Chronic neutrophilic inflammation is a manifestation of a variety of lung diseases including cystic fibrosis (CF). There is increasing evidence that fragments of extracellular matrix proteins, such as collagen and elastin, play an important role in inflammatory cell recruitment to the lung in animal models of airway inflammation. Unfortunately, the association of these peptides with human disease and the identification of therapeutic targets directed toward these inflammatory pathways have remained elusive. In this study, we demonstrate that a novel extracellular matrix-derived neutrophil chemoattractant, proline-glycine-proline (PGP), acts through CXC receptors 1 and 2 on neutrophils, similar to N-acetylated proline-glycine-proline (N-α-PGP). We describe the specific multistep proteolytic pathway involved in PGP generation from collagen, involving matrix metalloproteases 8 and 9 and prolyl endopeptidase, a serine protease for which we identify a novel role in inflammation. PGP generation correlates closely with airway neutrophil counts after administration of proteases in vivo. Using CF as a model, we show that CF sputum has elevated levels of PGP peptides and that PGP levels decline during the course of CF inpatient therapy for acute pulmonary exacerbation, pointing to its role as a novel biomarker for this disease. Finally, we demonstrate that CF secretions are capable of generating PGP from collagen ex vivo and that this generation is significantly attenuated by the use of inhibitors directed toward matrix metalloprotease 8, matrix metalloprotease 9, or prolyl endopeptidase. These experiments highlight unique protease interactions with structural proteins regulating innate immunity and support a role for these peptides as novel biomarkers and therapeutic targets for chronic, neutrophilic lung diseases.

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63 + /CD66b + nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to a1-antitrypsin (a1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and a1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.

Leukotriene A4 Hydrolase (LTA4H) is a pro-inflammatory enzyme which generates the inflammatory mediator leukotriene B4 (LTB4). LTA4H also possesses aminopeptidase activity with unknown substrate and physiological significance. We identified the neutrophil chemoattractant, Pro-Gly-Pro (PGP), as this physiological substrate. PGP is a biomarker for chronic obstructive pulmonary disease (COPD), and is implicated in neutrophil persistence in the lung. In acute neutrophil driven inflammation, PGP was degraded by LTA4H, which facilitated the resolution of inflammation. In contrast, cigarette smoke, a major risk factor for the development of COPD, selectively inhibited LTA4H aminopeptidase activity, which led to the accumulation of PGP and neutrophils. These studies imply that therapeutic strategies that inhibit LTA4H to prevent LTB4 generation may not reduce neutrophil recruitment because of elevated PGP.