Patricia L. Jackson

A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation

We describe the tripeptide neutrophil chemoattractant N-acetyl Pro-Gly-Pro (PGP), derived from the breakdown of extracellular matrix (ECM), which shares sequence and structural homology with an important domain on alpha chemokines. PGP caused chemotaxis and production of superoxide through CXC receptors, and administration of peptide caused recruitment of neutrophils (PMNs) into lungs of control, but not CXCR2-deficient mice. PGP was generated in mouse lung after exposure to lipopolysaccharide, and in vivo and in vitro blockade of PGP with monoclonal antibody suppressed PMN responses as much as chemokine-specific monoclonal antibody. Extended PGP treatment caused alveolar enlargement and right ventricular hypertrophy in mice. PGP was detectable in substantial concentrations in a majority of bronchoalveolar lavage samples from individuals with chronic obstructive pulmonary disease, but not control individuals. Thus, PGPs activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases.

A Novel Proteolytic Cascade Generates an Extracellular Matrix-Derived Chemoattractant in Chronic Neutrophilic Inflammation

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.

A Critical Role for LTA4H in Limiting Chronic Pulmonary Neutrophilic Inflammation

Smoke Gets in Your Lungs Chronic obstructive pulmonary disease (COPD) is a leading cause of death in the United States, primarily caused by cigarette smoking. The chronic inflammation that leads to tissue damage and organ dysfunction in COPD is mediated in large part by neutrophils, a type of granulocytic immune cell. Snelgrove et al. (p. 90, published online 2 September; see the Perspective by Barnes) now provide an explanation for why neutrophils stick around in the lung during COPD. The neutrophil chemoattractant Pro-Gly-Pro (PGP) is a biomarker for COPD and promotes neutrophil accumulation. The enzyme leukotriene A4 hydrolase degrades PGP in mice, and its activity was reduced by cigarette smoke both in vivo and in vitro. In contrast, during acute influenza infection in mice, leukotriene A4 hydrolase functioned normally, allowing for PGP degradation and the resolution of inflammation. Thus, in COPD, cigarette smoking may lead to the accumulation PGP—which, in turn, could keep neutrophils in the lung to drive inflammation and subsequent lung damage and dysfunction. Cigarette smoke promotes lung inflammation by hindering an enzyme that degrades an immune cell chemoattractant. Leukotriene A4 hydrolase (LTA4H) is a proinflammatory enzyme that generates the inflammatory mediator leukotriene B4 (LTB4). LTA4H also possesses aminopeptidase activity with unknown substrate and physiological importance; we identified the neutrophil chemoattractant proline-glycine-proline (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 inhibiting LTA4H to prevent LTB4 generation may not reduce neutrophil recruitment because of elevated levels of PGP.

Matrix Metalloproteinase 9 Activity Enhances Host Susceptibility to Pulmonary Infection with Type A and B Strains of Francisella tularensis

A striking feature of pulmonary infection with the Gram-negative intracellular bacterium Francisella tularensis, a category A biological threat agent, is an intense accumulation of inflammatory cells, particularly neutrophils and macrophages, at sites of bacterial replication. Given the essential role played by host matrix metalloproteinases (MMPs) in modulating leukocyte recruitment and the potentially indiscriminate destructive capacity of these cells, we investigated whether MMP-9, an important member of this protease family released by neutrophils and activated macrophages, plays a role in the pathogenesis of respiratory tularemia. We found that F. tularensis induced expression of MMP-9 in FVB/NJ mice and that the action of this protease is associated with higher bacterial burdens in pulmonary and extrapulmonary tissues, development of more extensive histopathology predominated by neutrophils, and increased morbidity and mortality compared with mice lacking MMP-9 (MMP-9−/−). Moreover, MMP-9−/− mice were able to resolve infection with either the virulence-attenuated type B (live vaccine strain) or the highly virulent type A (SchuS4) strain of F. tularensis. Disease resolution was accompanied by diminished leukocyte recruitment and reductions in both bacterial burden and proinflammatory cytokine production. Notably, neutrophilic infiltrates were significantly reduced in MMP-9−/− mice, owing perhaps to limited release of Pro-Gly-Pro, a potent neutrophil chemotactic tripeptide released from extracellular matrix through the action of MMP-9. Collectively, these results suggest that MMP-9 activity plays a central role in modulating the clinical course and severity of respiratory tularemia and identifies MMPs as novel targets for therapeutic intervention as a means of modulating neutrophil recruitment.

Matrix metalloproteinase-8 facilitates neutrophil migration through the corneal stromal matrix by collagen degradation and production of the chemotactic peptide Pro-Gly-Pro.

