematoxylin and eosin staining of representative histological images of lung sections (bar: 50 mm for ×40 and 200 mm for ×200). B: lung injury scores. Statistical analysis was performed using 1-way ANOVA (P < 0.0001) with Tukey’s multiple-comparison posttest; n = 6–10 mice/group. All values represent means ± SD.

Fig. 5.

Proline-glycine-proline (PGP) levels were elevated in acute respiratory distress syndrome (ARDS) compared with cardiogenic edema and nonlung disease patients. Mini-bronchoalveolar lavage (BAL) was collected from patients with ARDS (n = 9), cardiogenic edema (n = 4), and nonlung disease control (n = 5) for PGP measurement by electrospray ionization-liquid chromatography-tandem mass spectrometry (A). Myeloperoxidase (MPO, B) and albumin (C) levels in mini-BAL were detected by ELISA. Statistical analysis was performed using the Kruskal-Wallis test (P < 0.002) among groups and post hoc Dunn’s test of ARDS patients compared with the patients with cardiogenic edema and the nonlung disease control group. All values represent means ± SD.

Fig. 6.

A model of proline-glycine-proline (PGP)-mediated acute lung injury. Infection and tissue injury leads to activation of matrix-degrading enzymes from either structural cells or immune cells and subsequent breakdown of extracellular matrix. As a result, several bioactive matrikines are released. PGP and its acetylated form (Ac-PGP) mediate neutrophil chemotaxis and increase endothelial permeability through the CXCR2 pathway, leading to profound inflammation and tissue injury. ARDS, acute respiratory distress syndrome.