Lung transplantation is a therapeutic modality frequently utilized in end-stage lung disease. Unfortunately, lung transplant recipients have poor clinical outcomes, often due to the development of chronic rejection in the transplanted allograft. This process is characterized by neutrophil influx and extracellular matrix remodeling leading to luminal obstruction and airway inflammation. The molecular mechanisms underlying chronic rejection are poorly understood and disease-specific biomarkers are lacking. We report that in addition to increased levels of interleukin-8 (IL-8), the level of neutrophil chemoattractant proline-glycine-proline (PGP) is elevated in chronic rejection patient bronchoalveolar lavage (BAL) fluid and correlates with loss of lung function. The enzymes responsible for generating PGP, matrix metalloproteases-8 and -9 (MMP-8, -9) and prolyl endopeptidase (PE), are also elevated in chronic rejection samples. Together, IL-8 and PGP account for most of the neutrophil chemoattractant capacity seen in chronic rejection BAL fluid. Using specific neutralizing antibodies to both IL-8 and PGP, we demonstrate that PGP is the major neutrophil chemoattractant found in BAL fluid from individuals at the time of chronic rejection. These findings highlight the influence of a matrix-derived neutrophil chemoattractant in post-transplantation organ rejection and provide opportunities for the development of unique diagnostics and therapeutics to potentially improve disease outcomes.
Extensive variations in human surfactant protein D (SP-D) levels in circulation as measured by ELISA exist in the published literature. In order to determine the source of these variations, factors influencing the measurement by ELISA were explored.
Materials and Methods
Peripheral blood from healthy individuals was collected into various vacutainers during the same blood draw. Recombinant SP-D was diluted into different matrices and used for a standard curve. Samples were analyzed by capture ELISA using one of two distinct detection antibodies.
The type of matrix had some effects on detection of recombinant SP-D. The type of anticoagulant used and dilution factor had very little effect, except for in plasma collected in EDTA vacutainers. The extent of variation in published values seemed to be due to the ELISA configuration employed, and, in agreement with this, we found that by switching the detection antibody, there was a 50% decrease in the extrapolated SP-D value of serum and plasma samples. Storage of samples resulted in slight changes in measured SP-D levels.
The ELISA configuration employed to measure circulating levels of SP-D has a significant effect on the extrapolated values. In both configurations tested, the use of EDTA as a coagulant resulted in inconsistent values, and we, therefore, suggest the avoidance of this anticoagulant when assaying for SP-D by ELISA. While the demonstrated effects of several factors on measurement of SP-D may not account for all the disparities amongst the previous studies, they stress that variations in methodologies for measuring the same protein can result in very inconsistent results.
Cases of Ralstonia colonization/infection occasionally reported by hospitals has generated increased interest in an organism previously little known to most clinicians. Our goal was to determine the incidence of respiratory colonizations and infections involving Ralstonia and the association of mechanical ventilation (limited to reports on respiratory-related occurrences in the USA).
We performed a secondary analysis of published clinical reports of Ralstonia to determine the potential risks for respiratory colonization and infection in the USA and if being on mechanical ventilation (MV) had an influence on colonization and conversion to infection (symptomatic).
The odds of developing colonization with Ralstonia were eight times higher and the likelihood of developing infection with Ralstonia was twelve times higher in those mechanically ventilated compared to those not mechanically ventilated.
Our results suggest that individuals who are currently on mechanical ventilation and are Ralstonia culture-positive have an increased risk for colonization and may have increased propensity to the development of infection (two decision trees for approaching diagnosis and therapy included).
Ralstonia; Gram-Negative Bacterial Infections; Cross Infection; Ventilators, Mechanical; Equipment Contamination; Infection; Equipment, Disposable
RSV infection is a potent stimulus for airway epithelial expression of MMP-9, and MMP-9 activity in vivo is a predictor of disease severity in children with RSV-induced respiratory failure (RSV-RF).
Human airway epithelial cells were infected with RSV A2 strain, and analyzed for MMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1, a natural inhibitor of MMP-9) release. In addition, endotracheal samples from children with RSV-RF and controls (non-RSV pneumonia and non-lung disease controls) were analyzed for MMP-9, TIMP-1, human neutrophil elastase (HNE) and myeloperoxidase (MPO) activity.
RSV infection of airway epithelia was sufficient to rapidly induce MMP-9 transcription and protein release. Pulmonary MMP-9 activity peaked at 48 hours in infants with RSV-RF compared to controls. In the RSV group, MMP-9 activity and MMP-9:TIMP-1 ratio imbalance predicted higher oxygen requirement and worse Pediatric Risk of Mortality scores. Highest levels of HNE and MPO were measured in the RSV cohort but unlike MMP-9, these neutrophil markers failed to predict disease severity.