Matrix metalloproteinase (MMP)-8 and MMP-9 play several roles in inflammation, including degradation of extracellular matrix (ECM) components and regulation of cytokine activity. To determine the roles of MMP-8 and MMP-9 in a neutrophil-dependent inflammatory response, we used a murine model of corneal inflammation in which LPS is injected into the corneal stroma. In contrast to wild-type mice, we found that i) lipopolysaccharide (LPS)-injected CXCR2(-/-) corneas had impaired neutrophil infiltration and did not express either MMP-8 or MMP-9; ii) neutrophil migration through the central cornea was impaired in Mmp8(-/-), but not Mmp9(-/-), mice; iii) neutrophil migration was inhibited in collagenase-resistant mice; iv) the chemotactic Pro-Gly-Pro (PGP) tripeptide that binds CXCR2 was decreased in CXCR2(-/-) mice; v) PGP production was impaired in Mmp8(-/-) corneas; and vi) neutralizing anti-PGP antibody did not inhibit neutrophil infiltration in Mmp8(-/-) mice. We found no effects of MMP-8 on LPS-induced CXC chemokine (LIX, or CXCL5)-induced neutrophil recruitment or on LPS-induced CXC chemokine production. Together, these studies indicate that neutrophils contribute to the production of both MMP-8 and MMP-9 in LPS-injected corneas and that MMP-8 regulates neutrophil migration through the dense collagenous ECM of the corneal stroma by generating chemotactic PGP during inflammation.

Potential Role of High-Mobility Group Box 1 in Cystic Fibrosis Airway Disease

RATIONALE High-mobility group box 1 (HMGB1) is a potent inflammatory mediator elevated in sepsis and rheumatoid arthritis, although its role in cystic fibrosis (CF) lung disease is unknown. OBJECTIVES To determine whether HMGB1 contributes to CF lung inflammation, including neutrophil chemotaxis and lung matrix degradation. METHODS We used sputum and serum from subjects with CF and a Scnn1b-transgenic (Scnn1b-Tg) mouse model that overexpresses beta-epithelial Na(+) channel in airways and mimics the CF phenotype, including lung inflammation. Human secretions and murine bronchoalveolar lavage fluid (BALF) was assayed for HMGB1 by Western blot and ELISA. Neutrophil chemotaxis was measured in vitro after incubation with human neutrophils. The collagen fragment proline-glycine-proline (PGP) was measured by tandem mass spectroscopy. MEASUREMENTS AND MAIN RESULTS HMGB1 was detected in CF sputum at higher levels than secretions from normal individuals. Scnn1b-Tg mice had elevated levels of HMGB1 by Western blot and ELISA. We demonstrated that dose-dependent chemotaxis of human neutrophils stimulated by purified HMGB1 was partially dependent on CXC chemokine receptors and that this could be duplicated in CF sputum and BALF from Scnn1b-Tg mice. Neutralization by anti-HMGB1 antibody, in both the sputum and BALF-reduced chemotaxis, which suggested that HMGB1 contributed to the chemotactic properties of these samples. Intratracheal administration of purified HMGB1 induced neutrophil influx into the airways of mice and promoted the release of PGP. PGP was also elevated in Scnn1b-Tg mice and CF serum. CONCLUSIONS HMGB1 expression contributes to pulmonary inflammation and lung matrix degradation in CF airway disease and deserves further investigation as a biomarker and potential therapeutic target.

N-α-PGP and PGP, potential biomarkers and therapeutic targets for COPD

BackgroundChronic obstructive pulmonary disease (COPD) is a common respiratory disorder for which new diagnostic and therapeutic approaches are required. Hallmarks of COPD are matrix destruction and neutrophilic airway inflammation in the lung. We have previously described two tri-peptides, N-α-PGP and PGP, which are collagen fragments and neutrophil chemoattractants. In this study, we investigate if N-α-PGP and PGP are biomarkers and potential therapeutic targets for COPD.MethodsInduced sputum samples from COPD patients, healthy controls and asthmatics were examined for levels of N-α-PGP and PGP using mass spectrometry and for the ability to generate PGP de novo from collagen. Proteases important in PGP generation in the lung were identified by the use of specific inhibitors in the PGP generation assay and by instillation of proteases into mouse lungs. Serum levels of PGP were compared between COPD patients and controls.ResultsN-α-PGP was detected in most COPD sputum samples but in no asthmatics or controls. PGP was detected in a few controls and in all COPD sputum samples, where it correlated with levels of myeloperoxidase. COPD sputum samples had the ability to generate N-α-PGP and PGP de novo from collagen. PGP generation by COPD sputum was blocked by inhibitors of matrix metalloproteases (MMPs) 1 and 9 and prolyl endopeptidase. MMPs 1 and 9 and prolyl endopeptidase acted synergistically to generate PGP in vivo when instilled into mouse lungs. Serum levels of PGP were also significantly higher in COPD patients than in controlsConclusionN-α-PGP and PGP may represent novel diagnostic tests and biomarkers for COPD. Inhibition of this pathway may provide novel therapies for COPD directed at the chronic, neutrophilic, airway inflammation which underlies disease progression.

Cigarette smoke induces systemic defects in cystic fibrosis transmembrane conductance regulator function.

RATIONALE Several extrapulmonary disorders have been linked to cigarette smoking. Smoking is reported to cause cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the airway, and is also associated with pancreatitis, male infertility, and cachexia, features characteristic of cystic fibrosis and suggestive of an etiological role for CFTR. OBJECTIVES To study the effect of cigarette smoke on extrapulmonary CFTR function. METHODS Demographics, spirometry, exercise tolerance, symptom questionnaires, CFTR genetics, and sweat chloride analysis were obtained in smokers with and without chronic obstructive pulmonary disease (COPD). CFTR activity was measured by nasal potential difference in mice and by Ussing chamber electrophysiology in vitro. Serum acrolein levels were estimated with mass spectroscopy. MEASUREMENTS AND MAIN RESULTS Healthy smokers (29.45 ± 13.90 mEq), smokers with COPD (31.89 ± 13.9 mEq), and former smokers with COPD (25.07 ± 10.92 mEq) had elevated sweat chloride levels compared with normal control subjects (14.5 ± 7.77 mEq), indicating reduced CFTR activity in a nonrespiratory organ. Intestinal current measurements also demonstrated a 65% decrease in CFTR function in smokers compared with never smokers. CFTR activity was decreased by 68% in normal human bronchial epithelial cells exposed to plasma from smokers, suggesting that one or more circulating agents could confer CFTR dysfunction. Cigarette smoke-exposed mice had decreased CFTR activity in intestinal epithelium (84.3 and 45%, after 5 and 17 wk, respectively). Acrolein, a component of cigarette smoke, was higher in smokers, blocked CFTR by inhibiting channel gating, and was attenuated by antioxidant N-acetylcysteine, a known scavenger of acrolein. CONCLUSIONS Smoking causes systemic CFTR dysfunction. Acrolein present in cigarette smoke mediates CFTR defects in extrapulmonary tissues in smokers.

A Self-Propagating Matrix Metalloprotease-9 (MMP-9) Dependent Cycle of Chronic Neutrophilic Inflammation

Background Chronic neutrophilic inflammation is a poorly understood feature in a variety of diseases with notable worldwide morbidity and mortality. We have recently characterized N-acetyl Pro-Gly-Pro (Ac-PGP) as an important neutrophil (PMN) chemoattractant in chronic inflammation generated from the breakdown of collagen by the actions of MMP-9. MMP-9 is present in the granules of PMNs and is differentially released during inflammation but whether Ac-PGP contributes to this ongoing proteolytic activity in chronic neutrophilic inflammation is currently unknown. Methodology/Principal Findings Utilizing isolated primary blood PMNs from human donors, we found that Ac-PGP induces significant release of MMP-9 and concurrently activates the ERK1/2 MAPK pathway. This MMP-9 release is attenuated by an inhibitor of ERK1/2 MAPK and upstream blockade of CXCR1 and CXCR2 receptors with repertaxin leads to decreased MMP-9 release and ERK 1/2 MAPK activation. Supernatants obtained from PMNs stimulated by Ac-PGP generate more Ac-PGP when incubated with intact collagen ex vivo; this effect is inhibited by an ERK1/2 pathway inhibitor. Finally, clinical samples from individuals with CF demonstrate a notable correlation between Ac-PGP (as measured by liquid chromatography-tandem mass spectrometry) and MMP-9 levels even when accounting for total PMN burden. Conclusions/Significance These data indicate that ECM-derived Ac-PGP could result in a feed-forward cycle by releasing MMP-9 from activated PMNs through the ligation of CXCR1 and CXCR2 and subsequent activation of the ERK1/2 MAPK, highlighting for the first time a matrix-derived chemokine (matrikine) augmenting its generation through a discrete receptor/intracellular signaling pathway. These findings have notable implications to the development unrelenting chronic PMN inflammation in human disease.

Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease

Objective Proline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD). Design In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies. Results In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine. Conclusions The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.