These results support the hypothesis that RSV is a potent stimulus for MMP-9 expression and release from human airway epithelium, and that MMP-9 is an important biomarker of disease severity in mechanically ventilated children with RSV lung infection.
Significant airway remodeling is a major component of the increased morbidity and mortality observed in cystic fibrosis (CF) patients. These airways feature ongoing leukocytic inflammation and unrelenting bacterial infection. In contrast to acute bacterial pneumonia, CF infection is not cleared efficiently and the ensuing inflammatory response causes tissue damage. This structural damage is mainly a result of free proteolytic activity released by infiltrated neutrophils and macrophages. Major proteases in this disease are serine and matrix metalloproteases (MMPs). While the role of serine proteases, such as elastase, has been characterized in detail, there is emerging evidence that MMPs could play a key role in the pathogenesis of CF lung disease. This review summarizes studies linking MMPs with CF lung disease and discusses the potential value of MMPs as future therapeutic targets in CF and other chronic lung diseases.
Cystic fibrosis; lung disease; elastase; neutrophil; proteases; antiproteases
A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP), has been recently characterized in chronic obstructive pulmonary disease (COPD). This peptide is derived via the proteolytic activity of matrix metalloproteases (MMP's)-8/9 and PE, enzymes produced by neutrophils and present in COPD serum and sputum. Valproic acid (VPA) is an inhibitor of PE and could possibly have an effect on the severity of chronic inflammation. Here the interaction site of VPA to PE and the resulting effect on the secondary structure of PE is investigated. Also, the potential inhibition of PGP-generation by VPA was examined in vitro and in vivo to improve our understanding of the biological role of VPA. UV- visible, fluorescence spectroscopy, CD and NMR were used to determine kinetic information and structural interactions between VPA and PE. In vitro, PGP generation was significantly inhibited by VPA. In vivo, VPA significantly reduced cigarette-smoke induced neutrophil influx. Investigating the molecular interaction between VPA and PE showed that VPA modified the secondary structure of PE, making substrate binding at the catalytic side of PE impossible. Revealing the molecular interaction VPA to PE may lead to a better understanding of the involvement of PE and PGP in inflammatory conditions. In addition, the model of VPA interaction with PE suggests that PE inhibitors have a great potential to serve as therapeutics in inflammatory disorders.
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.
Surfactant Protein D (SP-D) is a multifunctional protein present in the lung and in respiratory secretions. In the process of developing new experimental approaches to examine SP-D function, we observed that SP-D adsorbs to polypropylene tubes to a great extent, thereby depleting SP-D from the solution. Although it is well known that proteins adsorb nonspecifically to plastic, this effect is usually diminished by treatments to make the plastic “low-retention” or “low-binding”. However, these treatments actually increased the binding of SP-D to the plastic. In addition, this adsorption affected the results of several assays, including proteolytic cleavage assays. In order to block SP-D from adsorbing to polypropylene and the effects caused by this adsorption, we coated the tubes with bovine serum albumin (BSA), as is commonly performed for ELISAs. This coating greatly diminished the amount of SP-D sticking to the plastic, providing an inexpensive and effective method for preventing adsorption and the artifacts resulting from this adsorption.
Cystic fibrosis (CF) is characterized by acute pulmonary exacerbations (APE). The CF nasal airway exhibits a similar ion transport defect as the lung, and colonization, infection, and inflammation within the nasal passages are common among CF patients. Nasal lavage fluid (NLF) is a minimally invasive means to collect upper airway samples.
We collected NLF at the onset and resolution of CF APE and compared a 27-plex cytokine profile to stable CF outpatients and normal controls. We also tested IP-10 levels in the bronchoalveolar lavage fluid (BALF) of CF patients. Well-differentiated murine sinonasal monolayers were exposed to bacterial stimulus, and IP-10 levels were measured to test epithelial secretion.
Subjects hospitalized for APE had elevated IP-10 (2582 pg/mL [95% CL of mean: 818,8165], N=13) which significantly decreased (647 pg/mL [357,1174], P<0.05, N =13) following antimicrobial therapy. Stable CF outpatients exhibited intermediately elevated levels (680 pg/mL [281,1644], N=13) that were less than CF inpatients upon admission (P=0.056) but not significantly different than normal controls (342 pg/mL [110,1061]; P=0.3, N=10). IP-10 was significantly increased in CF BALF (2673 pg/mL [1306,5458], N=10) compared to healthy post-lung transplant patients (8.4 pg/mL [0.03,2172], N=5, P<0.001). IP-10 levels from well-differentiated CF murine nasal epithelial monolayers exposed to Pseudomonas PAO-1 bacteria-free prep or LPS (100 nM) apically for 24 hours were significantly elevated (1159 ± 147, P<0.001 for PAO-1; 1373 ± 191, P<0.001 for LPS vs. 305 ± 68 for vehicle controls). Human sino-nasal epithelial cells derived from CF patients had a similar response to LPS (34% increase, P<0.05, N=6).
IP-10 is elevated in the nasal lavage of CF patients with APE and responds to antimicrobial therapy. IP-10 is induced by airway epithelia following stimulation with bacterial pathogens in a murine model. Additional research regarding IP-10 as a potential biomarker is warranted.
Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of iNOS, arginase, and NADPH oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of their infiltration into the lung. The nitric oxide-producing cells were Ly-6C+Ly-6G− and down-modulated T cell activation, recruited Treg cells, and dramatically down-regulated antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C−Ly-6G+ and expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a superoxide-dependent fashion. A smaller population of Ly-6C+Ly-6G+ cells also suppressed T cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells represent important targets for asthma therapy.
Acute respiratory distress syndrome (ARDS) due to sepsis has a high mortality rate with limited treatment options. High density lipoprotein (HDL) exerts innate protective effects in systemic inflammation. However, its role in ARDS has not been well studied. Peptides such as L-4F mimic the secondary structural features and functions of apolipoprotein (apo)A-I, the major protein component of HDL. We set out to measure changes in HDL in sepsis-mediated ARDS patients, and to study the potential of L-4F to prevent sepsis-mediated ARDS in a rodent model of lipopolysaccharide (LPS)-mediated acute lung injury, and a combination of primary human leukocytes and human ARDS serum. We also analyzed serum from non-lung disease intubated patients (controls) and sepsis-mediated ARDS patients. Compared to controls, ARDS demonstrates increased serum endotoxin and IL-6 levels, and decreased HDL, apoA-I and activity of anti-oxidant HDL-associated paraoxanase-1. L-4F inhibits the activation of isolated human leukocytes and neutrophils by ARDS serum and LPS in vitro. Further, L-4F decreased endotoxin activity and preserved anti-oxidant properties of HDL both in vitro and in vivo. In a rat model of severe endotoxemia, L-4F significantly decreased mortality and reduces lung and liver injury, even when administered 1 hour post LPS. Our study suggests the protective role of the apoA-I mimetic peptide L-4F in ARDS and gram-negative endotoxemia and warrant further clinical evaluation. The main protective mechanisms of L-4F are due to direct inhibition of endotoxin activity and preservation of HDL anti-oxidant activity.
Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype.
The contribution of nitric oxide (NO) to the pathophysiology of asthma remains incompletely defined despite its established pro- and anti-inflammatory effects. Induction of the inducible nitric oxide synthase (iNOS), arginase and superoxide pathways is correlated with increased airway hyperresponsiveness (AHR) in asthmatic subjects. To determine the contributions of these pathways in proximal and distal airways, we compared bronchial wash (BW) to traditional bronchoalveolar lavage (BAL) for measurements of reactive nitrogen/oxygen species, arginase activation, and cytokine/chemokine levels in asthmatic and normal subjects. Levels of NO were preferentially elevated in the BAL, demonstrating higher-level NOS activation in the distal airway compartment of asthmatic subjects. In contrast, DHE+ cells which have the potential to generate reactive oxygen species were found to be increased in both proximal and distal airway compartments of asthmatics compared to controls. Different patterns of cytokines and chemokines were observed, with a predominance of epithelial cell-associated mediators in the BW as compared to macrophage/monocyte-derived mediators in the BAL of asthmatic subjects. Our study demonstrates differential production of reactive species and soluble mediators within the distal airways as compared to the proximal airways in asthma. These results indicate that cellular mechanisms are activated in the distal airways of asthmatics and must be considered in the development of therapeutic strategies for this chronic inflammatory disorder.
Nitric oxide; superoxide; arginase; bronchoalveolar lavage; bronchial wash; distal airway
The present review discusses the role of tri-peptide Proline –Glycine –Proline (PGP) as a potential player, biomarker and therapeutic target in this process.
Chemotactic factors; Cystic fibrosis; Chronic obstructive pulmonary disease; Extracellular matrix; Neutrophil activation praline; Interleukin-8 A and B; Serine ednopeptidases
Chronic rhinosinusitis has a major impact on the quality of life of patients with cystic fibrosis (CF) and may contribute to progression of chronic lung disease. Despite multiple sinus surgeries, maxillary sinus involvement is a recurrent problem. The modified endoscopic medial maxillectomy (MEMM) permits debridement in the clinic, improves mucus clearance with nasal irrigations, and increases access for topical delivery of therapeutics. However, clinical outcomes of aggressive sinus surgery with regimented postoperative medical treatment have not been systematically evaluated.
CF patients completed the 22-Item Sinonasal Outcome Test questionnaires before sinus surgery (and bilateral MEMM) and at sequential postoperative visits. Objective measures included Lund-Kennedy endoscopic score and pulmonary function tests (forced expiratory volume at 1 second percent [FEV1%] predicted). Culture-directed antibiotic therapy, prednisone, and topical irrigations were initiated postoperatively.
Twenty-two patients (mean age, 26.5 years; 4.9 prior sinus operations) underwent MEMM and sinus surgery. Symptom scores were significantly reduced at 60 days (primary outcome, 64.7 ± 18.4 presurgery versus 27.5 ± 15.3 postsurgery; p < 0.0001) and up to a year postoperatively (27.6 ± 12.6; p < 0.0001). Endoscopic scores were also reduced after surgery (10.4 ± 1.1 presurgery versus 5.7 ± 2.4 [30 days], 5.7 ± 1.4 [60 days], 5.8 ± 1.3 [120 days], and 6.0 ± 1.1 [1 year]; p < 0.0001)]. There were no differences in FEV1% predicted up to 1 year postoperatively, but hospital admissions secondary to pulmonary exacerbations significantly decreased (2.0 ± 1.4 versus 3.2 ± 2.4, respectively; p < 0.05).
Prospective evaluation indicates sinus surgery with MEMM is associated with marked improvement in sinus disease outcomes. Additional studies are necessary to confirm whether this treatment paradigm is associated with improved CF pulmonary disease.
Antibiotics; chronic sinusitis; cystic fibrosis; endoscopic medial maxillectomy; mucociliary clearance; sinonasal outcomes; sinus surgery; SNOT-22
Matrix metalloproteinases (MMP) -8 and -9 may play key roles in the modulation of neutrophilic lung inflammation seen in pediatric Acute Respiratory Distress Syndrome (ARDS). We aimed to perform a comprehensive analysis of MMP-8 and MMP-9 activity in tracheal aspirates of pediatric ARDS patients compared with non-ARDS controls, testing whether increased MMP-8 and -9 activities were associated with clinical outcomes.
Tracheal aspirates were collected from 33 pediatric ARDS patients and 21 non-ARDS controls at 48 hours of intubation, and serially for those who remained intubated greater than five days. MMPs, tissue inhibitor of metalloproteinases (TIMPs), human neutrophil elastase (HNE) and myeloperoxidase (MPO) activity were measured by ELISA, and correlated with clinical indicators of disease severity such as PRISM (Pediatric Risk of Mortality) scores, oxygen index (OI), multi-organ system failure (MOSF) and clinical outcome measures including length of intubation, ventilator-free days (VFDs) and mortality in the Pediatric Intensive Care Unit (PICU).
Active MMP-9 was elevated early in pediatric ARDS subjects compared to non-ARDS controls. Higher MMP-8 and active MMP-9 levels at 48 hours correlated with a longer course of mechanical ventilation (r = 0.41, p = 0.018 and r = 0.75, p<0.001; respectively) and fewer number of VFDs (r = −0.43, p = 0.013 and r = −0.76, p<0.001; respectively), independent of age, gender and severity of illness. Patients with the highest number of ventilator days had the highest levels of active MMP-9. MMP-9 and to a lesser extent MMP-8 activities in tracheal aspirates from ARDS subjects were sensitive to blockade by small molecule inhibitors.
Higher MMP-8 and active MMP-9 levels at 48 hours of disease onset are associated with a longer duration of mechanical ventilation and fewer ventilator-free days among pediatric patients with ARDS. Together, these results identify early biomarkers predictive of disease course and potential therapeutic targets for this life threatening disease.
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.
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.
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.
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.
Prolyl endopeptidase (PE), a protease that cleaves after proline residues in oligopeptides, is highly active in brain and degrades neuropeptides in vitro. We have recently demonstrated that PE, in concert with MMP's, can generate PGP (prolineglycine-proline), a novel, neutrophil chemoattractant, from collagen. In this study, we demonstrate that human peripheral blood neutrophils contain PE, which is constitutively active, and can generate PGP de novo from collagen after activation with LPS. This novel, pro-inflammatory role for PE raises the possibility of a self-sustaining pathway of neutrophilic inflammation and may provide biomarkers and therapeutic targets for diseases caused by chronic, neutrophilic inflammation.
neutrophil; prolyl endopeptidase; PGP; collagen; inflammation
Cystic fibrosis (CF) is a lethal genetic disorder characterized by airway remodeling and inflammation, leading to premature death. Recent evidence suggests the importance of protease activity in CF pathogenesis. One prominent protease, matrix metalloprotease (MMP)-9, demonstrates increased activity in CF individuals undergoing acute pulmonary exacerbation. This is thought to be mediated by both direct MMP-9 activation and the degradation of its natural inhibitor, tissue inhibitor of metalloprotease-1 (TIMP-1). To examine if this relationship exists in nonexacerbating CF individuals, we examined protease activity in sputum from these individuals compared with nondisease controls. We demonstrated increased gelatinolytic activity in CF sputum. These samples had elevated human neutrophil elastase (HNE) levels which correlated with an increased MMP-9/TIMP-1 ratio. To determine if HNE could discretely cleave and activate MMP-9, these enzymes were coincubated and two specific cleavage sites, between Valine38 and Alanine39, and between Alanine 39 and glutamic acid40 were observed. These sites corresponded with appropriate molecular weight for the activated MMP-9 isoform in CF sputum. Using N-terminal sequencing of cleavage fragments obtained with TIMP-1 incubation with HNE, we confirmed the TIMP-1 cleavage site for HNE is at Valine69-Cysteine70. We also show for the first time that human neutrophils were capable of degrading TIMP-1 ex vivo and that a 16 kDa TIMP-1 fragment was identified in CF sputum, consistent with the expected cleavage of TIMP-1 by HNE. These results demonstrate increased MMP-9 activity in stable CF lung disease, and the presence of specific protease products in CF sputum highlights that HNE-mediated activity plays a role in this dysregulation.
Cystic fibrosis (CF) is chronic lung disease characterized by an unrelenting neutrophil-predominant airway inflammatory response. This inflammation leads to extracellular matrix (ECM) remodeling and eventually to the development of bronchiectasis. While many components of the immune response in CF have been well-characterized, recent data suggests that small molecules may play an important and underappreciated role in this inflammation. This review will examine two novel molecules: proline-glycine-proline (PGP) and high mobility group box protein-1 (HMGB1), and their potential impact in CF lung disease. This review will provide a brief overview of CF lung disease and background on both HMGB1 and PGP. It will then focus on these molecules in a murine model of CF-like airway disease and in human biological specimens from CF individuals. Finally, this manuscript will address possible mechanisms for therapeutic targeting of these bioactive mediators.
Inflammation; cystic fibrosis; neutrophils; airway; therapeutics.
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 β-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.
cystic fibrosis; HMGB1; inflammation; collagen;; fragmentation; proline-glycine-proline
Summary of recent advances
Chemoattractant properties of matrix proteins, like collagen and elastin, for neutophils and monocytes in vitro have long been recognized. This activity often resides in fragments of these proteins. These peptides may play a role in diseases of the lung matrix, such as chronic obstructive pulmonary disease. Recent advances include the elucidation of the structure of chemotactic collagen fragments and the demonstration that their activity may reside in a structural relatedness to CXC chemokines. Collagen and elastin fragments have been demonstrated to have a role in in vivo lung pathophysiology and have been quantified in patients with chronic lung diseases where they may activate autoimmune pathways. Elucidation of these pathways may provide novel biomarkers and therapeutic targets for chronic lung diseases.
Chronic 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.
Induced 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.
N-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 (MMP's) 1 and 9 and prolyl endopeptidase. MMP's 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 controls
N-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.
Pediatric Acute Lung Injury (ALI) is associated with a high mortality and morbidity, and dysregulation of matrix metalloproteinases (MMPs) may play an important role in the pathogenesis and evolution of ALI. Here we examined MMP expression and activity in pediatric ALI compared with controls. MMP-8, -9, and to a lesser extent, MMP-2, -3, -11 and -12 were identified at higher levels in lung secretions of pediatric ALI patients compared with controls. Tissue Inhibitor of Matrix metalloproteinase-1 (TIMP-1), a natural inhibitor of MMPs was detected in most ALI samples, but MMP-9:TIMP-1 ratios were high relative to controls. In subjects who remained intubated for ≥10 days, MMP-9 activity decreased, with > 80% found in the latent form. In contrast, almost all MMP-8 detected at later disease course was constitutively active. Discriminating MMP-9:TIMP-1 ratios were found in those who had a prolonged ALI course. These results identify a specific repertoire of MMP isoforms in the lung secretions of pediatric ALI patients, and demonstrate inverse changes in MMPs -8 and -9 with protracted disease.
matrix metalloproteinases; acute lung injury; pediatric; viral infection